【摘要】 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。更多还原

【Abstract】 Objective To express the carboxyl terminus of adhesion P1 of Mycoplasma pneumoniae(MP) in prokaryotic cells and purify the expressed product.Methods Genomic DNA of MP type FH was extracted and used as a template,with which the carboxyl terminal gene of MP was amplified by PCR and inserted into prokaryotic expressed vector pET-32a(+).The constructed recombinant plasmid pET-32a-MP-P1C was transformed to E.coli Rosetta for expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and identified by SDS-PAGE,Western blot and ELISA.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32a-MP-P1C was constructed correctly.The recombinant protein with a relative molecular mass of about 55 900 was expressed in a soluble form,and contained 47% of total somatic protein.The purified recombinant protein,at a concentration of 3.77 mg/ml,reached a purity of more than 94% and showed specific reaction with the sera of patients infected with MP,even at a dilution of 1 ∶ 3 000.Conclusion The carboxyl terminus of adhesion P1 of MP was expressed in prokaryotic cells and purified,which laid a foundation of further study on pathogenic mechanism of MP as well as deve-lopment of diagnostic kit and DNA vaccine.更多还原