摘要
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 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.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第9期1102-1105,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金项目资助(NSFC 30972585)
关键词
产毒素大肠杆菌
菌毛蛋白质类
FasA亚单位
原核细胞
基因表达
免疫原性
Enterotoxigenic Escherichia coli（ETEC）
Fimbrial proteins
FasA subunit
Prokaryotic cells
Gene expression
Immunogenicity
作者简介
通讯作者：马永平，E—mail：ypmaOg09@yahoo．com．cn
