A群链球菌四价重组蛋白的表达、纯化及其免疫原性

Expression,Purification and Immunogenicity of Tetravalent Recombinant Group A Streptococcus Protein

目的原核表达并纯化A群链球菌M1、3、6、18型四价重组蛋白,并检测其免疫原性。方法以克隆质粒pUC18-Strep4为模板,PCR法扩增四价重组蛋白基因,克隆至表达载体pQE30中,构建重组表达质粒,转化E.coilM15,筛选阳性克隆,IPTG诱导表达。采用Ni2+-NTA凝胶亲和层析纯化重组蛋白。以该蛋白免疫家兔,ELISA法检测血清抗体滴度;间接免疫荧光法(IFA)检测A群链球菌型特异性抗体;体外杀菌试验检测杀菌抗体活性。结果四价重组蛋白表达质粒pQE30-Strep4经双酶切和测序,证明构建正确。重组蛋白相对分子质量与预期相符,表达量约占菌体总蛋白的30%,为可溶性表达。纯化后蛋白纯度可达90%以上,并可诱导家兔产生高滴度的M1、3、6型特异性杀菌抗体。结论已成功表达了A群链球菌四价重组蛋白,纯化后的蛋白可诱导家兔产生针对A群链球菌M1、3、6三个血清型菌株的特异性杀菌抗体。

Objective To express tetravalent recombinant protein against group A streptococcus（GAS）of serotypes M1,3,6 and 18 in prokaryotic cells,purify the expressed product and determine its immunogenicity.Methods The target gene was amplified by PCR using cloning vector pUC18-Strep4 as a template and cloned into expression vector pQE30.The constructed recombinant plasmid was transformed to E.coli M15,and positive clones were screened for expression under induction of IPTG.The expressed recombinant protein was purified by Ni^2＋-NTA gel affinity chromatography.Rabbits were immunized with the expressed protein,and their serum antibody titers were determined by ELISA.The type-specific antibodies against M1,M3,M6 and M18 of GAS were determined by IFA,and serum bactericidal antibody activity by in vitro bactericidal test.Results Restriction analysis and sequencing proved that recombinant plasmid pQE30-Strep4 was constructed correctly.The expressed product in a soluble form,with a relative molecular mass consistent with that expected,contained about 30% of total somatic protein and reached a purity of more than 90%.Specific bactericidal antibody titers against serotypes M1,3 and 6 of GAS were induced in rabbits.Conclusion The tetravalent recombinant protein against GAS of serotypes M1,3,6 and 18 was successfully expressed.The purified recombinant protein induced specific bactericidal antibodies against serotypes M1,3 and 6 of GAS.