弓形虫棒状体蛋白2和膜表面蛋白1重组嵌合抗原免疫血清的制备与鉴定  

Preparation and Identification of Immune Serum against Recombinant Fusion Protein of Rhoptry 2 and Major Surface Protein 1 from Toxoplasma gondii

作　　者：李文姝[1] 朱珊丽[1] 王鹏飞[1] 张丽芳[1] 闵太善[2] 黄伟达[2] 

机构地区：[1]温州医学院微生物学免疫学教研室,温州325032 [2]复旦大学生化与分子生物学实验室,上海200433

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第5期397-400,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：浙江省自然科学基金(No.Y205567);浙江省教育厅科技项目(No.20051155)~~

摘　　要：目的制备与鉴定弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)重组嵌合抗原的特异性免疫兔血清。方法将已构建的重组质粒pET28b/ROP2-P30转化大肠埃希菌BL21-Codon Plus(DE3)-RIL菌株,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,分离上清液和包涵体,包涵体用2mol/L和4mol/L尿素梯度洗涤去杂蛋白,最后用8mol/L尿素溶解洗涤后的包涵体,进行十二烷基磺酸钠-聚丙稀酰胺凝胶电泳(SDS-PAGE),割胶回收目的蛋白,研磨成乳浊状后以背部多点注射免疫大白耳兔,制备免疫兔血清,以免疫扩散试验检测生成的特异性抗体,间接ELISA法测定免疫血清的抗体效价,蛋白质印迹(Western blotting)分析免疫血清的抗体特异性。结果免疫结束后,通过对流免疫扩散试验检测到了针对rROP2-P30的特异性抗体,间接ELISA法测定到其抗体滴度最高可达1∶12800,Western blotting分析结果表明,此免疫血清与可溶性的重组蛋白、包涵体形式的重组蛋白均能特异性结合。结论得到了针对rROP2-P30重组嵌合抗原的特异性免疫血清。Objective To prepare and identify immune serum against the recombinant fusion protein of rhoptry 2 （ROP2） and major surface protein 1 （P30） from Toxoplasma gondii. Methods The constructed recombinant plasmid of pET28b/ROP2-P30 was transformed to a bacterium BI21-Codon Plus （DE3）-RIL strain and was expressed under IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body respectively. The inclusion body was washed with 2 mol/L and 4 mol/L urea to remove the nonspecific protein. The washed products dissolved in 8 mol/L urea were received by SDS-PAGE. Two rabbits were immunized with the fusion protein rROP2-P30 and sera from the rabbits were collected. Immune diffusion test, indirect ELISA and Western-blot were used to detect antibody titer and specificity of the immune serum against rROP2-P30. Results Immune diffusion test demonstrated that specific immune serum were obtained. Indirect ELISA confirmed that the antibody titer in the serum reached 1：12 800 and the rROP2-P30 was recognized by specific IgG in this serum by Western-blot analysis. Conlusion Specific immune serum against the recombinant fusion protein rROP2-P30 has been prepared.

关 键 词：弓形虫 重组嵌合抗原 ROP2-P30 免疫血清 

分 类 号：R382.5[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]