本文選題：致密顆粒蛋白1、7 + 受試者工作特征曲線�。� 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：弓形蟲病(Toxoplasmosis)由剛地弓形蟲(Toxoplasma gondii)感染引起,是一種可導(dǎo)致人及動物流產(chǎn)、畸形或死胎的人獸共患寄生蟲病,嚴重危害公共衛(wèi)生安全和畜牧業(yè)生產(chǎn)。貓是其終末宿主,在弓形蟲的傳播過程中起重要作用。因此,建立快速、準(zhǔn)確的弓形蟲檢測方法對人類健康具有重大意義。弓形蟲致密顆粒蛋白(GRAs)是一類分泌排泄蛋白,其中GRA1與弓形蟲速殖子侵染宿主細胞的信號轉(zhuǎn)導(dǎo)有關(guān),而GRA7在弓形蟲各個時期均能分泌,是一種潛在的疫苗侯選分子。證明GRA1、GRA7作為診斷抗原具有良好的免疫原性,可作為血清學(xué)診斷弓形蟲病的基因標(biāo)志物。本研究利用原核表達的弓形蟲致密顆粒蛋白GRA1、GRA7,建立了GRA1-ELISA與GRA7-ELISA檢測方法。利用構(gòu)建原核表達載體pET-28a-GRA1、pET-28a-GRA7,轉(zhuǎn)化大腸桿菌,IPTG誘導(dǎo)表達,經(jīng)SDS-PAGE驗證,重組蛋白GRA1與GRA7分子量分別為25、27 kDa;Western blotting分析表達產(chǎn)物,結(jié)果表明,該產(chǎn)物具有較強的免疫反應(yīng);應(yīng)用Ni-NTA對表達蛋白進行純化,蛋白純度高達98%;GRA1與GRA7的濃度分別為200μg/mL和600μg/mL。利用純化重組蛋白GRA1和GRA7分別作為診斷抗原,建立GRA1-ELISA和GRA7-ELISA檢測方法。確立了GRA1、GRA7最佳抗原包被濃度5μg/mL、5μg/mL,血清一抗最佳稀釋度1:64,HRP標(biāo)記兔抗貓IgG二抗最佳工作濃度1:20000。試驗表明GRA1-ELISA與GRA7-ELISA方法檢測靈敏度高,重復(fù)性好。應(yīng)用GRA1-ELISA和GRA7-ELISA方法檢測旋毛蟲、包蟲、豬附紅細胞體和貓弓形蟲陽性血清,表明建立的GRA1-ELISA與GRA7-ELISA檢測方法具有特異性。建立GRA1-ELISA和GRA7-ELISA檢測方法以MAT/IFA法檢測結(jié)果為標(biāo)準(zhǔn)進行比較評價,GRA1-ELISA檢測方法假陽性率為10.8%,假陰性率為4.1%,而GRA7-ELISA檢測方法假陽性率為7.5%,假陰性率為1.4%,說明GRA7為診斷抗原檢測弓形蟲病的假陽性率和假陰性率較低。將差異部分通過McNemar卡方檢驗均無顯著性差異(P0.05);將一致部分通過Kappa檢驗,GRA1:Kappa=0.83,結(jié)果一致率為94.6%;GRA7:Kappa=0.92,結(jié)果一致率為97.2%;表明GRA7-ELISA檢測方法一致率較高。ROC曲線分析出建立的GRA7-ELISA穩(wěn)定性較好。GRA1-ELISA的cut-off值為0.11時,敏感性為83.6%,特異性為87%;GRA7-ELISA的cut-off值為0.26時,敏感性為89.7,特異性為92.5%。試驗表明GRA1作為診斷抗原建立GRA1-ELISA方法檢測效果不明顯,敏感性與特異性較低、穩(wěn)定性較弱;GRA7為檢測貓弓形蟲病的優(yōu)質(zhì)診斷抗原,所建立的GRA7-ELISA檢測方法具有較高的敏感性與特異性、較強的穩(wěn)定性。本研究建立的GRA7-ELISA診斷方法,解決了弓形蟲病不易準(zhǔn)確、快速診斷的難題,對貓弓形蟲病的臨床診斷、疾病防控具有重要意義,篩選出GRA7優(yōu)質(zhì)診斷抗原為我國貓弓形蟲病的快速診斷及試劑盒的研發(fā)提供依據(jù)。
[Abstract]:Toxoplasmosis (Toxoplasmosis) is caused by Toxoplasma gondii (Toxoplasma gondii) infection. It is a kind of human zoonosis which can cause human and animal abortion, deformity or stillbirth. It seriously endangers public health safety and animal husbandry. The cat is its terminal host and plays an important role in the transmission of Toxoplasma gondii. Therefore, it is fast and accurate. The detection method of Toxoplasma gondii is of great significance to human health. Toxoplasma dense granule protein (GRAs) is a class of excretory protein, in which GRA1 and Toxoplasma gondii infect host cell signal transduction, and GRA7 can be secreted at all stages of Toxoplasma gondii. It is a potential candidate for vaccine candidate. It is proved that GRA1 and GRA7 are used as diagnosis. The broken antigen has good immunogenicity and can be used as a gene marker for the diagnosis of toxoplasmosis in serology. In this study, the detection methods of GRA1-ELISA and GRA7-ELISA were established by using the dense granular protein GRA1 and GRA7 of the prokaryotic expression of Toxoplasma gondii. The prokaryotic expression vector pET-28a-GRA1, pET-28a-GRA7, Escherichia coli and IPTG were used to induce expression. SDS-PAGE showed that the molecular weight of recombinant protein GRA1 and GRA7 was 25,27 kDa, respectively, and Western blotting was used to analyze the expression products. The results showed that the product had strong immune response, and the purity of the protein was 98%, and the concentration of GRA1 and GRA7 was 200 mu g/mL and 600 micron, respectively. GRA1-ELISA and GRA7-ELISA detection methods were established respectively as diagnostic antigens. The optimum antigen inclusion concentration of GRA1, GRA7 was 5 u g/mL, 5 micron g/mL, the best dilution of serum 1:64, HRP labeled Rabbit anti cat IgG two anti optimal working concentration 1:20000. test showed that GRA1-ELISA and GRA7-ELISA methods were highly sensitive and reproducible. GRA7-ELISA method was used to detect Trichinella, echinococcosis, porcine eoeosis and Toxoplasma gondii positive serum, which showed that the method of detecting GRA1-ELISA and GRA7-ELISA was specific. The determination of GRA1-ELISA and GRA7-ELISA was compared with the test results of MAT/IFA, and the false positive rate of GRA1-ELISA detection method was 10.8%, false negative. The rate of sex was 4.1%, while the false positive rate of GRA7-ELISA detection method was 7.5% and the false negative rate was 1.4%. The false positive rate and false negative rate of GRA7 as diagnostic antigen were low. There was no significant difference (P0.05) by the McNemar Chi test. The same part passed the Kappa test, GRA1:Kappa=0.83, the result concordance rate was 94.6. %; GRA7:Kappa=0.92, the result consistent rate was 97.2%, indicating that the GRA7-ELISA detection method with high consistency rate.ROC curve showed that the cut-off value of.GRA1-ELISA with better GRA7-ELISA stability was 0.11, the sensitivity was 83.6%, the specificity was 87%, and the cut-off value of GRA7-ELISA was 0.26, the sensitivity was 89.7, and the specificity of 92.5%. test showed GRA1 as a GRA1. The diagnostic antigen based GRA1-ELISA method has no obvious effect, low sensitivity and specificity and weak stability. GRA7 is a good diagnostic antigen for the detection of toxoplasmosis in cats. The established GRA7-ELISA detection method has high sensitivity, specificity and strong stability. The GRA7-ELISA diagnosis method established in this study solves the Toxoplasma gondii It is very important for the clinical diagnosis of toxoplasmosis and prevention and control of toxoplasmosis. Screening out the GRA7 diagnostic antigen for the rapid diagnosis of toxoplasmosis and the research and development of the kit of toxoplasmosis in China.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.293

【共引文獻】

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2 闞松鶴;劉俠;林青;;一種弓形蟲單管套式PCR檢測方法的建立及其在奶山羊應(yīng)用[J];動物醫(yī)學(xué)進展;2015年05期

3 徐珊珊;朱潔瑩;胡增杰;陳俊;李文姝;;溫州地區(qū)育齡期婦女及孕婦弓形蟲感染情況調(diào)查[J];檢驗醫(yī)學(xué)與臨床;2013年18期

4 王正蓉;包懷恩;;孕晚期感染弓形蟲prugniaud株對子代的影響[J];黑龍江畜牧獸醫(yī);2013年17期

5 吳升偉;王正蓉;包懷恩;;氟康唑抗弓形蟲速殖子作用的超微結(jié)構(gòu)觀察[J];黑龍江畜牧獸醫(yī);2015年16期

6 魯燕飛;;弓形蟲病的免疫診斷研究進展[J];熱帶病與寄生蟲學(xué);2013年01期

7 李志偉;張晗;;弓形蟲眼病小鼠模型的建立及其眼底特征[J];山東大學(xué)耳鼻喉眼學(xué)報;2015年02期

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2 陶青;豬弓形蟲體內(nèi)誘導(dǎo)抗原的鑒定及功能分析[D];華中農(nóng)業(yè)大學(xué);2013年

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1 王璐;弓形蟲感染誘導(dǎo)小膠質(zhì)細胞的活化以及米諾環(huán)素的抑制作用[D];安徽醫(yī)科大學(xué);2013年

2 張義華;小膠質(zhì)細胞活化介導(dǎo)神經(jīng)元損傷在弓形蟲腦炎中的研究[D];安徽醫(yī)科大學(xué);2013年

3 程健曦;IFN-γ抑制弓形蟲緩殖子向速殖子轉(zhuǎn)化的研究[D];華中農(nóng)業(yè)大學(xué);2013年

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5 蔣姝婷;基于石墨烯及金磁納米粒子的弓形蟲IgM抗體免疫傳感器的研究[D];重慶醫(yī)科大學(xué);2013年

6 紀劍峰;弓形蟲SAG1蛋白的原核表達及其初步應(yīng)用[D];揚州大學(xué);2013年

7 李銳;雞弓形蟲診斷抗原的篩選[D];南京農(nóng)業(yè)大學(xué);2012年

8 孟德娣;中國西南地區(qū)貓源弓形蟲株基因分型及毒力研究[D];安徽醫(yī)科大學(xué);2013年

9 劉欣超;我國部分地區(qū)雞肉、羊肉弓形蟲感染及土壤污染情況調(diào)查[D];南京農(nóng)業(yè)大學(xué);2013年

10 方琨;弓形蟲C-18、MIC11、PI等體內(nèi)外差異表達基因?qū)F-κB信號通路的影響[D];華中農(nóng)業(yè)大學(xué);2014年

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本文編號：1797767