携带猪呼吸道冠状病毒S基因片段的重组腺病毒的构建及鉴定

Construction of recombinant adenovirus expressing PRCV S gene fragment

利用RT-PCR技术扩增猪呼吸道冠状病毒(Porcine respiratory coronavirus,PRCV)AR310毒株的S基因主要抗原位点片段,构建了重组腺病毒中间载体pDNR-1r-S1和表达质粒pInt.AV1.SpaT-S1。采用Cre-LoxP同源重组腺病毒系统和共转染技术构建了携带PRCV S基因片段的复制缺陷型重组腺病毒,经目的基因PCR检测和序列测定,结果表明:PRCV S基因片段已正确地插入到腺病毒基因组中;Western blot和间接免疫荧光实验证明目的片段在HEK293细胞中成功地获得了表达;子代重组腺病毒Ad5-PRCVS1的滴度为7×107 TCID50/mL。携带PRCV S基因片段重组腺病毒的构建为PRCV和TGEV重组腺病毒新型疫苗的研究奠定了基础。

Recombinant adenovirus expressing porcine respiratory coronavirus （PRCV） S gene fragment （S1） was constructed. PRCV S gene fragment was amplified by RT-PCR, sequenced and then inserted into vector pint.AV1.SpaT to produce plnt.AV1. SpaT-S1. The recombinant pint.AV1,SpaT-S1 was co-transfected with pint.B.B plasmid in the HEK293 cells and cytopathogenic effects were developed at 7th day post transfection. Insertion of PRCV S fragment in the adenovirus genome was confirmed by PCR and nucleotide sequencing. Western blot and indirect immuno-fluorescent assay showed that PRCV S1 protein was properly expressed in HEK293 cell. The titer of the recombinant adenovirus propagated in HEK293 cells was 7 × 10^7 TCID50/mL. This recombinant adenovirus expressing PRCV S1 gene paves the way for a new vaccine candidate of PRCV or TGEV.