【作者】 龙福泉；

【导师】 王千秋；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2012， 博士

【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。更多还原

【Abstract】 Background and ObjectiveT. pallidum subsp. pallidum (T. pallidum) is the causative agent of syphilis. The membrane proteins of T. pallidum play an important role in the intercellular contact, surface identification, signal transduction, enzymatic activity and material transportation. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis, and have significance in intensive study the pathogenesis of syphilis and the interaction of host-pathogen during infection. Previous studies have shown that such research was difficult for many reasons, and the results of research were also disputable. We use genetic engineering technique to produce recombinant membrane proteins Tp0965, Tp0136and Tp0608via vector bacteria E. coli, and then assess their antigenicity and immunoreactivity, also to evaluate the possibility of recombinant Tp0965as coating antigen in detecting syphilis.MethodsT. pallidum subsp. pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted. The genes of Tp0965and Tp0608were amplified by PCR from the genomic DNA. The complete gene of Tp0136was synthesized. Then the genes were subcloned into the expression vector pET28a to construct a recombinant plasmid, which were subsequently transfected into E. coli Rosetta for protein expression. These three recombinant proteins were expressed and purified by Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. The reactivities of protein Tp0965were examined by immunoblot analysis. New Zealand rabbits were immunized with the recombinant Tp0965and Tp0136, and the polyclonal antibodies in sera of immunized rabbits were examined by indirect enzyme-linked immunosorbent assay (ELISA) to evaluate its immunogenicity. Also, the positive sera of patients with syphilis were examined by Western blot to identify the ability of their immunoreactions. At last, we conduct the indirect ELISA which was coated with recombinant Tp0965as the antigen to detect syphilis serum.ResultsThe E. coli expression vectors of pET28with target genes were constructed successfully, and the recombinant proteins Tp0965,Tp0136and Tp0608were also successfully expressed with a higher purity and concentration. The higher titre of antibodies of ant-Tp0136in immuned rabbits was detected after the second time of immunization, however, only a low titer of antiserum against Tp0965in immune rabbits was detected after the third time of immunization. Western blot showed that there were specific bands appeared in gel which demonstrated the specific reactions of these three recombinant proteins with positive sera of syphilis. Furthermore, immunoblot assay showed that the recombinant protein Tp0965could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only4of74human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965to all28uninfected sera.ConclusionsThe recombinant membrane protein Tp0965,Tp0136and Tp0608with complete genes could be successfully expressed through E. coli expression system, and the recombinant Tp0965could be expressed with high level, however, the others expressed with lower level. The recombinant Tp0136has strong antigenicity, but Tp0965showed weak antigenicity during the rabbit immune test. All these three proteins showed immunoreactivity with human serum infected with T. pallidum. And the recombination Tp0965could be a candidate antigen which could be used to detect syphilitic serum.更多还原