鸡传染性贫血病毒VP2基因的表达及其免疫活性分析  

Expression of the VP2 gene of chicken infectious anemia virus in E.coli and analysis of immunogenicity

作　　者：王晓艳[1] 王笑梅[1] 高玉龙[1] 高宏雷[1] 陆桂丽[2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室,哈尔滨150001 [2]新疆农业大学,乌鲁木齐830052

出　　处：《微生物学报》2006年第5期841-843,共3页Acta Microbiologica Sinica

摘　　要：利用特异性引物从组织病料中提取的鸡传染性贫血病毒核酸经PCR扩增得到VP2基因,BamHⅠ和SalⅠ双酶切处理,纯化后,克隆至BamHⅠ和SalⅠ双酶切处理的表达载体pET-28a中,构建了原核表达质粒pET-28-VP2。将pET-28-VP2转化至感受态E.coliBL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约31kDa的目的蛋白以可溶性形式表达于上清中。Western blot分析发现,表达产物与鸡传染性贫血的阳性血清发生特异性反应。纯化蛋白免疫小鼠后制得的多抗可以与全病毒发生反应,证明其具有免疫原性,为进一步研究VP2蛋白的功能及开展CIAV疫苗及诊断试剂的研制奠定了基础。The coding region of VP2 gene from Chicken Infectious Anemia was amplified from genome extracted from chicken liver tissue by PCR. PCR product was double digested with restriction enzymes BamH Ⅰ and Sal Ⅰ and cloned into pET-28a digested with BamH Ⅰ and Sal Ⅰ . Subsequently, the recombinant plasmid pET-28-VP2 was extracted and double digested with restriction enzymes Bam H Ⅰ and Sal Ⅰ After confirming its rightness by PCR and analysis of restriction endonucleases,the recombinant plasmid pET-28-VP2 was transformed into E. coli BL21 （DE3）strain. The culture was induced by 1mmol/L IPTG at 37℃ for three hours and analyzed with SDS-PAGE. The result shows that gene encoding VP2 of CIAV was expressed successfully in E. coli and the fusion protein existed in supernatant, which was about 31kDa and showed specific immunoreactivity with anti-CIAV sera in Western blot. The fusion protein was purified by Ni^2＋ -affinity chromatography and quantitated by Bradford method. Then BALb/c mice were immunized with purified protein emulsified with Freund＇s complete adjuvant on day 0 and boosted twice on day 14 and 28 with the same dose of antigens emulsified with Freund＇s incomplete adjuvant, respectively. The serum isolated were examined by an enzymelinked immunosorbant assay （ELISA） using the purified VP2 and CIAV as coating antigens and the serum could react with target protein and CIAV in ELISA detection test.

关 键 词：鸡传染性贫血病毒 VP2基因 克隆 表达 

分 类 号：S852.4[农业科学—基础兽医学]