猪链球菌H因子结合蛋白截短体的表达、纯化及其结合人血清IgG的活性分析  

Expression,purification and hIgG-binding activity identification of truncated Streptococcus suis factor H-binding protein

作　　者：李雪琴[1,2] 刘鹏[2] 骈亚亚[2] 郑玉玲[2] 姜永强[1,2] 袁媛[2] 霍春月[3] 

机构地区：[1]安徽医科大学研究生学院,安徽合肥230032 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [3]首都医科大学燕京医学院,北京101300

出　　处：《细胞与分子免疫学杂志》2014年第5期537-541,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30870091)

摘　　要：目的构建带His标签的2型猪链球菌H因子结合蛋白(Fhb)截短表达片段Fhb-N(45-344aa)和Fhb-C(345-664aa)的重组质粒,并在大肠杆菌中表达,获得高纯度的重组蛋白,鉴定其与人血清IgG(hIgG)结合的活性。方法以05ZYH33基因组为模板并设计引物扩增目的片段,构建His-Fhb-N和His-Fhb-C重组表达质粒,利用亲和层析纯化目的蛋白并用Western blot法进行验证。蛋白G亲和层析柱从健康人血清中纯化人IgG(hIgG),Western blot法和生物膜干涉(BLI)鉴定和分析His-Fhb与hIgG结合的活性。结果成功构建了带His标签的原核表达质粒并获得了His-Fhb-N和His-Fhb-C截短表达蛋白,验证出Fhb特异性地与hIgG结合,且结合区域位于Fhb-N(45-344aa)。结论 Fhb能够结合hIgG,结合区域位于Fhb-N。Objective To construct a prokaryotic expression vector of the His-tagged truncated factor H-binding protein （Fhb） fragments, Fhb-N （amino acids 45-344aa） and Fhb-C （amino acids 345-644aa）, of Streptococcus suis serotype 2, express it in E. coli BI_21 （DE3） in order to acquire high-purity recombinant protein, and finally identify the binding activity with human serum IgG （hlgG）. Methods Fhb-N gene and Fhb-C gene were amplified using the primers designed according to 05ZYH33 genome sequences and cloned into the expression vector pET28a （ ＋ ） to construct recombinant plasmids. The plasmids were transformed into E. coli B1_21 （DE3） and induced to express by IPTG. The recombinant proteins were purified by nickel affinity chromatography and identified by Western blotting. The hlgG was purified from human serum by HiTrap protein G HP column in accordance with the manufacturer＇s instructions. In addition, the specific binding to hlgG was identified by Western blotting and biolayer Interferometry （BLI）. Results The prokaryotic expression vector of His-Fhb-N and His-Fhb-C was constructed, and the target proteins were expressed, purified and identified. The specific binding activity with hlgG was identified and the binding region was found located on the Fhb-N （45-344aa）. Conclusion His-Fhb-N can specifically bind to hlgG, which will help us to further study the role of Fhb-hlgG interaction in the pathogenesis of Streptococcus suis.

关 键 词：2型猪链球菌 Fhb 原核表达 人血清IgG 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]