【作者】 鲜凌瑾；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 微生物学， 2007， 硕士

【摘要】 本研究采用本实验室pIBV—S1质粒(DQ288927)构建自杀性DNA疫苗。通过PCR扩增在S1基因两端引入BamHⅠ酶切位点，经酶切插入自杀性DNA疫苗载体pSCA1，构建了自杀性DNA疫苗pSCA1-S1质粒，经由菌落PCR、酶切及测序鉴定目的基因被成功克隆至自杀性DNA疫苗载体pSCA1，且方向正确。以1：20的配比加入脂质体作佐剂，制备了IBV S1基因的自杀性DNA疫苗。为证实所构建自杀性DNA疫苗pSCA1-S1质粒的正确表达，分别取等量的重组质粒pSCA1-S1、常规IBV S1基因DNA疫苗质粒pcDNA-S1、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-S1以及pcDNAS1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。证实自杀性DNA疫苗载体能成功表达S1基因。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-S1、常规DNA疫苗pcDNAS1、空载体pSCA1和IBV灭活疫苗、PBS液对照，免疫非免疫健康雏鸡。自免疫后每周采血分离血清连续五周，采用间接ELISA试验检测血清特异性抗体IgG，结果表明：自杀性DNA疫苗产生针对IBV的特异性血清抗体，免疫后第二周上升到峰值，随后开始下降，其抗体水平与常规IBV DNA疫苗差异不显著(P＞0.05)。用流式细胞仪(FACS)测定各组CD3~+、CD4~+、CD8~+动态变化，结果显示：自杀性DNA疫苗pSCA1-S1组CD3~+水平在14d和28d差异显著高于常规IBV DNA疫苗组(P＜0.05)；CD4~+水平在21d、35 d差异显著高于常规IBV DNA疫苗组(P＜0.05)；CD8~+水平只在35d差异显著高于常规IBV DNA疫苗组(P＜0.05)。表明自杀性DNA疫苗能诱导机体产生高水平的细胞免疫。攻毒保护试验结果表明：pSCA1-S1组攻毒相对保护率为60％；pcDNAS1组免疫鸡只相对保护率低于pSCA1-S1，为50％。本研究采用IBV S1基因构建自杀性DNA疫苗对鸡进行免疫，并对其免疫效果进行评价，在国内外还未见报道。本研究为研制IBV“自杀性”DNA疫苗提供了基础材料和科学依据。更多还原

【Abstract】 In this reseach, the plamid pIBV—S1 (DQ288927)constructed by our lab was used toconstruct suicide DNA vaccine. The restrict site BamHⅠwas added by PCR. Then the S1gene of IBV was cloned and inserted into pSCA1. The recombinant plasmid pSCA1-S1was confirmed correct by PCR、restriction digestion and sequence analysis.Thelipospme-DNA IB DNA vaccines has been made at the ratio of 20/1.Recombinant plasmid pSCA1-S1、conventional DNA vaccine pcDNA3.1、emptyvector pSCA1 was transfected into Vero cells.48 hours later, expression of S1 in vitro wasverified by indirect immunofluorescence analysis. Recombinant plasmid pSCA1-S1、conventional DNA vaccine pcDNA3.1 can both be observed by green fluorescence, whilethe empty vector pSCA1 can not. The result confirm that the recombinant plasmidpSCA1-S1 can correttly express the S1 protein.7-day-old healthy chicken were injected intramuscularly with pSCA1-S1、conventional DNA vaccine pcDNA3.1、empty vector pSCA1、inactivated vaccine、PBScontral comparison. Picked the blood and separated the blood serum regularly afterimmunization continuously five weeks. Indirect ELISA assays were employed to evaluatehumoral immunity. The result showed that IBV-specific antibodies were induced at 7d afterimmunization. The data was maximum at the second week after immunization, thendeclined. The data between pSCA1-Sland conventional DNA vaccine pcDNA3.1 wassimilar. A significant difference was observed (P＜0.05), contrasting pSCA1-S1 with emptyvector. Fluorescence Activated Cell Sorter (FACS) were employed to evaluate cellimmunity. At 14d and 28d, the percent of CD3~+ was significant different by conventionalDNA vaccine pcDNA3.1(P＜0.05); at 21d、35 d, CD4~+ was significant different byconventional DNA vaccine pcDNA3.1(P＜0.05); CD8~+ was significant different byconventional DNA vaccine pcDNA3.1(P＜0.05 ) at 35d. The result shows that pSCA1-S1can induce a high level of cell immunity. Furthermore, the protected rate of pSCA1-S1、 pcDNA3.1 was 60% and 50%. The result appraisal IBV suicide DNA vaccineimmunogenicity.This study constructed the infectious bronchitis virus S1 gene suicidal DNA vaccineplasmid, and assessed the preliminary immunogenicity. There is no report about that. Thisstudy should encourage further work towards the development of a new kind of DNAvaccine against IBV.更多还原