A族链球菌表面蛋白Fba保护性区段的鉴定  

Identification of protective truncated domain of Fba anchored on the surface of group A streptococcus

作　　者：魏澎[1] 马翠卿[1] 郭奕阳[1] 顾海燕[1] 冯惠东[1] 王秀荣[1] 魏林[1] 

机构地区：[1]河北医科大学免疫教研室,石家庄050017

出　　处：《中华传染病杂志》2010年第5期257-261,共5页Chinese Journal of Infectious Diseases

基　　金：基金项目：国家自然科学基金资助项目（30872399、30901350）;河北省自然科学基金资助项目（C2009001091）河北省卫生厅资助项目（08054）

摘　　要：目的将A族链球菌表面蛋白Fba分段克隆、表达，免疫动物并攻毒，以鉴定各区段诱导的保护性免疫并确定保护性最强的区段。方法根据Fba蛋白的结构特点将其划分为4个重叠区域，以A族链球菌（GAS）的SSI-9为模板，PCR扩增4段蛋白的基因，克隆、表达后，Western印迹鉴定蛋白表达。4段蛋白纯化后分别免疫小鼠，同时设PBS对照组。ELISA检测各免疫组血清IgG水平，并用致死量的GAs（M＋Fba＋）攻击免疫3次后的小鼠，计算各组保护率。数据行方差检验和Fisher精确概率法。结果成功构建原核表达质粒pGEX-2T／FbaA1、pGEX-2T／FbaA2、pGEX2T／FhaA3和pGEX2T／FbaA4，成功表达了4段蛋白。Fba蛋白免疫小鼠后，实验组血清IgG随免疫次数的增加呈逐渐增高趋势。第3次免疫后，与PBS对照组比较，FbaA2蛋白组效价升高最为明显，达1：102400（F=1290．2，P〈0．01）。攻毒后，FbaA2蛋白组8只小鼠中有4只得到保护，FbaA1、FbaA3及FbaA4蛋白组各8只小鼠中，仅2只得到保护（P〈0．05）。结论成功构建、表达了Fba分段蛋白；初步确定FbaA2段可诱导较强的保护性免疫应答。Objective The truncated fibronectin-binding proteins A （Fba protein） were cloned and expressed, then animals were immunized with Fba protein and subsequently challenged with group A streptococcus （GAS） to further investigate protective antibodies induced by each domain and determine the most immunogenie domain. Methods Fba proteins, which were divided into four overlaps based on the structural domains, were truncated and expressed. The fba truncated genes were amplified by polymerase chain reaction （PCR） with SSI 9 of GAS as template, and cloned into prokaryotic expression plasmid pGEX 2T and expressed in E. coli BL- 21. The products were confirmed by Western blot and purified by affinity chromatography column. Female BALB/c mice were immunized with the four truncated proteins respectively, with phosphate buffered solution （PBS） as control. The serum IgG of mice was detected by enzyme-linked immunosorbent assay （ELISA）. After the third immunization, mice were challenged with fatal dose of GAS （M＋ Fba＋ ） to evaluate the protective rates in each group. The data were compared by analysis of variance and Fisher＇s exact test. Results Prokaryotic expression plasmids of pGEX2T/FbaA1, pGEX-2T/FbaA2, pGEX 2T/FbaA3 and pGEX-2T/FbaA4 were successfully constructed and the four truncated proteins were expressed and purified successfully. Serum levels of IgG in each experimental group gradually increased with immunization with Fba protein more times. After the third immunization, the IgG titer against FbaA2 protein group （1 ： 102 400） increased most significantly compared with PBS control group （F = 1290.2, P〈0. 01）. After GAS challenge, four out of eight mice were protected in FbaA2 protein group, while two out of eight mice in FbaA1 protein, FbaA3 protein and FbaA4 protein groups, respectively （P〈 0. 05）. Conclusions Four truncated Fba proteins are constructed and expressed successfully. Truncated FbaA2 protein could be able to induce strongest protective immune response.

关 键 词：A族链球菌 纤连蛋白类 基因表达 质粒 印迹法 蛋白质 

分 类 号：R392[医药卫生—免疫学]