重组腺病毒介导的猪瘟E0基因在山羊乳腺中的表达

Recombiant Adenoviral-mediated Transfer of CSFV E0 Gene into Goat Mammary Gland

为使猪温病毒(CSFV)E0蛋白在奶山羊乳腺中得以合成和分泌,一段上游带有信号肽和His标签序列的CSFVE0基因通过腺病毒穿梭载体被插入到腺病毒质粒中,将此重组质粒转染293A细胞包装得到重组腺病毒Ad-hisE0。为证明其有效性,用Ad-hisE0感染293A细胞,实时定量PCR检测显示E0基因的表达显著提高。用Ad-hisE0分别感染牛乳腺上皮细胞和泌乳期山羊乳腺,SDS-PAGE和Western blot分析结果显示出E0蛋白分别位于26ku和48ku处的2条特异性条带。证实构建的腺病毒Ad-hisE0可以介导重组融合蛋白CSFV E0在山羊乳腺中的表达和分泌。

In order to synthesize the goat mammary gland and CSFV EO gene,a CSFV EO gene with a signal peptide and a His tag sequence in its upstream was inserted into the recombinant adenovirus plasmid by an adenovirus shuttle vector. And then the recombinant plasmid was transfected into 293A cell line to package the recombinant adenovirus Ad-hisE0. To verify the availability of the virus,293A cells were infected by the resulting recombinant adenovirus （Ad-hisE0）. The result of quantitative re- al-time PCR showed that the expression of CSFV EO increased dramatically after infected with the ad- enovirus. And then the confirmed Ad-hisE0 was used to infect bovine mammary epithelial cells in vitro and goat mammary ceils in vivo. The expression products were analyzed by SDS-PAGE and Western blot. The result revealed that there are two different molecular mass o{ the recombinant fusion protein 26 ku and 48 ku. These results demonstrated that the recombinant adenovirus Ad-hisE0 can introduce the CSFV EO gene into the goat mammary gland,and enable the secretion of the recombinant protein.