产肠毒素性大肠埃希菌K99-987P-F41原核表达载体的构建、表达及其纯化

Construction of prokaryotic expression vector for fimbrial subunit gene K99-987P-F41 of enterotoxigenic Escherichia coli as well as expression and purification of target protein

目的构建产肠毒素性大肠埃希菌(enterotoxigenic Escherichia coli,ETEC)K99-987P-F41原核表达载体,诱导表达后进行纯化。方法利用PCR技术,从ETEC基因组DNA中扩增出K99、987P、F41部分基因片段,将3段目的基因融合亚克隆至大肠埃希菌原核表达载体p ET30a(+),构建重组质粒p ET30a(+)-K99-987P-F41,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达,表达的重组蛋白经镍离子亲和层析纯化。结果重组表达质粒p ET30a(+)-K99-987P-F41经PCR、酶切及测序鉴定证明构建正确。表达的重组蛋白相对分子质量约60 000,主要以包涵体形式存在,表达量约占菌体总蛋白的63.8%,纯度为81.2%,浓度为8.953 mg/ml,可同时被天然和重组抗原多抗识别,具有良好的反应原性。结论 ETEC重组质粒p ET30a(+)-K99-987P-F41可在大肠埃希菌BL21(DE3)中表达,利用镍离子亲和层析柱纯化可获得融合蛋白。

Objective To construct a prokaryotic expression vector for fimbraial subunit gene K99-987P-F41 of Enterotoxigenic Escherichia coli（ETEC）, and express and purify the target protein. Methods The K99, 987 P and F41 gene fragments were partly amplified by PCR from the genomic DNA of ETEC, and subcloned into prokaryotic expression vector p ET30a（＋）in fusion. The constructed recombinant plasmid p ET30a（＋）-K99-987P-F41 was transformed to E. coli BL21（DE3）and induced by IPTG. The expressed recombinant protein K99-987P-F41 was purified by nickel ion affinity chromatography. Results PCR, restriction analysis and sequencing proved that recombinant plasmid p ET30a（＋）-K99-987P-F41 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 60 000,mainly existed in a form of inclusion body and contained about 63. 8% of total somatic protein, of which the purity and concentration were 81. 2% and 8. 953 mg / ml respectively. The protein was recognized by polyclonal antibodies against natural and recombinant antigens, which showed good reactogenicity. Conclusion Recombinant plasmid p ET30a（＋）-K99-987P-F41 may be expressed in E. coli BL21（DE3）, and the fusion protein may be obtained by purification by nickel ion affinity chromatography.