【作者】 刘林；

【导师】 朱应；

【作者基本信息】 武汉大学 ， 微生物学， 2021， 硕士

【摘要】 流感病毒由八段序列编码和复制而成,包括10种蛋白和8个v RNA片段。通过构建八质粒系统,运用反向遗传学技术就可拯救流感病毒。目前我们对各个片段的序列及包装信号越发了解,在此基础上,人为改造流感病毒且表达特定蛋白成为可能。本文即构建了一种以流感病毒为载体表达Ⅱ型单纯疱疹病毒(HSV-2)蛋白的重组流感病毒,结果表明,重组流感病毒免疫小鼠后可保护小鼠免受HSV-2感染或死亡。本研究首先运用相关服务器,在HSV-2的包膜蛋白D蛋白中筛选一段抗原表位(g D2),通过质粒构建的方法将抗原表位插入至流感病毒NS片段间,连接至PHW2000。运用反向遗传学技术,拯救出带有g D2的重组流感病毒(IAV-g D2)并对其进行噬斑纯化。其次,在细胞水平上我们测试了IAV-g D2的十代遗传稳定性,发现IAV-g D2遗传稳定性良好,未出现片段丢失的情况;随后我们对IAV-g D2进行细胞扩增,浓缩纯化,测定病毒滴度,同时在MDCK细胞和Vero细胞中测定其生长曲线,发现IAV-g D2生长复制基本不受影响。接下来,我们用不同滴度对小鼠进行了免疫实验,并检测g D2的表达,发现外源片段(g D2)在病毒复制过程中正常表达;同时取出免疫过IAV-g D2的小鼠脾脏分出免疫细胞,以灭活过的HSV-2进行刺激,用q PCR测定特定的细胞因子水平,发现细胞因子表达明显上调,也表明接种IAV-g D2的小鼠体内已形成记忆T细胞。最后以HSV-2攻毒小鼠,结果表明IAV-g D2免疫过的小鼠感染HSV-2后死亡率明显降低或症状得到减轻,其病毒载量也明显低于空白组,表明IAV-g D2可有效保护小鼠并抑制HSV-2的复制。由于目前HSV-2引发的生殖器疱疹尚无疫苗可预防,且广泛的传播以及持续的治疗不断加重个人或社会的卫生医疗负担,本研究可为该领域病毒载体疫苗、亚单位疫苗以及IAV与HSV-2联合疫苗的研发提供新思路。更多还原

【Abstract】 Influenza virus is encoded and replicated by eight segments,including 10 proteins and 8 v RNA segments.By constructing an eight-plasmid system,the influenza virus can be rescued by using reverse genetics technology.At present,we have a better understanding of the sequence and packaging signal of each fragment.On this basis,it is possible to artificially modify the influenza virus and express a specific protein.This article constructed a recombinant influenza virus expressing type Ⅱ herpes simplex virus(HSV-2)protein with influenza virus as a vector.The results showed that the recombinant influenza virus can protect mice from HSV-2 infection or death after immunizing mice。In this study,we first used relevant servers to screen an epitope(g D2)from the envelope protein D of HSV-2,insert the epitope into the NS fragments of influenza virus through the method of plasmid construction,and connect to PHW2000.Using reverse genetics technology,the recombinant influenza virus with g D2(IAV-g D2)was rescued and plaque-purified.Secondly,at the cellular level,we tested the tenth generations genetic stability of IAV-g D2,and found that IAV-g D2 was genetically stable,and there was no fragment loss;then we performed cell expansion,concentration,purification,and determination of IAV-g D2.The virus titer was measured in MDCK cells and Vero cells at the same time,and it was found that the growth and replication of IAV-g D2 were basically unaffected.Next,we performed immunization experiments on mice with different titers,and detected the expression of g D2,and found that the foreign fragment(g D2)was normally expressed during the virus replication process;at the same time,we took out the spleens of the mice that had been immunized with IAV-g D2.Immune cells were stimulated with inactivated HSV-2,and the levels of specific cytokines were measured by q PCR.It was found that the expression of cytokines was significantly up-regulated,which also showed that memory T cells had been formed in mice vaccinated with IAV-g D2.Finally,the mice were challenged with HSV-2.The results showed that IAV-g D2 immunized mice were significantly reduced in mortality or symptoms after being infected with HSV-2,and their viral load was also significantly lower than that of the blank group,indicating that IAV-g D2 can Effectively protect mice and inhibit HSV-2 replication.Since there is no vaccine to prevent genital herpes caused by HSV-2,and the widespread transmission and continuous treatment continue to increase the health and medical burden of individuals or society,this study can be used for viral vector vaccines,subunit vaccines,and IAV and HSV in this field.-2 The development of a combined vaccine provides new ideas.更多还原