本文選題：弓形蟲 + SAG1基因��； 參考：《西南大學(xué)》2006年碩士論文

【摘要】：弓形蟲是一種機(jī)會性致病原蟲，分布十分廣泛，由其導(dǎo)致的弓形蟲病也是危害嚴(yán)重的人畜共患病之一。本文針對診斷方法和診斷所用抗原展開研究，以期建立快速簡便的診斷方法和獲得特異性強(qiáng)的診斷抗原。 一、PAPS(Polyaledehyde Polystyrene,PAPS)弓形蟲抗體診斷液的制備研究。 為了滿足基層現(xiàn)場疾病診斷和動物檢疫的需要，對用聚苯乙烯做載體的乳膠凝集試驗(yàn)(Latex agglutination test,LAT)進(jìn)行了優(yōu)化。通過對聚苯乙烯乳膠載體的改造，使其在較溫和的化學(xué)反應(yīng)條件下表面被聚醛化，聚苯乙烯載體攜帶功能團(tuán)后，載體變得更加疏松且易混勻，顆粒直徑變大，改變了以往聚苯乙烯乳膠以物理吸附的方式結(jié)合蛋白質(zhì)，使聚醛化后的聚苯乙烯可以化學(xué)吸附的方式結(jié)合蛋白質(zhì)；對PAPS微球載體進(jìn)行保存期試驗(yàn)，結(jié)果發(fā)現(xiàn)，于4℃保存九個(gè)月其功能團(tuán)和結(jié)合蛋白的穩(wěn)定性沒有發(fā)生變化；對PAPS弓形蟲抗體診斷液進(jìn)行保存期試驗(yàn)，結(jié)果表明，于4℃保存九個(gè)月其敏感性、穩(wěn)定性沒有發(fā)生變化；以市售標(biāo)準(zhǔn)間接血凝試劑盒作對照，利用所購買試劑盒內(nèi)的陽性血清和陰性血清對PAPS弓形蟲抗體診斷液進(jìn)行敏感性檢測，結(jié)果顯示，PAPS弓形蟲抗體診斷液敏感性比間接血凝試劑高出兩個(gè)稀釋度以上；利用血吸蟲、錐蟲、旋毛蟲、肺吸蟲、肝片吸蟲、囊蟲陽性血清與PAPS弓形蟲抗體診斷液進(jìn)行凝集試驗(yàn)，結(jié)果均呈陰性，未見發(fā)生交叉反應(yīng)；運(yùn)用鏡檢見蟲的弓形蟲陽性豬血清和經(jīng)小鼠傳代接種方法證實(shí)無弓形蟲的健康豬血清進(jìn)行PAPS凝集試驗(yàn)，結(jié)果發(fā)現(xiàn)陰陽性符合率均為100％；對上海市某區(qū)的九個(gè)地方2800只犬進(jìn)行弓形蟲抗體調(diào)查，總的陽性犬?dāng)?shù)485只，平均陽性率為17.3％。本方法可以使聚苯乙烯乳膠載體更穩(wěn)定地結(jié)合蛋白質(zhì)，可延長診斷液的保存時(shí)間，實(shí)驗(yàn)結(jié)果也更容易通過肉眼觀察，且具有特異性強(qiáng)、靈敏度高、重復(fù)性好、快速、簡便、無需特殊儀器設(shè)備等優(yōu)點(diǎn)。 二、截短弓形蟲SAGI(surface antigen 1)表面抗原的原核表達(dá)研究。 SAG1作為弓形蟲速殖子的表面抗原，已被證明能有效的刺激機(jī)體產(chǎn)生抗體，也是目前公認(rèn)最有潛力的免疫診斷抗原，試驗(yàn)將速殖子SAG1的基因片段截短使其在大腸桿菌中表達(dá)，，以求獲得具有良好抗原活性的重組抗原。本研究利用小白鼠進(jìn)行腹腔傳代收獲弓形蟲速殖子，然后直接利用它提取基因組DNA進(jìn)行PCR，將PCR產(chǎn)物回收后通過T-A克隆的方式直接與pGEM-T-easy載體相連，將連接物轉(zhuǎn)化到DH5α感受態(tài)細(xì)胞中，經(jīng)過藍(lán)白篩選和抗性篩選，選挑取陽性克隆，構(gòu)建pGEM-T-easy+SAGI重組質(zhì)粒；將pGEM-T-easy+SAG1重組質(zhì)粒經(jīng)EcoRI和HindⅢ雙酶切，回收目的片斷，連接到pGEX-2T上，將連接物轉(zhuǎn)化到DH5α感受態(tài)細(xì)胞中，經(jīng)過抗性篩選，選挑取陽性克隆，構(gòu)建重組質(zhì)粒pGEX-2T+SAG1；再將pGEX-2T+SAG1轉(zhuǎn)入E.coli(BL21)中用IPTG誘導(dǎo)進(jìn)行表達(dá)，經(jīng)過確定最佳誘導(dǎo)時(shí)間和最適IPTG濃度來優(yōu)化表達(dá)條件，經(jīng)過一系列緩沖液的處理，離心純化包涵體，將純化產(chǎn)物進(jìn)行SDS-PAGE電泳，然后將其轉(zhuǎn)移到NC膜上進(jìn)行Western-Blotting雜交來分析抗原活性。結(jié)果顯示，在DH5α感受態(tài)細(xì)胞中成功構(gòu)建pGEM-T-easy+SAG1和pGEX-2T+SAG1重組質(zhì)粒，通過IPTG誘導(dǎo)，此片斷在大腸桿菌中成功高效表達(dá)，相對分子量約61.5kD，據(jù)Western-Blotting顯示，表達(dá)產(chǎn)物能被
[Abstract]:Toxoplasma gondii is an opportunistic protozoan, is widely distributed, which causes toxoplasmosis is serious zoonotic disease. According to the diagnostic methods and diagnostic antigen used research, in order to establish a rapid diagnosis method is simple and highly specific diagnostic antigen.
A study on the preparation of PAPS (Polyaledehyde Polystyrene, PAPS) antibody diagnostic solution for Toxoplasma gondii.
In order to meet the needs of primary site disease diagnosis and animal quarantine, the latex agglutination test using polystyrene as carrier (Latex agglutination test, LAT) were optimized. Through modification of polystyrene latex carrier, the chemical reactions in mild conditions by surface aldehyde, polystyrene carrier group. The carrier has become more loose and easy to mix, larger particle diameter, changed polystyrene latex by physical adsorption method combined with protein, poly formaldehyde after the polystyrene chemical adsorption way binding protein; PAPS microspheres for preservation test, results showed that at 4 DEG C for nine months the functional group and combined with the protein stability did not change; the PAPS of Toxoplasma gondii antibody in the diagnosis of liquid storage test, results showed that at 4 DEG C for nine months the sensitivity, no stability A change to a commercially available standard; indirect hemagglutination test kit were purchased by the kit in positive and negative sera sensitivity detection, Toxoplasma antibody diagnostic reagents on PAPS results showed that PAPS of Toxoplasma gondii antibody diagnostic sensitivity than indirect hemagglutination test was higher than two dilution degrees above; the use of Schistosoma japonicum, Trypanosoma, Trichinella, paragonimiasis, Fasciola hepatica, cysticercosis and Toxoplasma PAPS positive serum antibody in the diagnosis of liquid agglutination test results were negative, no cross reaction; using microscopic insect Toxoplasma gondii positive pig and PAPS agglutination test method isolated mice confirmed that Toxoplasma free healthy pigs results serum, positive and negative coincidence rate was 100%; for Toxoplasma antibody survey of a district of Shanghai city in nine places in 2800 dogs, the total number of positive dogs was 485, the average positive rate was 17.3%. The method can make the polystyrene latex carrier bind to the protein more stable, prolong the storage time of the diagnostic solution, and the experimental result is more easily observed through the naked eye. It has the advantages of high specificity, high sensitivity, good reproducibility, fast and simple, and no special instrument and equipment are needed.
Two, study on the prokaryotic expression of the surface antigen of SAGI (surface antigen 1) of truncated Toxoplasma gondii.
SAG1 as a surface antigen of Toxoplasma gondii tachyzoites, has been shown to be effective in stimulating the body to produce antibodies, immune antigen is currently recognized as the most promising test, the gene fragment truncated tachyzoites. The expression of SAG1 in Escherichia coli, in order to obtain good immunological activity. In this study, the recombinant antigen intraperitoneal passage harvest of Toxoplasma gondii in mice, and then directly use it to extract genomic DNA by PCR, the product of PCR was cloned by T-A after the way is directly connected with the pGEM-T-easy carrier, will be connected into DH5 alpha feeling state in the cell, through the blue white screening and resistance screening, selection of positive clones pGEM-T-easy+SAGI, construction of recombinant plasmid; recombinant plasmid pGEM-T-easy+SAG1 by EcoRI and Hind III restriction enzyme digestion, amplified fragments, connected to the pGEX-2T, the connection is transformed into DH5 competent cells by alpha. After resistance screening, selected positive clones, recombinant plasmid pGEX-2T+SAG1; then pGEX-2T+SAG1 into E.coli (BL21) induced by IPTG for expression. After determining the optimal induction time and the optimal concentration of IPTG to optimize the expression conditions, through a series of processing buffer, centrifugation and purification of inclusion bodies, the purified product was SDS-PAGE. And then transferred to NC membrane by Western-Blotting hybridization analysis of antigenic activity. The results showed that the DH5 alpha feeling pGEM-T-easy+SAG1 was successfully constructed and recombinant plasmid pGEX-2T+SAG1 cells, induced by IPTG, the fragment in E.coli successfully expressed the relative molecular weight of about 61.5kD, according to Western-Blotting, the expression product can be

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392.1

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