口服鼠疫F1-V重组融合基因DNA活菌疫苗的构建及免疫原性研究

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Y. pestis F1-V fusion gene

目的构建携带鼠疫耶尔森菌F1-V融合基因的重组减毒沙门菌苗,口服免疫Balb/c小鼠检测其免疫原性,为口服鼠疫活载体DNA疫苗研究打下基础。方法将F1-V融合基因克隆到真核表达载体asd-pVAX1,进一步依次将重组质粒转化减毒沙门菌X3730、X4550得到重组沙门菌X4550(asd-pVAX1/F1-V),提取重组质粒转染COS-7细胞并做免疫组化和Western-blot检测F1-V融合蛋白在细胞中的表达。以1×109CFU/只的剂量3次口服免疫Balb/c小鼠,ELISA方法检测血清中抗体水平。结果构建的重组减毒沙门菌转染COS-7细胞后,免疫组化和Western-blot试验证明F1-V融合蛋白在细胞中得到了瞬时表达,ELISA检测到免疫小鼠血清有特异性抗体IgG产生。结论构建的重组沙门菌能运送DNA疫苗到体内并成功释放质粒刺激机体产生特异性免疫应答,为口服鼠疫活载体DNA疫苗的黏膜免疫研究打下了基础。

Objective To construct a recombinant attenuated Salmonella tyhimurium DNA vaccine carrying Y. pestis F-V gene and to detect its inenunogenicity. 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 eukaryotie 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 immunogenictiy of the vaccine in vitro, the recombinant asd-pVAX1/F1-V was transfected to COS-7 cells using lipefectamine^TM2000, and then the immunngenicity 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-basc 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 tyhimurium 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 attenusted Salmonella typimurium DNA vaccine strain expressing F1- V fusion protein with immunogenicity can be constructed , which may be helpful for further investgating the immune action of DNA vaccine in vivo.