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本研究利用基因工程技术探索B型流感病毒NS1蛋白羧基端缺失对病毒致病性的影响,并利用反向遗传学技术从体外拯救NS1蛋白羧基端缺失的B型流感病毒,评估去除NS1蛋白羧基端对B型流感病毒致病性的影响,探究B型流感病毒致病性变异的分子基础。通过全基因合成和反向遗传学技术从体外拯救NS1蛋白羧基端发生171个氨基酸缺失的B型流感病毒B/Yamagata/16/88株,命名为B-L5。将实验室之前拯救的母本株B/Yamagata/16/88(以下简称B-S9)和B-L5以3×105 EID5 0的攻毒剂量分别人工感染BALB/c小鼠,通过体重变化、生存率、肺脏病毒滴度等方面进行致病性分析。成功构建了B型流感病毒B/Yamagata/16/88NS1蛋白羧基端缺失株反向遗传平台。母本株B-S9和NS1蛋白羧基端缺失株B-L5均能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠均呈现低致病性;B-S9感染小鼠后,从攻毒后第3d至第7d,体重持续下降,相反,B-L5感染小鼠后,只在攻毒后第2d出现体重下降,攻毒后第3d体重开始回升;B-S9和B-L5感染小鼠后均能在小鼠肺部进行复制,但攻毒后第3d,B-L5感染小鼠肺脏滴度要比B-S9低7 900倍,攻毒后第6d,B-L5感染小鼠肺脏已检测不到病毒复制。实验结果表明,NS1蛋白羧基端缺失可以明显降低B型流感病毒对小鼠的致病性。B型流感病毒致病性变异的分子基础还有待继续研究,该反向遗传操作系统的建立为B型流感病毒分子机制的研究奠定了基础,同时也为B型流感减毒活疫苗的研究开辟了新途径。
In this study, genetic engineering was used to explore the influence of the carboxyl terminal deletion of the NS1 protein of the influenza B virus on the pathogenicity of the virus and the reverse genetics technique was used to rescue the B-type influenza virus deleted at the carboxyl terminus of the NS1 protein in vitro to assess the effect of removing the carboxyl group End on the pathogenicity of influenza B virus to explore the molecular basis of pathogenic mutation of influenza B virus. A total of 171 amino acids deleted type B influenza B / Yamagata / 16/88 strains were obtained from the carboxyl end of NS1 protein by in vitro synthesis and reverse genetics technology and named as B-L5. BALB / c mice were artificially infected with the rescued female parent strain B / Yamagata / 16/88 (hereinafter referred to as B-S9) and B-L5 with a challenge dose of 3 × 10 5 EID 50, respectively, Survival rate, lung virus titer and other aspects of pathogenicity analysis. The reverse genetics platform of B / Yamagata / 16 / 88NS1 protein carboxyl terminal deletion mutant was successfully constructed. BALB / c mice were artificially infected with B-S9 and B-S5, but not lethal to BALB / c mice. B-S9 infected mice , The body weight continued to decline from the 3rd day to the 7th day after challenge. On the contrary, after the mice were infected with B-L5, the body weight decreased only on the 2nd day after challenge and the third day after the challenge, the body weight began to rise. The B-S9 and B L5 infected mice could replicate in the lungs of mice, but the lungs of mice infected with B-L5 were 7 900 times lower than those of B-S9 on the 3rd day after challenge, Virus replication was undetectable in lungs of L5-infected mice. The experimental results show that the deletion of the carboxyl terminal of NS1 protein can significantly reduce the pathogenicity of influenza B virus in mice. The molecular basis of the pathogenic mutation of influenza B virus remains to be further studied. The establishment of this reverse genetic operation system lays a foundation for the study of the molecular mechanism of influenza B virus, and also opens up the research of live attenuated influenza B vaccine A new way.