發(fā)布時(shí)間：2018-01-22 05:27

  本文關(guān)鍵詞： 梅毒螺旋體 重組蛋白 蛋白純化 Gpd 免疫活性　出處：《南華大學(xué)》2006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Gpd(Glycerophosphodiester Phosphodiesterase)1～353位氨基酸編碼基因的重組表達(dá)載體，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Gpd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)功能提供一定的實(shí)驗(yàn)依據(jù)。 方法：通過生物信息學(xué)分析，篩選并挑選Gpd基因優(yōu)勢抗原表位片段，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pET28b(+)中、構(gòu)建重組質(zhì)粒pET28b(+)／Gpd，然后轉(zhuǎn)化至表達(dá)宿主菌RIL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法測定純化蛋白濃度。用純化的Gpd重組蛋白包被微孔板，建立間接ELISA方法，檢測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性血清的反應(yīng)情況，評價(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Gpd重組蛋白免疫新西蘭兔，間接ELISA方法檢測免疫兔血清中Gpd多克隆抗體的效價(jià)，對Gpd重組蛋白的免疫原性進(jìn)行分析。 結(jié)果：軟件分析Gpd基因的抗原表位，選擇了Gpd基因的1～1059bp位堿基序列為目的基因片段(片段長度為1059bp，編碼353個(gè)氨基酸)；PCR擴(kuò)增得到以大小約為1059bp的目的片斷；構(gòu)建的重組質(zhì)粒經(jīng)酶切鑒定和測序鑒定證明其中插入片斷為Gpd目的基因，測序結(jié)果與Genbank上登錄序列完全一致；
[Abstract]:Objective: to construct Treponema pallidum containing Treponema pallidum. TP) outer membrane protein Gpd(Glycerophosphodiester Phosphodiesterase. The recombinant expression vector of amino acid coding gene at position 1 ~ (353). The expression was induced in Escherichia coli, the expression product was purified and the immunogenicity and immunoreactivity were analyzed. To explore the application value and biological function of Gpd recombinant protein in the serological diagnosis of syphilis. Methods: the epitopes of Gpd gene were screened and selected by bioinformatics analysis. The genomic DNA of TP Nichols strain was used as template. The target fragment was amplified by high fidelity polymerase chain reaction and subcloned into prokaryotic expression vector pET28b. the recombinant plasmid pET28bwas constructed. Then it was transformed into the expression host strain RIL to induce the expression, and the expression product was analyzed and identified by SDS-PAGE and Western-Blot. The recombinant protein was purified by Ni-NTA affinity chromatography and the concentration of purified protein was determined by dilution dialysis renaturation. The purified Gpd recombinant protein was coated with micropore plate to establish an indirect ELISA method. To detect syphilis reference serum and clinical syphilis patient serum, and compare with TPPA method, according to the reaction of recombinant protein and syphilis yin-yang serum. To evaluate the application value of recombinant antigen in the serological diagnosis of syphilis and immunize New Zealand rabbits with purified Gpd recombinant protein. Indirect ELISA method was used to detect the titer of polyclonal antibody against Gpd in sera of immunized rabbits. The immunogenicity of Gpd recombinant protein was analyzed. Results: the antigenic epitopes of Gpd gene were analyzed by software, and the target gene fragment (1059bp) was selected as the target gene sequence of Gpd gene (1: 1059bp). Encoding 353 amino acids; The target fragment of 1059bp was obtained by PCR amplification. 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)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R377

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 杜蘭英,莊輝,何軍,周育森,王海濤;梅毒螺旋體TPN17重組抗原的表達(dá)及其在獻(xiàn)血篩查中的應(yīng)用[J];中國輸血雜志;2001年01期

相關(guān)碩士學(xué)位論文 前1條

1 劉雙全;梅毒螺旋體Tp0453重組蛋白的表達(dá)、純化及免疫活性研究[D];南華大學(xué);2005年

本文編號：1453813