梅毒螺旋体膜蛋白的表达、纯化及其免疫活性的研究

发布时间：2018-05-18 17:50

本文选题：梅毒螺旋体 + 膜蛋白　；参考：《北京协和医学院》2012年博士论文

【摘要】：研究背景及目的梅毒螺旋体膜蛋白在细胞间接触、表面识别、信号转导、酶活性和运输等方面都扮演着重要的角色,通过对梅毒螺旋体重组抗原的研究有利于鉴别并筛选可用于梅毒早期实验室诊断及疫苗研制的候选抗原,同时对于深入研究梅毒的发病机制、了解在梅毒螺旋体感染过程中宿主-病原菌的相互关系,具有重要意义。然而,资料表明,对梅毒螺旋体膜蛋白的研究一直较为困难,研究结果也存在着争议,为此我们利用基因工程技术,采用大肠杆菌表达系统,分别对Tp0965、Tp0136及Tp0608三种蛋白进行表达,对表达出的蛋白进行相关免疫活性的检测,并探讨重组蛋白Tp0965作为包被抗原建立间接ELISA的方法应用于梅毒诊断的可行性。研究方法使用Tp Nichols标准株接种家兔睾丸,制备Tp Nichols菌株DNA, PCR扩增Tp0965、Tp0608基因,Tp0136基因采用全基因合成的方法；构建重组质粒pET-28a/Tp0965、Tp0136及Tp0608,分别进行BamHI/SaI1、BamHI/Hindlll及Ndel/Xhol双酶切鉴定,鉴定正确后大肠杆菌转化,IPTG诱导表达,用SDS-PAGE和Western blot鉴定,并纯化重组蛋白,BCA法检测重组蛋白浓度。用纯化的重组蛋白Tp0965、p0136免疫新西兰兔,并以该重组蛋白作为包被抗原建立间接ELISA法以检测免疫兔的多克隆抗体效价；Western blot检测重组的Tp0965、Tp0136及Tp0608与梅毒阳性血清的免疫反应。以重组蛋白Tp0965为包被抗原建立间接ELISA检测各期梅毒患者血清。研究结果 (1)成功构建目的基因与E. coli表达质粒载体pET28,成功表达和纯化出具有较高纯度和浓度的膜蛋白Tp0965、Tp0136及Tp0608。(2)用重组蛋白Tp0136免疫新西兰兔,在免疫2周后即能检测到抗体的产生,免疫3次后产生高效价抗体；而重组蛋白Tp0965只有在免疫3次以后,才能刺激兔产生较低效价的抗体。(3) Westernblot实验显示三种重组蛋白Tp0965、Tp0136及Tp0608均能与梅毒阳性血清发生特异性反应,在电泳图上有特异性条带出现。(4)以重组蛋白Tp0965作为包被抗原建立间接ELISA诊断各期梅毒,阳性率高达94.6%,而与梅毒阴性血清不能结合。结论 (1)能够通过大肠杆菌表达系统重组出具有全基因片段长度的膜蛋白Tp0965、 Tp0136及Tp0608; Tp0965可以高表达,但Tp0136及Tp0608纯化较为困难。(2)Tp0136能刺激机体产生多克隆抗体,具有较强的免疫原性,而Tp0965的免疫原性较弱。(3) Tp0965、Tp0136及Tp0608均具有一定的免疫反应性,能够与梅毒阳性血清反应。(4)重组蛋白Tp0965有作为候选抗原应用于梅毒血清学检测的可能,需要进一步扩大样本验证。
[Abstract]:Research background and purpose Treponema pallidum membrane proteins play an important role in cell contact, surface recognition, signal transduction, enzyme activity and transport. The study of Treponema pallidum recombinant antigen is helpful for the identification and screening of candidate antigens for early laboratory diagnosis and vaccine development of syphilis, and for the further study of the pathogenesis of syphilis. It is important to understand the relationship between host and pathogenic bacteria during Treponema pallidum infection. However, the data showed that the study of Treponema pallidum membrane protein was always difficult, and the results were controversial. Therefore, we expressed Tp0965Tp0136 and Tp0608 by using the gene engineering technique and E. coli expression system, respectively. To detect the immunological activity of the expressed protein, and to explore the feasibility of establishing indirect ELISA with recombinant protein Tp0965 as a coated antigen in the diagnosis of syphilis. Research method The standard strain of TP Nichols was inoculated into rabbit testis, the strain of TP Nichols was prepared, the Tp0608 gene of Tp0608 was amplified by PCR and the recombinant plasmid pET-28a / Tp0965Tp0136 and Tp0608 were constructed and identified by BamHI / SaI1 BamHIP / Hindlll and Ndel/Xhol double enzyme digestion respectively, and the recombinant plasmid pET-28a / Tp0965Tp0136 and Tp0608 were constructed, respectively, and the recombinant plasmids pET-28a / Tp0965Tp0608 were digested. The recombinant protein was identified by SDS-PAGE and Western blot, and the concentration of recombinant protein was detected by BCA method. New Zealand rabbits were immunized with the purified recombinant protein Tp0965p0136, and the recombinant protein was used as coating antigen to establish an indirect ELISA assay to detect the polyclonal antibody titer of immunized rabbits. Western blot was used to detect the immunoreaction of recombinant Tp0965Tp0136 and Tp0608 with syphilis positive serum. Indirect ELISA was established with recombinant protein Tp0965 as coating antigen to detect serum of syphilis patients. Research results 1) the target gene and E. coli expression plasmid pET28 were successfully constructed, and the membrane proteins Tp0965Tp0136 and Tp0608.02 were successfully expressed and purified. The recombinant protein Tp0136 was used to immunize New Zealand rabbits. The antibody production was detected 2 weeks after immunization. The high titer antibody was produced after three times of immunization, and the recombinant protein Tp0965 could only stimulate rabbit to produce a low effective antibody. The results of Westernblot test showed that the three recombinant proteins Tp0965Tp0136 and Tp0608 could react specifically with syphilis positive serum, and the recombinant protein Tp0965Tp0136 and Tp0608 could react with syphilis positive serum. Using recombinant protein Tp0965 as the coating antigen, indirect ELISA was established for the diagnosis of syphilis in different stages. The positive rate was as high as 94.6, but could not be combined with syphilis negative serum. Conclusion The membrane proteins Tp0965, Tp0136 and Tp0608 with full gene fragment length can be recombined by E. coli expression system. Tp0965 can be overexpressed, but the purification of Tp0136 and Tp0608 is difficult. Tp0136 can stimulate the production of polyclonal antibodies and has strong immunogenicity. However, the immunogenicity of Tp0965 is weak. 3) Tp0965Tp0136 and Tp0608 have some immunoreactivity, and can react with syphilis positive serum. The recombinant protein Tp0965 can be used as a candidate antigen for serological detection of syphilis, which needs to be further expanded.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R377

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