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目的原核表达并纯化肺炎支原体(Mycoplasma pneumoniae,MP)黏附蛋白P1羧基末端。方法提取MP FH型基因组DNA,以其为模板,PCR扩增P1羧基末端基因,插入原核表达载体pET-32a(+),构建重组表达质粒,转化E.coli Rosetta,IPTG诱导表达。表达的重组蛋白经镍离子亲和层析纯化后,进行SDS-PAGE、Western blot和ELISA鉴定。结果重组表达质粒pET-32a-MP-P1C经PCR、双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约55 900,以可溶性形式表达,表达量占菌体总蛋白的47%;纯化的重组蛋白纯度达94%以上,浓度为3.77 mg/ml,可与支原体肺炎患者血清特异性反应,经3 000倍稀释后,仍可与支原体肺炎患者血清发生阳性反应。结论原核表达并纯化了MP黏附蛋白P1羧基末端,为进一步研究MP的致病机制及研发诊断试剂和基因疫苗奠定了基础。
Objective To prokaryotic express and purify the carboxyl terminal of adhesion protein P1 of Mycoplasma pneumoniae (MP). Methods The MP FH genomic DNA was extracted and used as a template to amplify the P1 carboxyl terminal gene. The recombinant plasmid was inserted into prokaryotic expression vector pET-32a (+) to construct a recombinant plasmid. The recombinant plasmid was transformed into E.coli Rosetta and induced by IPTG. The expressed recombinant protein was purified by nickel ion affinity chromatography and identified by SDS-PAGE, Western blot and ELISA. Results The recombinant plasmid pET-32a-MP-P1C was constructed correctly by PCR, double enzyme digestion and sequencing. The recombinant protein was expressed in soluble form with a relative molecular mass of about 55 900, accounting for 47% of the total bacterial proteins. Purified recombinant protein purity of 94% or more, a concentration of 3.77 mg / ml, mycoplasma pneumonia patients with serum-specific reaction after 3000 dilution, still with mycoplasma pneumonia patients serum positive reaction. Conclusion Prokaryotic expression and purification of the carboxyl terminus of MP adhesion protein P1 lay the foundation for further study on the pathogenesis of MP and the development of diagnostic reagents and gene vaccines.