猪瘟病毒非结构蛋白NS2基因不同片段的克隆及在原核系统中的高效表达

参照猪瘟病毒兔化弱毒疫苗株（HCLV）的基因组序列，设计并合成一组引物。从已构建含有猪瘟病毒兔化弱毒株全基因组质粒pMCfT1—6中分别扩增出NS2长1389bp和876bp的片段。将扩增的不同长度的NS2基因序列克隆至表达载体pGEX—KG中，经酶切鉴定后，转化大肠杆菌BL21（DE3），于37℃，1．0mmol／L IPTG条件下诱导表达。大肠杆菌菌体裂解产物经SDS—PAGE分析，在分子量约为40ku处出现和预期的目的蛋白分子量相符的条带。Western—blotting检测表明，表达产物能与猪瘟病毒阳性血清发生特异性反应，出现单一反应带，该表达产物主要存在于包涵体中。上述结果为NS2基因表达产物在免疫检测中的进一步应用奠定了基础。

Cloning and expression of NS2 gene in different lengths of classical swine fever virus in E. coli

With a serious ofprimers which were designed and synthesized accordingtothe sequence of HCLV,different fragments in length of NS2 were amplified from a recombinant plasmid pMCfT1-6 Which consists of NS2 gene of Classical Swine Fever Virus.Then the fragments were cloned into the expression vector pGEX-KG to construct the recombinant plasmids.The recombinant plasmids were proved to be true by restriction endonuclease analysis,transformed into E.coli BL21(DE3) competent cells.Expression of the protein was induced with IPTG.SDS-PAGE results showed that the part protein of NS2 was highly expressed in E.coli.Molecular weight of the expressed protein was about 40 ku.The expressed protein was specific to antisera against CSFV by Western-blot analysis,beingpresented as a single reaction band.The expressed protein was laid a foundation for diagnostic reagent development.