本文選題：梅毒螺旋體 + 膜蛋白�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：研究背景及目的 梅毒螺旋體膜蛋白在細胞間接觸、表面識別、信號轉(zhuǎn)導(dǎo)、酶活性和運輸?shù)确矫娑及缪葜匾慕巧?通過對梅毒螺旋體重組抗原的研究有利于鑒別并篩選可用于梅毒早期實驗室診斷及疫苗研制的候選抗原,同時對于深入研究梅毒的發(fā)病機制、了解在梅毒螺旋體感染過程中宿主-病原菌的相互關(guān)系,具有重要意義。然而,資料表明,對梅毒螺旋體膜蛋白的研究一直較為困難,研究結(jié)果也存在著爭議,為此我們利用基因工程技術(shù),采用大腸桿菌表達系統(tǒng),分別對Tp0965、Tp0136及Tp0608三種蛋白進行表達,對表達出的蛋白進行相關(guān)免疫活性的檢測,并探討重組蛋白Tp0965作為包被抗原建立間接ELISA的方法應(yīng)用于梅毒診斷的可行性。 研究方法 使用Tp Nichols標(biāo)準(zhǔn)株接種家兔睪丸,制備Tp Nichols菌株DNA, PCR擴增Tp0965、Tp0608基因,Tp0136基因采用全基因合成的方法；構(gòu)建重組質(zhì)粒pET-28a/Tp0965、Tp0136及Tp0608,分別進行BamHI/SaI1、BamHI/Hindlll及Ndel/Xhol雙酶切鑒定,鑒定正確后大腸桿菌轉(zhuǎn)化,IPTG誘導(dǎo)表達,用SDS-PAGE和Western blot鑒定,并純化重組蛋白,BCA法檢測重組蛋白濃度。用純化的重組蛋白Tp0965、p0136免疫新西蘭兔,并以該重組蛋白作為包被抗原建立間接ELISA法以檢測免疫兔的多克隆抗體效價；Western blot檢測重組的Tp0965、Tp0136及Tp0608與梅毒陽性血清的免疫反應(yīng)。以重組蛋白Tp0965為包被抗原建立間接ELISA檢測各期梅毒患者血清。 研究結(jié)果 (1)成功構(gòu)建目的基因與E. coli表達質(zhì)粒載體pET28,成功表達和純化出具有較高純度和濃度的膜蛋白Tp0965、Tp0136及Tp0608。(2)用重組蛋白Tp0136免疫新西蘭兔,在免疫2周后即能檢測到抗體的產(chǎn)生,免疫3次后產(chǎn)生高效價抗體；而重組蛋白Tp0965只有在免疫3次以后,才能刺激兔產(chǎn)生較低效價的抗體。(3) Westernblot實驗顯示三種重組蛋白Tp0965、Tp0136及Tp0608均能與梅毒陽性血清發(fā)生特異性反應(yīng),在電泳圖上有特異性條帶出現(xiàn)。(4)以重組蛋白Tp0965作為包被抗原建立間接ELISA診斷各期梅毒,陽性率高達94.6%,而與梅毒陰性血清不能結(jié)合。 結(jié)論 (1)能夠通過大腸桿菌表達系統(tǒng)重組出具有全基因片段長度的膜蛋白Tp0965、 Tp0136及Tp0608; Tp0965可以高表達,但Tp0136及Tp0608純化較為困難。(2)Tp0136能刺激機體產(chǎn)生多克隆抗體,具有較強的免疫原性,而Tp0965的免疫原性較弱。(3) Tp0965、Tp0136及Tp0608均具有一定的免疫反應(yīng)性,能夠與梅毒陽性血清反應(yīng)。(4)重組蛋白Tp0965有作為候選抗原應(yīng)用于梅毒血清學(xué)檢測的可能,需要進一步擴大樣本驗證。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R377

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