本文選題：弓形蟲(chóng) + 重組蛋白質(zhì)�。� 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：弓形蟲(chóng)病是由剛地弓形蟲(chóng)(Toxoplasma gondii)引起的一種人畜共患寄生蟲(chóng)病,廣泛的分布于世界各地。弓形蟲(chóng)對(duì)宿主無(wú)嚴(yán)格的選擇性,在眾多溫血?jiǎng)游锛叭巳褐袕V泛存在。貓科動(dòng)物為弓形蟲(chóng)的終末宿主也是重要的傳染源,哺乳類、鳥(niǎo)類都是其中間宿主。弓形蟲(chóng)作為一種細(xì)胞內(nèi)寄生的機(jī)會(huì)性致病原蟲(chóng),對(duì)免疫功能不全者及妊娠動(dòng)物的危害嚴(yán)重。豬感染了弓形蟲(chóng)病后造成大量死亡,造成懷孕母豬流產(chǎn)、早產(chǎn)、死胎、畸形胎等,對(duì)豬場(chǎng)危害嚴(yán)重。本研究利用生物信息學(xué)進(jìn)行分析,對(duì)豬弓形蟲(chóng)診斷抗原進(jìn)行了篩選,目的是為了尋找和確定弓形蟲(chóng)高免疫原性的蛋白質(zhì),建立一種快速靈敏的弓形蟲(chóng)病診斷方法。本實(shí)驗(yàn)在確定弓形蟲(chóng)分泌蛋白質(zhì)的基礎(chǔ)上,將篩選出的基因克隆到表達(dá)載體,表達(dá)出2個(gè)重組蛋白質(zhì)。通過(guò)親和層析法純化得到His標(biāo)簽重組蛋白質(zhì)和GST標(biāo)簽重組蛋白質(zhì)。利用酶聯(lián)免疫吸附試驗(yàn)(ELISA)和免疫印跡法(Western-blot)篩選出了 TGME49__223140基因重組蛋白質(zhì)的特異性單克隆抗體,通過(guò)間接免疫熒光(IFA)確定抗原在蟲(chóng)體上的表達(dá)部位。最后,以該重組蛋白質(zhì)作為抗原,利用ELISA方法對(duì)54份疑似感染弓形蟲(chóng)的豬血清進(jìn)行了檢測(cè),檢出率為98.15%,結(jié)果與標(biāo)準(zhǔn)檢測(cè)方法具有非常好的吻合。高抗原的確定對(duì)建立弓形蟲(chóng)病臨床檢測(cè)/診斷方法具有重要意義。
[Abstract]:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. It is widely distributed all over the world. Toxoplasma gondii has no strict selectivity to the host and is widely found in many warm-blooded animals and people. Cat is the terminal host of Toxoplasma gondii, and mammals and birds are the intermediate hosts of Toxoplasma gondii. Toxoplasma gondii is a kind of opportunistic protozoa parasitic in cells, which is harmful to the immune insufficiency and pregnant animals. Pigs infected with Toxoplasma gondii caused a large number of deaths, resulting in pregnant sows miscarriage, premature delivery, stillbirth, abnormal fetus, and so on, which caused serious harm to pig farms. In this study, the diagnostic antigens of Toxoplasma gondii were screened by bioinformatics analysis. The aim of this study was to find and identify the proteins with high immunogenicity of Toxoplasma gondii and to establish a rapid and sensitive diagnostic method for toxoplasmosis. On the basis of determining the secreted protein of Toxoplasma gondii, the selected gene was cloned into the expression vector and two recombinant proteins were expressed. His tag recombinant protein and GST label recombinant protein were purified by affinity chromatography. The specific monoclonal antibody of recombinant protein of TGME49__223140 gene was screened by Elisa and Western-blot, and the expression site of the antigen was determined by indirect immunofluorescence assay (IFA). Finally, the recombinant protein was used as antigen to detect 54 sera of pigs suspected to be infected with Toxoplasma gondii by ELISA method. The detection rate was 98.15. The results were in good agreement with the standard detection method. The determination of high antigen is of great significance for the establishment of clinical detection / diagnosis of toxoplasmosis.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.7

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