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来源：百度知道 编辑：UC知道 时间：2024/07/06 21:58:31
M基因是传染性支气管炎病毒重要的免疫原基因之一，M基因上可能存在重要的抗原决定簇，且在进化过程中具有高度保守性。本研究在对IBV结构蛋白基因分析研究的基础上，选取IBV的M基因，通过PCR扩增在两端引入BamHⅠ酶切位点，经酶切插入“自杀性”DNA疫苗载体pSCA1 CMV IE启动子之下的克隆位点，构建了“自杀性”DNA疫苗表达质粒pSCA1-M。经由菌落PCR、酶切及测序鉴定目的基因被成功克隆至载体pSCA1，且方向正确。分别取等量的重组质粒pSCA1-M、常规IBV M基因DNA疫苗pcDNAM及空载体pSCA1转染Vero细胞。48h后经间接免疫荧光试验观察发现，pSCA1-M以及pcDNAM转染组细胞浆均可见特异性亮绿色荧光，pSCA1空载体组无荧光为阴性。这证实“自杀性”DNA疫苗载体能成功表达M基因。而pSCA1-M组所发荧光细胞数比pcDNAM组少，说明的“自杀性”DNA疫苗转染率低于常规DNA疫苗。
为评价IBV “自杀性”DNA疫苗的免疫效果，分别用构建的“自杀性”DNA疫苗pSCA1-M、常规DNA疫苗pcDNAM、空载体pSCA1和IBV灭活疫苗免疫非免疫健康雏鸡，并设对照组注射等量的PBS液。自免疫后每隔一周各组鸡只采血分离血清连续五周，采用流式细胞仪(FACS)和间接ELISA试验分别对宿主外周血T淋巴细胞亚类CD3+、CD4+、CD8+和特异性抗IBV血清IgG的动态变化进行检测，并在第五周后以IBV强毒株进行攻毒保护实验。间接ELISA试验检测结果表明，我们构建的“自杀性”DNA疫苗能刺激机体产生针对IBV的特异性血清抗体，免疫后第三周上升到峰值，随后开始下降，其抗体水平与常规IBV DNA疫苗相当，低于灭活疫苗。T淋巴细胞亚类动态变化结果显示，“自杀性”DNA疫苗pSCA1-M组CD3+、CD4+、CD8+各T淋巴细胞数量均高于其余各组，表现为差异显著（P＜0.01或P＜0.05）。但在第4周左右有一个明显的下降，之后又略有回升。这说明“自杀性”DNA疫苗能诱导机体产生高于常规DNA疫苗和灭活疫苗的细胞免疫，但随着宿主细胞的凋亡迅速下降。攻毒保护试验结果统计，pSCA1-M组鸡只发病率为12%，保护率为50%；pcDNAM组鸡只发病率为13%，保护率略低于pSCA1-M，为40%；空载体组发病率（75%）＜对照组（85%）

The M gene is one of infectious bronchitis virus important immunity original genes, the M gene possibly has the important antigen decision bunch, also has highly the conservative nature in the evolution process. This research selects the M gene of IBV , on the foundation of researching the IBV structure protein gene, expands the PCR increases introduces the BamH I enzyme in the beginnings and ends to cut the position spot, cuts the insertion after the enzyme "the suicide" DNA vaccine carrier pSCA1 CMV the IE start under clone position spot, constructed "the suicide" the DNA vaccine to express material particle pSCA1-M. By the way of colony PCR, the enzyme cut and measured the foreword appraisal goal gene succeeds the clone to carrier pSCA1, also the direction is correct. Separately takes the isometric reorganization material particle pSCA1-M, conventional IBV M gene DNA vaccine pcDNAM and the spatial carrier pSCA1 extension dyes the Vero cell. After 48h, by the