狂犬病病毒CTN181株糖蛋白的真核表达及初步应用

发布时间：2018-06-15 16:51

本文选题：狂犬病 + 病毒　；参考：《中国疾病预防控制中心》2009年硕士论文

【摘要】： 狂犬病是由狂犬病病毒(Rabies Virus,RV)引起的一种烈性传染病,目前尚无有效的治疗手段,病死率几乎为100%,而接种狂犬疫苗是唯一有效的预防措施。评价狂犬疫苗效果的途径是检测免疫后血清中和抗体水平,目前主要是利用小鼠中和试验和细胞中和实验来检测,但这种方法操作较为复杂,不宜推广。狂犬病病毒糖蛋白(Glycoprotein,GP)是5种病毒蛋白中唯一能刺激机体产生中和抗体的抗原,所以狂犬病病毒GP在疫苗研制、抗体检测等方面有广泛的应用。除此之外,狂犬病病毒GP还与病毒毒力、致病性及病毒滴度等密切相关,本研究通过杆状病毒表达系统所表达的狂犬病病毒GP以及所建立的稳定表达狂犬病病毒GP的细胞系,不仅有助于探索更为安全易行的狂犬病抗体检测方法,还可为进一步研究狂犬病病毒GP结构和功能奠定基础。由于狂犬病病毒GP的已知抗原位点主要位于膜外区,本研究首先利用Bac-to-Bac杆状病毒表达系统分别表达狂犬病病毒GP及狂犬病病毒GP膜外区,利用重组表达的狂犬病病毒GP及狂犬病病毒GP膜外区蛋白做抗原,探索建立间接ELISA法检测人免疫后血清抗狂犬病病毒GP IgG水平。首先利用RT-PCR方法扩增得到狂犬病病毒CTN-181株GP基因编码区段和GP基因膜外区编码区段,然后利用杆状病毒表达系统将两段编码基因在昆虫细胞Sf9中进行表达。结果显示:克隆有G基因编码区段的重组杆状病毒表达质粒转染Sf9细胞后24小时出现细胞病变,48小时细胞病变明显;而在转染克隆有GP基因膜外区段的重组杆状病毒表达质粒转染Sf9细胞36小时后出现细胞病变,60小时细胞病变明显;间接免疫荧光及Western Blot结果显示表达的这两种蛋白均具有抗原性,并且完整GP的抗原性要优于GP膜外区的抗原性。进一步分别利用表达的GP及其膜外区做抗原,初步探索建立ELISA检测方法,检测人免疫后血清中抗GP IgG水平。结果显示,HRP标记的抗人IgG最佳稀释度为1:60000,通过棋盘滴定的方法获得了包被抗原的最佳稀释度为1:320,待测血清的最佳稀释度为1:100。在设定的实验条件下,GP膜外区做抗原用于ELISA检测方法(P/N值4.15:)优于以完整的GP做抗原(P/N值:3.12),但是存在的问题是背景值偏高,仍需进一步优化。本研究进一步利用IRES(内部核糖体进入位点)介导的双表达载体pIRES2-ZsGreen1,分别将CTN-181株G基因、增强型绿色荧光蛋白(ZsGreen)基因插入,构建可以同时表达狂犬病病毒CTN-181株G基因及增强型绿色荧光蛋白(ZsGreen)基因的双表达重组质粒,通过脂质体转染BHK-21细胞后,用新霉素类似物G418进行细胞株的筛选,建立稳定表达狂犬病病毒GP及ZsGreen的细胞系。结果所获得的细胞株可以高效并同时表达狂犬病病毒GP和ZsGreen,稳定进行细胞传代21代次,狂犬病病毒CTN-181 GP和ZsGreen两个蛋白仍可在已经获得的细胞系中稳定表达,所获得的细胞株ZsGreen检测阳性率在80%左右,生长周期为6天左右。该细胞系可以应用于狂犬病病毒GP抗体检测,也可以应用于建立狂犬病病毒反向遗传系统反式提供狂犬病病毒GP以期提高重组病毒滴度。狂犬病病毒GP及GP膜外区在杆状病毒中的成功表达以及表达狂犬病病毒GP及绿色荧光蛋白ZsGreen细胞系的建立为狂犬病特异性抗体的检测和评价奠定了基础,也为优化已有的狂犬病病毒反向遗传系统提供了新的科研思路和技术路线。
[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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

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