刚地弓形虫主要抗原B细胞表位的筛选及鉴定  

Screening and identification of B cell epitopes of Toxoplasma gondii

作　　者：曹利民[1] 潘宇红[1] 卢志贤[1] 陈江[1] 陈蓉芳[1] 程华莉[1] 姜东林[1] 司进[2] 章辉[2] 朱荫昌[2] 

机构地区：[1]江苏省无锡市第三人民医院,无锡214041 [2]江苏省寄生虫病防治研究所

出　　处：《中国血吸虫病防治杂志》2007年第1期24-27,共4页Chinese Journal of Schistosomiasis Control

基　　金：江苏省寄生虫分子生物学重点实验室;江苏省寄生虫病学重点学科开放课题(WK005-008)

摘　　要：目的筛选刚地弓形虫主要抗原B细胞表位并在原核表达载体中表达,对纯化的表位重组蛋白进行免疫反应性鉴定。方法用计算机软件BioSun、DNAstar综合分析6个弓形虫主要抗原的亲水性、可及性、柔韧性、二级结构、极性参数等,每一抗原分子预测2个最佳表位,设计合成24条共12对寡核苷酸,两端分别引入NocI、XhoI酶切位点,退火形成的双链定向克隆至原核表达载体pET-32c中,EcoRI单酶切鉴定重组质粒Epitope/pET-32c。将含有Epitope/pET-32c的BL21单菌落接种至LB肉汤培养基中培养,导入表达。表达产物用Ni2+螯合柱亲和纯化,免疫印迹试验(Westernblot)分析重组表位蛋白与弓形虫抗原免疫血清的免疫反应性。结果成功构建了12个原核表达质粒Epitope/pET-32c,并经测序证实,在E.coliBL21有11个表位基因能有效表达重组融合蛋白;纯化的表位蛋白大小均在20.0kDa左右;Westernblot结果显示表位蛋白SAG2-A、SAG3-B能被弓形虫抗原免疫血清识别,且反应较强,SAG2-B为弱阳性,其余的则不能被识别。结论成功筛选到3个弓形虫B细胞表位基因,为进一步构建弓形虫复合表位抗原诊断弓形虫病奠定了基础。Objective To screen and identify B cell epitopes in SAG1, SAG2, SAG3, GRA1, GRA6 and P35 antigens of Toxoplasma gondii. Methods The indexes such as hydrophilicity, ac cessibility, flexibility, secondary structure and polarity of the 6 antigen moleculars above mentioned were analyzed by BioSun system. Two B cell epitopes with high antigenicity from each antigen molecular were selected, and the total twelve pairs of oligonucleotide chains were designed accord- ing to the 12 B cell epitopes＇ sequence and synthesized, then cloned into plasmid pET-32c. The 12 fragment B cell epitopes were expressed and the expressed fusion proteins were identified with Western blot. Results Twelve B cell epitopes from 6 Toxoplasma antigens （two from each antigen） were predicted and selected. The epitope genes were successfully cloned into pET-32c and expressed. Western blot results showed that 3 of 12 expressed fusion proteins could be recognized by the immunized rabbit sera with soluble antigen of Toxoplasma gondii, but not by the unimmunized rabbit sera. Conclusion Three B cell epitopes of Toxoplasma with potential diagnostic value are obtained.

关 键 词：弓形虫 抗原 B细胞表位 鉴定 

分 类 号：R382.5[医药卫生—医学寄生虫学]