本文選題：狂犬病病毒 + L蛋白　； 參考：《華中農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：狂犬病是由狂犬病病毒(RABV)引起的一種古老的人畜共患傳染病,全世界每年約有55000人死于狂犬病,主要發(fā)生在非洲和亞洲一些發(fā)展中國家,我國是狂犬病高發(fā)國家之一。RABV為單股負(fù)鏈RNA病毒,其基因組共編碼五個(gè)結(jié)構(gòu)蛋白,分別是N、P、M、G和L蛋白,其中L蛋白的研究甚少。通過對(duì)其它單股負(fù)鏈RNA病毒的L蛋白的相關(guān)研究,尤其是與RABV同屬于彈狀病毒科的水皰性口炎病毒(VSV),表明L蛋白含有K-D-K-E的保守催化四聚體區(qū)域,該區(qū)域主要在病毒mRNA加帽過程中行使N-7和2’-O甲基轉(zhuǎn)移酶(MTase)的功能,預(yù)計(jì)會(huì)在病毒的致病性及病毒逃逸宿主的先天性免疫中起到重要作用。本研究首先利用在線軟件ClustalW2對(duì)RABV、VSV、麻疹病毒、仙臺(tái)病毒、新城疫病毒和尼帕病毒的L蛋白羧基端進(jìn)行序列比對(duì),結(jié)果表明RABV的L蛋白中也存在K-D-K-E四聚體區(qū)域,且四個(gè)關(guān)鍵氨基酸K、D、K、E是完全保守的。此外,進(jìn)一步通過比較RABV不同毒株之間的L蛋白中的K-D-K-E四聚體區(qū)域,發(fā)現(xiàn)該保守區(qū)域在不同的RABV毒株間是高度保守的。為了探索該K-D-K-E四聚體區(qū)域在RABV的致病性及逃逸宿主先天性免疫方面的作用,我們利用反向操作技術(shù)將該保守四聚體區(qū)域中的四個(gè)關(guān)鍵氨基酸位點(diǎn)K(第1685位)、D(第1797位)、K(第1829位)、E(第1867位)分別進(jìn)行突變,構(gòu)建一系列的重組狂犬病病毒(rRABV),并將這些重組病毒與親本病毒在致病性上進(jìn)行體外和體內(nèi)的比較。體外實(shí)驗(yàn)表明,D1797和E1867突變的rRABV(rB2c-K1797A和r B2c-K1867A)體外擴(kuò)增2代時(shí)便會(huì)快速回復(fù)突變?yōu)橛H本型,而K1685和K1829突變的rRABV(r B2c-K1685A和rB2c-K1829A)的遺傳性狀則比較穩(wěn)定,細(xì)胞傳代擴(kuò)增至15代仍未發(fā)生回復(fù)突變,因此,本研究將以rB2c-K1685A和rB2c-K1829A兩個(gè)突變毒株為主要研究對(duì)象。病毒體外生長曲線結(jié)果表明兩個(gè)突變毒株的病毒滴度和在細(xì)胞中的擴(kuò)散能力都要明顯低于親本毒株;在對(duì)小鼠的致病性實(shí)驗(yàn)中發(fā)現(xiàn),無論是以后肢肌肉注射方式還是腦內(nèi)注射方式進(jìn)行感染,rB2c-K1685A和r B2c-K1829A都不能致成年小鼠發(fā)病;且其對(duì)乳鼠的致病性(體重變化、臨床癥狀、死亡率)顯著低于親本毒株rB2c。為了研究這些突變了K-D-K-E四聚體區(qū)域的重組病毒是否對(duì)病毒逃逸宿主先天性免疫方面也產(chǎn)生影響,我們將這些突變的重組病毒與親本病毒在誘導(dǎo)I型干擾素(interferon,IFN)的產(chǎn)生及對(duì)其敏感性方面進(jìn)行了比較,結(jié)果表明與親本病毒相比,rB2c-K1685A和r B2c-K1829A對(duì)I型IFN敏感性增加,但其在感染RAW264.7細(xì)胞后并未誘導(dǎo)更多的I型IFN的產(chǎn)生。研究表明IFIT1/2等干擾素刺激因子(interferon stimulated gene,ISG)可以對(duì)一些2’-O MTase缺陷的病毒復(fù)制產(chǎn)生影響,為了研究突變的rRABV是否也受這些ISG的影響,我們分別構(gòu)建了過表達(dá)IFIT1和IFIT2的細(xì)胞系,用重組病毒感染這些細(xì)胞,結(jié)果表明過表達(dá)IFIT1并不能明顯抑制重組病毒的復(fù)制,而過表達(dá)IFIT2則能顯著抑制重組病毒的復(fù)制。為了進(jìn)一步研究K-D-K-E四聚體區(qū)域的突變是否對(duì)病毒的免疫原性產(chǎn)生影響,我們將重組病毒和親本病毒分別免疫小鼠,并于免疫后第2周進(jìn)行病毒中和抗體(VNA)滴度的測(cè)定,發(fā)現(xiàn)重組病毒和親本病毒誘導(dǎo)產(chǎn)生的VNA均值都在10 IU/ml,在免疫后第3周用CVS-24進(jìn)行攻毒實(shí)驗(yàn),重組病毒rB2c-K1829A可以達(dá)到90%的保護(hù)率,而rB2c-K1685A和親本病毒都能達(dá)到100%的免疫保護(hù)率。以上結(jié)果表明K-D-K-E四聚體區(qū)域的突變對(duì)病毒的免疫原性未有顯著影響。綜上所述,K-D-K-E四聚體區(qū)域的突變對(duì)可以顯著降低RABV的致病性,使其對(duì)I型IFN以及IFIT2更為敏感,但并未顯著影響病毒的免疫原性。該研究將對(duì)進(jìn)一步理解L蛋白的K-D-K-E四聚體區(qū)域在RABV的致病性和逃逸宿主先天性免疫方面的作用機(jī)制奠定基礎(chǔ),同時(shí)也為RABV弱毒疫苗的研發(fā)提供了新的思路。
[Abstract]:Rabies is an ancient zoonotic infectious disease caused by rabies virus (RABV). About 55000 people die from rabies every year in the world, mainly in Africa and some developing countries in Asia. Our country is one of the high incidence countries of rabies,.RABV is a single strand negative chain RNA virus, and its genome COCODES five structural proteins, which are N, P, M, respectively. The study of G and L proteins, including L protein, is very small. Through the study of the L protein of other single stranded negative chain RNA viruses, especially with RABV belonging to the bullous stomatitis virus (VSV), which belongs to the family of the family of elastic viruses, it is indicated that L protein contains the conserved catalytic four polymer region of K-D-K-E, and this region is mainly made of N-7 and 2 '-O methyl during the virus mRNA adding process. The function of transferase (MTase) is expected to play an important role in the virulence of the virus and the innate immunity of the host of the virus. In this study, the sequence alignment of the L protein carboxyl ends of the L protein of RABV, VSV, measles virus, Sendai virus, Newcastle disease virus and NPV virus was compared with the online software ClustalW2. The results showed that the L protein of RABV was also found to be the same. There is a K-D-K-E four polymer region, and four key amino acids, K, D, K, and E are completely conserved. Furthermore, the conserved region is highly conservative among the different RABV strains by comparing the K-D-K-E four polymer region of the L protein between RABV different strains. In order to explore the K-D-K-E four polymer region in RABV pathogenicity and escape. The host's innate immune function, we use reverse operation technology to transform the four key amino acid sites K (1685th bits), D (1797th), K (1829th), and E (1867th)) in the conservative four polymer region to construct a series of recombinant rabies virus (rRABV), and the virulence of these recombinant viruses to the parent virus. In vitro and in vivo comparison, in vitro experiments showed that D1797 and E1867 mutations of rRABV (rB2c-K1797A and R B2c-K1867A) could quickly return to parental type when the 2 generation of amplification in vitro, and the genetic traits of rRABV (R B2c-K1685A and rB2c-K1829A) of K1685 and K1829 mutations were more stable than that of the 15 generation. Therefore, two mutant strains of rB2c-K1685A and rB2c-K1829A will be studied in this study. The results of the virus in vitro growth curve show that the virus titer of the two mutant strains and the ability to spread in the cells are significantly lower than those of the parent strain; in the pathogenicity test of the mice, it is found that it is the side muscle injection side. RB2c-K1685A and R B2c-K1829A did not cause infection in adult mice, and the pathogenicity (body weight, clinical symptoms, mortality) of the mice was significantly lower than that of the parent strain rB2c. in order to study whether the recombinant virus of the K-D-K-E four polymer region had a congenital immune response to the escape host of the virus. In addition, we compared the production of these mutant recombinant viruses with parental viruses in inducing I type interferon (interferon, IFN) and their sensitivity to their sensitivity. The results showed that compared with parental viruses, rB2c-K1685A and R B2c-K1829A increased the sensitivity to I IFN, but they did not induce more I after infected RAW264.7 cells. The production of type IFN. Studies have shown that IFIT1/2 and interferon stimulated gene (ISG) can affect the replication of some 2 '-O MTase defects. In order to study whether the mutant rRABV is also affected by these ISG, we respectively constructed the cell lines that express IFIT1 and IFIT2, and infect these cells with the recombinant virus. The results showed that overexpression of IFIT1 did not significantly inhibit the replication of recombinant virus, while overexpression of IFIT2 could significantly inhibit the replication of recombinant virus. In order to further study whether the mutation of the K-D-K-E four polymer region has an effect on the immunogenicity of the virus, we immunized mice with the recombinant virus and the parent virus respectively, and second weeks after the immunization. The detection of the titer of virus neutralizing antibody (VNA) showed that the VNA average of the recombinant virus and the parent virus was 10 IU/ml, and CVS-24 was used to attack the virus at third weeks after the immunization. The recombinant virus rB2c-K1829A could reach the 90% protection rate, and the rB2c-K1685A and parent virus could reach 100% of the immune protection rate. The above results showed that K- Mutations in the D-K-E four polymer region have no significant effect on the immunogenicity of the virus. To sum up, the mutation in the K-D-K-E four polymer region can significantly reduce the pathogenicity of RABV, making it more sensitive to I IFN and IFIT2, but does not significantly affect the immunogenicity of the virus. The study will further understand the K-D-K-E four polymer region of the L protein. It laid the foundation for the pathogenicity and the mechanism of escape immunity of RABV, and provided a new idea for the development of RABV attenuated vaccine.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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