【作者】 焦洋；

【导师】 唐青；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 硕士

【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。更多还原

【Abstract】 Rabies is a fatal zoonosis caused by rabies virus (RV). Inspite of no any effective therapy methods have been available to cure the disease, the pre-exposure and/or post-exposure prophylaxis with rabies vaccine is always the only way for rabies prevention. The method to evaluate the effectivity of vaccination is to detect the neutralization antibody (NA) in the serum after immunization. Ideally, the neutralization test is commonly used for the NA detection. However, the operation of neutralization test is too complicated to extend for routine use. Because only the rabies virus glycoprotein (GP) has the ability to be as an antigen to stimulate the production of NA in vaccinees, in a way, the anti-GP immunoglobulin (IgG) could be used to reflect the NA level indirectly. The established Enzyme-Linked ImmunoSorbent Assay (ELISA) method is potential to evaluate the effectivity of vaccination indirectly through detection of the anti-GP immunoglobulin (IgG) level. Additionly, the established ELISA method is so convenient and time-save that can be widely used in the related laboratories, basic CDCs to evaluate the effectivity of vaccination rapidly and precisely. Moreover, the RV GP also has close connection with viral toxicity, pathogenicity, and virus titer, so the established RV GP-stable-expressing BHK-21 cell line is helpful for the study of protein structure and function.In the first section, the GP coding gene (GP CD) and GP extramembrane region coding gene (GP EX) was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and then both were cloned into and expressed by the Baculovirus Expression System in insect cell line Sf9. The results observed initial, mild cytopathic effect (CPE) of Sf9 cells 24 hours after transfection with recombinant GP CD Baculovirus plasmid and obvious CPE after 48 hours, while the silimar CPE was observed after 36 and 60 hours after transfection with recombinant GP EX Baculovirus plasmid. In addition, the antigenicity of both the expressed proteins was identified by Immunofluorescence Assay (IFA) and Western Blot. Both had the property of antigenicity and the antigenicity of GP CD was superior compared with GP EX. In the further study, both the expressed proteins were used as antigens to established ELISA method for detection the serum anti-GP IgG level in the immunized population. The results showed that the ELISA method established using GP EX was superior compared with ELISA using GP CD (the P/N value of the former was higher than the latter). However, the established ELISA methods should be optimized because of the presence of impure background.In the second section, recombinant double expression plasmid (RDEP) mediated by IRES (internal ribosome entry sites) was constructed using pIRES2-ZsGreenl vector, which can express RV GP and enhanced GFP (green fluorescent protein) ZsGreen simultaneously. Then the RDEP was transfected into BHK-21 cells using lipofectamine 2000, and the transfected cells was screened by G418 (a kind of neomycin analog) to establish the cell line stablely expressing both RV GP and ZsGreen. The results demonstrated that the screened BHK-21 cell line has the ability to stalbely express both RV GP and ZsGreen with high performance, and the ratio of ZsGreen-expressed cells is higher and higher with the cell passage, and finally the ratio can reached up to 80%. This cell line can also be helpful for the enhancement of rescued rabies virus titer through reverse genetics and for the study of property and function of RV GP.In conclusion, both the successful expression of RV GP and GP extramembrane region, and the establishment of cell line with stably expressing RV GP and ZsGreen are not only helpful for the detection and evaluation of specific RV antibody, but also beneficial for the optimization of the established RV CTN-181 strain-based reverse genetics.更多还原