表达小反刍兽疫病毒F基因重组腺病毒的构建及对小鼠的免疫原性

摘要

为构建表达小反刍兽疫病毒(PPRV)F蛋白的重组腺病毒,本实验合成密码子优化的PPRV F基因,将其克隆至腺病毒穿梭载体pShuttle-CMV中,并以Pme I线性化后转化含有人5型腺病毒骨架的BJ5183感受态细胞,通过细菌内同源重组构建表达PPRV F蛋白的重组腺病毒质粒pAdV-F,经Pac I线性化后转染AD-293细胞,获得重组腺病毒rHAdV-F.经PCR和Dot-ELISA鉴定,F蛋白在AD-293细胞中获得表达.该重组腺病毒与野生型腺病毒复制能力相近,但病毒滴度略低.间接ELISA方法检测显示rHAdV-F可以诱导小鼠产生抗PPRV特异性体液免疫应答.%To construct recombinant adenovims expressing pests des petits ruminants virus (PPRV) F protein, a codon optimized truncated PPRV F gene was chemically synthesized and cloned into shuttle plasmid pShuttle-CMV. The recombinant shuttle plasmid was linerized by Pme Ⅰ and transformed into E. coli BJ5183 to construct the pAdV-F by homologous recombination with bone pAdEasy-1. The recombinant adenovirus rAdV-F was generated by transfection of the Pac Ⅰ linerized pAdV-F into AD-293 cells and identified by PCR and Dot-ELISA. The rAdV-F replication was similar as the wild type adenovirus type 5 (wtAd5), and the titer of rAdV-F was slightly lower comparing with wtAd5. The BALB/c mice were inoculated with the rAdV-F by intramuscular routes. The results showed that rAdV-F induced effective humoral immune responses in mice.