小鼠IFN-γ基因的克隆及其分子佐剂作用的研究  

Cloning of Murine IFN-γ and its Effect as a Molecular Adjuvant

作　　者：刘勇[1] 王海燕[1] 张甲[1] 王雨舟[1] 张冰[1] 

机构地区：[1]中国农业大学动物医学院,北京100094

出　　处：《实验动物与比较医学》2008年第6期367-371,共5页Laboratory Animal and Comparative Medicine

摘　　要：目的通过融合表达小鼠IFN-γ(MuIFN-γ)与猪脑心肌炎病毒(EMCV)结构蛋白VP1研究MuIFN-γ的分子佐剂作用。方法通过RT-PCR克隆IFN-γ的成熟肽基因,从pGEX-6p-1/vp1中亚克隆猪脑心肌炎病毒(EMCV)抗原基因VP1,分别构建原核表达菌株pET32-a/VP1/Rosetta(DE3)和pET32-a/VP1/MuIFN-γ,将VP1及VP1与MuIFN-γ在宿主菌Rosetta(DE3)中进行表达与共表达。将表达的VP1及MuIFN-γ/VP1蛋白分别免疫BALB/c小鼠,每次免疫2周后采血,分离血清,利用ELISA测定抗体效价。结果表达的VP1蛋白分子量为60kD,共表达蛋白MuIFN-γ/VP1分子量为73kD,VP1及MuIFN-γ/VP1蛋白免疫小鼠测定的抗体效价分别为1:32000和1:128000。结论表达的VP1蛋白具有良好的免疫原性,可刺激免疫小鼠产生明显的体液免疫反应。MuIFN-γ/VP1免疫小鼠后产生的佐剂效果要强于弗氏佐剂。MuIFN-γ/VP1蛋白能增强小鼠的体液免疫水平,MuIFN-γ具有一定的分子佐剂效应。Objective Study the molecular adjuvant effect of MuIFN-γ by co-expressing MuIFN-γ and EMCV structural protein VP1. Method Mature peptide gene of MuIFN-γ was cloned by using RT- PCR. VP1 of pig EMCV was subcloned from pGEX-6p-1/vp1, and constructed into the express plasmid pET32-a/VP1/Rosetta（DE3） and co-expression plasmid pET32-a/VP1/MuIFN-γ respectively. BALB/c mice were inoculated with the VP1 and MuIFN-γ/VP1 proteins in E.coli, respectively, for three times at two week intervals, serum IgG were detected by ELISA every two weeks post-inoculation. Result In E.coli Rosetta（DE3）, expression and co-expression were succeeded IPTG （Isopropyl β-D-1- Thiogalactopyranoside） induction, with 60 MW of VP1 protein and 73 MW of co-expressed protein. The serum IgG of mice post-inoculated by VP1 and MuIFN-γ/VP1 are 1 ： 32000 and 1 ： 128000. Conclusions VP1 had good antigenicity which could stimulated humoral immune response in inoculated mice, the detected serum IgG of MuIFN-γ/VP1 inoculation is higher than VP1. Moreover, immune enhancing effects was found in mice inoculated with co-expressed MuIFN-γ/VP1 .￡,MuIFN-γ was a satisfactory immune adjuvant.

关 键 词：MuIFN—γ EMCV VP1 原核表达 免疫佐剂 

分 类 号：Q78[生物学—分子生物学]