本文選題：梅毒螺旋體　切入點(diǎn)：重組蛋白　出處：《南華大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:構(gòu)建含梅毒螺旋體(Treponema pallidum,Tp)外膜蛋白Tp0453的優(yōu)勢(shì)表位(28-288aa)基因的重組表達(dá)體,在大腸桿菌中進(jìn)行誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物并進(jìn)行免疫原性和免疫反應(yīng)性分析,為探索Tp0453重組蛋白在梅毒血清學(xué)診斷中的應(yīng)用價(jià)值和其生物學(xué)功能提供實(shí)驗(yàn)依據(jù)。 方法:通過生物信息學(xué)分析,篩選并挑選Tp0453基因優(yōu)勢(shì)抗原表位,以Tp Nichols株基因組DNA為模板,高保真聚合酶鏈反應(yīng)擴(kuò)增目的片斷, 將其亞克隆進(jìn)原核表達(dá)載體pQE32中、構(gòu)建重組質(zhì)粒pQE32/Tp0453,然后轉(zhuǎn)化至表達(dá)宿主菌M15中進(jìn)行誘導(dǎo)表達(dá),利用SDS-PAGE和Western-Blot進(jìn)行分析和鑒定表達(dá)產(chǎn)物:Ni-NTA親和層析柱純化重組蛋白,并進(jìn)行稀釋透析復(fù)性,BCA法測(cè)定純化蛋白濃度。用純化的Tp0453重組蛋白包被微孔板,建立間接ELISA方法,檢測(cè)梅毒參比血清和臨床梅毒患者血清,同時(shí)與TPPA法進(jìn)行比較,根據(jù)重組蛋白與梅毒陰陽(yáng)性血清的反應(yīng)情況,評(píng)價(jià)重組抗原在梅毒血清學(xué)診斷中的應(yīng)用價(jià)值。同時(shí)用純化的Tp0453重組蛋白免疫新西蘭兔,間接ELISA方法檢測(cè)免疫兔血清中Tp0453多克隆抗體的效價(jià),對(duì)Tp0453重組蛋白的免疫原性進(jìn)行分析。 結(jié)果:軟件分析Tp0453基因的抗原表位選擇了Tp0453基因的86-849bp位堿基序列為目的表位(片段長(zhǎng)度為764bp,編碼255個(gè)氨基酸);PCR擴(kuò)增得到以大小約為800bp的目的片斷;構(gòu)建的重組質(zhì)粒經(jīng)酶切鑒定和測(cè)序鑒定證明其中插入片斷為Tp0453目的基因,測(cè)序結(jié)果與Genbank上登錄序列完全一致;
[Abstract]:Objective: to construct the recombinant expression of the outer membrane protein Tp0453 containing Treponema pallidum TpP, and express it in Escherichia coli, purify the expressed product and analyze its immunogenicity and immunoreactivity. To explore the application value and biological function of Tp0453 recombinant protein in syphilis serological diagnosis. Methods: the dominant epitopes of Tp0453 gene were screened and selected by bioinformatics analysis. Using the genomic DNA of TP Nichols strain as template, the target fragment was amplified by high fidelity polymerase chain reaction and subcloned into prokaryotic expression vector pQE32. The recombinant plasmid pQE32 / Tp0453 was constructed and transformed into M15 to induce expression. The recombinant protein was purified by SDS-PAGE and Western-Blot. The concentration of purified protein was determined by dilution dialysis renaturation method. The purified Tp0453 recombinant protein was coated with micropore plate. Indirect ELISA method was established to detect syphilis reference serum and clinical syphilis serum, and compared with TPPA method. According to the reaction between recombinant protein and syphilis yin-positive serum, the application value of recombinant antigen in syphilis serological diagnosis was evaluated. New Zealand rabbits were immunized with purified recombinant Tp0453 protein. Indirect ELISA method was used to detect the titer of Tp0453 polyclonal antibody in serum of immunized rabbits and the immunogenicity of Tp0453 recombinant protein was analyzed. Results: the epitope of Tp0453 gene was analyzed by software. The 86-849bp base sequence of Tp0453 gene was selected as the target epitope (the fragment length was 764 BP, encoding 255 amino acids) to amplify the target fragment of 800 BP. The recombinant plasmid was identified by restriction endonuclease digestion and sequencing. The result of sequencing was identical with that of Genbank.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R377

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 嚴(yán)加林;梅毒螺旋體Gpd重組蛋白的表達(dá)、純化及免疫活性研究[D];南華大學(xué);2006年

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本文編號(hào)：1585383