小反刍兽疫病毒H基因的真核表达  

Eukaryotic expression of the H gene from peste des petits ruminants virus

作　　者：阮洋[1,2] 秦峻岭[2,3] 高玉伟[2] 黄海楠[2,3] 孙玮[2,3] 杨松涛[2] 王承宇[2] 王铁成[2] 黄耕[2] 王志亮[4] 夏咸柱[1,2,3] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室 [3]吉林大学畜牧兽医学院 [4]中国动物卫生与流行病学中心 国家外来动物疫病诊断中心

出　　处：《中国病原生物学杂志》2011年第2期97-100,F0003,共5页Journal of Pathogen Biology

基　　金：公益性行业科研专项资助项目(No.200903037)

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。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.

关 键 词：小反刍兽疫 H基因 pCAGGS真核表达载体 转染 WESTERN BLOT 

分 类 号：S852.65[农业科学—基础兽医学]