猪瘟病毒囊膜糖蛋白E2的克隆表达

为了研究不同时间段细胞MHC分子表达的变化情况，从分子水平上说明E2基因与NHC之间的关系，本研究克隆了中国猪瘟病毒强毒石门毒株E2基因，构建了pcDNA3.0真核表达质粒P-E2基因。限制性内切酶进行双酶切和序列分析鉴定E2基因插入位置、方向和读码框架的正确性。构建成功的真核表达质粒P-E2，用脂质体转染方法瞬时转染进行体外细胞表达，转染后24，48 h后用Western-blot和间接免疫荧光技术检测P-E2基因质粒在PK-15细胞内的表达情况。结果表明：用His抗体进行间接免疫荧光检测发现E2基因得到表达，并定位于细胞浆，同时Western-blot检测后发现重组质粒P-E2转染后也在PK-15细胞中得到了表达。为进一步研究猪瘟病毒与宿主细胞之间的关系，病毒的致病机理提供了基础材料。

Molecular Cloning and Expression of E2 Gene of the Classical Swine Fever Virus

In order to study the relationship between E2 and MHC at different time point.The E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain was cloned,then inserted into the expression vector pcDNA 3.0,eukaryotic recombinant expression plasmid P-E2 was generated.The P-E2 was identified that the position of E2 gene of CSFV insert,the size and the reading frame were all right by restriction digestion and the sequence analysis.The P-E2 was used to transfect PK-15 cells in vitro by liposome infection protocol.The results of Western-blot showed that the recombinant E2 protein was CSFV-specific.The fluorescence microscope detection indicated expressed E2 protein mainly located in plasma of PK-15 cells,which was the same as the results when PK-15 cells were infected by CSFV.These results above would provided basis for the pathogenesis of Classical swine fever virus.