畜牧兽医学报2012，Vol.43Issue(8)：1273-1280,8.
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
Construction and Identification of Attenuated Salmonella typhimurium Harbouring S/N Double Fusion Genes of Porcine Transmissible Gastroenteritis Virus
摘要
Abstract
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.关键词
减毒沙门氏菌/猪传染性胃肠炎病毒/融合双基因/构建/生物学特性Key words
attenuated Salmonella typhimurium/ transmissible gastroenteritis virus/ double fusion gene/ construction/ biological characteristics分类
农业科技引用本文复制引用
黄小波,李春松,杨恒,曹三杰,文心田,廖晓丹,张鑫淼..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[J].畜牧兽医学报,2012,43(8):1273-1280,8.基金项目
国家自然科学基金项目(30901084) （30901084）
教育部高等学校校博士点基金项目(20095103120006) （20095103120006）
教育部《长江学者和创新团队发展计划》创新团队项目(IRTO848) （IRTO848）
