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目的:克隆tp15、tp17、tp47、tp0453基因,构建原核表达质粒,进行蛋白的表达、纯化并用于检测梅毒病人血清抗体。方法:PCR扩增4个基因,构建原核表达质粒pET28a(+)/tp15、pET28a(+)/tp17、pGEX-6P-1/tp47和pGEX-6P-1/tp0453,经双酶切及测序鉴定,将获得的阳性克隆转化到大肠杆菌BL21(DE3)中进行诱导表达,对表达产物进行SDS-PAGE电泳及Western blot分析鉴定,利用亲和层析方法对4个蛋白进行纯化。将四个纯化的蛋白固定到NC膜上,利用immuno-slot blot方法对梅毒病人血清进行检测。结果:经鉴定表明成功构建四个原核表达质粒。SDS-PAGE电泳及Western blot分析结果显示四个重组蛋白大小正确。immuno-slot blot实验显示四个蛋白能与梅毒病人血清特异性结合。结论:成功克隆、表达和纯化了tp15、tp17、tp47、tp0453蛋白,四个蛋白可以与梅毒病人血清特异性结合,为进一步建立梅毒病人血清检测试剂盒奠定基础。
OBJECTIVE: To clone the tp15, tp17, tp47 and tp0453 genes, construct prokaryotic expression plasmids, express and purify proteins and use them to detect serum antibodies in syphilis patients. Methods: Four genes were amplified by PCR. The prokaryotic expression plasmids pET28a (+) / tp15, pET28a (+) / tp17, pGEX-6P-1 / tp47 and pGEX-6P-1 / tp0453 were constructed and identified by double enzyme digestion and sequencing The obtained positive clones were transformed into E. coli BL21 (DE3) for inducing expression. The expressed products were identified by SDS-PAGE electrophoresis and Western blot analysis. The four proteins were purified by affinity chromatography. Four purified proteins were immobilized on NC membrane and the serum of syphilis patients was detected by immuno-slot blot. Results: Four prokaryotic expression plasmids were constructed successfully. SDS-PAGE electrophoresis and Western blot analysis showed that the four recombinant protein size is correct. Immuno-slot blot experiments showed that four proteins could specifically bind to the serum of syphilis patients. CONCLUSION: The successful cloning, expression and purification of tp15, tp17, tp47 and tp0453 proteins and the four proteins can specifically bind with the serum of syphilis patients, which will lay the foundation for further establishment of the serum test kit for syphilis patients.