間日瘧原蟲醛縮酶的抗原和單克隆抗體制備及其在瘧疾快速診斷中的應(yīng)用
發(fā)布時間：2018-08-31 07:58
【摘要】：瘧疾是全球死亡的一個主要病之一。大部分針對瘧疾的快速診斷試劑不能區(qū)分來自于不同瘧原蟲的感染。開發(fā)靈敏度高且能區(qū)分兩種最常見瘧原蟲感染的快速診斷試劑對于疾病的控制和治療有重要的意義。本文以提取的間日瘧原蟲DNA為模板,通過PCR擴(kuò)增了醛縮酶基因(PvALDO)并成功構(gòu)建pET30a-Aldo原核表達(dá)質(zhì)粒,成功在大腸桿菌BL21(DE3)中表達(dá)并使用親和層析法純化得到了純度高于95%的重組間日瘧原蟲醛縮酶蛋白。用重組蛋白對小鼠和兔子進(jìn)行免疫用于生產(chǎn)抗體。將生產(chǎn)得到的兔多克隆抗體使用在一種新型抗體捕獲ELISA法進(jìn)行雜交瘤細(xì)胞的篩選,一共篩選得到九個穩(wěn)定的單克隆抗體(1C3,1G3,1H3,3F1,9A1,9B5,9G6,11G7,12F10),將得到的單克隆抗體用作包被抗體或者標(biāo)記抗體在膠體金免疫層析平臺進(jìn)行評價。 把實(shí)驗(yàn)結(jié)果最好的抗體對(1C3-12F10)用于開發(fā)商業(yè)化的間日瘧原蟲檢測快速診斷試劑并對其檢測效果進(jìn)行評估。對來自云南和廣州的臨床血液樣本(n=503)的評估結(jié)果表明我們的快速診斷試劑針對間日瘧原蟲的特異性為100%(95%Confidence Interval (CI):99.12to100.00%),與顯微鏡鏡檢法相比靈敏度為98.75%(95%CI:93.20to99.79%)且其中僅出現(xiàn)一例假陰性。同時對惡性瘧原蟲樣本(n=33)和健康人血液樣本(n=390)的檢測沒有發(fā)應(yīng)。我們的快速診斷試劑的陽性預(yù)測值(PPV)以及陰性預(yù)測值(NPV)分別為100.00%和99.76%,檢測的置信區(qū)間為95%,與顯微鏡鏡檢法有很好的相關(guān)性(kappa值,K=0.992530).針對重組間日瘧原蟲醛縮酶蛋白,膠體金檢測試紙條的靈敏度能達(dá)到6.25ng/ml。本文闡明了以醛縮酶蛋白作為檢測指標(biāo)的快速診斷試劑區(qū)分不同瘧原蟲引起的瘧疾感染是可行的。開發(fā)得到的快速診斷試劑能有效區(qū)分間日瘧原蟲感染和惡性瘧原蟲感染,同時本文采用的新的抗體篩選方法可以在其他抗原高特異性單克隆抗體的篩選過程中得到運(yùn)用。
[Abstract]:Malaria is one of the leading causes of death worldwide. Most rapid diagnostic kits for malaria do not distinguish between infections from different malaria parasites. It is of great significance to develop a rapid diagnostic reagent with high sensitivity and ability to distinguish the two most common Plasmodium infections for the control and treatment of the disease. Based on the extracted DNA of Plasmodium vivax, the aldolase gene (PvALDO) was amplified by PCR and the pET30a-Aldo prokaryotic expression plasmid was constructed successfully. The recombinant plasmodium vivax aldolase protein was successfully expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Mice and rabbits were immunized with recombinant proteins to produce antibodies. Rabbit polyclonal antibodies were used in a novel antibody capture ELISA method to screen hybridoma cells. A total of nine stable monoclonal antibodies (1C3G3G3H3H3F1F1F1F1A1A1A1F10) were obtained. The monoclonal antibodies were used as coated or labeled antibodies for evaluation on a colloidal gold immunochromatographic platform. The best antibody pair (1C3-12F10) was used to develop a commercial rapid diagnostic reagent for Plasmodium vivax and to evaluate its effectiveness. The evaluation results of clinical blood samples from Yunnan and Guangzhou showed that the specificity of our rapid diagnostic reagent for Plasmodium vivax was 100% (95%Confidence Interval (CI): 99.12 to 100.00%), and the sensitivity was 98.75% (95CIW 93.20 to 99.79%) compared with microscopic examination. At the same time, the detection of Plasmodium falciparum (nm33) and healthy blood samples (nm390) was not detected. The positive predictive value (PPV) and negative predictive value (NPV) of our rapid diagnostic reagent were 100.00% and 99.76%, respectively, and the confidence interval was 95, which had a good correlation with microscopic examination (kappa value: 0.992530). For recombinant Plasmodium vivax aldolase protein, the sensitivity of colloidal gold test strip was 6.25 ng / ml. It is feasible to distinguish malaria infection caused by different malaria parasites by using aldolase protein as a rapid diagnostic reagent. The developed rapid diagnostic reagent can effectively distinguish the infection of Plasmodium vivax from that of Plasmodium falciparum. At the same time, the new antibody screening method used in this paper can be used in the screening process of other highly specific monoclonal antibodies against Plasmodium vivax and Plasmodium falciparum.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R531.3;Q78
本文編號：2214374
[Abstract]:Malaria is one of the leading causes of death worldwide. Most rapid diagnostic kits for malaria do not distinguish between infections from different malaria parasites. It is of great significance to develop a rapid diagnostic reagent with high sensitivity and ability to distinguish the two most common Plasmodium infections for the control and treatment of the disease. Based on the extracted DNA of Plasmodium vivax, the aldolase gene (PvALDO) was amplified by PCR and the pET30a-Aldo prokaryotic expression plasmid was constructed successfully. The recombinant plasmodium vivax aldolase protein was successfully expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Mice and rabbits were immunized with recombinant proteins to produce antibodies. Rabbit polyclonal antibodies were used in a novel antibody capture ELISA method to screen hybridoma cells. A total of nine stable monoclonal antibodies (1C3G3G3H3H3F1F1F1F1A1A1A1F10) were obtained. The monoclonal antibodies were used as coated or labeled antibodies for evaluation on a colloidal gold immunochromatographic platform. The best antibody pair (1C3-12F10) was used to develop a commercial rapid diagnostic reagent for Plasmodium vivax and to evaluate its effectiveness. The evaluation results of clinical blood samples from Yunnan and Guangzhou showed that the specificity of our rapid diagnostic reagent for Plasmodium vivax was 100% (95%Confidence Interval (CI): 99.12 to 100.00%), and the sensitivity was 98.75% (95CIW 93.20 to 99.79%) compared with microscopic examination. At the same time, the detection of Plasmodium falciparum (nm33) and healthy blood samples (nm390) was not detected. The positive predictive value (PPV) and negative predictive value (NPV) of our rapid diagnostic reagent were 100.00% and 99.76%, respectively, and the confidence interval was 95, which had a good correlation with microscopic examination (kappa value: 0.992530). For recombinant Plasmodium vivax aldolase protein, the sensitivity of colloidal gold test strip was 6.25 ng / ml. It is feasible to distinguish malaria infection caused by different malaria parasites by using aldolase protein as a rapid diagnostic reagent. The developed rapid diagnostic reagent can effectively distinguish the infection of Plasmodium vivax from that of Plasmodium falciparum. At the same time, the new antibody screening method used in this paper can be used in the screening process of other highly specific monoclonal antibodies against Plasmodium vivax and Plasmodium falciparum.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R531.3;Q78
【共引文獻(xiàn)】
相關(guān)期刊論文 前5條
1 陳國春;張鎖才;邵幼林;王安平;張曉霞;;惡性瘧患者血清腺苷脫氨酶和乳酸脫氫酶的測定及意義[J];廣東醫(yī)學(xué);2013年22期
2 郝文波;王萍;陳白虹;高洋;李明;;間日瘧原蟲部分乳酸脫氫酶基因的克隆及序列分析[J];熱帶醫(yī)學(xué)雜志;2006年08期
3 周志成;孫莉;吳英松;廖小青;郝文波;;間日瘧原蟲乳酸脫氫酶GST融合表達(dá)載體的構(gòu)建及表達(dá)[J];分子診斷與治療雜志;2010年02期
4 江莉;王真瑜;馬曉疆;張小萍;蔡黎;;間日瘧原蟲和惡性瘧原蟲乳酸脫氫酶基因的序列和重組抗原表位分析[J];中國寄生蟲學(xué)與寄生蟲病雜志;2010年02期
5 方強(qiáng);夏惠;王雪梅;齊文娟;常雪蓮;高琪;;間日瘧原蟲乳酸脫氫酶編碼區(qū)全長基因的克隆、序列分析及線性B細(xì)胞表位預(yù)測[J];中國人獸共患病學(xué)報;2010年06期
相關(guān)博士學(xué)位論文 前1條
1 方強(qiáng);間日瘧原蟲全長cDNA文庫的構(gòu)建與PvGDH的克隆表達(dá)、表位鑒定及單抗研究[D];蘇州大學(xué);2010年
相關(guān)碩士學(xué)位論文 前2條
1 孫莉;間日瘧原蟲乳酸脫氫酶的表達(dá)、純化及單克隆抗體的制備[D];南方醫(yī)科大學(xué);2008年
2 王錦明;莫氏巴貝斯蟲在我國的流行狀況調(diào)查及馬達(dá)復(fù)合物組件蛋白基因的初步研究[D];中國農(nóng)業(yè)科學(xué)院;2013年
，本文編號：2214374
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