猪圆环病毒2型ORF2截短基因片段真核表达载体的构建及其DNA疫苗的免疫效果  

Construction of eukaryotic expression vectors with truncated ORF2 gene of porcine circovirus type 2 and observation of immune efficacies

作　　者：张宁[1] 李茂宁[1] 侯韶毅[1] 兰家暖[1] 谢丽华[2] 黄伟坚[1] 冯励[1] 韦英益[1] 刘芳[1] 李军[1] 

机构地区：[1]广西大学动物科学技术学院,广西南宁530005 [2]梧州市动物疫病预防控制中心,广西梧州543002

出　　处：《中国兽医科学》2009年第4期327-332,共6页Chinese Veterinary Science

基　　金：广西科学研究与技术开发项目(桂科攻0537008-3);广西科技基础条件平台建设项目(08-42-01C)

摘　　要：为研究猪圆环病毒2型(PCV-2)ORF2截短基因片段核酸疫苗的免疫效果,以PCV-2 GXWZ-1株为模板,用PCR方法扩增出ORF2基因及其截短基因共8个片段[A(ORF2)、B(51～100 aa)、C(101～150 aa)、D(181～235 aa)、E(151～200 aa)、F(51～150 aa)、G(101～235 aa)、H(51～235 aa)],将其插入pcD-NA3.0载体中,成功构建了pcDNA-A、pcDNA-B、pcDNA-C、pcDNA-D、pcDNA-E、pcDNA-F、pcDNA-G、pcDNA-H。将纯化后的真核表达质粒转染PK-15细胞后,用间接免疫荧光试验检测各个质粒在细胞中的瞬时表达情况。分别用单独免疫和组合免疫的方法免疫小鼠,每只0.15 mg,用ELISA试剂盒检测小鼠血清中的抗体效价,并分析这两种方法的免疫效果。结果显示,6个片段较短的重组质粒(pcDNA-B、pcDNA-C、pcDNA-D、pcDNA-E、pcDNA-F、pcDNA-G)转染的细胞中均有明显且较亮的荧光出现,而2个片段较长的重组质粒(pcDNA-A、pcDNA-H)及空载体pcDNA3.0转染的细胞中没有荧光出现。在单独免疫组中,这8个片段的真核表达质粒都能刺激机体产生特异性抗体,其中片段较短的pcDNA-C、pcDNA-D、pcDNA-E的免疫效果比较理想;在组合免疫中,除pcDNA-C-F外,各个真核表达质粒都能产生较高水平且稳定的抗体,其中pcDNA-D-H、pcDNA-B-F的免疫效果最好。表明组合免疫要比单独免疫的效果好。To study the immune efficacy of DNA vaccines with truncated ORF2 gene of porcine circovirus type 2（PCV-2）,the ORF2 gene fragments of A（ORF2）,B（51-100 aa）,C（101-150 aa）,D（181-235 aa）,E（151-200 aa）,F（51-150 aa）,G（101-235 aa） and H（51-235 aa） were amplified from PCV-2 GXWZ-1 strain by PCR, respectively, and cloned into pcDNA3.0 vector to construct the eukaryotic expression plasmids pcDNA-A, pcDNA-B, pcDNA-C, pcDNA-D, pcDNA-E, pcDNA-F, pcDNA-G and pcDNA-H. The eight purified recombinant plasmids were transfected into PK-15 cells, respectively, and their expressed products were examined by indirect immunoftuorescence assay. Female mice were inoculated respectively with 0.15 mg purified recombinant plasmids by method of single plasmid or combined plasmids. The levels of antibody against PCV2 in sera were detected by ELISA Kits and the immune efficacy was analyzed. The results showed that fluorescence were only observed in the cells transfected with pcDNA-B, pcDNA-C, pcDNA-D,pcDNA-E,pcDNA-F, and pcDNA G. In single plasmid immune group, the mice respectively immunized with pcDNA-C, pcDNA-D, pcDNA E had the higher antibody levels lasting longer time. In combined plasmid immunization group, the mice immunized with combined plasmids except pcDNA-C-F had also the higher antibody levels for a long time and the mice immunized with the combined plasmids pcDNA-D-H or pcDNA-B-F all reached the best immune efficacy. So the mice immunized with combined plasmids had a better immune efficacy.

关 键 词：猪圆环病毒2型 ORF2截短基因 真核表达 重组质粒 免疫效果 

分 类 号：S852.659.2[农业科学—基础兽医学]