犬细小病毒VP2基因的克隆表达与免疫印迹分析

根据基因库已发表犬细小病毒序列设计合成了VP2基因的1对特异引物,以细小病毒感染的犬粪样品中提取的总DNA为模板,进行PCR扩增。把扩增产物克隆至pMD18-T载体进行测序、鉴定。将克隆的VP2基因片段克隆至原核表达载体pGEX-4T-2,再将构建成功的原核表达质粒载体pGEX-4T-2-VP2转化至大肠杆菌BL21(DE3)中,进行鉴定、测序、表达。结果表明,克隆的VP2基因片段全长1 755 bp,与13个VP2基因序列的同源性为98.4%～99.8%。系统进化树分析表明,所克隆的VP2 CSN830611序列同日本株(AB054222)、意大利株(AF306445)的进化途径一致。Western-blotting分析显示,表达产物为90 kD的融合蛋白,可被犬细小病毒阳性血清所识别,具有良好的反应原性。

Cloning Expression of Canine Parvovirus VP2 Gene and Analysis on Immune Blot

One pair of the special primers were designed to compose the VP2 protein gene sequences of ca- nine parvovirus （CPV） published in GenBank. The VP2 protein gene fragment in CPV was amplified by PCR from the genomic DNA in the ill dog＇s stool samples infected with CPV and was cloned into a pMD18-T vector in order to construct pMD18-CPV VP2 recombination plasmid and then transformed into the E. coil DHSa after sequencing analysis. The cloned CPV VP2 gene was subcloned into expression vec- tor pGEX-4T-2 and the resultant constract was transformed into E. coil BL21（DE3）cells sequencing, iden- tifying and induced by IPTG for expression. Sequence analysis was shown the total length of CPV VP2 gene was 1 755 bp. It shared 98.4~--99.8% with the homology of 13 VP2 genetic sequence. The phylo- genetic evolutionary tree was shown that the cloned VP2 CSN830611 （AB054222） sequence showed no difference with other strains from Japan,ltalia. Western-blotting analysis were shown that a fussion protein of 90 kDa was expressed which could be recognised by canine antiserum against CPV. This result was indi- cated the fussion protein （GST-CPV VP2） possessed strong antigenicity which could be used as a recombi- nant antigen to develop a subunit vaccine.