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

【摘要】：目的:構(gòu)建含梅毒螺旋體(Treponema pallidum, TP)外膜蛋白TP 0993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)行免疫原性和抗原性分析,為探索TP 0993重組蛋白在梅毒血清學(xué)診斷中的應(yīng)用價值和其生物學(xué)功能提供實(shí)驗(yàn)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(kuò)增目的片段,將其亞克隆至原核表達(dá)載體pET28a(+)中構(gòu)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(dá)宿主菌E.coli Rosseta中,IPTG誘導(dǎo)表達(dá)。采用SDS-PAGE和Western-blot分析和鑒定表達(dá)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(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,評價重組抗原在梅毒血清學(xué)診斷中的應(yīng)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(jìn)行分析。 結(jié)果:通過Genbank獲取選TP 0993全基因序列;PCR擴(kuò)增得到其目的片段;構(gòu)建的重組質(zhì)粒經(jīng)酶切和測序鑒定證實(shí)插入片段為目的基因;SDS-PAGE結(jié)果顯示,在IPTG誘導(dǎo)下,重組表達(dá)菌分別表達(dá)了一相對分子量(Mr)約為34KDa的重組蛋白,目的蛋白在菌體細(xì)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(jìn)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(jié)論: 1、成功構(gòu)建了pET28a(+)/TP 0993原核表達(dá)體,并能在大腸桿菌中表達(dá)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(jié)合,具有良好的抗原性,可應(yīng)用于梅毒血清學(xué)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(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】：2011
【分類號】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前10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

2 徐樹良,趙源源,蔣理,趙旺勝;金標(biāo)法檢測梅毒螺旋體特異性抗體的適用性分析[J];中國輸血雜志;2004年03期

3 莫健民;梅毒血清學(xué)USR、RPR、TRUST三種篩選試驗(yàn)與TPHA的比較觀察[J];廣東醫(yī)學(xué);1997年05期

4 劉瑋;梅毒實(shí)驗(yàn)室診斷研究進(jìn)展[J];國外醫(yī)學(xué).皮膚性病學(xué)分冊;1994年06期

5 劉雙全;吳移謀;;梅毒螺旋體4種主要膜蛋白分子的研究現(xiàn)狀[J];國外醫(yī)學(xué)(微生物學(xué)分冊);2004年01期

6 李鈺;兩種梅毒血清學(xué)試驗(yàn)的檢測結(jié)果比較[J];廣西預(yù)防醫(yī)學(xué);2001年02期

7 王福俊,賀堅(jiān)慧,王琳琳;梅毒螺旋體抗體ELISA測定方法的建立和應(yīng)用[J];上海醫(yī)學(xué)檢驗(yàn)雜志;2000年04期

8 鄭中波;我國的性病流行狀況及其分析[J];中國性病艾滋病防治;1996年02期

9 宋繼權(quán),盛晚香,馮誼,夏琳,吳東方;ELISA在梅毒血清學(xué)診斷中的應(yīng)用[J];中國皮膚性病學(xué)雜志;2000年04期

10 董雅榮，李桂娥，馬季，，李曉春，木克達(dá)斯，金雁，張昆;TRUST與USR、RPR在梅毒篩選試驗(yàn)中的對比觀察[J];中國皮膚性病學(xué)雜志;1994年02期

本文編號：1732734