本文關(guān)鍵詞：蝙蝠感染絲狀病毒的血清學(xué)調(diào)查　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蝙蝠 絲狀病毒 核蛋白 間接ELISA 交叉反應(yīng)

【摘要】：絲狀病毒(Filovirus)屬于單股負(fù)鏈RNA病毒目(Mononegavirales),目前包括三個(gè)屬,分別為埃博拉病毒屬(Ebolavirus)、馬爾堡病毒屬(Marburgvirus)和庫瓦病毒屬(Cuevavirus),前二個(gè)屬主要分布于非洲,后一個(gè)屬分布于歐洲。絲狀病毒,對人具有高度的傳染性和致死性,所以國際上將該科一些成員列為潛在的生物戰(zhàn)劑,例如扎伊爾埃博拉病毒(Zaire Ebola virus,ZEBOV)和馬爾堡病毒(Marburgvirus,MARV)。除了雷斯頓埃博拉病毒(Reston Ebolavirus,REBOV)的自然宿主是豬以外,其余已知的埃博拉病毒、馬爾堡病毒以及庫瓦病毒的自然宿主是蝙蝠。我們實(shí)驗(yàn)室于2015年首次在亞洲(中國云南省)發(fā)現(xiàn)了新型蝙蝠絲狀病毒,說明亞洲地區(qū)的蝙蝠也有絲狀病毒的分布,但其種類、感染率以及公共衛(wèi)生意義尚不明確,而我國蝙蝠種類和數(shù)量繁多,因而建立一種快速、高效和準(zhǔn)確的蝙蝠絲狀病毒感染血清的檢測方法并以此開展血清流行病學(xué)調(diào)查具有十分重要的科學(xué)意義。本研究為了建立檢測MARV、REBOV、ZEBOV、庫瓦病毒(Lloviu cuevavirus,LLOV)以及新型絲狀病毒DH04株的血清抗體間接ELISA方法,首先構(gòu)建以上五種病毒核蛋白(NP)羧基端的原核表達(dá)質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21(DE3)進(jìn)行原核表達(dá),結(jié)果MARV、REBOV、ZEBOV和DH04四種病毒NP蛋白表達(dá)成功,鑒定為可溶性蛋白,并可與Anti-His單抗進(jìn)行特異性反應(yīng),說明蛋白表達(dá)正確。然后,分別以純化的目的蛋白為抗原包被96孔ELISA板,建立了檢測四種病毒血清抗體的間接ELISA方法:優(yōu)化抗原包被量為2μg/ml、封閉液為5%脫脂奶粉、二抗稀釋度為1:20000。建立的ELISA方法與一種蝙蝠輪狀病毒、三種蝙蝠和人的布尼亞病毒(來賓病毒、漢城病毒、春山病毒)蝙蝠感染血清均無交叉反應(yīng),證明特異性良好;并能檢測1∶1 600稀釋的陽性血清,說明敏感性良好;試驗(yàn)的批內(nèi)及批間變異系數(shù)均小于10%,重復(fù)性良好。用建立的間接ELISA方法檢測了云南、廣西以及浙江3個(gè)地區(qū)的蝙蝠血清,總體陽性率為45.2%(141/312),其中云南陽性率明顯高于其它兩個(gè)省份(P0.05),浙江與廣西的陽性率無明顯的統(tǒng)計(jì)學(xué)差異。云南陽性率為65.7%(94/143),其中與DH04、REBOV、ZEBOV和MARV反應(yīng)的血清分別占46.9%、36.4%、29.4%和21.0%;廣西陽性率26.2%(32/122),與REBOV反應(yīng)的血清最多,占16.4%,其次是DH04和ZEBOV,分別占10.7%和9.8%,MARV最少,僅占4.1%;浙江陽性率為31.9%(15/47),有27.7%的血清針對DH04反應(yīng),6.4%的血清針對ZEBOV反應(yīng),4.3%的血清針對MARV反應(yīng),2.1%的血清針對REBOV反應(yīng)。根據(jù)ELISA檢測結(jié)果,挑選75份蝙蝠血清,其中59份陽性血清,16份陰性血清,進(jìn)行Western blot(WB)檢測,發(fā)現(xiàn)ELISA檢測為陽性的蝙蝠血清與WB檢測的符合度在80%-100%之間,ELISA檢測為陰性的血清與WB檢測的符合度在78%-94%之間。使用單抗進(jìn)行交叉反應(yīng)確定,證明DH04不與REBOV、ZEBOV、MARV的抗體反應(yīng),MARV與REBOV、ZEBOV之間有較弱的交叉反應(yīng),REBOV和ZEBOV之間有很強(qiáng)的交叉反應(yīng)。本研究成功表達(dá)了DH04、MARV、REBOV和ZEBOV的NP羧基端蛋白,在此基礎(chǔ)上建立了檢測四種絲狀病毒感染血清抗體的間接ELISA方法,并對我國南方蝙蝠血清進(jìn)行了絲狀病毒的血清流行病學(xué)調(diào)查,結(jié)果表明我國蝙蝠存在比較廣泛的絲狀病毒感染,而且可能存在多種絲狀病毒混合感染的情況,同時(shí)不同病毒在不同地域和蝙蝠種類上具有一定的特征:在云南和浙江DH04流行最廣,而在廣西REBOV流行最廣;棕果蝠陽性率顯著高于其它四種蝙蝠,馬鐵菊頭蝠中針對DH04反應(yīng)的血清最多,小黃蝠中針對REBOV反應(yīng)的血清最多,而在南長翼蝠中未檢測到陽性血清。本研究提示我國面臨蝙蝠將自身攜帶的絲狀病毒傳播給其它動物的潛在風(fēng)險(xiǎn),為了防范蝙蝠源絲狀病毒病未來需要加大對蝙蝠絲狀病毒病原的監(jiān)測和防范措施的研究力度。
[Abstract]:Filamentous virus (Filovirus) belongs to the single stranded RNA (Mononegavirales), virales currently includes three genera, respectively (Ebolavirus), the Ebola virus belongs to the genus of Marburg virus (Marburgvirus) and Curval virus (Cuevavirus), the first two genera are mainly distributed in Africa, after a genus distributed in Europe. Filamentous virus, is highly contagious and fatal to people, so the family members of international admiral as potential biological agents, such as the Zaire Ebola virus (Zaire Ebola, virus, ZEBOV) and Marburg virus (Marburgvirus, MARV). In addition to Reston Ebola virus (Reston Ebolavirus REBOV) is the natural host of pigs and the rest of the known Ebola virus, Marburg virus and Curval virus natural host bats. Our laboratory in 2015 for the first time in Asia (Yunnan, China) discovered a new type of bat filamentous virus, that of Asia. The bats are also distributed filamentous viruses, but the species, infection rate and public health significance is not clear, and our country and there are a large number of bat species, and to establish a rapid, efficient and accurate detection method of bat filoirus infections serum and to carry out epidemiological investigation has very important scientific significance. The purpose of this study is to establish the detection of MARV, REBOV, ZEBOV, Curval (Lloviu cuevavirus, LLOV) virus, a novel filamentous virus strain DH04 serum antibody by indirect ELISA method, firstly, the above five kinds of virus nucleoprotein (NP) prokaryotic expression plasmid C-terminal, transformed into Escherichia coli BL21 (DE3) for prokaryotic expression results MARV, REBOV, four kinds of virus NP protein ZEBOV and DH04 expression, identified as soluble protein, and specific reaction with Anti-His monoclonal antibody, indicating correct expression of the protein. Then, with the purpose of purification Protein antigen ELISA 96 hole plate, an indirect ELISA was developed to detect four kinds of virus antibody: optimization of antigen amount is 2 g/ml, the closed solution of 5% skim milk, two anti dilution ELISA method established by 1:20000. and a bat rotavirus, three species of bats and people bunyavirus (guest virus, Seoul virus, spring Hill virus) infection of bat serum had no cross reaction demonstrated good specificity; and detection of 1: 1600 diluted serum showed good sensitivity; the intra batch and inter batch variation coefficient test were less than 10%, good repeatability. The developed indirect ELISA method the detection of Yunnan, the 3 areas of Guangxi and Zhejiang bat serum, the total positive rate was 45.2% (141/312), the positive rate of Yunnan was higher than that of the other two provinces (P0.05), no significant statistical positive rate in Zhejiang and Guangxi, Yunnan difference. The positive rate was 65 .7% (94/143), with DH04, REBOV, ZEBOV and MARV in serum were 46.9%, 36.4%, 29.4% and 21% in Guangxi; the positive rate was 26.2% (32/122), serum reacted with REBOV the most, accounting for 16.4%, followed by DH04 and ZEBOV, respectively accounted for 10.7% and 9.8% MARV, at least, accounted for only 4.1%; in Zhejiang, the positive rate was 31.9% (15/47), serum 27.7% response to DH04 6.4%, the serum response to ZEBOV, 4.3% to 2.1% of the serum MARV reaction, serum response to REBOV. According to the results of ELISA, selected 75 bat serum, 59 positive sera and 16 negative serum samples (blot, Western WB) detection, ELISA detected for serum and WB were detected with the bat between 80%-100%, ELISA and WB for the detection of serum test negative coincidence rate between 78%-94%. Using monoclonal antibody cross reactivity to determine, proof of DH04 and REBOV, ZEBOV, MARV and MARV antibody response, RE BOV, a weak cross reaction between ZEBOV, a strong cross reaction between REBOV and ZEBOV. The expression of DH04, MARV, NP and REBOV protein carboxyl terminal ZEBOV, established on the basis of indirect ELISA for serum antibody detection of four species of filamentous virus infection, and the South China bat serum a seroepidemiological survey of filamentous viruses, the results show that China's bat more extensive filamentous virus infection, but there may be a variety of filamentous virus mixed infection, while different virus has certain characteristics in different regions and bat species: Zhejiang DH04 in Yunnan and the most popular, the most popular REBOV in Guangxi; the positive rate of Rousettus was significantly higher than that of other four species of bats, Rhinolophus ferrumequinum in serum response to DH04 most, in response to REBOV yellow bat serum most, while in the South long winged bats in unghecded The positive serum was detected. This study indicates that our country is facing the potential risk that bats transmit their filamentous virus to other animals. In order to prevent bat's filamentous disease, we need to intensify the research on monitoring and prevention measures of bat's filamentous virus.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 蔣麗香;邢超;羅靜;史秋梅;何宏軒;;野生動物在埃博拉病毒維持和傳播中的作用[J];科學(xué)通報(bào);2015年20期

2 蓋微微;鄭學(xué)星;薛向紅;高玉偉;趙永坤;王鐵成;王化磊;黃耕;馮娜;楊松濤;夏咸柱;;埃博拉病毒檢測與分型Real-time PCR方法的建立[J];中國病原生物學(xué)雜志;2013年03期

3 Herve Raoul;;Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays[J];Virologica Sinica;2012年05期

4 ;Laboratory Detection and Diagnosis of Filoviruses[J];Virologica Sinica;2011年02期

相關(guān)博士學(xué)位論文 前1條

1 何彪;蝙蝠病毒組學(xué)及其新病毒的發(fā)現(xiàn)與鑒定[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2014年

相關(guān)碩士學(xué)位論文 前2條

1 李興宇;我國東北三省蝙蝠病毒組學(xué)及其新病毒的鑒定[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2016年

2 夏樂樂;蝙蝠輪狀病毒的分離與鑒定[D];吉林農(nóng)業(yè)大學(xué);2014年

，

本文編號：1416724