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Prokaryotic Expression of IBV N Protein and Development of Indirect IBV N Protein-mediated ELISA
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作者 Li Wei-qun Wang Xin +6 位作者 Zhong Ming Sun Xiao-qi Zhao Lei Huang Xiao-dan Zhang Rui-li Li Guang-xing 《Journal of Northeast Agricultural University(English Edition)》 CAS 2020年第1期69-79,共11页
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
关键词 InFECTIOUS BROnCHITIS virus n protein prokaryotic expression indirect ELISA
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Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti 被引量:2
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作者 李文学 李海峰 金清洙 《Agricultural Science & Technology》 CAS 2010年第5期96-100,共5页
[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surf... [Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti. 展开更多
关键词 Theileria sergenti P23 major surface protein gene prokaryotic expression
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Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli
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作者 廖芳 宋启发 万沐芬 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2004年第5期417-420,共4页
In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot... In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed. 展开更多
关键词 neisseria gonorrhoeae porin B porin A prokaryotic expression plasmid recombinant fusion protein
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Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus
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作者 袁野 王秀利 +1 位作者 郭设平 仇雪梅 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2011年第5期952-957,共6页
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer m... Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies. 展开更多
关键词 vibrio parahaemolyticus outer membrane protein (OmpW) CLOnInG prokaryotic expression protein characterization recombinant proteins
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Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation
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作者 Liu Xueguang Zheng Huaidong +3 位作者 Guo Xinshuo Luo Jin Lin Cuicui Wang Qiuyu 《Animal Husbandry and Feed Science》 CAS 2014年第2期55-58,共4页
[Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoie... [Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip. 展开更多
关键词 Infectious hematopoietic necrosis virus(IHnV) prokaryotic expression Inclusion body G protein AnTIBODY
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Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr. 被引量:1
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作者 黄琼林 何瑞 +1 位作者 詹若挺 陈蔚文 《Agricultural Science & Technology》 CAS 2012年第6期1211-1214,共4页
[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein (EBP1), which plays important roles in regulating plant organ size from Nervilia fordii (Hance) Schltr. [Methods] PCR produ... [Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein (EBP1), which plays important roles in regulating plant organ size from Nervilia fordii (Hance) Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function. 展开更多
关键词 nervilia fordii (Hance) Schltr. Coding gene of Erb3-binding protein (EBP1) prokaryotic expression vector
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Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression 被引量:3
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作者 LI Jun Lü Chang-rong DOU Lin DOU Zhong-ying 《Agricultural Sciences in China》 CAS CSCD 2007年第4期487-492,共6页
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... 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 TG Ⅰ. 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 TG Ⅰ and was transformed into BL21(DE3) for expression. After induction by isopropyl-β-D-thiogalactoside (IPTG) at 37℃, 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
关键词 nanog gene prokaryotic expression glutathione-S-transferase (GST) fusion protein MOUSE
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Construction and Expression of Prokaryotic Expression Vector of MPT-64 Gene
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作者 Long PENG Linbo ZHANG 《Agricultural Biotechnology》 CAS 2014年第3期11-13,17,共4页
[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
关键词 Mycobacterium tuberculosis Protective antigen genes Secreted protein MPT64 prokaryotic expression Fusion protein
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Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product
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作者 Xi-jun CHEN Xiao-wei LIU +4 位作者 Si-min ZUO Yu-yin MA Yun-hui TONG Xue-biao PAN Jing-you XU 《Rice science》 SCIE 2011年第4期250-256,共7页
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
关键词 Ospgip1 gene polygalacturonase-inhibiting protein prokaryotic expression bioinformatic analysis RICE Rhizoctonia solani
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Efficient and Soluble Expression of N Protein of Peste Des Petits Ruminants Virus and Development of Indirect ELISA
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作者 Sun Yu Zhao Bolin +7 位作者 Wang Xiaoying Dong Hao Zhai Xinyan Qu Ping Hu Dongmei Yang Tianyi Shi Hui Song Xiaohui 《Animal Husbandry and Feed Science》 CAS 2017年第1期15-18,共4页
[ Objective] The paper was to get effective soluble N protein to establish indirect ELISA method for Peste des petits ruminants (PPR). [ Method] Soluble N protein with high expression was obtained from Escherichia c... [ Objective] The paper was to get effective soluble N protein to establish indirect ELISA method for Peste des petits ruminants (PPR). [ Method] Soluble N protein with high expression was obtained from Escherichia coli expression system through codon optimization and optimization of expression conditions, and indirect ELISA detection method based on N protein was further established. [ Result] The assay had no cross reaction with other sheep pathogens. The intra- and inter-batch variation coefficients were less than 9%, indicating the method had good repeatability. Furthermore, totally 480 clinical serum samples were detec- ted by the assay, and the agreement rate with commercial ELISA kit (IDVET) was 98.33%. [ Conclusion] The study laid a foundation for further development of mature PPRV antibody detection kits. 展开更多
关键词 Pestedes petits ruminants n active protein Soluble expression and purification Indirect ELISA
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cDNA Cloning, Prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide Response
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作者 Zhongming Dai Zanguo Nie +2 位作者 Liang He Lina Guan Yunsheng Yang 《International Journal of Clinical Medicine》 2015年第11期867-875,共9页
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
关键词 mLRG LIPOPOLYSACCHARIDE RESPOnSE GEnE cDnA Cloning prokaryotic protein expression
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Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies
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作者 Dong-Mei Zhang Han-Shuo Yang +7 位作者 Xin-Yu Zhao Wen Zhu Zhi-Hua Feng Yang Wan Zhi-Wei Zhao Ming-Hai Tang Nong-Yu Huang Yu-Quan Wei 《Journal of Biomedical Science and Engineering》 2010年第4期397-404,共8页
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 &#215;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
关键词 FUS1 POLYCLOnAL Antibody prokaryotic expression RECOMBInAnT protein Tumor SUPPRESSOR Gene
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High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells 被引量:14
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作者 Lan-lan ZHANG Jin-yu SHEN +2 位作者 Cheng-feng LEI Xiao-ming LI Qin FANG 《Virologica Sinica》 SCIE CAS CSCD 2008年第1期51-56,共6页
Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a... Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells. 展开更多
关键词 Grass carp reovirus (GCRV) VP7 protein prokaryotic expression
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Cloning,sequencing and prokaryotic expression of cDNAs for antifreeze protein family from Beetle Tenebrio molitor
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作者 Zhongyuan LIU Yun WANG +3 位作者 Guodong LÜ Xianlei WANG Fuchun ZHANG Ji MA 《Frontiers in Biology》 CSCD 2008年第3期279-286,共8页
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
关键词 Tenebrio molitor antifreeze proteins cDnA fragment sequence analysis prokaryotic expression
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Characterization of ST13 Protein Expression in Human Colorectal Cancer Tissues 被引量:4
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作者 郑树 邵吉民 +2 位作者 董琦 彭佳萍 张苏展 《The Chinese-German Journal of Clinical Oncology》 CAS 2005年第1期2-7,65,共7页
Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombi... Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery. 展开更多
关键词 colorectal cancer related gene ST13 prokaryotic expression monoclonalantibody preparation immunocytochemical staining western blot protein/characterization/expressionlevel
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Gene cloning,expression analysis of JcACP (Acyl Carrier Protein) in Jatropha curcas L. and its prokaryotical expression
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作者 JIANG Lu-ding1,2,LI Xiao-hui1(1.Institute of Materia Medica,Third Military Medical University,Chongqing 400038,China 2.School of Life Sciences,Sichuan University,Chengdu 610064,China) 《沈阳药科大学学报》 CAS CSCD 北大核心 2008年第S1期128-129,共2页
Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(... Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism. 展开更多
关键词 JATROPHA curcas L. ACYL Carrier protein(ACP) Clone expression ananlysis prokaryotical expression IMMUnOHISTOCHEMICAL
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Construction of Recombinant Expression Plasmids Containing H and F Protein Genes of Canine Distemper Virus Isolated from a Mink and Their Expression in Prokaryotic Cells
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作者 Fengyan SU Cunfa LIU +2 位作者 Tiefeng WEN Ying ZONG Quankai WANG 《Agricultural Biotechnology》 CAS 2013年第1期38-41,共4页
[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well... [Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids(pMD-18T-H and pMD-18T-F) and recombinant expression plasmids(pET28a-H and pET28a-F) ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV's infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines. 展开更多
关键词 Canine distemper virus H protein gene F protein gene expression in prokaryotic ceils
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Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta(Busck) 被引量:11
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作者 SONG Yue-qin DONG Jun-feng +1 位作者 QIAO Hui-li WU Jun-xiang 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2014年第12期2709-2720,共12页
Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the orien... Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12:Ac and Z8-12:Ac, as well as weaker binding afifnities to Z8-12:OH and 12:OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth. 展开更多
关键词 Grapholita molesta pheromone-binding proteins molecular cloning mRnA expression prokaryotic expression lfuorescence competitive binding assays
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Construction and Identification of siRNA Expression Vector Targeting Nucleocapsid Protein N gene of PRRSV 被引量:1
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作者 曹素芳 李明 +4 位作者 王岩 朱赞梅 刘长斗 唐桂芬 肖松云 《Agricultural Science & Technology》 CAS 2009年第4期171-174,共4页
[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucl... [ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique . 展开更多
关键词 PRRSV nucleocapsid protein n SIRnA expression vector
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Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée) 被引量:3
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作者 QIAO Qi LI Hai-chao YUAN Guo-hui GUO Xian-ru LUO Mei-hao 《Agricultural Sciences in China》 CAS CSCD 2008年第2期187-192,共6页
The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guen6e) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis sho... The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guen6e) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame (ORF) is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights (MW) and isoelectric point (PI) are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific. 展开更多
关键词 Helicoverpa assulta G protein α subunit gene cloning prokaryotic expression expression pattern
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