抗弓形虫β微管蛋白小分子抗原肽多克隆抗体的制备及鉴定  

Preparation and Identification of Polyclonal Antibody against Small Peptides of β-tubulin of Toxoplasma gondii

作　　者：毛小勇[1] 华倩倩[2] 李相志[2] 姚丽丽[2] 谭峰[2] 

机构地区：[1]温州医科大学附属诸暨医院,诸暨311800 [2]温州医科大学基础医学院,温州325035

出　　处：《中国寄生虫学与寄生虫病杂志》2014年第4期322-323,共2页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：浙江省自然科学基金(No.LY12H19004;LQ14H190003)~~

摘　　要：对弓形虫GT1株、ME49株和人的β微管蛋白的氨基酸序列进行生物信息学分析。根据生物信息学分析结果,人工合成弓形虫β微管蛋白氨基酸序列C端小分子抗原肽。用合成的抗原肽免疫日本大耳兔,每2周免疫1次,0.5 mg/(只·次),共5次。末次免疫后2周,收集兔血清,ELISA检测血清中特异性抗体效价,蛋白质印迹(Western blotting)检测该多克隆抗体与弓形虫RH株、ME49株和PRU株速殖子蛋白的免疫反应。生物信息学分析结果表明,弓形虫GT1株与ME49株的β微管蛋白的氨基酸序列完全一致,与人的β微管蛋白氨基酸序列同源性为98%,差异氨基酸主要集中在C端。经5次免疫后,ELISA证实兔血清中抗体效价为1∶52 800。Western blotting分析结果显示,该多克隆抗体可分别与弓形虫RH株、ME49株和PRU株的β微管蛋白特异性结合。The amino acid sequences of β-tubulin from Toxoplasma gondii stains（GT1 and ME49） and human were aligned by ClustalW2 software. Based on the alignment result, the C-terminal peptides of β-tubulin of T. gondii were artificially synthesized. Rabbits were immunized with 0.5 mg synthesized peptides for five times at 2-week intervals.Serum samples were collected at the second week after the final immunization, and were analyzed for specific antibodies by ELISA. Finally, the specific-β-tubulin polyclonal antibody was evaluated by Western blotting with the total protein of RH strain, ME49 strain, and PRU strain of T. gondii, respectively. The results showed that β-tubulin of T. gondii stains（GT1 and ME49） shared 100% amino-acid sequence identity, and there was 98% amino acid homology between T. gondii and human. The main variable region was the C-terminus. After the fifth immunization, the titers of polyclonal antibody reached 1 ∶ 52 800. Western blotting result indicated that the specific-β-tubulin polyclonal antibody reacted withβ-tubulin in all the three strains（RH, ME49, and PRU）, respectively.

关 键 词：刚地弓形虫 Β-微管蛋白 多克隆抗体 

分 类 号：R382.5[医药卫生—医学寄生虫学]