高致病性PRRS VORF3基因在昆虫细胞中的表达与免疫原性研究  

Expression of highly pathogenic PRRSV ORF3 gene in insect cells

作　　者：徐明明[1,2] 蔡雪辉[1] 刘永刚[1] 王淑杰[1] 王洪峰[1] 石文达[1] 李丽琴[1] 荣福龙[1,2] 

机构地区：[1]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/猪传染病研究室,黑龙江哈尔滨150001 [2]东北农业大学,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2010年第6期455-459,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：十一五国家支撑计划(2006BAD06A07)

摘　　要：为进一步研究猪繁殖与呼吸综合征病毒(PRRSV)GP3的结构、功能和免疫学特性,本研究根据GenBank登录的高致病性PRRSV变异株(HP-PRRSV) HuN4株ORF3基因序列,经序列分析去除其N端的一部分疏水性区域(1aa～49aa),设计合成引物。将扩增的基因连接于pFastBac HTb杆状病毒载体中,构建重组质粒pF-ORF3,转化至感受态DH10Bac中,获得重组杆粒rBac-ORF3。再将其转染昆虫细胞Sf9,获得重组杆状病毒rAc-ORF3。用western blot和间接免疫荧光试验(IFA)分析表明GP3在昆虫细胞Sf9中获得正确表达,而且具有良好的反应活性。用纯化的GP3免疫小鼠,制备抗GP3多克隆抗血清,用酶联免疫吸附试验(ELISA)测定其效价。本研究利用昆虫杆状病毒表达系统表达了重组GP3,经GP3免疫的小鼠体内产生了较高滴度的特异性抗体,证明表达的GP3具有特异的免疫原性。To study the structure, function and immunogenicity of the GP3 protein of highly pathogenic porcine reproductive and respiratory syndrome virus （PRRSV）, the ORF3 gene of PRRSV was amplified by RT-PCR from PRRSV HuN4 strains and cloned into the baculovirus donor vector pFastBac HTb. The recombinant plasmid pF-ORF3 DNA was transformed into E.coli competent cells DH10Bac and the bacmid rBac-ORF3 was transfected into Sf9 insect cells. The recombinant baculovirus rAc-ORF3 was obtained and confirmed by PCR. Western blot and IFA confirmed that the GP3 was expressed in Sf9 cells. The expressed GP3 was purified and inoculated to BALB/c mice to prepare anti-GP3 serum, the specificity and titer of the antiserum were evaluated by ELISA. High titer of specific antibodies was obtained from the vaccinated mice. The results showed that the GP3 expressed by recombinant baculovirus had strong immunogenicity and could induce high titer antibody against GP3.

关 键 词：猪繁殖与呼吸综合征病毒 ORF3 杆状病毒表达 免疫原性 

分 类 号：S852.4[农业科学—基础兽医学]