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
In this paper,we studied the structure,expression and function of black carp insulin gene.The complete Mylopharyngodon piceus insulin(Mp-Ins)gene is 1,965 bp long and includes a 1,499 bp 5ʹ-untranslated region(UTR),a ...In this paper,we studied the structure,expression and function of black carp insulin gene.The complete Mylopharyngodon piceus insulin(Mp-Ins)gene is 1,965 bp long and includes a 1,499 bp 5ʹ-untranslated region(UTR),a 139 bp 3′-UTR with a poly(A)tail,and an open reading frame(ORF)of 327 bp.The predicted molecular weight of the recombinant Mp-Ins(rMp-Ins)protein is 11.87 kDa.The mRNA expression of Mp-Ins is upregulated in the brain and liver.After the injection of rMp-Ins,Mp-Ins mRNA transcript abundance was significantly upregulated in the liver.The rMp-Ins protein could inhibit the concentration of glycogen phosphorylase(GP),growth hormone(GH),fatty acid synthase(FAS),and insulin-like growth factors-1(IGF-1),and it also significantly increased the concentration of PI3K.Additionally,the injection of rMp-Ins did not have a significant impact on the glucose-6-phosphatase(G6Pase)content in blood.In situ hybridization results showed that the positive signal of the Mp-Ins gene was mainly concentrated in the cell nucleus of brain tissue and the cell membrane of liver tissue and muscle tissue.Together,these results demonstrated that Mp-Ins plays an important role in growth and metabolism in M.piceus.展开更多
Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein ge...Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.展开更多
Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at lo...Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.展开更多
AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcript...AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry in CRC and colorectal normal tissues.PDGFRL prokaryotic expression vector was carried out in Escherichia coli(E.coli),and purified by immobilized metal affinity chromatography.The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT),clone counting,cell cycle,and wound healing assay.RESULTS:Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues.Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E.coli,and the target protein was expressed in the form of inclusion bodies.After purification and refolding,recombinant human PDGFRL(rhPDGFRL)could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT,clone counting and wound healing assay.Moreover,rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase.CONCLUSION:PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.展开更多
Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed...Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene(zein) from maize endosperm protein with lysine-rich gene(Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector p HT43, and the recombinant plasmid p HT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.展开更多
The NAC(NAM,ATAF,and CUC)superfamily is one of the largest plant-specific families containing transcription factors.An increasing number of studies suggest that NAC1 is involved in plants response to different biotic ...The NAC(NAM,ATAF,and CUC)superfamily is one of the largest plant-specific families containing transcription factors.An increasing number of studies suggest that NAC1 is involved in plants response to different biotic and abiotic stimulis.Nicotiana benthamiana is a widely used system for evaluating plant-pathogen interactions.In order to study the biochemical function of NbNAC1,NbNAC1 protein and antibody are essential.Therefore,we focused on developing a prokaryotic expression system for producing the Nicotiana benthamiana NbNAC1 protein of in Escherichia coli and the preparation of its polyclonal antibody.Firstly,we constructed two different molecular weight prokaryotic expression vectors:pGE vector with GST tag(pGEX4T-1–NbNAC1)and pET expression vector with His tag(pET28a-NbNAC1).The NbNAC1 protein can be successfully expressed in both vectors.The His-tagged NbNAC1 proteins are insoluble,while the GST-tagged NbNAC1 proteins are partially soluble.We then successfully purified and enriched both proteins.The His-tagged NbNAC1 was chosen to immunize rabbits owing to an unknown protein accompanying the GST-tagged NbNAC1.The anti-NbNAC1 polyclonal antibody had good specificity and could be used in subsequent protein-related studies.展开更多
The outer membrane protein, omp A, of Aeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, omp AⅠ of Aeromonas veronii strain WA106 was cl...The outer membrane protein, omp A, of Aeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, omp AⅠ of Aeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of omp AⅠ gene was 1 023 base pairs(Gen Bank Accession NO.KC748024), which showed 100% homology with that of A. veronii(NO.AB290200.1). This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that omp AⅠ protein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of omp AⅠ protein.展开更多
Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ templa...Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220,and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3'terminal of the signal and leader peptides of NT4,and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.展开更多
The open reading frame(ORF)of hAPOA1 was inserted into the prokaryotic expression vector pGEX-4T-1to construct the recombinant plasmid pGEX-4T-1-hAPOA1,which was then transformed into Escherichiacolistrain BL21.The ex...The open reading frame(ORF)of hAPOA1 was inserted into the prokaryotic expression vector pGEX-4T-1to construct the recombinant plasmid pGEX-4T-1-hAPOA1,which was then transformed into Escherichiacolistrain BL21.The expression of target fusion protein was induced with isopropylβ-D-1-thiogalactopyranoside(IPTG).The purified fusion protein in inclusion bodies was used to immunize New Zealand white rabbits to prepare hAPOA1 antiserum and the antibody titer was detected with indirect enzyme-linked immunosorbent assay(ID-ELISA).ID-ELISA and Western Blot proved that rabbit polyclonal antibody with a high titer of 1∶40 000 was produced,which may bring considerable economic benefits.展开更多
[Objectives]This study was conducted to construct a pColdI-HSP70 recombinant prokaryotic expression vector.[Methods]With rice‘N22’as the test variety,hydroponic experiments were set up for rice seedlings with normal...[Objectives]This study was conducted to construct a pColdI-HSP70 recombinant prokaryotic expression vector.[Methods]With rice‘N22’as the test variety,hydroponic experiments were set up for rice seedlings with normal growth(CK),high temperature treatment(H),drought treatment(D)and drought-high temperature cross treatment(DH).The recombinant prokaryotic expression vector was constructed by the method of prokaryotic expression,and its induction expression time,IPTG concentration and temperature were optimized.[Results]The pColdI-HSP70 expression vector was successfully constructed,and the fusion protein was highly expressed in host strain BL21,and the expressed proteins were all in a soluble form.By optimizing the induction expression conditions,it was found that the optimal expression conditions were the IPTG concentration of 0.5 mmol/L and induction at 20℃for 36 h.The expression analysis of the rice HSP70 gene under different stress treatments was carried out by qRT-PCR technology,and it was found that H,D and DH stresses all could induce its expression,and its expression levels were 4.65,1.40 and 17.66 times higher than that of the CK group,respectively.[Conclusions]This study lays a solid foundation for the isolation,purification and functional study of rice HSP70 proteins.展开更多
ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(...ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.展开更多
A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicte...A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.展开更多
Using MEDDF cDNA fragment in plasmid pBS-SK-MEDDF as template the coding sequence was cloned into pGEM-T-Easy plasmid by PCR method to delete non-coding sequence. After DNA sequencing it was confirmed that the clone s...Using MEDDF cDNA fragment in plasmid pBS-SK-MEDDF as template the coding sequence was cloned into pGEM-T-Easy plasmid by PCR method to delete non-coding sequence. After DNA sequencing it was confirmed that the clone sequence was correct, the coding region then was inserted into the vector pET-30a between BamH I and Hind III to construct eukaryotic expression vector. It was found that the specific protein was up to 40% of total bactorial proteins in certain high-expression E. coli. High titer of anti-sera was detected by inoculating New Zealand rabbits with purified MEDDF protein as an antigen. By using immunocytochemical staining it was demonstrated that the expression of MEDDF was exhibited in a developmental stage-specific manner, suggesting that MEDDF may play a certain role in the initiation of murine erythroid terminal differentiation and nuclear condensation. As for the expression of MEDDF appearing in granulocytes and megakaryocyter in murine bone marrow, it may indicate that there is an original relationship between the proteins and differentiation of murine myelogenous lineage.展开更多
Uridine diphosphate(UDP)-glycosyltransferases(UGTS)are widely distributed within living organisms and share roles in biotransformation of various lipophilic endo-and xenobiotics with activated UDP sugars.In this study...Uridine diphosphate(UDP)-glycosyltransferases(UGTS)are widely distributed within living organisms and share roles in biotransformation of various lipophilic endo-and xenobiotics with activated UDP sugars.In this study,it was found that the activity of UGTs in abamectin-resistant(AbR)strain was significantly higher(2.3-fod)than that in susceptible strain(SS)of Tetramychus cinnabarinus.Further analysis showed that 5-nitrouracil,the inhibitor ofUGTs,could enhance the lethal effect of abamectin on mites.From the previous microarray results,we found an UGT gene(UGT201D3)overexpressed in AbR strain.Quantitative PCR analysis showed that UGT20ID3 was highly expressed and more inducible with abamectin exposure in the AbR strain.After silencing the tran-scription of UGT201D3,the activity of UGTs was decreased and the susceptibility to abamectin was increased in AbR strain whereas it was not in sS.Furthermore,UGT201D3 gene was then succefully expressed in Escherichia coli.The recombinant UGT20ID3 exhibited a-naphthol activity(2.810.43 nmol mg protein/min),and the enzyme activity could be inhibited by abamectin(inhibitory concentration at 50%:57.50±3.54 μmol/L).High-performance liquid chromatography analysis demonstrated that the recombinant UGT201D3 could efctively deplete abamectin(15.77%±3.72%)incubating with 150 ug protein for 6 h.These results provided direct evidence that UGT201D3 was involved in abamectin resistance in T cinnabarinus.展开更多
Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sR...Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.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 research was supported by China’s Agricultural Research System(CARS-45-03).
文摘In this paper,we studied the structure,expression and function of black carp insulin gene.The complete Mylopharyngodon piceus insulin(Mp-Ins)gene is 1,965 bp long and includes a 1,499 bp 5ʹ-untranslated region(UTR),a 139 bp 3′-UTR with a poly(A)tail,and an open reading frame(ORF)of 327 bp.The predicted molecular weight of the recombinant Mp-Ins(rMp-Ins)protein is 11.87 kDa.The mRNA expression of Mp-Ins is upregulated in the brain and liver.After the injection of rMp-Ins,Mp-Ins mRNA transcript abundance was significantly upregulated in the liver.The rMp-Ins protein could inhibit the concentration of glycogen phosphorylase(GP),growth hormone(GH),fatty acid synthase(FAS),and insulin-like growth factors-1(IGF-1),and it also significantly increased the concentration of PI3K.Additionally,the injection of rMp-Ins did not have a significant impact on the glucose-6-phosphatase(G6Pase)content in blood.In situ hybridization results showed that the positive signal of the Mp-Ins gene was mainly concentrated in the cell nucleus of brain tissue and the cell membrane of liver tissue and muscle tissue.Together,these results demonstrated that Mp-Ins plays an important role in growth and metabolism in M.piceus.
文摘Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.
基金This work was supported by the National Science Foundation of China(31572220,31272633,31201760)the National High Technology Research and Development Program of China(863 Program)(2011AA100403)the Shanghai University Knowledge Service Platform(ZF1206).
文摘Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.
基金Supported by The National Natural Science Foundation of China,No.30672352
文摘AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry in CRC and colorectal normal tissues.PDGFRL prokaryotic expression vector was carried out in Escherichia coli(E.coli),and purified by immobilized metal affinity chromatography.The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT),clone counting,cell cycle,and wound healing assay.RESULTS:Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues.Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E.coli,and the target protein was expressed in the form of inclusion bodies.After purification and refolding,recombinant human PDGFRL(rhPDGFRL)could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT,clone counting and wound healing assay.Moreover,rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase.CONCLUSION:PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.
基金Supported by the Funding of High Technology Project of Ministry of Science and Technology of China(863 Project,2013AA102504-03)
文摘Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene(zein) from maize endosperm protein with lysine-rich gene(Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector p HT43, and the recombinant plasmid p HT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.
基金This work was supported by the National Natural Science Foundation of China(31500209)Natural Science Foundation of Jiangsu Province of China,(BK20201431)+2 种基金Agricultural science and technology independent innovation Foundation of Jiangsu Province of China(CX(20)3128)Open Project Program of Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China,Yangzhou University(JILARKF202006)Qing Lan Project of Yangzhou University and Yangzhou University of“High-end Talent Support Program”.
文摘The NAC(NAM,ATAF,and CUC)superfamily is one of the largest plant-specific families containing transcription factors.An increasing number of studies suggest that NAC1 is involved in plants response to different biotic and abiotic stimulis.Nicotiana benthamiana is a widely used system for evaluating plant-pathogen interactions.In order to study the biochemical function of NbNAC1,NbNAC1 protein and antibody are essential.Therefore,we focused on developing a prokaryotic expression system for producing the Nicotiana benthamiana NbNAC1 protein of in Escherichia coli and the preparation of its polyclonal antibody.Firstly,we constructed two different molecular weight prokaryotic expression vectors:pGE vector with GST tag(pGEX4T-1–NbNAC1)and pET expression vector with His tag(pET28a-NbNAC1).The NbNAC1 protein can be successfully expressed in both vectors.The His-tagged NbNAC1 proteins are insoluble,while the GST-tagged NbNAC1 proteins are partially soluble.We then successfully purified and enriched both proteins.The His-tagged NbNAC1 was chosen to immunize rabbits owing to an unknown protein accompanying the GST-tagged NbNAC1.The anti-NbNAC1 polyclonal antibody had good specificity and could be used in subsequent protein-related studies.
基金Supported by the Science&Technology Department of Sichuan Province(2013FZ0014)the Construction Project of Postgraduate Academic Degree in Southwest University for Nationalities(2015XWD-S071007)
文摘The outer membrane protein, omp A, of Aeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, omp AⅠ of Aeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of omp AⅠ gene was 1 023 base pairs(Gen Bank Accession NO.KC748024), which showed 100% homology with that of A. veronii(NO.AB290200.1). This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that omp AⅠ protein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of omp AⅠ protein.
基金supported by the National Natural Science Foundation of China (No.30471877)
文摘Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220,and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3'terminal of the signal and leader peptides of NT4,and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.
基金Supported by the Agricultural Science Independent Innovation Fund Jiangsu Province[CX(16)1326]
文摘The open reading frame(ORF)of hAPOA1 was inserted into the prokaryotic expression vector pGEX-4T-1to construct the recombinant plasmid pGEX-4T-1-hAPOA1,which was then transformed into Escherichiacolistrain BL21.The expression of target fusion protein was induced with isopropylβ-D-1-thiogalactopyranoside(IPTG).The purified fusion protein in inclusion bodies was used to immunize New Zealand white rabbits to prepare hAPOA1 antiserum and the antibody titer was detected with indirect enzyme-linked immunosorbent assay(ID-ELISA).ID-ELISA and Western Blot proved that rabbit polyclonal antibody with a high titer of 1∶40 000 was produced,which may bring considerable economic benefits.
基金Supported by Anhui Provincial Education Department Project(KJ2019A0213)National Natural Science Foundation of China(31501245)Undergraduate Innovation and Enterpreneurship Training Program of Anhui Province(S202010364185,XJDC2020541,202110364788)。
文摘[Objectives]This study was conducted to construct a pColdI-HSP70 recombinant prokaryotic expression vector.[Methods]With rice‘N22’as the test variety,hydroponic experiments were set up for rice seedlings with normal growth(CK),high temperature treatment(H),drought treatment(D)and drought-high temperature cross treatment(DH).The recombinant prokaryotic expression vector was constructed by the method of prokaryotic expression,and its induction expression time,IPTG concentration and temperature were optimized.[Results]The pColdI-HSP70 expression vector was successfully constructed,and the fusion protein was highly expressed in host strain BL21,and the expressed proteins were all in a soluble form.By optimizing the induction expression conditions,it was found that the optimal expression conditions were the IPTG concentration of 0.5 mmol/L and induction at 20℃for 36 h.The expression analysis of the rice HSP70 gene under different stress treatments was carried out by qRT-PCR technology,and it was found that H,D and DH stresses all could induce its expression,and its expression levels were 4.65,1.40 and 17.66 times higher than that of the CK group,respectively.[Conclusions]This study lays a solid foundation for the isolation,purification and functional study of rice HSP70 proteins.
基金supported by National Natural Science Foundation of China(31500247)Key Project of Department of Education of Sichuan Province(14ZA0016)Natural Science Foundation of Zhejiang Province(LZ15C150001)
文摘ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.
基金supported by the National Science Foundation for Distinguished Young Scholars of China(Grant No.31325024)Innovation Team Project of Shandong Modern Agricultural Industry Technology System(SDAIT-03-022-03)
文摘A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.
文摘Using MEDDF cDNA fragment in plasmid pBS-SK-MEDDF as template the coding sequence was cloned into pGEM-T-Easy plasmid by PCR method to delete non-coding sequence. After DNA sequencing it was confirmed that the clone sequence was correct, the coding region then was inserted into the vector pET-30a between BamH I and Hind III to construct eukaryotic expression vector. It was found that the specific protein was up to 40% of total bactorial proteins in certain high-expression E. coli. High titer of anti-sera was detected by inoculating New Zealand rabbits with purified MEDDF protein as an antigen. By using immunocytochemical staining it was demonstrated that the expression of MEDDF was exhibited in a developmental stage-specific manner, suggesting that MEDDF may play a certain role in the initiation of murine erythroid terminal differentiation and nuclear condensation. As for the expression of MEDDF appearing in granulocytes and megakaryocyter in murine bone marrow, it may indicate that there is an original relationship between the proteins and differentiation of murine myelogenous lineage.
基金by Chongqing Research Program of Basic Research and Frontier Technology(No.cstc2017jcyjBX0061)the National Natural Science Foundation of China(31672085).
文摘Uridine diphosphate(UDP)-glycosyltransferases(UGTS)are widely distributed within living organisms and share roles in biotransformation of various lipophilic endo-and xenobiotics with activated UDP sugars.In this study,it was found that the activity of UGTs in abamectin-resistant(AbR)strain was significantly higher(2.3-fod)than that in susceptible strain(SS)of Tetramychus cinnabarinus.Further analysis showed that 5-nitrouracil,the inhibitor ofUGTs,could enhance the lethal effect of abamectin on mites.From the previous microarray results,we found an UGT gene(UGT201D3)overexpressed in AbR strain.Quantitative PCR analysis showed that UGT20ID3 was highly expressed and more inducible with abamectin exposure in the AbR strain.After silencing the tran-scription of UGT201D3,the activity of UGTs was decreased and the susceptibility to abamectin was increased in AbR strain whereas it was not in sS.Furthermore,UGT201D3 gene was then succefully expressed in Escherichia coli.The recombinant UGT20ID3 exhibited a-naphthol activity(2.810.43 nmol mg protein/min),and the enzyme activity could be inhibited by abamectin(inhibitory concentration at 50%:57.50±3.54 μmol/L).High-performance liquid chromatography analysis demonstrated that the recombinant UGT201D3 could efctively deplete abamectin(15.77%±3.72%)incubating with 150 ug protein for 6 h.These results provided direct evidence that UGT201D3 was involved in abamectin resistance in T cinnabarinus.
基金The author would like to acknowledge the National Science Fund for Distinguished Young Scholars(21425624)the National Natural Science Foundation of China(21476026,21376028).
文摘Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.
