鸡堆形艾美球虫3-1E基因真核表达质粒的构建及其抗球虫免疫保护作用  

Construction of eukaryotic expression plasmid with Eimeria acervulina 3-1E gene and its immune protection efficacy against homologous Eimeria challenge

作　　者：马德星[1] 潘龙[1] 杨静红[1] 蔡浩璠 李广兴[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第10期900-904,共5页Chinese Veterinary Science

基　　金：黑龙江省自然科学基金重点项目(ZNJ0702-01)

摘　　要：应用反转录-聚合酶链式反应(RT-PCR)技术从堆形艾美球虫SH株子孢子中扩增3-1E基因片段,将其克隆入真核表达载体pcDNA3.1(+)中,构建重组表达质粒pcDNA3.1(+)-3-1E。质粒纯化后体外转染293T细胞,通过间接免疫荧光技术和免疫组织化学技术检测3-1E蛋白在转染细胞中的表达情况。将构建的重组质粒以及空载体质粒分别于14、21日龄分2次经腿部肌肉注射免疫SPF雏鸡,28日龄除非免疫非感染对照组外各组攻击性感染5×104个堆形艾美球虫孢子化卵囊,观察真核表达质粒对球虫感染鸡的免疫保护作用并探讨其免疫保护机理。结果显示,与载体对照组相比,质粒pcDNA3.1(+)-3-1E 2次免疫后可显著提高脾CD8+T淋巴细胞亚型数量,并具有一定的抗球虫免疫保护作用,可显著减少每克粪便的卵囊排出量,降低十二指肠病变记分,减少增重下降等。A 3-1E gene fragment was amplified from Eimeria acervulina SH strain sporozoite by RT- PCR,and it was cloned into eukaryotic expression vector pcDNA3. 1 （＋） to construct the recombinant plasmid pcDNA3.1 （q-）-3-1E. The purified plasmid pcDNA3.1（-k） 3-1E was transfected into 293T cells in vitro. The expressed 3-1E protein was detected by indirect immunofluorescence assay and immunohisto-chemistry. The plasmid pcDNA3.1 （＋）-3-1E and peDNA3. 1 （＋） was respectively used to immunize SPF chicks by injection intra-muscularly in thigh muscle at 14 and 21 days of age. Chicks in each group except that in the unchallenged control group were challenged with 5×10^4 E. acervulina sporulated oocysts at 28 days post-inoculation,and the immune protective efficacy was observed. The results showed that DNA vaccine pcDNA3, 1（＋）-3-1E could remarkably increase the number of CD8＋ T lymphocyte after two immunizations,and also could provide partial immune protection against homologous Eimeria challenge, such as significantly decreasing oocyst shedding, reducing the average lesion score in duodenum, improving body weight gain and so on.

关 键 词：堆形艾美球虫 3-1E基因 真核表达质粒 免疫保护 

分 类 号：S852.723[农业科学—基础兽医学]