A族链球菌表面蛋白Fba的原核表达及免疫原性分析

Prokaryotic expression and immunogenicity of Fba, a novel fibronectin-binding protein of group A streptococcus

目的 研究A族链球菌（GAS）表面新型纤连蛋白Fba的免疫原性，探讨GAS感染机体的免疫应答机制。方法 PCR扩增fba基因，测序正确后克隆至原核表达质粒pGEX4T-2，并在E．coli BL21中表达，应用ELISA和Western印迹法鉴定目的蛋白表达，以该蛋白作为抗原，包被酶联板．俭测GAS全菌免疫鼠血清、33份抗链球菌“0”阳性患者血清。同时，将该蛋白免疫Balb／C小鼠，3次免疫后收集血清，检测IgG效价。结果 ELISA和Western印迹证实，表达的Fba重组蛋白可与已知兔抗Fba阳性血清产生特异性反应；且Fba蛋白可与GAS全菌免疫鼠血清、抗链球菌“0”阳性患者血清发生特异性结合。Fba蛋白免疫小鼠后抗血清IgG效价达1：4800。结论 GAS感染或Fba蛋白免疫动物后均可诱导动物体产生Fba抗体，该抗体与Fba重组蛋白可产生特异性反应，提示Fba蛋白具有良好的免疫原性和抗原性。Fba蛋白可作为GAS的候选疫苗及检测GAS感染患者血清效价的重要工具。

Objective To express the novel fibronectin-binding protein Fba of groupA streptococcus （GAS） and analyze its immunogenicity, so that to evaluate the immune responses to GAS infection. Methods fba gene was amplified by polymerase chain reaction （PCR） and confirmed by sequencing. Then it was cloned into pGEX4T-2 vector and Fba protein was expressed in E. coli BL21. The protein expression was identified by enzyme-linked immunosorbent assay（ELISA） and Westernblot. The sera from mice infected with GAS and anti-streptolysin-O positive patients were detected using microtiter plates coated with purified Fba protein as antigen. Afterward Balb/C mice were immunized with this purified protein and the sera were collected after the third immunization for the detection of IgG titer. Results It was confirmed by ELISA and Western blot that the recombinant Fba protein had a specific affinity with anti-Fba sera of rabbit. The anti-serum IgG titer of mice immunized with Fba protein was up to 1 ： 4 800, Conclusions GAS infection or Fba protein immunization are able to induce high serum titer of anti-Fba which could react specifically with the recombinant Fba protein. It indicates that Fba protein has good immunogenicity and antigenicity. So Fba protein could be a GAS candidate vaccine and an important tool to detect anti-GAS titer in GAS infected patients.