表达传染性法氏囊病毒VP2蛋白的重组火鸡疱疹病毒的构建  

Construction of recombinant herpesvirus of turkeys expressing the VP2 protein of infectious bursal disease virus

作　　者：李奇蒙[1,2] 陈普成[2] 柳金雄[2] 姜永萍[2] 王笑梅[2] 田雨[2] 胡永浩[1] 陈化兰[1,2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第7期515-518,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重大科技专项(2012ZX10004214)

摘　　要：为构建表达传染性法氏囊病毒(IBDV)VP2蛋白的重组火鸡疱疹病毒(rHVT-VP2),本研究利用RT-PCR方法扩增IBDV的VP2基因构建了重组真核表达质粒pCAGG-VP2,并采用限制性内切酶将携带Pec复合启动子和CMV增强子的VP2基因表达盒(Pec-VP2)切下,连接于Gateway系统入门质粒pENTR构建pENTR-VP2。采用Gateway LR克隆技术将pENTR-VP2与构建的重组粘粒f5354ccdBK+共同参与基因片段互换反应,构建重组粘粒f5354-VP2。f5354-VP2与其他4个相互重叠覆盖HVT全基因组的粘粒经酶切线性化后共同转染CEF细胞,并拯救出重组病毒rHVT-VP2。将重组病毒在CEF细胞中连续传代20次,每隔5代采用PCR、western blot和间接免疫荧光进行检测,结果表明VP2蛋白均得到稳定地表达。rHVT-VP2的构建为进一步研究其免疫学特性奠定了基础。In order to construct recombinant herpesvirus of turkeys （HVT） expressing the VP2 gene of infectious bursal disease virus （IBDV）, the VP2 gene was amplified from IBDV strain HLJ-0504 by RT-PCR and cloned into expression vector pCAGGs. The VP2 expression cassette was constructed which containing the Pec promoter and poly （A） signal sequence and cloned into pENTR plasmid to yield the entry clone pENTR-VP2. The insertion of Pec-VP2 cassette into the desired destination fosmid f5354ccdBK^＋ was done by mixing the destination fosmid with entry clone and treated with LR Clonase Ⅱ enzyme. The recombinant virus, designated rHVT-VP2, was generated by co-transfection of 5 overlapping fosmid DNAs which cover the complete HVT genome. PCR, western blot and immunofluorenscence assays indicated that the IBDV VP2 gene was stable existed and expressed over 20 passages. These data showed that the recombinant HVT expressing the VP2 gene of IBDV was successfully constructed which provided a basis for evaluation in chicks for immune protection against IBDV.

关 键 词：传染性法氏囊病毒 火鸡疱疹病毒 VP2 

分 类 号：S852.65[农业科学—基础兽医学]