Construction of a prokaryotic expression vector containing the EV71 VP1-VP4 fusion gene and detection of its expressions

Based on splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,theag85aandmpb70were amplified and the fusion gene ag85a-mpb70 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-85a-70. pMD-85a-70 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified ag85a-mpb70 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-85a-70 was constructed. Plasmid containing pET-85a-70 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 49 kDa fusion protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that Ag85A-MPB70 was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of novel vaccine against bovine tuberculosis.

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Fusion and Prokaryotic Expression on the mpb83 and mpb63 Genes of Mycobacterium Bovis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.791-793.212 ◽

2013 ◽

Vol 791-793 ◽

pp. 212-215

Author(s):

Jia Ning Guan ◽

Chun Fang Wang ◽

Jia Ming Lin ◽

Hong Xia Ma ◽

Shuang Hou ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Prokaryotic Expression ◽

Expression Vector ◽

Fusion Gene ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Antigenic Activity ◽

Sds Page ◽

Prokaryotic Expression Vector ◽

Single Antigen

Based on the (Gly4Ser)3 linker, thempb83andmpb63gene were fused for raising the antigenicity of single antigen. The DNA fragments ofmpb83andmpb63were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,and the fusion gene mpb83-63 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-83-63. pMD-83-63 and pET28a (+) were digested byBamH I andEcoR I double enzymes. The purified mpb83-63 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-83-63 was constructed. Plasmid containing pET-83-63 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thioga-lactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 38 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

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Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene

Chinese Science Bulletin ◽

10.1007/bf02898917 ◽

1998 ◽

Vol 43 (3) ◽

pp. 223-230

Author(s):

Shengjun Ren ◽

Huiqiu Jiang ◽

Jianren Gu

Keyword(s):

Prokaryotic Expression ◽

Expression Vector ◽

Cytosine Deaminase ◽

Ofescherichia Coli ◽

Prokaryotic Expression Vector ◽

Cytosine Deaminase Gene

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Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium bovis in Escherichia coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.421.354 ◽

2013 ◽

Vol 421 ◽

pp. 354-358

Author(s):

Jia Ming Lin ◽

Chun Fang Wang ◽

Jia Ning Guan ◽

Hong Xia Ma ◽

Shuang Hou ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Bovis ◽

Expression Vector ◽

Fusion Gene ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Antigenic Activity ◽

Sds Page ◽

Prokaryotic Expression Vector ◽

Single Antigen

Based on the (Gly4Ser)3 linker, theesat-6andcfp-10gene were fused for raising the antigenicity of single antigen. The DNA fragments ofesat-6andcfp-10were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR),and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

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Specific T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese HIV Positive Individuals

Infection International ◽

10.1515/ii-2017-0034 ◽

2013 ◽

Vol 2 (1) ◽

pp. 6-12

Author(s):

Bin Zhang ◽

Wen-hui Lun ◽

Xing-wang Li ◽

Qi Wang ◽

Jun Cheng ◽

...

Keyword(s):

T Cell ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Prokaryotic Expression ◽

Expression Vector ◽

T Cell Responses ◽

Hiv Positive ◽

E Coli ◽

Cell Responses ◽

Prokaryotic Expression Vector

Abstract Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity; the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94% in total bacterial protein of E. coli and the molecular weight is 31 kD; Western blot confirmed the recombinant proteins had high antigenicity; our in-house ELISpot-IFN-γ assay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

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Construction of Prokaryotic Expression Vector Containing Flocculation Gene FLO5 and Expression of FLO5 in E. coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.310.157 ◽

2013 ◽

Vol 310 ◽

pp. 157-161 ◽

Cited By ~ 2

Author(s):

Jie Xue ◽

Wen Qiang Wei ◽

Dong Yan Zhang ◽

Yong Li Li ◽

Xin Zhang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Prokaryotic Expression ◽

Genomic Dna ◽

Expression Vector ◽

Gene Fragment ◽

Form Expression ◽

Protein Fragments ◽

E Coli ◽

Sds Page ◽

Prokaryotic Expression Vector

FLO5 has been identified as a dominant flocculation gene. The goal of this study is to clone the FLO5 gene from Saccharomyces cerevisiae and express it in E. coli. In this study, the FLO5 gene amplified by PCR from S. cerevisiae was cloned into prokaryotic expression vector pET-28a to form expression vector pET28a-FLO5, finally, transferred into E.coli BL21. Methods: FLO5 gene was amplified by PCR from genomic DNA extracted from Saccharomyces cerevisiae. The amplified FLO5 gene fragment was then recombined with clone vector pMD18-T to form clone vector pMD18-T-FLO5 amplified in E.coli JM109. After confirmed with sequencing, FLO5 fragment cut out from pMD18-T-FLO5 by enzyme EcoRI and NotI was recombined into expression vector pET-28a to form vector pET28a-FLO5. Vector pET28a-FLO5 was then transferred into E. coli BL21 and protein FLO5 was expressed in E. coli BL21 by the induction with IPTG. Expressed protein fragments separated by SDS-PAGE showed a band with the size of protein FLO5 suggesting the expression of gene FLO5. with the expected This study will lay the foundation for further research in studying flocculating effect of exogenous protein expressed by genetic engineering and making new flocculating agent through recombinant engineering.

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Cloning, Expression and Characterization of β-Glucosidase from L. Delbrueckii Subsp. Delbrueckii in Escherichia coli

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.301-303.347 ◽

2011 ◽

Vol 301-303 ◽

pp. 347-351

Author(s):

Xiu Hong Zhao ◽

Jie Zeng ◽

Hai Yan Gao ◽

Chang Biao Li ◽

Chang Jiang Liu

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Gel Electrophoresis ◽

Prokaryotic Expression ◽

Expression Vector ◽

Polyacrylamide Gel Electrophoresis ◽

Gene Encoding ◽

Prokaryotic Expression Vector ◽

Strain Bl21

Gene encoding β-glucosidase was amplified through PCR by using the genome DNA extracted from L .delbrueckii subsp. delbrueckii as a template. The gene encoding β-glucosidase was inserted into a prokaryotic expression vector pET-28a(+) and expressed in E.coli strain BL21(DE3). The gene encoding β-glucosidase was of 1380bp. The nucleotide sequence of the gene encoding β-glucosidase from L. delbrueckii subsp. delbrueckii showed as high as 97.9% homology comparing with that from L. delbrueckii subsp. bulgaricus indicating that the gene encoding β-glucosidase is highly conservative. The enzyme activity was about 34U/mg and the molecular weight of β-glucosidase is about 51 kDa analyzed by SDS-polyacrylamide gel electrophoresis.

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Construction and expression of a prokaryotic expression vector for the goat sry gene

Indian Journal of Animal Research ◽

10.18805/ijar.b-963 ◽

2019 ◽

Author(s):

Xiao Wang ◽

Guang-Xin E ◽

Shu-Zhu Cheng ◽

Wei-Wei Ni ◽

Yue-Hui Ma ◽

...

Keyword(s):

Recombinant Protein ◽

Prokaryotic Expression ◽

Expression Vector ◽

Target Gene ◽

Rabbit Antiserum ◽

Rabbit Serum ◽

Molecular Sexing ◽

Coding Region ◽

Sry Gene ◽

Prokaryotic Expression Vector

Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

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