本文選題：基孔肯雅病毒(CHIKV) + 雙抗體夾心ELISA　； 參考：《中國(guó)疾病預(yù)防控制中心》2017年碩士論文

【摘要】：基孔肯雅病毒(Chikungunyavirus,CHIKV)是單股正鏈RNA病毒,為披膜病毒科,甲病毒屬;病毒顆粒呈T=4正二十面體結(jié)構(gòu),直徑約60-70nm,在電鏡下呈圓形。病毒主要有五個(gè)結(jié)構(gòu)蛋白(C、E2、E3、6K、E1),E1與E2形成異二聚體,每三個(gè)E1/E2異二聚體形成一個(gè)微小的凸起,每個(gè)病毒顆粒的表面共有80個(gè)相同的凸起。在包膜之下E1/E2異二聚體同C蛋白構(gòu)成的衣殼相連。除結(jié)構(gòu)蛋白外,還有四個(gè)非結(jié)構(gòu)蛋白(NS1、NS2、NS3、NS4)主要在病毒復(fù)制過(guò)程中起輔助調(diào)節(jié)作用。人感染CHIKV后可引起以關(guān)節(jié)痛、發(fā)熱、皮疹為主要癥狀的基孔肯雅熱。大多數(shù)患者癥狀輕,其他癥狀數(shù)周之內(nèi)可以自行痊愈,但關(guān)節(jié)痛往往持續(xù)存在,影響正常的生活、產(chǎn)生經(jīng)濟(jì)負(fù)擔(dān)。少數(shù)患者會(huì)并發(fā)腦炎等重癥危及生命。CHIKV通過(guò)伊蚊傳播,伊蚊分布范圍廣泛;在2014年CHIKV傳入美洲大陸后,在全球大部分地區(qū)均有流行報(bào)道。同我國(guó)臨近的東南亞地區(qū)一直是基孔肯雅熱的主要流行地區(qū)之一。我國(guó)于1986年首次成功分離CHIKV,2008年首次報(bào)道輸入性基孔肯雅熱,2010年首次由于輸入性病例導(dǎo)致的本土基孔肯雅熱的暴發(fā)流行。隨著CHIKV在全球范圍內(nèi)擴(kuò)大流行,我國(guó)仍有再次暴發(fā)基孔肯雅熱的可能性。在疾病流行的預(yù)防控制工作當(dāng)中,早期診斷十分重要,本研究基于以上目的擬建立血清CHIKV抗原檢測(cè)的方法。通過(guò)桿狀病毒表達(dá)系統(tǒng)生產(chǎn)CHIKV病毒樣顆粒(VLP),該VLP包含CHIKV的全部結(jié)構(gòu)蛋白,在電鏡下可以見到圓形顆粒。VLP與滅活的CHIKV有一致的抗原性,可以與抗CHIKV抗體良好結(jié)合。VLP由于不含有核酸不具有感染性,相比滅活病毒更加安全,可以在生物安全一級(jí)實(shí)驗(yàn)室中進(jìn)行操作,降低了研究的生物安全風(fēng)險(xiǎn)。使用VLP免疫小鼠和家兔制備抗CHIKV的鼠免疫腹水和兔免疫血清。通過(guò)免疫熒光方法檢測(cè),顯示抗體與病毒結(jié)合良好;通過(guò)ELISA方法檢測(cè)抗體效價(jià)均在1:10萬(wàn)以上。1、以純化鼠免疫腹水作為包被抗體,兔免疫血清作為捕獲抗體,建立了檢測(cè)CHIKV抗原的雙抗體夾心ELISA方法。,該方法法具有較好的特異性和敏感性,在0.1ml中含50TCID50以上CHIKV的樣本均可以用本方法檢測(cè)出;模擬病人血清可被成功檢出;登革熱、腎綜合征出血熱病人及健康人血清進(jìn)行CHIKV抗原檢測(cè),均為陰性;重復(fù)性良好板間變異小于10%,板內(nèi)變異小于5%。2、用VLP為抗原建立了檢測(cè)抗CHIKV IgG抗體的間接法ELISA,VLP擁有的良好抗原性可以替代滅活病毒作為抗原對(duì)IgG進(jìn)行捕獲檢測(cè)。完善抗CHIKV IgM抗體的捕獲法ELISA,檢測(cè)病人急性期血清檢測(cè)均呈陽(yáng)性。兩種方法重復(fù)性良好板間變異小于10%,板內(nèi)變異小于5%。本研究建立了 CHIKV抗原和抗體檢測(cè)方法,并進(jìn)行初步評(píng)價(jià),對(duì)于臨床診斷以及流行病學(xué)調(diào)查有積極意義。
[Abstract]:Chikungunya virus of Chikungunya virus (Chikungunya virus) is a single-stranded RNA virus belonging to the genus Arbovirus belonging to the family Erythroviridae. The virus particles have a Tap-4 regular icosahedron structure with a diameter of about 60-70 nm and are circular under electron microscope. The virus mainly has five structural proteins, Con E2E _ 3, E _ 3N _ 6K, E _ 1, E _ 1, and E _ 2. Each three E1/E2 heterodimers form a tiny protuberance, and each virus particle has 80 identical protuberances on the surface. Under the capsule, E1/E2 heterodimer is connected to the capsid of C protein. In addition to structural proteins, there are four nonstructural proteins, NS1, NS2, NS3, NS4, which play a coregulatory role in viral replication. Human infection with CHIKV can cause Kikungunya fever with arthralgia, fever and rash as the main symptoms. Most patients have mild symptoms and other symptoms can heal themselves within a few weeks, but joint pain often persists, affecting normal life and creating financial burdens. A few patients with encephalitis and other serious life-threatening. CHIKV spread through the Aedes mosquito, Aedes is widely distributed. After CHIKV was introduced to the American continent in 2014, there are popular reports in most parts of the world. Southeast Asia, which is close to our country, has been one of the main endemic areas of Kikungunya fever. CHIKV was isolated successfully in China in 1986, the imported Kikungunya fever was first reported in 2008, and the outbreak of local Kikungunya fever caused by imported cases was first reported in 2010. With the spread of CHIKV in the world, there is still the possibility of a further outbreak of Kikungunya fever in China. Early diagnosis is very important in the prevention and control of epidemic diseases. This study aims to establish a method for detection of serum CHIKV antigen. CHIKV virus-like particles were produced by baculovirus expression system. The VLP contains all the structural proteins of CHIKV. Under electron microscope, circular particles. VLP have the same antigenicity as inactivated CHIKV. It is more safe than inactivated virus because it does not contain nucleic acid. It can be operated in biosafety primary laboratory and reduce the risk of biosafety. VLP was used to immunize mice and rabbits to prepare anti-CHIKV ascites and rabbit immune serum. Immunofluorescence assay showed that the antibody was well bound to the virus, and the titers of the antibody were all above 1: 100,000 by ELISA method. The purified mouse ascites were used as the coated antibody, and the rabbit immune serum was used as the capture antibody. A sandwich ELISA method for the detection of CHIKV antigen was established. The method has good specificity and sensitivity. All samples containing CHIKV above 50TCID50 in 0.1ml can be detected by this method; the simulated patient serum can be successfully detected; dengue fever can be detected successfully. Serum CHIKV antigen was negative in patients with hemorrhagic fever with renal syndrome (HFRS) and healthy subjects. The good inter-plate variation was less than 10% and the intraplate variation was less than 5.2%. Using VLP as antigen, an indirect method for detection of anti CHIKV IgG antibodies, ELISAN VLP with good antigenicity, could be used as antigen instead of inactivated virus to capture and detect IgG. The capture method of anti-CHIKV IgM antibody was improved to detect positive serum of patients in acute phase. The reproducibility of the two methods was better than 10, and the intra-plate variation was less than 5. In this study, a method for the detection of CHIKV antigen and antibody was established and evaluated, which has positive significance for clinical diagnosis and epidemiological investigation.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6;R511

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鄭夔;袁帥;梁潔怡;黃吉城;李小波;戴俊;師永霞;張顯光;宋衛(wèi);吳惠明;;我國(guó)口岸首例輸入性寨卡病毒感染病例的實(shí)驗(yàn)室檢測(cè)[J];中國(guó)國(guó)境衛(wèi)生檢疫雜志;2016年02期

2 劉起勇;;寨卡病毒媒介伊蚊控制策略和措施展望[J];中國(guó)媒介生物學(xué)及控制雜志;2016年02期

3 胡慧瓊;劉斌;王紅;吳增輝;;類風(fēng)濕因子對(duì)酶聯(lián)免疫吸附試驗(yàn)和電化學(xué)發(fā)光免疫分析檢測(cè)甲型肝炎IgM的影響探討[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2016年03期

4 楊明東;姜進(jìn)勇;鄭宇婷;周紅寧;;云南省邊境地區(qū)埃及伊蚊分布調(diào)查[J];中國(guó)媒介生物學(xué)及控制雜志;2015年04期

5 仇書興;霍玉奇;黃曉媛;楊曉明;;包膜病毒樣顆粒疫苗研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2015年03期

6 李建東;張全福;張碩;李川;劉琴芝;梁米芳;李德新;;基于病毒樣顆粒的檢測(cè)基孔肯雅病毒IgM抗體的MacELISA方法的建立與評(píng)價(jià)[J];病毒學(xué)報(bào);2014年06期

7 王艷紅;寶福凱;柳愛華;;云南基孔肯雅熱研究概況[J];中國(guó)熱帶醫(yī)學(xué);2013年09期

8 龍遺芳;郭中敏;陸家海;;病毒樣顆粒技術(shù)——現(xiàn)代生物醫(yī)學(xué)應(yīng)用的新平臺(tái)[J];中國(guó)病毒病雜志;2013年04期

9 戈勝?gòu)?qiáng);王志亮;;禽流感病毒樣顆粒研究進(jìn)展[J];病毒學(xué)報(bào);2013年02期

10 鄭夔;丁國(guó)允;周惠瓊;謝雪妹;李小波;師永霞;蘇錦坤;黃吉城;;應(yīng)用含內(nèi)參的多重實(shí)時(shí)熒光RT-PCR方法快速檢測(cè)登革病毒和基孔肯雅病毒[J];中國(guó)人獸共患病學(xué)報(bào);2013年03期

相關(guān)博士學(xué)位論文 前1條

1 龐正;病毒性出血熱多重?zé)晒舛縍T-PCR檢測(cè)方法的建立[D];中國(guó)疾病預(yù)防控制中心;2013年

相關(guān)碩士學(xué)位論文 前2條

1 王肖yN;雞白細(xì)胞介素18雙抗體夾心ELISA檢測(cè)方法的建立及初步應(yīng)用[D];山東農(nóng)業(yè)大學(xué);2013年

2 凡敏;基孔肯亞與辛德畢斯病毒檢測(cè)基因芯片的建立及初步應(yīng)用[D];吉林農(nóng)業(yè)大學(xué);2012年

，

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