梅毒螺旋体TP 0993重组蛋白的表达、纯化及免疫活性研究

发布时间：2018-04-10 20:12

本文选题：梅毒螺旋体 + 重组蛋白　；参考：《南华大学》2011年硕士论文

【摘要】：目的:构建含梅毒螺旋体(Treponema pallidum, TP)外膜蛋白TP 0993的重组表达载体,在大肠杆菌中进行诱导表达,纯化表达产物并进行免疫原性和抗原性分析,为探索TP 0993重组蛋白在梅毒血清学诊断中的应用价值和其生物学功能提供实验依据。方法:通过Genbank获取选TP 0993基因序列,以TP Nichols株基因组DNA为模板,PCR扩增目的片段,将其亚克隆至原核表达载体pET28a(+)中构建重组质粒pET28a(+)/TP 0993,pET28a(+)/TP 0993经PCR、双酶切、测序鉴定后将其转化至表达宿主菌E.coli Rosseta中,IPTG诱导表达。采用SDS-PAGE和Western-blot分析和鉴定表达产物;Ni-NTA亲和层析柱纯化重组蛋白,BCA法测定蛋白浓度。用纯化的TP 0993重组蛋白包被微孔板,建立间接ELISA方法,检测临床梅毒患者血清和健康体检正常血清,同时与TPPA法进行比较,根据重组蛋白与梅毒阴阳性血清的反应情况,评价重组抗原在梅毒血清学诊断中的应用价值。同时用纯化的TP 0993重组蛋白免疫新西兰兔,间接ELISA方法检测免疫兔血清中TP 0993多克隆抗体的效价,对TP 0993重组蛋白的免疫原性进行分析。结果:通过Genbank获取选TP 0993全基因序列;PCR扩增得到其目的片段;构建的重组质粒经酶切和测序鉴定证实插入片段为目的基因;SDS-PAGE结果显示,在IPTG诱导下,重组表达菌分别表达了一相对分子量(Mr)约为34KDa的重组蛋白,目的蛋白在菌体细胞内主要以包涵体形式存在,经Ni-NTA亲和纯化后,纯度在85%以上;以重组蛋白为包被抗原建立间接ELISA法;对TPPA法检测的480份阴阳性血清进行检测,与TPPA法比较ELISA法的灵敏度为88.3%,特异度为85.8%,ELISA法与TPPA法符合率为86.5%;利用纯化的TP 0993重组蛋白免疫新西兰兔,间接ELISA法测定兔免疫血清特异性抗体效价在1: 640以上。结论: 1、成功构建了pET28a(+)/TP 0993原核表达体,并能在大肠杆菌中表达出TP 0993重组蛋白。 2、TP 0993重组蛋白能刺激新西兰兔产生较高效价的特异性抗体,具有较好的免疫原性。 3、TP 0993重组蛋白能与梅毒阳性血清特异性结合,具有良好的抗原性,可应用于梅毒血清学诊断。
[Abstract]:Objective: to construct a recombinant expression vector containing Treponema pallidum (TPTP) outer membrane protein TP 0993, and express it in Escherichia coli, purify the expressed product and analyze its immunogenicity and antigenicity.To explore the application value and biological function of TP 0993 recombinant protein in syphilis serological diagnosis.Methods: the selected TP 0993 gene sequence was obtained by Genbank. The target fragment was amplified by genomic DNA of TP Nichols strain and subcloned into prokaryotic expression vector pET28a (pET28a) to construct the recombinant plasmid pET28a (pET28a).After sequencing, it was transformed into E.coli Rosseta, which was induced by IPTG.The recombinant protein was purified by SDS-PAGE and Western-blot, and the protein concentration was determined by Ni-NTA affinity chromatography.To evaluate the value of recombinant antigen in the serological diagnosis of syphilis.New Zealand rabbits were immunized with purified TP 0993 recombinant protein. The titers of TP 0993 polyclonal antibody were detected by indirect ELISA method, and the immunogenicity of TP 0993 recombinant protein was analyzed.Results: the whole gene sequence of TP 0993 was amplified by Genbank, and the recombinant plasmid was confirmed by restriction endonuclease digestion and sequencing. The result of SDS-PAGE showed that the recombinant plasmid was induced by IPTG.The recombinant expression strain expressed a relative molecular weight of 34KDa protein. The target protein was mainly in the form of inclusion body in the cell. After Ni-NTA affinity purification, the purity of the protein was more than 85%.Indirect ELISA assay was established with recombinant protein as coating antigen, and 480 serum samples of yin and yang were detected by TPPA method.Conclusion:1. The prokaryotic expression of pET28a (P / TP 0993) was successfully constructed, and the recombinant protein of TP 0993 was expressed in E. coli.The recombinant protein TP 0993 could stimulate New Zealand rabbits to produce specific antibodies with high titer and had good immunogenicity.The recombinant protein of TP 0993 can specifically bind to syphilis positive serum and has good antigenicity. It can be used in the serological diagnosis of syphilis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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