新型沙门菌载体递送H9N2亚型禽流感病毒HA2抗原的分子免疫机制研究

发布时间：2018-06-02 10:47

本文选题：禽流感 + 延迟裂解型沙门菌　；参考：《吉林农业大学》2017年硕士论文

【摘要】：禽流感(Avian influenza,AI)因其高致病性导致禽类大批量死亡,不但给我国养禽业造成了巨大的经济损失,而且严重威胁人类的公共卫生安全。迄今为止,控制禽流感的最有效的方法仍是接种疫苗,但由于该病毒的高突变性,普通的传统疫苗很难达到理想的免疫效果,所以,对禽流感病毒(avian influenza virus,AIV)疫苗的研究成为国内外学者的研究热点。本研究选择AIV的血凝素(Hemagglutinin,HA)颈部的一段高度保守的序列HA2片段,并将其与粘膜免疫佐剂大肠杆菌不耐热肠毒素B亚单位(Escherichia coli heat-labile enterotoxin,LTB)基因融合,分别将HA2基因与HA2-LTB融合基因导入到延迟裂解型沙门菌载体中,构建两组重组沙门菌并对其免疫效果进行初步评价。具体研究内容及结果如下:(1)重组沙门菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)制备及反应原性分析本试验运用现代分子生物学技术,经PCR扩增目的基因、目的基因与载体连接转化、双酶切鉴定等方法构建了重组质粒pYA4545-HA2和pYA4545-HA2-LTB;通过脂质体转染细胞和Western-Blot等方法验证了HA2及HA2-LTB蛋白能够成功表达,并能够与抗体特异性结合,具有良好的免疫原性;再将重组质粒最终导入到沙门菌χ11218中,获得重组沙门菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB),并验证其对阿拉伯糖具有依赖性,为下一步实验奠定基础。(2)重组沙门菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)免疫保护效果比较与评价将实验小鼠分为5组,每组15只。分别将成功制备的χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)两组沙门菌和本实验室已经构建的χ11218(pYA4545)沙门菌免疫小鼠,共口服免疫4次,同时设置对照组与H9N2亚型禽流感灭活疫苗组。最后一次加强免疫后一周,通过流式细胞术(Flow cytometry,FCM)和间接ELISA方法检测免疫效果;通过滴鼻攻毒实验,观察小鼠的生命体征、体重变化及生存情况;攻毒两周后,取各组小鼠肺组织,制成病理切片,通过HE染色比较各组病理变化。进一步对两组重组沙门菌的免疫保护作用进行评价。结果显示,小鼠口服免疫新型沙门菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB),能够增加CD3+CD4+和CD3+CD8+细胞数量,促进T细胞分化,提高CD3+CD4+T细胞分泌的IL-4、IL-17和IFN-r的水平,对CD3+CD8+T细胞分泌IFN-γ也有一定的促进作用,还能提高树突细胞CD40、CD80和MHCⅡ的表达量,而且对B细胞分泌型IgA的表达也有一定的影响。与Control组及χ11218(pYA4545)组相比较,重组沙门菌组和灭活疫苗组,血清中细胞因子及特异性抗体IgG浓度较高,肠道灌洗液中的特异性抗体IgA含量也相对较高。针对沙门菌LPS特异性抗体的检测中,重组沙门菌χ11218(pYA4545-HA2)、χ11218(pYA4545-HA2-LTB)及χ11218(pYA4545)组均有较高浓度的LPS抗体。感染流感病毒后,各组小鼠体重变化趋势基本相同,攻毒的前7天,各组体重均不同程度下降,在第8天达到最低,从第9天起,各组小鼠体重均逐渐恢复,最后趋于稳定。通过对小鼠肺指数及肺指数抑制率的计算,说明重组沙门菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)对H9N2型禽流感具有抵抗作用。肺脏病理切片结果显示χ11218(pYA4545-HA2)组、χ11218(pYA4545-HA2-LTB)组和灭活疫苗组肺组织相对完整,病理症状较轻。以上结果中,虽然重组沙门菌组的免疫效果较疫苗组差,但同样证实了递送HA2及HA2-LTB基因的延迟裂解型沙门菌对H9N2亚型流感病毒具有较好的保护作用。说明HA2能够作为作为禽流感病毒疫苗的靶标基因,同时发现HA2融合LTB能够增强机体对禽流感病毒的抵抗作用,以上结果为延迟裂解性沙门菌作为疫苗活载体提供了理论依据,为禽流感分子免疫机制的研究奠定了基础。
[Abstract]:Avian influenza (AI), because of its high pathogenicity, causes large poultry deaths, not only causes huge economic losses to poultry industry in China, but also poses a serious threat to human health safety. So far, the most effective way to control avian influenza is still vaccinated, but because of the high mutagenicity of the virus, conventional traditional vaccines. It is difficult to achieve the ideal immune effect, so the research on the avian influenza virus (AIV) vaccine has become a hot spot of research at home and abroad. This study selected a highly conservative sequence of HA2 fragment in the neck of AIV (Hemagglutinin, HA), and used it with the mucosal immune adjuvant of Escherichia coli. Escherichia coli heat-labile enterotoxin (LTB) gene fusion was used to import the HA2 gene and HA2-LTB fusion gene into the carrier of the delayed lysate Salmonella. Two groups of recombinant Salmonella were constructed and their immune effects were evaluated. The specific contents and results were as follows: (1) the recombinant Salmonella Chi 11218 (pYA4545-HA2) and Chi 11218 (pYA4). 545-HA2-LTB) preparation and reactivity analysis, the recombinant plasmid pYA4545-HA2 and pYA4545-HA2-LTB were constructed by using modern molecular biology technology, PCR amplification of the target gene, the target gene and the vector, and the double enzyme digestion method. The HA2 and HA2-LTB proteins were verified by liposome transfected cells and Western-Blot. The recombinant plasmid was finally introduced into the Salmonella Chi Chi 11218, and the recombinant Salmonella Chi Chi 11218 (pYA4545-HA2) and Chi 11218 (pYA4545-HA2-LTB) were obtained. The recombinant plasmid was proved to be dependent on the Arabia sugar, which was the basis for the next experiment. (2) the recombinant Salmonella x 11218 (pYA4545-HA2) and the comparison and evaluation of the effect of Chi 11218 (pYA4545-HA2-LTB) immune protection, the experimental mice were divided into 5 groups, with 15 mice in each group. The mice were successfully prepared by Chi 11218 (pYA4545-HA2), Chi 11218 (pYA4545-HA2-LTB) two group and the already constructed Chi 11218 (pYA4545) Salmonella mice were immune to 4 times and set up at the same time. In the control group and the H9N2 subtype avian influenza inactivated vaccine group, the immune effect was detected by flow cytometry (Flow cytometry, FCM) and indirect ELISA after the last week of immunization. The vital signs, weight change and survival of the mice were observed by the intranasal attack test. After two weeks of attack, the lung tissue of each group was taken to make a pathological cut. The immunological protection of two groups of recombinant Salmonella was evaluated by HE staining. The results showed that the oral immunization of new Salmonella x 11218 (pYA4545-HA2) and Chi 11218 (pYA4545-HA2-LTB) could increase the number of CD3+CD4+ and CD3+CD8+ cells, promote the differentiation of T cells and improve the I of CD3+CD4+T cells. The level of L-4, IL-17 and IFN-r also promoted the secretion of IFN- gamma by CD3+CD8+T cells, and also increased the expression of CD40, CD80 and MHC II in dendritic cells, and also had a certain influence on the expression of B cell secretory IgA. Compared with the Control group and the Chi 11218 (pYA4545) group, the recombinant Salmonella and inactivated vaccine group, and the serum cell factors in the serum. The concentration of sub and specific antibody IgG was high, and the specific antibody IgA content in the intestinal lavage fluid was relatively high. In the detection of Salmonella LPS specific antibody, the recombinant Salmonella Chi 11218 (pYA4545-HA2), Chi 11218 (pYA4545-HA2-LTB) and Chi 11218 (pYA4545) group had a higher concentration of LPS antibody. After infection of influenza virus, the weight of mice in each group The change trend was basically the same. The weight of each group declined in the first 7 days and reached the lowest in the eighth day. From the ninth day, the weight of the mice in each group gradually recovered and finally tended to be stable. Through the calculation of the lung index and the lung index inhibition rate of the mice, the recombinant Salmonella x 11218 (pYA4545-HA2) and the X 11218 (pYA4545-HA2-LTB) to the H9N2 type were calculated. The results of lung pathology showed that the lung pathological sections showed the Chi 11218 (pYA4545-HA2) group, the lung tissue of the Chi 11218 (pYA4545-HA2-LTB) group and inactivated vaccine group was relatively complete and the pathological symptoms were lighter. The results of the above results showed that the immune effect of the recombinant Salmonella group was worse than the vaccine group, but it also confirmed the delayed lysis of the delivery of HA2 and HA2-LTB genes. The Salmonella type has a good protective effect on H9N2 subtype influenza virus. It shows that HA2 can be used as a target gene for avian influenza virus vaccine, and that HA2 fusion LTB can enhance the body's resistance to avian influenza virus. The above results provide a theoretical basis for the delayed lysis of Salmonella as a live carrier of the vaccine, for avian influenza. The research of molecular immune mechanism has laid the foundation.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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