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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。
Objective To express and purify the 987P pili protein FasA subunit of Enterotoxigenic Escherichia coli (ETEC) and test its immunogenicity. Methods The fasA gene with 5 ’signal peptide sequence was cloned into the prokaryotic expression vector pQE-30. The recombinant plasmid pQE-30-fasA was constructed and transformed into E. coli M15 and induced with 0.5 mmol / L IPTG. The expressed fusion protein 6 × His-FasA was purified and refolded by Ni-Agarose His. The BALB / c mice were immunized subcutaneously with multiple injections at a dose of 100 μg for three times at intervals of 2 weeks. After the last immunization for 10 days , Remove the mouse eyeball blood, serum was separated, indirect ELISA method to detect antiserum titer. Results The recombinant plasmid pQE-30-fasA was confirmed by PCR, restriction enzyme digestion and sequencing. The relative molecular mass of the expressed 6 × His-FasA fusion protein was about 18 500, which was mainly expressed in inclusion bodies, 30% of the total protein; Purified protein purity of up to 95%, a concentration of 0.6 mg / ml, immune mouse serum titer obtained 1: 125 000. Conclusion The 6 × His-FasA fusion protein has been successfully expressed in E. coli M15. The purified fusion protein has good immunogenicity, which lays the foundation for the further development of a novel ETEC subunit vaccine with Bifidobacterium expression system.