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摘要：H9N2亚型的禽流感毒株近些年变异的非常快，毒力有所增强，不仅对养殖业带来巨大的损失，而且有研究表明，H9N2亚型的禽流感病毒还可以传染给人。因此对H2N9本课题以在genebank公布的HA、NA基因核酸序列为参考序列，设计合成引物、扩增到HA、NA基因分别为480bp和690bp，克隆到T载体中，进行测序分析，并应用分子生物学软件，对HA和NA基因序列进行特性分析。然后以该H9N2禽流感灭活抗原乳化进行疫苗免疫试验，对免疫后动物的抗体水平、T细胞亚型及淋巴细胞的增殖进行检测。结果显示，HA、NA基因与genebank里的同源基因比较未发生明显变异。HA和NA基因的二级结构预测发现HA和NA基因有多个B细胞表位优势位点。注射H9N2禽流感疫苗可以能显著提高免疫小鼠血清特异性抗体水平，能提高免疫小鼠体内CD3+CD4+和CD3+CD8+T淋巴细胞比例，促进树突状细胞CD40的表达，显著提高淋巴细胞活力。该研究结果为解析我国禽流感流行毒株的特性及禽流感疫病防控提供了重要的参考依据。29693
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毕业论文关键词：HA基因； NA基因；基因扩增；同源性分析；动物免疫
Cloning and characterization of the HA and NA genes of avian influenza H9N2
Abstract: H9N2 subtype avian influenza strain mutated very fast in recent years, virulence increased, not only bring huge losses to aquaculture, and studies have shown that H9N2 subtype avian influenza virus can infect people. So on H2N9 released this topic in the genebank HA and NA genes nucleic acid sequence for the reference sequence, design and synthesis of primers, swell to HA, NA gene 480 bp and 690 bp respectively, cloned into T vector, sequencing analysis, molecular biology and application software, on the HA and NA genes sequence characteristics analysis. And then to the H9N2 avian influenza inactivated antigen emulsified vaccine trials, after the immune antibody levels, T cell subtype and lymphocyte proliferation were detected. The results showed that the HA, NA and genebank genes did not mutate significantly. The HA and the NA gene's secondary structure predicted that HA and NA genes had multiple b-cells. H9N2 avian influenza vaccine can significantly improve immune serum specific antibody level in mice, and can improve immune mice CD3 +, CD4 + and CD3 + CD8 + T lymphocytes percentage, promote dendritic cells the expression of CD40, improve lymph cell vitality. The results of this study provide an important reference for the analysis of the characteristics of the epidemic strain in our country and the prevention and control of avian influenza.
Key words: The HA gene; NA genes; Gene amplification; Homology analysis; Animal immunination
目录
摘要1
关键词1
Abstract1
Keywords1
引言2
1 材料与方法2
1.1实验材料和仪器 2
1.1.1载体、分子克隆试剂及试剂盒 2
1.1.2主要试剂与器材 2
1.1.3培养基和溶液的配制 2
1.2 RNA的提取和反转录 3
1.2.1 mRNA的提取3
1.2.2 RNA的反转录 4
1.3 HA、NA基因的扩增 4
1.3.1 HA、NA基因的引物设计4
1.3.2PCR反应条件的探索4
1.3.3PCR大量扩增目的基因 4
1.3.4 PCR产物核酸凝胶电泳 4
1.3.5 PCR产物的凝胶回收 5
1.4目的基因的测定及分析6
1.4.1克隆载体HA-pMD-19T的构建8
1.5 用H9N2疫苗免疫小鼠 8
1.6 用ELASA 方法检测特异性抗体水平 9
1.7用流式细胞术的方法检测T细胞亚型、树突状细胞、和CD4010
1.8小鼠的淋巴细胞增殖 10
2 结果和分析11
2.1 HA、NA的基因扩增11
2.2 原核表达载体的构建12
2.3HA、NA基因同源性分析13
2.4同源序列的进化树分析 13
2.5 HA 基因和NA基因的二级结构的预测分析14
2.6 H9N2禽流感疫苗对小鼠血清中特异性抗体水平的检测 15