产肠毒素大肠杆菌987P菌毛蛋白FasA亚单位的原核表达及其免疫原性

目的表达、纯化产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)987P菌毛蛋白FasA亚单位,并检测其免疫原性。方法将去掉5′端信号肽序列的fasA基因克隆至原核表达载体pQE-30中,构建重组表达质粒pQE-30-fasA,转化E.coli M15,0.5 mmol/L IPTG诱导表达。表达的融合蛋白6×His-FasA经Ni-Agarose His纯化和复性后,经皮下多点注射免疫BALB/c小鼠,每次100μg,共免疫3次,间隔2周,末次免疫10 d后,摘除小鼠眼球取血,分离血清,采用间接ELISA法检测抗血清效价。结果重组表达质粒pQE-30-fasA经PCR、双酶切和测序证实构建正确;表达的6×His-FasA融合蛋白相对分子质量约为18 500,主要以包涵体形式表达,表达量占菌体总蛋白的30%;纯化的蛋白纯度可达95%,浓度为0.6 mg/ml,免疫小鼠所得抗血清效价为1∶125 000。结论已成功在E.coli M15中表达了6×His-FasA融合蛋白,纯化的融合蛋白免疫原性良好,为进一步研制以双歧杆菌为表达系统的新型ETEC亚单位口服疫苗奠定了基础。

Prokaryotic expression and immunogenicity of FasA subunit of 987P fimbrial protein of enterptpxogemoc E. coli

Objective To express and purify the FasA subunit of fimbrial protein of enterotoxigenic Escherichia coli(ETEC) and determine its immunogenicity.Methods The fasA gene without signal peptide at 5′-terminus was cloned into the prokaryotic expression vector pQE-30,and the constructed recombinant plasmid pQE-30-fasA was transformed to E.coli M15 and induced with 0.5 mmol / L IPTG.The expressed fusion protein 6 × His-FasA was purified by protein purification kit with Ni-Agarose His tag and refolded.BALB / c mice were immunized with the fusion protein by subcutaneous injection at several sites,at a dosage of 100 μg,for 3 times each at an interval of 2 weeks,of which the sera were collected 10 d after the last immunization and determined for titer by ELISA.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pQE-30-fasA was constructed correctly.The expressed fusion protein 6 × His-FasA,with a relative molecular mass of about 18 500,mainly existed in a form of inclusion body,contained 30% of total somatic protein,reached a purity of 95% and a concentration of 0.6 mg / ml,and induced a serum antibody titer of 1 ∶ 125 000 in mice.Conclusion Fusion protein 6 × FasA was successfully expressed in E.coli M15 and showed good immunogenicity after purification,which laid a foundation of further preparation of Bifidobacterium-based novel oral ETEC subunit vaccine.