本文選題：巨噬細胞感染增強蛋白 + 雙抗原夾心ELISA��； 參考：《中國農(nóng)業(yè)科學院》2015年碩士論文

【摘要】：流產(chǎn)衣原體(Chlamydia abortus,C.abortus)是一種嚴格細胞內(nèi)寄生的革蘭氏陰性細菌,可感染妊娠母畜導致流產(chǎn)、死產(chǎn)或者弱胎等;也可引起公畜發(fā)生睪丸炎、尿道炎等癥狀;孕婦與感染的動物直接接觸也會造成流產(chǎn)及全身感染等癥狀,因此流產(chǎn)衣原體是一種重要的人獸共患病病原。巨噬細胞感染增強蛋白(MacropHage Infectivity Potentiator,MIP)是一種脂蛋白,作為一種免疫顯性抗原,參與衣原體感染,引起炎癥反應等過程。本研究以MIP為研究對象,利用Gen Bank已公布的序列,設(shè)計引物,PCR擴增得到MIP蛋白的編碼基因,經(jīng)過酶切、連接、轉(zhuǎn)化將其插入原核表達載體pET30a(+),成功構(gòu)建表達載體pET-mip并轉(zhuǎn)化入E.coli BL21(DE3)中誘導表達,經(jīng)SDS-PAGE分析可見在27 kD處出現(xiàn)特異性條帶,表明插入的mip基因成功表達;Western-blot特異性實驗驗證表達的MIP蛋白具有反應原性。以純化的MIP蛋白做為包被抗原,HRP標記的MIP蛋白為酶標抗原,最適包被濃度為2.5μg/mL,血清最佳稀釋度為1:100,建立雙抗原夾心ELISA方法。建立的雙抗原ELISA與IHA相比,敏感性高,符合率為89.3%;與支原體、嗜肺軍團菌、口蹄疫等陽性血清無交叉反應;批間、批內(nèi)CV%均小于11%,表明建立的雙抗原夾心ELISA方法特異性強、靈敏度高、重復性好。以純化的MIP蛋白免疫Balb/c小鼠后篩選陽性細胞克隆并進行有限稀釋克隆化,建立了兩株能穩(wěn)定分泌抗MIP蛋白單克隆抗體的雜交瘤細胞株,分別命名為M1和M3。間接ELISA法鑒定單抗上清抗體效價為1:1600;染色體鑒定符合雜交瘤細胞的特性;兩株單抗50%最大結(jié)合濃度均為10μg/mL;Western-blot鑒定兩株單克隆抗體能特異識別MIP蛋白;Mouse單克隆抗體亞類檢測試劑盒鑒定2株單抗免疫球蛋白類型均為IgG1,輕鏈為κ鏈;雞胚接種鑒定2株單抗能有效中和衣原體感染性。綜上所述,本研究成功表達了流產(chǎn)衣原體MIP蛋白,建立了MIP蛋白雙抗原夾心ELISA診斷方法,制備了抗MIP單克隆抗體,為流產(chǎn)衣原體的準確診斷及相關(guān)致病機制的研究和奠定了基礎(chǔ)。
[Abstract]:Chlamydia abortus C. abortus is a strictly cell-parasitic gram-negative bacteria, which can infect pregnant females to cause abortion, stillbirth or weak fetus, and can also cause orchitis, urethritis and other symptoms in male animals. Direct contact between pregnant women and infected animals can also cause abortion and systemic infection, so chlamydia miscarriage is an important zoonotic pathogen. Macrophage infection Enhancement protein (MIP) is a lipoprotein, which is involved in the process of chlamydia infection and inflammation as an immune dominant antigen. In this study, MIP coding gene was amplified by PCR using the published sequence of GenBank. The gene was digested and ligated by enzyme. The expression vector pET-mip was successfully constructed and transformed into E. coli BL21 (DE3). SDS-PAGE analysis showed that there were specific bands at 27kD, and pET-mip was transformed into E. coli BL21 (DE3). The results showed that the inserted mip gene was successfully expressed by Western-blot assay. The purified MIP protein was labeled with HRP as the enzyme labeled antigen, the optimal coating concentration was 2.5 渭 g / mL, and the optimal dilution of serum was 1: 100. A double antigen sandwich Elisa was established. Compared with IHA, the established double-antigen Elisa had a higher sensitivity and a coincidence rate of 89.3%, and had no cross reaction with mycoplasma, Legionella pneumophila, foot-and-mouth disease and so on. High sensitivity and good repeatability. After immunizing Balb / c mice with purified MIP protein, two hybridoma cell lines were established, which secreted monoclonal antibodies against MIP protein stably, and were named M1 and M3, respectively. The titer of monoclonal antibody supernatant was 1: 1600 by indirect Elisa, and chromosome identification was consistent with the characteristics of hybridoma cells. The maximum binding concentration of the two McAbs was 10 渭 g / mL Western-blot. The two monoclonal antibodies could specifically recognize MIP protein mouse monoclonal antibody subclass detection kit. The two McAb immunoglobulin types were IgG1 and the light chain was 魏 chain. Chlamydia infection was effectively neutralized by two McAbs inoculated with chicken embryo. In conclusion, this study successfully expressed the MIP protein of Chlamydia abortus, established a sandwich Elisa method for the diagnosis of MIP protein, and prepared anti-MIP monoclonal antibody. It lays a foundation for the accurate diagnosis of chlamydia miscarriage and the study of related pathogenic mechanism.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.67

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