表达犬瘟热病毒H基因重组狂犬病病毒生物学特性的研究

发布时间：2018-05-03 07:57

本文选题：狂犬病病毒 + 犬瘟热病毒　；参考：《华南农业大学》2016年硕士论文

【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies virus,RABV)引起的一种人兽共患传染病,在全球范围内流行,是目前病死率最高的传染病之一,一旦感染出现临床症状,几乎100%的死亡。犬瘟热(Canine Distemper,CD)是由犬瘟热病毒(Canine Distemper vrus,CDV)引起的高度接触性传染病,它在全球范围内流行,死亡率高达80%,给养犬业和毛皮动物养殖业造成严重的危害。预防接种是控制狂犬病和犬瘟热的主要途径。目前,传统的狂犬病弱毒疫苗成本低廉,但存在毒力残留或致病性回复突变的安全隐患;进口疫苗安全性高,免疫效果好,但价格昂贵,难以广泛推广使用。研制安全、高效、成本低廉的动物狂犬病疫苗,仍具现实意义。市场上应用广泛的犬瘟热疫苗存在热稳定性差、免疫效果易受母源抗体干扰等多重问题。因此,研制安全、高效的新型CD疫苗具有重要意义。本研究利用已建立的狂犬病病毒(Rabies virus,RABV)反向遗传操作技术平台,在通过反向遗传拯救的狂犬病病毒弱毒株HEP-d G的基础上,运用分子生物学技术在狂犬病病毒(HEP-d G株)基因组G与L之间再插入犬瘟热病毒H基因,构建了重组全长c DNA质粒p HEP-d G(H),成功拯救出同时表达狂犬病病毒双G蛋白和犬瘟热病毒H蛋白的重组狂犬病病毒HEP-d G(H)。通过对重组病毒的生物学特性研究,该重组狂犬病毒HEP-d G(H)可以在BHK细胞上稳定的增殖,达到较高的病毒滴度。抽取该重组病毒第2、5、10代的RNA,进行RT-PCR扩增H、d G基因并测序,基因未发生突变。荧光定量PCR和Western blot验证表明外源基因H和d G能够表达。病毒的生长曲线表明HEP-d G(H)保持着与HEP-d G毒株相似的生长特性,在96h达到高峰,但重组病毒HEP-d G(H)的滴度在每个时间点略低于HEP-d G毒株。病毒的扩散感染实验显示在低MOI的情况下,重组病毒HEP-d G(H)的扩散能力不及HEP-d G毒株,这与生长曲线相吻合。致病性实验显示,重组病毒HEP-d G(H)与HEP-d G对6-7周龄成鼠没有致死性,且重组病毒HEP-d G(H)对成鼠体重的影响弱于HEP-d G;而对3日龄内的乳鼠,结果显示HEP-d G(H)的致命性高于HEP-d G。免疫原性实验显示,重组病毒HEP-d G(H)与HEP-d G均能诱导小鼠产生抗狂犬病的抗体,在7 d时抗体已达到保护水平(0.5 EU),且HEP-d G(H)可诱导产生CDV中和抗体。攻毒试验显示HEP-d G(H)与HEP-d G免疫3周后,诱导的RABV抗体水平能够抵御CVS-24的攻击,这些结果表明,重组的狂犬病病毒可以作为有潜力的疫苗候选株。综上所述,重组狂犬病病毒HEP-d G(H)具有良好的免疫原性,具有作为新型基因工程疫苗的潜质,本研究为犬的新型多价基因工程疫苗的研制奠定了基础。
[Abstract]:Rabies is a zoonotic infectious disease caused by rabies virus Rabies virus (Rabies virus). Rabies is one of the most fatal infectious diseases in the world. Once the infection appears clinical symptoms, almost 100% of the deaths occur. Canine distemper virus (Canine distemperus) is a highly contagious disease caused by canine distemper virus (Canine Distemper vrusus). It is prevalent in the world, and the mortality rate is as high as 80%. Vaccination is the main way to control rabies and canine distemper. At present, the traditional rabies attenuated virus vaccine has low cost, but it has the hidden danger of virulence residue or pathogenicity reverting mutation. The imported vaccine has high safety and good immune effect, but the price is expensive, so it is difficult to be widely used. It is still of practical significance to develop animal rabies vaccine which is safe, efficient and low cost. The widely used canine distemper vaccine in the market has many problems such as poor thermal stability, immune effect easily interfered by maternal antibody and so on. Therefore, it is of great significance to develop a safe and efficient CD vaccine. In this study, the rabies virus rabies virus rabies virus Rabies virus Rabies virus rabV reverse genetic operating technology platform was established, and the rabies virus attenuated strain HEP-d G was saved by reverse heredity. The gene of canine distemper virus H was inserted between the genome G and L of rabies virus (HEP-d G strain) by molecular biology technique. The recombinant c DNA plasmid, p HEP-d GnH, was constructed, and the recombinant rabies virus HEP-d GnH was successfully rescued, which simultaneously expressed the double G protein of rabies virus and the H protein of canine distemper virus. By studying the biological characteristics of the recombinant virus, the recombinant rabies virus (HEP-d GnH) could proliferate stably on BHK cells and reach a higher titer of the virus. The HG gene of the recombinant virus was amplified by RT-PCR and sequenced. No mutation was found in the gene. Fluorescence quantitative PCR and Western blot analysis showed that exogenous genes H and G could be expressed. The growth curve of the virus showed that the growth characteristics of the HEP-d G strain were similar to those of the HEP-d G strain, and reached the peak at 96 h, but the titer of the recombinant virus HEP-d G H was slightly lower than that of the HEP-d G strain at each time point. The results of virus diffusion test showed that the proliferation ability of recombinant virus HEP-d GnH was not as good as that of HEP-d G strain at low MOI level, which was consistent with the growth curve. The results of pathogenicity test showed that HEP-d G) and HEP-d G had no lethal effect on 6-7 week old adult rats, and the effect of recombinant virus HEP-d G on the body weight of adult rats was weaker than that of HEP-d G, but the results showed that HEP-d G G) was more lethal than HEP-d G in 3 days old neonatal mice. The immunogenicity test showed that both the recombinant virus HEP-d GnH) and HEP-d G could induce the production of anti-rabies antibody in mice, and the antibody reached the protective level of 0.5 EUG at 7 d, and HEP-d GnH) could induce the production of CDV neutralizing antibody. The results showed that the induced RABV antibody level could resist the attack of CVS-24 after 3 weeks of immunization with HEP-d G and HEP-d G. these results indicated that the recombinant rabies virus could be used as a potential vaccine candidate. In conclusion, the recombinant rabies virus (HEP-d) has good immunogenicity and potential as a novel genetic engineering vaccine. This study has laid a foundation for the development of a novel multivalent genetic engineering vaccine for dogs.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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