本文選題：森林腦炎病毒 + prM-E基因��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文

【摘要】：森林腦炎又稱蜱傳腦炎,是由森林腦炎病毒經(jīng)硬蜱媒介所致自然疫源性急性中樞神經(jīng)系統(tǒng)傳染病,主要分布于亞洲和歐洲一些國家。我國主要見于東北及西北原始森林地區(qū)。近年來隨著旅游事業(yè)的發(fā)展、外來人群的涌入、森林工業(yè)生產(chǎn)環(huán)境的改變和林區(qū)生態(tài)環(huán)境的破壞,使得森林腦炎流行范圍逐漸擴(kuò)大,病例數(shù)逐年上升。因此森林腦炎的防治受到高度重視。 中和試驗(yàn)(NT)和血凝抑制試驗(yàn)(HI)是診斷黃病毒感染的最特異方法,然而中和試驗(yàn)耗時(shí)較長,常需要幾天,且需要專門進(jìn)行活病毒操作的封閉實(shí)驗(yàn)室,因此應(yīng)用受到一定限制�，F(xiàn)在一般使用ELISA試驗(yàn)檢測血清和CSF中特異性IgM和IgG抗體。商品化ELISA試劑盒使用滅活的病毒作為抗原,此種病毒的培養(yǎng)和抗原的純化需要在生物安全實(shí)驗(yàn)室進(jìn)行,存在許多安全隱患,并且發(fā)現(xiàn)完整滅活病毒作為抗原的ELISA試驗(yàn)與其他黃病毒感染有交叉反應(yīng),因此許多學(xué)者針對替代抗原進(jìn)行了研究。 M和E蛋白是森林腦炎病毒的結(jié)構(gòu)蛋白,E蛋白能誘導(dǎo)保護(hù)性抗體,具有與受體結(jié)合、細(xì)胞膜融合的功能和血凝活性。prM-E基因在轉(zhuǎn)染細(xì)胞中表達(dá)時(shí),可牢固地錨定在胞內(nèi)內(nèi)質(zhì)網(wǎng)膜上,形成穩(wěn)定的空間結(jié)構(gòu),并保證prM與E間的共價(jià)結(jié)合。 本研究選擇我國森林腦炎病毒MDJ01株的prM-E基因作為靶基因,首先對其進(jìn)行了克隆表達(dá)。從TBE病毒MDJ01株感染的BHK21細(xì)胞培養(yǎng)液中提取總RNA,RT-PCR擴(kuò)增獲得含prM-E基因的DNA,利用NotⅠ和HindⅢ雙酶切后,將prM-E基因插入供體質(zhì)粒pFastBacl中,測序證實(shí)編碼基因插入正確。將該重組質(zhì)粒轉(zhuǎn)化含有Bacmid DNA和Help質(zhì)粒的DH10Bac菌株,利用Tn7轉(zhuǎn)座子為工具,使之發(fā)生同源轉(zhuǎn)座,通過藍(lán)白斑篩選和PCR擴(kuò)增,獲得含有prM-E基因的重組Bacmid DNA。 將重組Bacmid DNA轉(zhuǎn)染昆蟲細(xì)胞Sf9后獲得重組桿狀病毒(rvBACME),將rvBACME感染Sf9細(xì)胞,用RT-PCR對其轉(zhuǎn)錄產(chǎn)物進(jìn)行了鑒定,結(jié)果表明
[Abstract]:Forest encephalitis, also known as tick-borne encephalitis, is a natural infectious disease of central nervous system caused by tick-borne encephalitis virus. It mainly distributes in some countries in Asia and Europe. China is mainly found in the northeast and northwest of the primeval forest region. In recent years, with the development of tourism, the influx of foreign people, the change of the production environment of forest industry and the destruction of ecological environment in forest area, the epidemic range of forest encephalitis is gradually expanded, and the number of cases increases year by year. Therefore, the prevention and treatment of forest encephalitis is highly valued. Neutralization test (NTT) and hemagglutination inhibition test (HIH) are the most specific methods for the diagnosis of yellow virus infection. However, neutralization test takes a long time and usually takes a few days. ELISA tests are now commonly used to detect specific IgM and IgG antibodies in serum and CSF. Commercial ELISA kit uses inactivated virus as antigen. The culture and purification of this virus need to be carried out in biosafety laboratory. It was also found that the ELISA test of intact inactivated virus as antigen had cross reaction with other yellow virus infections, so many researchers have studied alternative antigens. M and E proteins, the structural proteins of TEV, can induce protective antibody, and have the function of binding to receptor, cell membrane fusion and hemagglutination activity. PrM-E gene is expressed in transfected cells. It can be firmly anchored on the endoplasmic omentum to form a stable spatial structure and to ensure the covalent binding between prM and E. In this study, the prM-E gene of MDJ01 strain of Chinese Forest Encephalitis virus was selected as the target gene. The DNA containing prM-E gene was amplified by RT-PCR from BHK21 cells infected with TBE virus MDJ01 strain. The prM-E gene was inserted into the donor plasmid pFastBacl by double enzyme digestion of Not 鈪，

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