人呼吸道合胞病毒融合糖蛋白非复制型重组腺病毒的构建和表达

Construction and identification of replication deficient recombinant adenovirus encoding F gene of subgroup A human respiratory syncytial virus

目的构建含有A亚型人呼吸道合胞病毒（Human Respiratory Syneytial Virus，RSV）融合糖蛋白（Fusion glycoprotein，F）基因的非复制型第一代重组腺病毒（First generation adenovirus vector，FGAd），并研究F基因在重组腺病毒中的表达。方法利用限制性内切酶Xho I和Hind Ⅲ从质粒pGEM3zf-F中切下目的基因F，克隆至穿梭质粒pShuttle-CMV，再与pAdeasy-1在大肠埃希菌BJ5183中进行同源重组，鉴定正确后，用脂质体法转染293细胞，Western Blot鉴定目的基因表达。结果获得了表达RSVF基因的非复制型重组腺病毒FGAd／F，Western Blot检测到F基因的表达。结论获得一株可表达A亚型RSVF的非复制型重组腺病毒FGAd／F，可用于体内研究观察其免疫效果及免疫保护作用。

Objective A strain of replication deficient recombinant adenvirus encoding fusion glycoprotein （F） of subgroup A human respiratory syncytial virus （RSV） was constructed and the expression of F was identified. Methods The F gene was obtained from pGEM3zf-F with Xho Ⅰ and Hind Ⅲ, cloned into adenovirnse shuttle vector pShuttle-CMV, and then the resulting pShuttle-CMV/F was transformed into E. coil BJ5183/p with pAdeasy- 1 to produce pre-adenoviral plasmid encoding F by homologous recombination. This resultant plasmid was linearized by digestion with Pac I and transfected into 293 packaging ceils to generate FGAd-F. Finally, the expression of F protein was identified by Western Blot analysis. Results FGAd/F was successfully constructed, and the expression of RSV F protein was identified by Western Blot. Conclusion We have obtained a strain of replication-defective adenovirus FGAd/F encoding RSV F protein, which can be used further to investigate its protective efficacy against RSV infection in vivo.