本文選題：狂犬病 + 病毒��； 參考：《中國疾病預(yù)防控制中心》2009年碩士論文

【摘要】： 狂犬病是由狂犬病病毒(Rabies Virus,RV)引起的一種烈性傳染病,目前尚無有效的治療手段,病死率幾乎為100%,而接種狂犬疫苗是唯一有效的預(yù)防措施。評價狂犬疫苗效果的途徑是檢測免疫后血清中和抗體水平,目前主要是利用小鼠中和試驗和細胞中和實驗來檢測,但這種方法操作較為復(fù)雜,不宜推廣。狂犬病病毒糖蛋白(Glycoprotein,GP)是5種病毒蛋白中唯一能刺激機體產(chǎn)生中和抗體的抗原,所以狂犬病病毒GP在疫苗研制、抗體檢測等方面有廣泛的應(yīng)用。除此之外,狂犬病病毒GP還與病毒毒力、致病性及病毒滴度等密切相關(guān),本研究通過桿狀病毒表達系統(tǒng)所表達的狂犬病病毒GP以及所建立的穩(wěn)定表達狂犬病病毒GP的細胞系,不僅有助于探索更為安全易行的狂犬病抗體檢測方法,還可為進一步研究狂犬病病毒GP結(jié)構(gòu)和功能奠定基礎(chǔ)。 由于狂犬病病毒GP的已知抗原位點主要位于膜外區(qū),本研究首先利用Bac-to-Bac桿狀病毒表達系統(tǒng)分別表達狂犬病病毒GP及狂犬病病毒GP膜外區(qū),利用重組表達的狂犬病病毒GP及狂犬病病毒GP膜外區(qū)蛋白做抗原,探索建立間接ELISA法檢測人免疫后血清抗狂犬病病毒GP IgG水平。首先利用RT-PCR方法擴增得到狂犬病病毒CTN-181株GP基因編碼區(qū)段和GP基因膜外區(qū)編碼區(qū)段,然后利用桿狀病毒表達系統(tǒng)將兩段編碼基因在昆蟲細胞Sf9中進行表達。結(jié)果顯示:克隆有G基因編碼區(qū)段的重組桿狀病毒表達質(zhì)粒轉(zhuǎn)染Sf9細胞后24小時出現(xiàn)細胞病變,48小時細胞病變明顯;而在轉(zhuǎn)染克隆有GP基因膜外區(qū)段的重組桿狀病毒表達質(zhì)粒轉(zhuǎn)染Sf9細胞36小時后出現(xiàn)細胞病變,60小時細胞病變明顯;間接免疫熒光及Western Blot結(jié)果顯示表達的這兩種蛋白均具有抗原性,并且完整GP的抗原性要優(yōu)于GP膜外區(qū)的抗原性。進一步分別利用表達的GP及其膜外區(qū)做抗原,初步探索建立ELISA檢測方法,檢測人免疫后血清中抗GP IgG水平。結(jié)果顯示,HRP標記的抗人IgG最佳稀釋度為1:60000,通過棋盤滴定的方法獲得了包被抗原的最佳稀釋度為1:320,待測血清的最佳稀釋度為1:100。在設(shè)定的實驗條件下,GP膜外區(qū)做抗原用于ELISA檢測方法(P/N值4.15:)優(yōu)于以完整的GP做抗原(P/N值:3.12),但是存在的問題是背景值偏高,仍需進一步優(yōu)化。 本研究進一步利用IRES(內(nèi)部核糖體進入位點)介導(dǎo)的雙表達載體pIRES2-ZsGreen1,分別將CTN-181株G基因、增強型綠色熒光蛋白(ZsGreen)基因插入,構(gòu)建可以同時表達狂犬病病毒CTN-181株G基因及增強型綠色熒光蛋白(ZsGreen)基因的雙表達重組質(zhì)粒,通過脂質(zhì)體轉(zhuǎn)染BHK-21細胞后,用新霉素類似物G418進行細胞株的篩選,建立穩(wěn)定表達狂犬病病毒GP及ZsGreen的細胞系。結(jié)果所獲得的細胞株可以高效并同時表達狂犬病病毒GP和ZsGreen,穩(wěn)定進行細胞傳代21代次,狂犬病病毒CTN-181 GP和ZsGreen兩個蛋白仍可在已經(jīng)獲得的細胞系中穩(wěn)定表達,所獲得的細胞株ZsGreen檢測陽性率在80%左右,生長周期為6天左右。該細胞系可以應(yīng)用于狂犬病病毒GP抗體檢測,也可以應(yīng)用于建立狂犬病病毒反向遺傳系統(tǒng)反式提供狂犬病病毒GP以期提高重組病毒滴度。 狂犬病病毒GP及GP膜外區(qū)在桿狀病毒中的成功表達以及表達狂犬病病毒GP及綠色熒光蛋白ZsGreen細胞系的建立為狂犬病特異性抗體的檢測和評價奠定了基礎(chǔ),也為優(yōu)化已有的狂犬病病毒反向遺傳系統(tǒng)提供了新的科研思路和技術(shù)路線。
[Abstract]:Rabies is a strong infectious disease caused by Rabies Virus (RV). At present, there is no effective treatment, the mortality rate is almost 100%, and the vaccination of rabies is the only effective preventive measure. The way to evaluate the effect of rabies vaccine is to detect the level of immunized blood and neutralization antibody. At present, the main method is to use the neutralization test in mice. Glycoprotein (GP) is the only antigen in the 5 virus proteins that can stimulate the body to produce neutralizing antibodies, so the rabies virus GP is widely used in vaccine development and anti physical examination. In addition, rabies disease, in addition to rabies disease. Toxic GP is also closely related to virulence, pathogenicity and viral titer. The rabies virus GP expressed in the baculovirus expression system and the cell lines that have been established to express the stable rabies virus GP are not only helpful for the exploration of more safe and easy detection methods of rabies antibody, but also for further study of rabies disease. The structure and function of toxic GP lay the foundation.
Because the known antigen loci of rabies virus GP are mainly located in the outer region of the membrane, this study first uses Bac-to-Bac baculovirus expression system to express rabies virus GP and the outer region of rabies virus GP membrane. The recombinant expressed rabies virus GP and the egg white of the rabies virus GP membrane are used as antigen to explore the indirect ELISA method for the detection of human beings. After immunization, serum anti rabies virus GP IgG level. First, RT-PCR method was used to amplify the coding region of GP gene of rabies virus CTN-181 strain and the encoding region of GP gene, and then the two segment encoding gene was expressed in the insect cell Sf9 by baculovirus expression system. The results showed that the clone had the weight of the G gene coding region. 24 hours after the transfection of the baculovirus expression plasmid to Sf9 cells, the cytopathic lesions were found, and the cytopathic lesions were obvious at 48 hours, while the recombinant baculovirus expressing the recombinant baculovirus transfected from the GP gene was transfected to Sf9 cells for 36 hours, and the cytopathic lesions were observed in 60 hours. The results of indirect immunofluorescence and Western Blot showed the expression. All the two proteins have antigenicity, and the antigenicity of the complete GP is better than the antigenicity of the GP outer region. Further using the expressed GP and its outer region as antigen, the ELISA detection method is initially explored to detect the anti GP IgG level in the serum of human immune system. The results show that the best dilution degree of the HRP labeled human IgG is 1:60000, through chess. The best dilution of the coated antigen was obtained by the method of disk titration, and the best dilution of the serum was 1:100. at the set of experimental conditions. The antigen used in the outer region of the GP membrane for the ELISA detection method (P/N value 4.15:) was superior to the complete GP (P/N value: 3.12), but the problem was that the background value was high and still need to be further optimized.
In this study, the double expression vector pIRES2-ZsGreen1 mediated by IRES (internal ribosome entry site) was used to insert the CTN-181 strain G gene and the enhanced green fluorescent protein (ZsGreen) gene to construct a double expression recombinant plasmid that could simultaneously express the G gene of the rabies virus CTN-181 strain and the enhanced green fluorescent protein (ZsGreen) gene. After transfection of BHK-21 cells with liposomes, the cell lines were screened with neomycin analogs G418 to establish a cell line that stably expressed rabies virus GP and ZsGreen. The results showed that the obtained cell lines could express rabies virus GP and ZsGreen at the same time, stabilize cell transmission for 21 generations, rabies virus CTN-181 GP and ZsGreen two The protein can still be expressed steadily in the obtained cell lines. The positive rate of the cell line ZsGreen is about 80% and the growth cycle is about 6 days. The cell line can be applied to the detection of rabies virus GP antibody and can be used to establish rabies virus reverse genetic system to provide rabies virus GP in order to increase the weight of the virus. Group virus titer.
The successful expression of rabies virus GP and GP membrane in baculovirus and the establishment of the expression of rabies virus GP and green fluorescent protein ZsGreen cell line have laid the foundation for the detection and evaluation of rabies specific antibodies, and also provide new scientific ideas and technical routes for optimizing the existing reverse genetic system of rabies virus.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373

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