犬冠状病毒部分纤突蛋白基因的分子克隆

Molecular Cloning of Partial Spike Protein Gene of Canine Coronavirus

以RT-PCR方法扩增出犬冠状病毒(CCV)YS1、CI1 2株国内分离病毒的部分纤突蛋白基因,经酶切鉴定为CCV特异性核苷酸片段。采用平端连接法将WizardTM PCR纯化试剂盒纯化的PCR产物克隆于pUC19Sma I酶切位点上,获得YS1pUC19、CI1pUC19重组质粒。琼脂糖凝胶电泳鉴定表明:重组质粒大于蓝斑菌质粒。以BarnH I、Kpn I双酶切重组质粒可获得比原PCR产物略大的核苷酸片段.并从中以PCR方法扩增出与原PCR产物大小相同的核苷酸片段。

Partial spike protein genes of canine coronavirus (CCV) strains CCV YS1 , CI1 2 first isolated in China were amplified with the method of reverse transcription polymerase chain reaction (RT- PCR) and the products were specific for CCV through the identification of restriction endocleuase cut. The PCR products purified with the Wizard?PCR purifying kit were cloned into the Sma I point of pUC19 vector and named YSlpUC19 and CIlpUC19.On the agarose gel of electro phoresis, the recombinant plasmids were bigger than the normal. The nucleotide fragments cut from the recombinant plasmids with the restriction endocleuase Bam H I and Kpn I were slightly longer than that of CCV YS1 strain PCR product. The same PCR products could be amplified from the templates of YSlpUC19, CIlpUC19 as that of reverse transcription templates of the 2 CCV isolates. The results showed that the CCV YS1 and CI1 PCR products were cloned successfully into the pUC19 vector. The results provided a basis for gene sequence analysis and gene engineer vaccine construction of CCV.