【摘要】 目的构建小反刍兽疫病毒(PPRV)H基因真核表达载体,并对其表达能力及免疫活性进行鉴定,为后期疫苗的研制提供基础。方法根据GenBank上公布的PPRV(China/Tib/Gej/07-30株)H基因的序列进行引物设计并进行扩增,纯化回收后将其克隆到pMD-18T载体中,将测序鉴定正确的目的基因克隆到真核表达载体pCAGGS上。结果成功构建了含有PPRV H基因的真核表达载体pCAGGS-H。通过脂质体介导质粒pCAGGS-H转染DK细胞,经间接免疫荧光及Western blot证实,表达蛋白分子质量单位为68.8 ku,能与PPRV阳性血清发生特异性反应。结论 pCAGGS-H在真核细胞内可有效表达。更多还原

【Abstract】 Objective To construct a eukaryotic expression vector for the H gene of the peste des petits ruminants virus(PPRV),determine its level of expression and immune activity,and therefore provide a basis for subsequent development of vaccines.Methods According to the encoding sequence of the PPRV H gene in GenBank(China/Tib/Gej/07-30 strain),appropriate primers specific to H gene of PPRV were designed.A target DNA fragment was amplified by PCR and then the amplicon was cloned into a pMD18-T vector.Sequencing was done to correctly identify the DNA fragment,which was cloned into the eukaryotic expression vector pCAGGS and then designated pCAGGS-H.Results The plasmid pCAGGS-H containing the H gene of PPRV was successfully constructed and was transfected into DK cells with liposomes.Indirect immunofluorescence(IFA) and Western blotting were used to confirm that the H protein from pCAGGS-H was efficiently expressed.The H protein was approximately 68.8 ku and produced a specific reaction to sera positive for PPRV.Conclusion pCAGGS-H can be efficiently expressed in mammalian cells.更多还原