PPV VP2-PCV2 ORF2真核表达载体的构建及对小鼠免疫效应试验  

Construction of eukaryotic expression vector with PPV VP2-PCV2 ORF2 gene and its immunation effect on the mice

作　　者：徐凯[1] 徐志文[1] 郭万柱[1] 朱玲[1] 王印[1] 陈燕凌[1] 唐玉香[1] 王小玉[1] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014

出　　处：《中国兽医学报》2011年第1期16-20,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30500019);"长江学者和创新团队发展计划"创新团队资助项目(IRT0848);四川省青年科技基金资助项目(200614-049)

摘　　要：将猪细小病毒(PPV)VP2基因和猪圆环病毒2型(PCV2)ORF2截短基因(47～207aa)克隆到真核表达载体pCI-neo,构建了含VP2和ORF2基因的融合表达质粒pCI-ORF2-VP2,经脂质体法转染PK15细胞后,用间接免疫荧光检测法测到融合蛋白能正常表达。将pCI-ORF2-VP2质粒肌注BALB/c小鼠(100μg/只),相同剂量免疫2次,间隔2周,同时设立圆环亚单位苗、细小灭活苗和空载体对照组,于免疫后7、14、21、28、42d无菌摘取脾脏作T淋巴细胞增殖试验,摘眼球采凝血和抗凝血,分别作PPV、PCV2抗体的监测和T淋巴细胞亚群的动态监测。结果显示,pCI-ORF2-VP2免疫小鼠脾脏T淋巴细胞对ConA刺激的反应性、诱导CD4+、CD8+T淋巴细胞的免疫功能和血清PPV/PCV2的抗体水平在不同时间段均显著高于空载体对照组。表明真核表达质粒pCI-ORF2-VP2能刺激小鼠产生良好的细胞免疫和体液免疫。The porcine parvovirus（PPV） VP2 gene and the porcine circorirus type 2（PCV2） ORF2 truncated gene（47-207 aa） were cloned into the expression vector pCI-neo respectively,to constructe the expression plasmid pCI-ORF2-VP2.After transfected to PK15 cells,the fusion protein can express normally by the indirect immunofluorescence.Then the pCI-ORF2-VP2 plasmids was injected into the BALB/c mice,100 μg each,two times in the same dose and two weeks interval.In the meantime,the PPV vaccine,PCV vaccine and empty vector were set up as the control groups.At 7,14,21,28,42 days after immunization,spleen samples were collected for T lymphocyte proliferation assay,and the blood samples which obtained from the eyes were used to detecting of the PPV,PCV2 antibody and the dynamic monitoring of the Tlymphocyte subsets respectively.The results showed that,after immunized with pCI-ORF2-VP2,the reaction of the splenic T lymphocytes to the ConA stimulation,immune function of inducing the CD4＋,CD8＋ T lymphocyte and the PPV/PCV2 antibody levels were significantly higher than those of the empty vector control group at different periods,indicating that the eukaryotic expression plasmid pCI-ORF2-VP2 can stimulate the mice to produce satisfactory cytoimmunity and humoral immunity.

关 键 词：细小病毒 圆环病毒2型 VP2基因 ORF2基因 免疫 

分 类 号：S852.65[农业科学—基础兽医学]