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- W2362484927 abstract "Objective To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Y.pestis F-V gene and to detect its immunogenicity. Methods Plasmid DNA of the EV76 strain Yersina pestis was isolated as the template. F1 and V gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pMD-18T vector. DNA sequence of the amplified F1-V fusion gene was assayed, and then cloned into the eukaryotic expression vector asd-pVAX1 through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli X6212, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant asd-pVAX1/F1-V was used to transform X3730 and the recombinant plasmid isolated from X3730 was finally used to transform X4550. The recombinant strain was grown repeatedly in vitro. In order to identify the immunogenicity of the vaccine in vitro, the recombinant asd-pVAX1/F1-V was transfected to COS-7 cells using lipofectamineTM2000, and then the immunogenicity of expressed F1-V fusion protein was detected with SDS-PAGE and Western blotting. Levels of serum antibodies in immunized Balb/c mice were detected by ELISA method. Results The amplified 1500-base pair F1-V fusion gene was consistent with the sequence of F1-V fusion gene on genebank confirmed by sequence analysis. PCR and restriction enzyme digestion confirmed that F1-V fusion gene was inserted into the eukaryotic asd-pVAX1 expression vector and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying F1-V fusion gene was constructed successfully. The specific strip of F1-V fusion protein expressed by asd-pVAX1/F1-V was detected by Western blotting. Specific serum antibody in immunized Balb/c mice was detected by ELISA method. Conclusion The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing F1-V fusion protein with immunogenicity can be constructed, which may be helpful for further investigating the immune action of DNA vaccine in vivo." @default.
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- W2362484927 date "2007-01-01" @default.
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- W2362484927 title "Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Y. pestis F1-V fusion gene" @default.
- W2362484927 hasPublicationYear "2007" @default.
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