本文選題：生殖支原體 + MgPa��； 參考：《南華大學(xué)》2006年碩士論文

【摘要】：目的：構(gòu)建生殖支原體(Mycoplasma genitalium，Mg)黏附蛋白MgPa的優(yōu)勢表位(1075～1364 aa)基因(MgPa′)的重組表達體，在大腸桿菌中進行誘導(dǎo)表達，純化表達產(chǎn)物并進行免疫原性和免疫反應(yīng)性分析，為探索MgPa重組蛋白在Mg血清學(xué)診斷中的應(yīng)用價值和其生物學(xué)功能提供實驗依據(jù)。 方法：通過生物信息學(xué)分析，篩選并挑選MgPa基因優(yōu)勢抗原表位，以Mg G-37標(biāo)準(zhǔn)株基因組DNA為模板，高保真聚合酶鏈反應(yīng)擴增目的片段，將其亞克隆到原核表達載體pET-30a(+)中，構(gòu)建重組質(zhì)粒pET-30a(+)／MgPa′，然后轉(zhuǎn)化至表達宿主菌E.coliRosett~(TM)2(DE3)中進行誘導(dǎo)表達，利用SDS-PAGE和Western-Blot進行分析和鑒定表達產(chǎn)物；用Ni-NTA親和層析柱純化重組蛋白，BCA法測定純化蛋白濃度。用純化的MgPa重組蛋白(rMgPa′)免疫新西蘭兔，間接ELISA方法檢測免疫兔血清中MgPa′多克隆抗體的效價，，對rMgPa′的免疫原性進行分析。同時用純化的rMgPa′包被微孔板，建立間接ELISA方法，檢測Mg感染者陽性血清及對照血清，根據(jù)重組蛋白與MS陰、陽性血清的反應(yīng)情況，對rMgPa′的免疫反應(yīng)性作出評價。 結(jié)果：Expasy軟件分析MgPa基因的抗原表位選擇了MgPa′基因的3223～4092bp位堿基序列為目的基因(MgPa′，片段長度為870bp，編碼290個aa)；PCR擴增得到大小約為870bp的目的片段；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段為MgPa′目的基因，測序結(jié)果與Genbank上登錄序列完全一
[Abstract]:Objective: to construct the recombinant expression of MgPa, a dominant epitope of Mycoplasma genitalium MgPa, 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 MgPa recombinant protein in mg serological diagnosis. Methods: the dominant epitopes of MgPa gene were screened and selected by bioinformatics analysis. The target fragment was amplified by high fidelity polymerase chain reaction using the genomic DNA of mg G-37 standard strain as template, and subcloned into prokaryotic expression vector pET-30a (). The recombinant plasmid pET-30a (pET-30a) was constructed, and then transformed into E. coli Rosettl TMT 2DE3 to induce the expression. The expression product was analyzed and identified by SDS-PAGE and Western-Blot, and the purified protein was determined by Ni-NTA affinity chromatography. New Zealand rabbits were immunized with purified recombinant MgPa protein rmg Pag. The immunogenicity of rMgPa' was analyzed by indirect ELISA method for detecting the titer of polyclonal antibody against MgPa' in the serum of immunized rabbits. At the same time, using purified rMgPa' coated with microporous plate, an indirect ELISA method was established to detect the positive serum of Mg-infected patients and the control serum. According to the reaction of recombinant protein with MS negative and positive serum, the immunoreactivity of rMgPa' was evaluated. Results the epitope of MgPa gene was analyzed by the: easy software. The 3223~4092bp base sequence of MgPa' gene was selected as the target gene. The length of the fragment was 870bp. the length of 870bp fragment was about 290 AAS. The recombinant plasmid was identified by restriction endonuclease digestion and sequencing. It was proved that the inserted fragment was a MgPa' target gene. The result of sequencing was identical to that of the Genbank login sequence.
【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】：R375

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