重组蝙蝠流感病毒的生物学特性的研究

发布时间：2018-06-21 16:15

本文选题：蝙蝠流感病毒 + 反向遗传操作系统　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：蝙蝠是世界上种类极为丰富的哺乳动物,将近有1200多个不同的种类,分布在除了两极外世界的各个地区。蝙蝠也是重要的病毒携带者,能够携带至少170种病毒。2012年研究人员在蝙蝠身上发现了H17N10亚型蝙蝠流感病毒。蝙蝠是否是流感病毒的宿主之一,尚不清楚。在基因组结构方面,蝙蝠流感病毒与其它甲型流感病毒有许多相同的地方,也有许多不同的地方。人们掌握了H17N10亚型流感病毒的序列结果,但H17N10亚型流感病毒还不能利用实验室的方法重组得到。本研究利用反向遗传操作技术将H17N10亚型流感病毒内部基因分别与H9N2亚型、H1N1亚型流感病毒携带蝙蝠流感病毒包装信号的表面基因进行重组,拯救出重组蝙蝠流感病毒,分别命名为cH9cN2/H17、cH1cN1/H17。为了研究拯救病毒在MDCK细胞和DF1细胞中的生长规律,将cH9cN2/H17、cH1cN1/H17以MOI(感染复数)为0.001病毒量分别在MDCK,DF1细胞上进行接种病毒。分别在接种病毒后12h、24h、36h、48h收取病毒液,之后进行病毒滴度(TCID50)测定。表明,重组蝙蝠流感病毒cH9cN2/H17、cH1cN1/H17在MDCK细胞、DF1细胞中生长36h时,病毒滴度达到最高。为了研究这两株病毒在小鼠体内的生物学特性,10TCID50的病毒量滴鼻感染小鼠,结果表明,将重组蝙蝠流感病毒cH9cN2/H17、cH1cN1/H17鼻腔接种五周龄小鼠,不会引起小鼠体重下降等异常,也不导致小鼠死亡。但在接种病毒后3天、5天剖杀的小鼠肺脏中检测到较高的病毒滴度。在小鼠体内接种重组蝙蝠流感病毒cH9cN2/H17、cH1cN1/H1715天后分离小鼠血清,可以检测到小鼠血清转阳。本研究成功利用反向遗传操作系统重组了蝙蝠流感病毒,对重组蝙蝠流感病毒在MDCK细胞、DF1细胞的相互适应提供一定的参考依据,为进一步研究蝙蝠重组流感病毒的致病性等奠定了基础。并对cH9cN2/H17、cH1cN1/H17在小鼠体内的生物学特性进行探索,为流感病毒的传染性,致病性研究提供了支持。
[Abstract]:Bats are very abundant mammals in the world, there are nearly 1200 different species, distributed in all parts of the world except the two poles. Bats are also important carriers of at least 170 viruses. Researchers found the H17N10 subtype of bat influenza virus in bats in 2012. It is unclear whether bats are one of the hosts of influenza viruses. In terms of genome structure, there are many similarities and differences between bat influenza virus and other influenza A viruses. The sequence of H17N10 subtype influenza virus has been obtained, but the H17N10 subtype influenza virus can not be recombined by laboratory method. In this study, reverse genetic manipulation technique was used to recombine the internal genes of H17N10 influenza virus and the surface gene of H9N2 subtype H1N1 influenza virus carrying the packaging signal of bat influenza virus, so as to save the recombinant bat influenza virus. They were named cH9cN2 / H17c1cN1 / H17. In order to study the growth pattern of saving virus in MDCK cells and DF1 cells, we inoculated cH9cN2 / H17cHH1cN1 / H17 on MDCK DF1 cells with moi (plural number of infection) as 0.001. The virus solution was collected at 12h, 24h, 36h and 48h after inoculation, and the titer of the virus was determined by TCID50. The results showed that the titer of recombinant bat influenza virus cH9cN2 / H17cHH1 / H17 in MDCK cells reached the highest level after 36 h growth. In order to study the biological characteristics of the two viruses in mice, the results showed that inoculating the recombinant bat influenza virus cH9cN2 / H17cH17cH1 / H17 into the nasal cavity of 5-week-old mice would not cause the mice to lose weight and other abnormalities. Nor did it kill the mice. However, a higher titer of the virus was detected in the lungs of mice killed 3 days and 5 days after inoculation. Mouse serum was isolated after inoculating recombinant bat influenza virus cH9cN2 / H17cN1 / H17cN1 / H1715 days after inoculation in mice. In this study, the bat influenza virus was successfully recombined using the reverse genetic operating system, which provides a certain reference for the adaptation of the recombinant bat influenza virus in MDCK cells and DF1 cells. It lays a foundation for further study on the pathogenicity of bat recombinant influenza virus. The biological characteristics of cH9cN _ 2 / H _ (17) H _ (17) C _ (1) N _ (1) / H _ (17) in mice were explored in order to provide support for the study of the infectivity and pathogenicity of influenza virus.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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