犬瘟热病毒重组囊膜糖蛋白(H/F)全长质粒的构建

The Construction of Recombinant Full-Length Plasmids withthe Double-Envelope Glycoproteins of Canine Distemper Virus

目的开发新型疫苗和改良目前使用的犬瘟热病毒(CDV)疫苗。方法以犬瘟热弱毒株CDV/R-20/8为模板,用PmeI单酶切pBluescript-F和pBluescript-H质粒中获得完整CDV-F和CDV-H基因片段,定向克隆到pCI-c1-EGFP质粒中,构建pCI-c1-F和pCI-c1-H质粒,正反向鉴定正确后,用SalI和MluI酶分别对pCI-c1-F、pCI-c1-H、pCI-CDV-EGFP质粒进行双酶切,分别回收目的片段c1-F、c1-H片段和载体片段pCI-CDV-,将c1-F和c1-H分别插入载体pCI-CDV-中,通过PCR和酶切的方法进行筛选、鉴定阳性克隆。结果成功构建重组双囊膜糖蛋白全长质粒,分别命名为pCI-CDV-F和pCI-CDV-H,为下一步重组犬瘟热病毒的拯救及免疫原性分析奠定基础。

Objective To develop new vaccines and improve the current vaccines of canine distemper virus （CDV）. Methods With the template of CDV/R-20/8 attenuated strain of canine distemper virus, the full length CDV-F and CDV-H gene fragments were got after the plasmids ofpBluescript-F and pBluescript-H were digested by Pme I alone, then the full-length CDV-F and CDV-H gene fragments were directly cloned into pCI-c 1-EGFP plasmid respectively and plasmids of pCI-c I-F and pCI-c 1-H were constructed, which were identified correctly by PCR and restriction enzyme digestion, than digested the plasmids ofpCI-cl-F, pCI-cl-H, pCI-CDV-EGFP respectively with Sal I and Mlu I, the fragments ofcl-F, cl-H and pCI-CDV-were recycled respectively; The cl-F and cl-H were inserted into the vector of pCI-CDV-,the positive clones were validated by PCR and restriction enzyme digestion. ~ The recombinant full-length double-glycoprotein plasmids were constructed successfully and named pCI-CDV-F and pCI-CDV-H, thus setting up basis for the further research of the rescue of recombinant canine distemper virus and the immunogenicity analysis.