表达番鸭细小病毒VP3基因重组鸡痘病毒转移载体的构建

Construction of recombinant fowlpox virus transfer vector expressing VP3 gene of muscovy duck parvovirus

根据Genbank上痘病毒TK基因序列分别设计左右同源臂引物TKL-F/R和TKR-F/R,以FPV基因组为模板,扩增左右同源重组臂(TKL、TKR);PCR连接左右同源臂,使中间带有多酶切位点MCS,然后连入pBluescriptIISK(+)骨架质粒,获得的阳性质粒命名为pTK;合成反向串联表达盒:p7.5-MCS1-SV40-MCS2-P11,将表达盒插入质粒pTK的MCS位点,筛选获得阳性质粒pTKS;在鸡痘病毒早期启动子P7.5后的MCS1处插入MDPVVP3基因,晚期启动子P11后的MCS2处插入EGFPBGHpA,从而构建MDPVVP3基因重组鸡痘病毒转移载体pTKS-VP3-EGFP,对构建好的转移载体进行酶切和测序鉴定。将转移载体pTKS-VP3-EGFP转染鸡痘病毒感染的鸡胚成纤维细胞(CEF)后,经RT-PCR和绿色荧光蛋白基因表达鉴定了MDPVVP3基因的表达,为进一步研制安全、高效的MDPV重组鸡痘病毒疫苗奠定了基础。

In order to construct the recombinant fowlpox virus vaccine against muscovy duck parvovirus （MDPV）, 2 pairs of primers were designed amplifying two arms of the thymidine kinase （TK） gene （TK left and TK right）,according to genome of fowlpox virus FM strain on Genbank. Segment of TK left and TK right were linked by overlap PCR, inserted a segment of muhiple clone site （MCS）. The TK left-MCS-TK right segment were inserted to vector pBluescript II SK （＋）, and the abtained positive plasmid was named as pTK. The synthetized reverse tandem expression cassettes p7.5-MCS 1-SV40-MCS 2-P11 were inserted to MCS of plasmid pTK, and the abtained positive plasmid was named as pTKS. VP3 gene of MDPV was cloned into downstream of early promoter P7.5 and reporter gene EGFP was cloned into downstream of late promoter Pll on plasmid pTKS. The recombinant fowlpox virus transfer vector pTKS-VP3-EGFP were constructed and confirmed by digestion and sequencing. Plasmid pTKS-VP3-EGFP was used to co-transfect into CEF with fowlpox virus vaccine strain. The expression of VP3 in the recombinant fowlpox virus was confirmed by RT-PCR and expression of green fluorescent protein. This study provided useful information for development of a safe and effective recombinant FPV vaccine against MDPV.