發(fā)布時間：2018-06-21 16:15

  本文選題：蝙蝠流感病毒 + 反向遺傳操作系統(tǒng)　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：蝙蝠是世界上種類極為豐富的哺乳動物,將近有1200多個不同的種類,分布在除了兩極外世界的各個地區(qū)。蝙蝠也是重要的病毒攜帶者,能夠攜帶至少170種病毒。2012年研究人員在蝙蝠身上發(fā)現(xiàn)了H17N10亞型蝙蝠流感病毒。蝙蝠是否是流感病毒的宿主之一,尚不清楚。在基因組結(jié)構(gòu)方面,蝙蝠流感病毒與其它甲型流感病毒有許多相同的地方,也有許多不同的地方。人們掌握了H17N10亞型流感病毒的序列結(jié)果,但H17N10亞型流感病毒還不能利用實驗室的方法重組得到。本研究利用反向遺傳操作技術(shù)將H17N10亞型流感病毒內(nèi)部基因分別與H9N2亞型、H1N1亞型流感病毒攜帶蝙蝠流感病毒包裝信號的表面基因進(jìn)行重組,拯救出重組蝙蝠流感病毒,分別命名為cH9cN2/H17、cH1cN1/H17。為了研究拯救病毒在MDCK細(xì)胞和DF1細(xì)胞中的生長規(guī)律,將cH9cN2/H17、cH1cN1/H17以MOI(感染復(fù)數(shù))為0.001病毒量分別在MDCK,DF1細(xì)胞上進(jìn)行接種病毒。分別在接種病毒后12h、24h、36h、48h收取病毒液,之后進(jìn)行病毒滴度(TCID50)測定。表明,重組蝙蝠流感病毒cH9cN2/H17、cH1cN1/H17在MDCK細(xì)胞、DF1細(xì)胞中生長36h時,病毒滴度達(dá)到最高。為了研究這兩株病毒在小鼠體內(nèi)的生物學(xué)特性,10TCID50的病毒量滴鼻感染小鼠,結(jié)果表明,將重組蝙蝠流感病毒cH9cN2/H17、cH1cN1/H17鼻腔接種五周齡小鼠,不會引起小鼠體重下降等異常,也不導(dǎo)致小鼠死亡。但在接種病毒后3天、5天剖殺的小鼠肺臟中檢測到較高的病毒滴度。在小鼠體內(nèi)接種重組蝙蝠流感病毒cH9cN2/H17、cH1cN1/H1715天后分離小鼠血清,可以檢測到小鼠血清轉(zhuǎn)陽。本研究成功利用反向遺傳操作系統(tǒng)重組了蝙蝠流感病毒,對重組蝙蝠流感病毒在MDCK細(xì)胞、DF1細(xì)胞的相互適應(yīng)提供一定的參考依據(jù),為進(jìn)一步研究蝙蝠重組流感病毒的致病性等奠定了基礎(chǔ)。并對cH9cN2/H17、cH1cN1/H17在小鼠體內(nèi)的生物學(xué)特性進(jìn)行探索,為流感病毒的傳染性,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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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