本文選題：梅毒螺旋體 + 重組蛋白��； 參考：《南華大學》2011年碩士論文

【摘要】：目的:構建含梅毒螺旋體(Treponema pallidum, TP)外膜蛋白TP 0993的重組表達載體,在大腸桿菌中進行誘導表達,純化表達產(chǎn)物并進行免疫原性和抗原性分析,為探索TP 0993重組蛋白在梅毒血清學診斷中的應用價值和其生物學功能提供實驗依據(jù)。 方法:通過Genbank獲取選TP 0993基因序列,以TP Nichols株基因組DNA為模板,PCR擴增目的片段,將其亞克隆至原核表達載體pET28a(+)中構建重組質(zhì)粒pET28a(+)/TP 0993,pET28a(+)/TP 0993經(jīng)PCR、雙酶切、測序鑒定后將其轉(zhuǎn)化至表達宿主菌E.coli Rosseta中,IPTG誘導表達。采用SDS-PAGE和Western-blot分析和鑒定表達產(chǎn)物;Ni-NTA親和層析柱純化重組蛋白,BCA法測定蛋白濃度。用純化的TP 0993重組蛋白包被微孔板,建立間接ELISA方法,檢測臨床梅毒患者血清和健康體檢正常血清,同時與TPPA法進行比較,根據(jù)重組蛋白與梅毒陰陽性血清的反應情況,評價重組抗原在梅毒血清學診斷中的應用價值。同時用純化的TP 0993重組蛋白免疫新西蘭兔,間接ELISA方法檢測免疫兔血清中TP 0993多克隆抗體的效價,對TP 0993重組蛋白的免疫原性進行分析。 結果:通過Genbank獲取選TP 0993全基因序列;PCR擴增得到其目的片段;構建的重組質(zhì)粒經(jīng)酶切和測序鑒定證實插入片段為目的基因;SDS-PAGE結果顯示,在IPTG誘導下,重組表達菌分別表達了一相對分子量(Mr)約為34KDa的重組蛋白,目的蛋白在菌體細胞內(nèi)主要以包涵體形式存在,經(jīng)Ni-NTA親和純化后,純度在85%以上;以重組蛋白為包被抗原建立間接ELISA法;對TPPA法檢測的480份陰陽性血清進行檢測,與TPPA法比較ELISA法的靈敏度為88.3%,特異度為85.8%,ELISA法與TPPA法符合率為86.5%;利用純化的TP 0993重組蛋白免疫新西蘭兔,間接ELISA法測定兔免疫血清特異性抗體效價在1: 640以上。 結論: 1、成功構建了pET28a(+)/TP 0993原核表達體,并能在大腸桿菌中表達出TP 0993重組蛋白。 2、TP 0993重組蛋白能刺激新西蘭兔產(chǎn)生較高效價的特異性抗體,具有較好的免疫原性。 3、TP 0993重組蛋白能與梅毒陽性血清特異性結合,具有良好的抗原性,可應用于梅毒血清學診斷。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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