传染性法氏囊病病毒VP2与C3d串联基因重组表达质粒的构建及其免疫功能研究  

Construction and immune responses of the tandemed gene of infectious bursal disease virus VP2 and chicken complement C3d

作　　者：马金荣[1,2] 欧阳伟[1] 王永山[1] 吴晓悠[1,2] 朱向东[1,2] 张海彬[2] 范红结[2] 王忠灿[3] 唐雨德[3] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]南京军区军事医学研究所南京军区疾病预防控制中心,江苏南京210002

出　　处：《中国预防兽医学报》2012年第6期471-475,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏省自然科学基金资助项目(BK2008065;BK2008352;BK2009041)

摘　　要：为提高传染性法氏囊病病毒(IBDV)VP2基因核酸疫苗的免疫效力,本研究根据已发表的鸡源补体C3d序列,设计并合成在5'端添加编码连接肽(Gly4Ser)2序列的C3d基因。用同尾酶BglⅡ和BamHⅠ构建含有3拷贝C3d与VP2基因融合的重组表达质粒pcDNA-VP2-3C3d。用脂质体法转染BHK21细胞,48 h后,westernblot分析表明,表达的重组蛋白为162 ku;间接免疫荧光试验检测转染细胞中具有特异性荧光。用pcDNA-VP2-3C3d与前期构建的pcDNA-VP2分别免疫2周龄SPF鸡,二免14 d后,间接ELISA法检测IBDV抗体效价,pcDNA-VP2-3C3d组抗体水平显著高于pcDNA-VP2组;MTT法检测鸡脾淋巴细胞增殖活性,pcDNA-VP2-3C3d组免疫诱导的特异性淋巴细胞增殖活性显著高于pcDNA-VP2组(p<0.05)。本研究表明C3d可以增强VP2基因免疫诱导的IBDV特异性体液和细胞免疫应答。To enhance the immune efficacy of the DNA vaccine against the challenge of infectious bursal disease virus （IBDV）, a three tandemed copies of chicken complement C3d gene （3C3d） with the linker sequence of （Gly4Ser）2 at 5＇ end was synthesized and inserting into pUC57 vector, pcDNA-VP2-3C3d was constructed by insertion of the 3C3d tandem gene into pcDNA-VP2 and transfected into BHK21 cells for expression. Western blot result showed that the expressed fusion protein of VP2-3C3d was about 162 ku and reacted positively with IBDV polyclonal antibodies. Expression of VP2-3C3d was also confirmed by the indirect immunofluorescence assay. In addition, pcDNA-VP2-3C3d and pcDNA-VP2 were used to immunize 2-week-old SPF chicken. The antibody titrations and lymphocyte proliferative responses indicated that immunized with ocDNA-VP2-3C3dinduced higher titers of IBDV antibodies and lymphocyte proliferation than that of immunized with pcDNA-VP2 （p〈0.05）, indicating that C3d enhanced both the humoral and cellular immune response against IBDV.

关 键 词：传染性法氏囊病病毒 VP2基因 鸡补体C3d 分子免疫佐剂 基因免疫 

分 类 号：S852.65[农业科学—基础兽医学]