梅毒螺旋体Tp0259假定蛋白的原核表达、纯化及活性研究

发布时间：2018-01-11 02:07

本文关键词：梅毒螺旋体Tp0259假定蛋白的原核表达、纯化及活性研究　出处：《中国病原生物学杂志》2017年02期 　论文类型：期刊论文

【摘要】：目的表达、纯化梅毒螺旋体(Tp)重组蛋白Tp0259(rTp0259),鉴定其免疫原性,并探讨其在梅毒血清诊断中的价值。方法 PCR扩增tp0259基因,构建pET-28a(+)/Tp0259原核表达载体,转化至E.coli BL21中诱导表达。采用Ni-NTA亲和层析法纯化重组蛋白,并进行Western blot鉴定。以重组蛋白Tp0259为抗原,采用Western blot方法检测梅毒患者血清(一期梅毒患者血清21份,二期梅毒患者血清13份,潜伏梅毒患者血清6份,并与梅毒诊断金标准比较)。结果 PCR扩增获得600bp左右大小的目的基因片段并成功构建原核表达载体pET28a-Tp0259,经诱导高效表达分子质量单位为28ku的重组蛋白。Western blot证实该重组蛋白为Tp0259假定蛋白,能被梅毒患者血清识别。以Tp0259为抗原采用Western blot检测梅毒患者血清特异性抗体结果与梅毒诊断金标准(临床症状、病史结合血清学诊断方法)间的κ值为0.944,表明这两种方法检测结果的一致性较好。结论成功表达、纯化了可溶性rTp0259,该蛋白具有良好的免疫反应性并具有较高的血清学诊断价值。
[Abstract]:Objective to express and purify the recombinant protein Tp0259rTp0259 from Treponema pallidum and identify its immunogenicity. Methods the tp0259 gene was amplified by PCR and the prokaryotic expression vector pET-28awas constructed. The recombinant protein was purified by Ni-NTA affinity chromatography. The recombinant protein Tp0259 was used as antigen to detect 21 sera of syphilis patients by Western blot method. There were 13 sera for secondary syphilis and 6 for latent syphilis. Results the target gene fragment of about 600bp was obtained by PCR amplification and the prokaryotic expression vector pET28a-Tp0259 was constructed successfully. The recombinant protein with a molecular weight unit of 28 ku was highly expressed. Western blot confirmed that the recombinant protein was assumed to be a Tp0259 protein. Using Tp0259 as antigen, Western blot was used to detect the serum specific antibody of syphilis patients and the diagnostic gold standard for syphilis (clinical symptoms). The 魏 value between the two methods was 0.944, which indicated that the two methods were in good agreement. Conclusion the soluble rTp0259 was successfully expressed and purified. The protein has good immunoreactivity and high serological diagnostic value.
【作者单位】：南华大学附属第二医院检验科;南华大学医学院病原生物学研究所特殊病原体防控湖南省重点实验室;南华大学附属第二医院消化内科;
【基金】：国家自然科学基金项目(No.81471576) 湖南省分子靶标与新药物研究协同创新中心项目(No.2015-351) 特殊病原体防控湖南省重点实验室项目(No.2014-5) 湖南省科技厅一般项目(No.2014TT2025)
【分类号】：R377.1
【正文快照】： *?**?梅毒是由梅毒螺旋体(Treponema　pallidum,Tp)感染引起的一种性传播疾病。目前,梅毒血清学检查仍然是梅毒诊断的重要依据。在过去几十年,性病研究实验室试验(VDRL)、快速血浆反应素试验(RPR)、梅毒螺旋体颗粒凝集试验(TPPA)和荧光密螺旋体抗体吸收试验(FTA-ABS)等方法已

【相似文献】