犬瘟热病毒水貂株H与F蛋白基因原核表达重组质粒的构建及表达

Construction and Expression of Protokaryon Expression Plasmid for H and F Genes of Canine Distemper Virus Isolated from Mink

摘要: 将克隆质粒pMD-18T-H和pMD-18T-F分别进行双酶切，获得纯化的H基因和F基因，在T₄ DNA连接酶的作用下，亚克隆到原核表达载体pET28ａ（＋）上, 获得原核重组质粒pET28a-H和pET28a-F.将原核重组质粒转化大肠埃希菌Escherichia coli Rosetta2(DE3)中进行表达,SDS-PAGE结果显示H 基因和F基因分别表达相对分子质量约为31 400和38 200的融合蛋白,Western-blot 检测结果显示,表达蛋白均可与CDV 标准阳性血清呈阳性反应，表明原核表达的CDV H蛋白和F蛋白在反应原性上具有与天然H蛋白和F蛋白同样的特性,可作为CDV诊断用抗原，为CDV的免疫预防研究奠定基础.

Abstract: H and F genes were obtained by digesting the cloning plasmid pMD-18T-H and pMD-18T-F respectively.The recombinant protokaryon expression plasmids pET28a-H and pET28a-F were constructed by sub-cloning the objective gene into pET28a (+) vector. Recombinant protokaryon expression plasmids pET28a-H and pET28a-F were transformed into Escherichia coli Rosetta2(DE3)cell. Target genes were successfully expressed by SDS-PAGE detection. The size of the recombinant fusion proteins was 31 400 (H) and 38 200 (F) respectively. The recombinant fusion proteins showed positive reaction to anti-CDV canine serum by Western-blot detection.