本文選題：狂犬病病毒 + 犬瘟熱病毒�。� 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies virus,RABV)引起的一種人獸共患傳染病,在全球范圍內(nèi)流行,是目前病死率最高的傳染病之一,一旦感染出現(xiàn)臨床癥狀,幾乎100%的死亡。犬瘟熱(Canine Distemper,CD)是由犬瘟熱病毒(Canine Distemper vrus,CDV)引起的高度接觸性傳染病,它在全球范圍內(nèi)流行,死亡率高達(dá)80%,給養(yǎng)犬業(yè)和毛皮動(dòng)物養(yǎng)殖業(yè)造成嚴(yán)重的危害。預(yù)防接種是控制狂犬病和犬瘟熱的主要途徑。目前,傳統(tǒng)的狂犬病弱毒疫苗成本低廉,但存在毒力殘留或致病性回復(fù)突變的安全隱患;進(jìn)口疫苗安全性高,免疫效果好,但價(jià)格昂貴,難以廣泛推廣使用。研制安全、高效、成本低廉的動(dòng)物狂犬病疫苗,仍具現(xiàn)實(shí)意義。市場(chǎng)上應(yīng)用廣泛的犬瘟熱疫苗存在熱穩(wěn)定性差、免疫效果易受母源抗體干擾等多重問(wèn)題。因此,研制安全、高效的新型CD疫苗具有重要意義。本研究利用已建立的狂犬病病毒(Rabies virus,RABV)反向遺傳操作技術(shù)平臺(tái),在通過(guò)反向遺傳拯救的狂犬病病毒弱毒株HEP-d G的基礎(chǔ)上,運(yùn)用分子生物學(xué)技術(shù)在狂犬病病毒(HEP-d G株)基因組G與L之間再插入犬瘟熱病毒H基因,構(gòu)建了重組全長(zhǎng)c DNA質(zhì)粒p HEP-d G(H),成功拯救出同時(shí)表達(dá)狂犬病病毒雙G蛋白和犬瘟熱病毒H蛋白的重組狂犬病病毒HEP-d G(H)。通過(guò)對(duì)重組病毒的生物學(xué)特性研究,該重組狂犬病毒HEP-d G(H)可以在BHK細(xì)胞上穩(wěn)定的增殖,達(dá)到較高的病毒滴度。抽取該重組病毒第2、5、10代的RNA,進(jìn)行RT-PCR擴(kuò)增H、d G基因并測(cè)序,基因未發(fā)生突變。熒光定量PCR和Western blot驗(yàn)證表明外源基因H和d G能夠表達(dá)。病毒的生長(zhǎng)曲線表明HEP-d G(H)保持著與HEP-d G毒株相似的生長(zhǎng)特性,在96h達(dá)到高峰,但重組病毒HEP-d G(H)的滴度在每個(gè)時(shí)間點(diǎn)略低于HEP-d G毒株。病毒的擴(kuò)散感染實(shí)驗(yàn)顯示在低MOI的情況下,重組病毒HEP-d G(H)的擴(kuò)散能力不及HEP-d G毒株,這與生長(zhǎng)曲線相吻合。致病性實(shí)驗(yàn)顯示,重組病毒HEP-d G(H)與HEP-d G對(duì)6-7周齡成鼠沒(méi)有致死性,且重組病毒HEP-d G(H)對(duì)成鼠體重的影響弱于HEP-d G;而對(duì)3日齡內(nèi)的乳鼠,結(jié)果顯示HEP-d G(H)的致命性高于HEP-d G。免疫原性實(shí)驗(yàn)顯示,重組病毒HEP-d G(H)與HEP-d G均能誘導(dǎo)小鼠產(chǎn)生抗狂犬病的抗體,在7 d時(shí)抗體已達(dá)到保護(hù)水平(0.5 EU),且HEP-d G(H)可誘導(dǎo)產(chǎn)生CDV中和抗體。攻毒試驗(yàn)顯示HEP-d G(H)與HEP-d G免疫3周后,誘導(dǎo)的RABV抗體水平能夠抵御CVS-24的攻擊,這些結(jié)果表明,重組的狂犬病病毒可以作為有潛力的疫苗候選株。綜上所述,重組狂犬病病毒HEP-d G(H)具有良好的免疫原性,具有作為新型基因工程疫苗的潛質(zhì),本研究為犬的新型多價(jià)基因工程疫苗的研制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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