Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmet...Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.展开更多
Using the reference sequences of pgip genes in GenBank,a fragment of 930 bp covering the open reading frame(ORF) of rice Ospgip1(Oryza sativa polygalacturonase-inhibiting protein 1) was amplified.The prokaryotic expre...Using the reference sequences of pgip genes in GenBank,a fragment of 930 bp covering the open reading frame(ORF) of rice Ospgip1(Oryza sativa polygalacturonase-inhibiting protein 1) was amplified.The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani,the causal agent of rice sheath blight,and reduced its polygalacturonase activity.Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point(pI) of 7.26.The protein is mainly located in the cell wall of rice,and its signal peptide cleavage site is located between the 17th and 18th amino acids.There are four cysteines in both the N-and C-termini of the deduced protein,which can form three disulfide bonds(between the 56th and 63rd,the 278th and 298th,and the 300th and 308th amino acids).The protein has a typical leucine-rich repeat(LRR) domain,and its secondary structure comprises α-helices,β-sheets and irregular coils.Compared with polygalacturonase-inhibiting proteins(PGIPs) from other plants,the 7th LRR is absent in OsPGIP1.The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi,such as polygalacturonase.展开更多
[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the ...[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.展开更多
BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have bee...BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera’s placenta tissue by the reverse transcriptase-polymerase chain reaction （RT-PCR） method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 （DE3） by the CaCl<sub>2</sub> transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane （CAM）. RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 （DE3）. SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.展开更多
Izumo1 is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumo1 was fractionated...Izumo1 is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumo1 was fractionated into 6 fragments (F0-F5) which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumo1, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumo1 ascetic polyclonal antibody with intraperitoneal injection of S180 cells. Subsequently, the anti-GST-Izumo1 polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumo1 fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumo1 proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumo1 in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumo1 to the egg surface.展开更多
Lipoxygenase (LOX, EC1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the...Lipoxygenase (LOX, EC1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+) and transformed into Escherichia coli BL21 (DE3). Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and the purified recombinant protein was obtained by His·Bind? Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer) and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.展开更多
The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin(CATH) PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH...The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin(CATH) PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low(3195.88 and 2838.34 Da, respectively). Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid p ET-32 a. This reconstructed plasmid was then transfected into the expression vector E. coliBL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The antimicrobial activity assay using Staphylococcus aureus(Song) and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.展开更多
To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was a...To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E.coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.展开更多
AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to expl...AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8％ of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.展开更多
The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E.coli.A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene s...The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E.coli.A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank.The DNA fragment of 918 bp was amplified by polymerase chain reaction(PCR)from the pNA992 recombinant plasmid with Nanog gene,then cloned into pGEX-KG and transformed into the host E.coli strain TGⅠ.The sequence of the fragment was matched with the original sequence of pNA992.It indicated that fusion expression vector,pGEX-KG- Nanog,was constructed successfully.The pGEX-KG-Nanog plasmid was extracted from E.coli strain TGⅠand was transformed into BL21(DE3)for expression.After induction by isopropy1-β-D-thiogalactoside(IPTG)at 37°C,the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and the expression condition was optimized.Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa.Meanwhile,the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L-1 IPTG for 5 h.The mouse Nanog gene was successfully expressed in E.coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.展开更多
[Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [Method] The complete ORF of glutamate dehydrogenase (GDH) gene was amplified from the genomic DNA of Streptococcus suis serotype ...[Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [Method] The complete ORF of glutamate dehydrogenase (GDH) gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by polymerase chain reaction (PCR). The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coli BL21 (DE3). The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot assay, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could react specifically with serum samples collected from five pigs experimentally infected by strain SC22. [Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.展开更多
mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression...mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression of mCLCA3 in asthmatic mouse lung,an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E.coli,purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated.Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen.Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3.The results demonstrate that the 90 kDa N-terminal peptide,neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.展开更多
In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) eff...In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli(E.coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E.coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5 % homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E.coli.展开更多
Objective: To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fc (hp75NTR-Fc) in prokaryotic expression system, and investigate the effect of the recombinant prot...Objective: To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fc (hp75NTR-Fc) in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia (DRG) neuron neurites. Methods: The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction (PCR) and subcloned into vector pET30a (+), in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results: The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a (+). SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein (MAG). Conclusion: The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a (+) correctly and expressed successfully in the prokaryotic expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.展开更多
Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to ...Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.展开更多
Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely...Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.展开更多
FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investig...FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.展开更多
WT5”HZ]The recombinant expression vector pGEMD fhit which contains full encoding region of fhit gene was constructed. The recombinant was introduced into the BL21(DE3) strain of E.coli and induced by 1 mmol/L IPTG to...WT5”HZ]The recombinant expression vector pGEMD fhit which contains full encoding region of fhit gene was constructed. The recombinant was introduced into the BL21(DE3) strain of E.coli and induced by 1 mmol/L IPTG to express a 29×10 3 polypeptide of fhit fusion protein. And the 29×10 3 protein was sensitive and specific in reaction with anti fhit antibody in Western blot.展开更多
Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the p...Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the protein utilizing methods of bioinformatics.Methods:fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR.The fbpB-esxA fusing gene Iigated by（Gly<sub>4</sub>Ser）<sub>3</sub> linker was gained by means of Gene Splicing by Overlapping Extension PCU（SOEPCR）, and fusing gene was cloned into expression vector pET-30a.The recombinant plasmid was sequenced and expressed in E.coli BL21（DE3）.The protein was identified by Western blot using anti-HIS antibody.Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.Results:The UNA sequences fbpB-esxA were identical with that published by GenBank.The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E.coli B1.21（DE3） under the induction of IPTG.Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.Conclusions:The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag.Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly.The existence of linker doesn’t affect immunogenicity of Ag85B and ESAT-6.It will allow lor characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.展开更多
文摘Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.
基金supported by the Programs of Natural Science Foundation of Jiangsu Province,China(Grant Nos.BK2008223and BK2010305)Transgenic Major Special Funds in China(Grant No.2009ZX08001-014B)
文摘Using the reference sequences of pgip genes in GenBank,a fragment of 930 bp covering the open reading frame(ORF) of rice Ospgip1(Oryza sativa polygalacturonase-inhibiting protein 1) was amplified.The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani,the causal agent of rice sheath blight,and reduced its polygalacturonase activity.Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point(pI) of 7.26.The protein is mainly located in the cell wall of rice,and its signal peptide cleavage site is located between the 17th and 18th amino acids.There are four cysteines in both the N-and C-termini of the deduced protein,which can form three disulfide bonds(between the 56th and 63rd,the 278th and 298th,and the 300th and 308th amino acids).The protein has a typical leucine-rich repeat(LRR) domain,and its secondary structure comprises α-helices,β-sheets and irregular coils.Compared with polygalacturonase-inhibiting proteins(PGIPs) from other plants,the 7th LRR is absent in OsPGIP1.The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi,such as polygalacturonase.
基金Supported by Project of Science and Technology Development of Jilin Province(20140204018YY)
文摘[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.
基金This work was supported by a grant from Science and Technology Fund of Shanxi Province, China (No. 042082).
文摘BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera’s placenta tissue by the reverse transcriptase-polymerase chain reaction （RT-PCR） method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 （DE3） by the CaCl<sub>2</sub> transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane （CAM）. RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 （DE3）. SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.
基金supported by the Natural Science Foun-dation of Inner Mongolia Autonomous Region, China(20080404MS0505)the National Training Foun-dation for Talents of Basic Sciences, China (J0730648)
文摘Izumo1 is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumo1 was fractionated into 6 fragments (F0-F5) which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumo1, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumo1 ascetic polyclonal antibody with intraperitoneal injection of S180 cells. Subsequently, the anti-GST-Izumo1 polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumo1 fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumo1 proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumo1 in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumo1 to the egg surface.
基金grants from the National Basic Research Program of China (Grant No. 2004CB2117204)the National High-tech Research and Development Program of China (Grant No. 2006AA100101)+1 种基金the National Program of Science Technology and Tackle Key Problem of Chinathe Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) of China
文摘Lipoxygenase (LOX, EC1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+) and transformed into Escherichia coli BL21 (DE3). Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and the purified recombinant protein was obtained by His·Bind? Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer) and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.
基金supported by the Industry-University-Research Project of Application of the Active Substances from Amphibian Skin from the Education Ministry of Guizhou (Q. J. HE and K. Y. ZHI [2013]121)
文摘The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin(CATH) PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low(3195.88 and 2838.34 Da, respectively). Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid p ET-32 a. This reconstructed plasmid was then transfected into the expression vector E. coliBL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The antimicrobial activity assay using Staphylococcus aureus(Song) and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.
基金This project was supported by grants from the National Natural Science Foundation of China ( No.30271242,30371396).
文摘To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E.coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.
文摘AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8％ of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.
文摘The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E.coli.A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank.The DNA fragment of 918 bp was amplified by polymerase chain reaction(PCR)from the pNA992 recombinant plasmid with Nanog gene,then cloned into pGEX-KG and transformed into the host E.coli strain TGⅠ.The sequence of the fragment was matched with the original sequence of pNA992.It indicated that fusion expression vector,pGEX-KG- Nanog,was constructed successfully.The pGEX-KG-Nanog plasmid was extracted from E.coli strain TGⅠand was transformed into BL21(DE3)for expression.After induction by isopropy1-β-D-thiogalactoside(IPTG)at 37°C,the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and the expression condition was optimized.Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa.Meanwhile,the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L-1 IPTG for 5 h.The mouse Nanog gene was successfully expressed in E.coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.
基金supported by the grants of the Independent Innovation Fund of Shandong Binzhou Animal Science & Veterinary Medicine Academy (200802)
文摘[Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [Method] The complete ORF of glutamate dehydrogenase (GDH) gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by polymerase chain reaction (PCR). The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coli BL21 (DE3). The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot assay, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could react specifically with serum samples collected from five pigs experimentally infected by strain SC22. [Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.
基金Supported by the Distinguished Young Scholars Fund of China(No.30325011)the National Natural Science Foundation of China(Nos.30470405and30670477)+1 种基金Distinguished Young Scholars Fund of Jilin Province,China(No.20030112)Excellent Young Teachers Program of MOE,China.
文摘mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression of mCLCA3 in asthmatic mouse lung,an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E.coli,purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated.Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen.Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3.The results demonstrate that the 90 kDa N-terminal peptide,neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.
文摘In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli(E.coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E.coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5 % homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E.coli.
基金Supported by the National Natural Science Foundation of China (30600665)the Natural Science Foundation Project of CQ CSTC (CSTC, 2008BB5107)+1 种基金the Youth Scientific Research Foundation of Third Military Medical University (06XG048)the Open Project Program of the State Key Laboratory of Trauma, Burns and Combined Injury (2006A-3)
文摘Objective: To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fc (hp75NTR-Fc) in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia (DRG) neuron neurites. Methods: The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction (PCR) and subcloned into vector pET30a (+), in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results: The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a (+). SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein (MAG). Conclusion: The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a (+) correctly and expressed successfully in the prokaryotic expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.
基金Supported by the National Natural Science Foundation of China(31172295,31272569)。
文摘Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.
文摘Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.
文摘FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.
基金Supported by the National Natural Science Foundation of China!(39770373)
文摘WT5”HZ]The recombinant expression vector pGEMD fhit which contains full encoding region of fhit gene was constructed. The recombinant was introduced into the BL21(DE3) strain of E.coli and induced by 1 mmol/L IPTG to express a 29×10 3 polypeptide of fhit fusion protein. And the 29×10 3 protein was sensitive and specific in reaction with anti fhit antibody in Western blot.
基金Supported by Research Program of The Health Department of Hainan Province(No.2007-44)Research Cultivation Program of Hainan Medical University(HY2010-006)+1 种基金Research Program in higher educational institutes of The Education Department of Hainan Province(No.Hj2010-21)Natural Science Fund of Hainan Province(No.2008~30837)
文摘Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the protein utilizing methods of bioinformatics.Methods:fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR.The fbpB-esxA fusing gene Iigated by（Gly<sub>4</sub>Ser）<sub>3</sub> linker was gained by means of Gene Splicing by Overlapping Extension PCU（SOEPCR）, and fusing gene was cloned into expression vector pET-30a.The recombinant plasmid was sequenced and expressed in E.coli BL21（DE3）.The protein was identified by Western blot using anti-HIS antibody.Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.Results:The UNA sequences fbpB-esxA were identical with that published by GenBank.The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E.coli B1.21（DE3） under the induction of IPTG.Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.Conclusions:The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag.Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly.The existence of linker doesn’t affect immunogenicity of Ag85B and ESAT-6.It will allow lor characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.
