携带猪传染性胃肠炎病毒S/N融合双基因的减毒沙门氏菌的构建与鉴定

摘要

旨在构建携带猪传染性胃肠炎病毒(TGEV)S/N融合双基因的减毒沙门氏菌,并鉴定该疫苗菌株的生物学特性,为开展TGEV口服免疫研究奠定材料基础.采用PCR方法从克隆质粒19T-S和19T-N中分别扩增了TGEV的S基因(含主要抗原位点,2.1kb)和N基因(1.2 kb),将S基因和N基因插入pVAX1载体,构建携带S/N融合双基因的真核表达质粒pVAX-S/N.将pVAX-S/N电转化减毒沙门氏菌SL7207,筛选获得重组菌株SL7207 (pVAX-S/N),并对重组菌株SL7207 (pVAX-S/N)的体外稳定性、目的基因在体内的转录、口服接种小鼠的安全性及在体内稳定性等特性进行了鉴定.结果表明,真核质粒pVAX-S/N构建成功,该质粒转染COS7中能表达2个目的蛋白,重组菌SL7207 (pVAX-S/N)在Kan+抗性下体外培养稳定性好,口服接种小鼠3d可从回肠组织检测到目的基因的转录,以0.5×10 9、1×10 9和2×109 CFU口服对小鼠均具有安全性,重组菌在接种小鼠的肝、脾于4周左右逐渐被机体清除.结果表明成功构建TGEV S/N双基因疫苗SL7207 (pVAX-S/N),该疫苗具有良好的稳定性与安全性等特点,为开展TGEV口服免疫研究奠定了基础.%To provide a new vaccine for oral immunization of transmissible gastroenteritis virus (TGEV) , attenuated Salmonella typhimurium harbouring S/N double fusion genes of (TGEV) was constructed and identified. The S gene fragment (2. 1 kb) and the N gene fragment (1. 2 kb) were respectively amplified from the recombinant plasmid 19T-S and 19T-N of TGEV by RT-PCR, and then the two gene fragments were successively inserted into the eukaryotic expression vector pVAXl to construction the recombinant eukaryotic expression plasmid pVAX-S/N that expressing the S-N double fusion gene. The plasmid pVAX-S/N was identified by PCR and restric-tive digestion, and then the pVAX-S/N was transfected into COS7 cells through liposome trans-fection to identify the expressions of the two target genes hy indirect immunofluorscence assay. The plasmid pVAX-S/N was transformed by electroporation into attenuate S. typhimurium SL7207, the stability of pVAX-S/N in SL7207 (pVAX-S/N) cultured in vitro was detected. The transcription of pVAX-S/N in mouse ileal tissue cells orally immunized with SL7207 (pVAX-S/ N) , BALB/c mice were inoculated orally with SL7207 (pVAX-S/N) at different dosages to detect the safety and stability in vivo. The results showed that the recombinant plasmid pVAX-S/N was constructed correctly and the expression of S gene and JV gene were detected in COS7 cells. The transcription and expression of S/N double fusion gene were detected in mouse ileal tissue cells after 3 days infected with SL7207 (pVAX-S/N). The recombinant bacteria SL7207 (pVAX-S/N) was highly stable cultured with Kanamycin Resistance medium in vitro. The recombinant SL7207 (pVAX-S/N) were all safe to mouse at dosage of 0. 5 × 109, 1 × 109 and 2 × 109 CFU. The SL7207 (pVAX-S/N) were eventually eliminated from the spleen and liver at about four weeks post-immunization. These results indicated that the recombinant bacteria SL7207 (pVAX-S/N) had good safety and stability, which lay a foundation for oral immunization of TGEV.