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目的原核表达梅毒螺旋体-15(Treponema pallidum-15,TP-15)脂蛋白基因,并进行纯化及鉴定。方法人工合成455 bp的TP-15基因序列,克隆至原核表达载体p ETDuet-1,构建原核表达质粒p ETDuet-1/TP-15,经双酶切及测序鉴定后,将鉴定正确的重组质粒转化至E.coli BL21(DE3),经IPTG诱导表达;表达产物经Ni2+亲和层析纯化,纯化产物进行SDS-PAGE和Western blot分析。结果重组质粒p ETDuet-1/TP-15经双酶切及测序鉴定证明构建正确;表达产物相对分子质量约27 000,主要以包涵体形式存在,表达量约占菌体总蛋白的2.34%,纯化后蛋白纯度达99%以上,可与HRP标记的梅毒螺旋体多克隆抗体发生特异性结合。结论构建了原核表达质粒p ETDuet-1/TP-15,并于E.coli中成功表达,纯化后获得了高纯度的TP-15蛋白,为进一步研究建立梅毒病人血清检测试剂盒奠定了基础。
Objective To prokaryotic express the lipoprotein gene of Treponema pallidum-15 (TP-15) and purify it. Methods The 455 bp TP-15 gene was synthesized and cloned into the prokaryotic expression vector p ETDuet-1. The prokaryotic expression plasmid p ETDuet-1 / TP-15 was constructed. After double digestion and sequencing, the correct recombinant plasmid The recombinant protein was transformed into E.coli BL21 (DE3) and induced by IPTG. The expressed product was purified by Ni2 + affinity chromatography. The purified product was analyzed by SDS-PAGE and Western blot. Results The recombinant plasmid pETDuet-1 / TP-15 was confirmed by double enzyme digestion and sequencing. The recombinant plasmid pETDuet-1 / TP-15 was constructed correctly. The relative molecular mass of the expressed product was about 27 000, which mainly existed in the form of inclusion bodies. Purified protein purity of 99% or more, with HRP-labeled Treponema pallidum polyclonal antibodies specifically binding. CONCLUSION: The prokaryotic expression plasmid p ETDuet-1 / TP-15 was constructed and expressed in E.coli successfully. After purification, TP-15 protein with high purity was obtained, which laid the foundation for further study on establishing a serum test kit for syphilis patients.