本文選題：巨噬細(xì)胞感染增強(qiáng)蛋白 + 雙抗原夾心ELISA　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：流產(chǎn)衣原體(Chlamydia abortus,C.abortus)是一種嚴(yán)格細(xì)胞內(nèi)寄生的革蘭氏陰性細(xì)菌,可感染妊娠母畜導(dǎo)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(dòng)物直接接觸也會(huì)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(xì)胞感染增強(qiáng)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(yīng)等過(guò)程。本研究以MIP為研究對(duì)象,利用Gen Bank已公布的序列,設(shè)計(jì)引物,PCR擴(kuò)增得到MIP蛋白的編碼基因,經(jīng)過(guò)酶切、連接、轉(zhuǎn)化將其插入原核表達(dá)載體pET30a(+),成功構(gòu)建表達(dá)載體pET-mip并轉(zhuǎn)化入E.coli BL21(DE3)中誘導(dǎo)表達(dá),經(jīng)SDS-PAGE分析可見(jiàn)在27 kD處出現(xiàn)特異性條帶,表明插入的mip基因成功表達(dá);Western-blot特異性實(shí)驗(yàn)驗(yàn)證表達(dá)的MIP蛋白具有反應(yīng)原性。以純化的MIP蛋白做為包被抗原,HRP標(biāo)記的MIP蛋白為酶標(biāo)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(tuán)菌、口蹄疫等陽(yáng)性血清無(wú)交叉反應(yīng);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(qiáng)、靈敏度高、重復(fù)性好。以純化的MIP蛋白免疫Balb/c小鼠后篩選陽(yáng)性細(xì)胞克隆并進(jìn)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(xì)胞株,分別命名為M1和M3。間接ELISA法鑒定單抗上清抗體效價(jià)為1:1600;染色體鑒定符合雜交瘤細(xì)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(shí)別MIP蛋白;Mouse單克隆抗體亞類(lèi)檢測(cè)試劑盒鑒定2株單抗免疫球蛋白類(lèi)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(dá)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(zhǔn)確診斷及相關(guān)致病機(jī)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(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.67

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 吳中明,彭理年,劉祖林,張蕾青,肖瑜;細(xì)胞培養(yǎng)法檢測(cè)沙眼衣原體的探討[J];貴州醫(yī)藥;1999年01期

2 姜天童;楊宜生;孟慶友;方雨玲;韓勝利;;間接血凝試驗(yàn)檢測(cè)豬衣原體抗體的研究初報(bào)[J];湖北畜牧獸醫(yī);1983年03期

3 吳亞安;鄭和平;吳志華;李燕娃;吳興中;;基于omp1基因VS1-VS2序列的沙眼衣原體RFLP基因分型[J];嶺南皮膚性病科雜志;2007年02期

4 孔君;劉箐;韓躍武;王天昌;劉金芝;;單克隆抗體制備技術(shù)的最新進(jìn)展及應(yīng)用前景[J];免疫學(xué)雜志;2011年02期

5 楊宜生;姜天童;孟慶友;方雨玲;韓勝利;;間接血凝試驗(yàn)檢測(cè)衣原體抗體的研究[J];畜牧獸醫(yī)學(xué)報(bào);1984年03期

6 王建科;程世鵬;易立;楊莘;羅彬;許紅麗;閆喜軍;武華;;水貂腸炎病毒雙抗體夾心ELISA檢測(cè)方法的建立[J];中國(guó)獸醫(yī)科學(xué);2011年02期

7 趙凱;孫立新;孫宇石;周東坡;;抗紫杉醇單克隆抗體的制備及ELISA檢測(cè)方法的建立[J];中國(guó)新藥雜志;2012年04期

8 張文通;魏鳳;李峰;苗立中;沈志強(qiáng);;豬流產(chǎn)嗜性衣原體病診斷方法研究進(jìn)展[J];中國(guó)豬業(yè);2014年07期

相關(guān)碩士學(xué)位論文 前1條

1 楊軍;動(dòng)物衣原體病通用LAMP方法的建立[D];四川農(nóng)業(yè)大學(xué);2013年

，

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