【作者】 张立成；

【导师】 杨增岐； 陈洪岩；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2003， 硕士

【摘要】 根据已发表的禽脑脊髓炎病毒1143株特异性结构蛋白VP0、VP1、VP3基因序列,分别设计三对引物,利用反转录—聚合酶链式反应(RT-PCR)技术,在体外从AEVVan-Roekel株感染的SPF鸡胚胚脑和内脏器官组织中成功扩增出各结构基因片段。将其回收、纯化后,以VP0、VP1、VP3基因连接到线状克隆载体PMD18-Teasy Vector,转化JM109感受态细胞。在含氨苄青霉素的LB平板上筛选阳性克隆,扩大培养,提取质粒,经酶切和PCR鉴定,取阳性质粒用Sangers双脱氧末端终止法对核酸序列进行测定,推算其氨基酸序列;并将其与已发表的AEV 1143株相关基因以及小RNA病毒科其它病毒属在对应位置的核苷酸和氨基酸的同源性进行比较、分析。 将VP0基因克隆到原核高效表达载体PET30α,构建重组表达质粒PET30α-VP0,转化PET30α表达宿主菌BL21;经IPTG和42℃升温诱导表达,收获菌体沉淀,经菌体全细胞裂解和提取包涵体后进行SDS-PAGE和Western-blot电泳分析表明,重组质粒能在大肠杆菌中获得表达,表达产物主要以包涵体形式存在于菌体中。蛋白印迹试验检测表达蛋白分子量PET-VP0为52Ku;经薄层扫描测定,蛋白相对含量为26.3%。上述表达产物能与AEV阳性血清发生特异性反应。更多还原

【Abstract】 According to Three pair of premier were designed according to specific structural protein VP0, VP1 and VP3 gene sequence of Avian Encephalomyelitis virus(AEV) 1143 strain in represented articles. By RT-PCR the gene fragment from brains and guts of SPF chicken embryos, which were infected with AEV Van-Roekel strain., were cloned in vitro. After harvesting and purifying, VP0、 VP1、 VP3 gene fragment were connected into PMD18-Teasy Vector linearized, and then these were transferred into JM109 cells. Positive clones were.selected by LB plates with Ampicillin and proliferated, and plasmids were extracted from Positive clones. By digestion with enzyme and PCR identification, nucleic acid sequence of positive plasmids were analyzed with Sangers deoxynucleotidyl termination, and relevant amino acid sequences were also calculated. So the homology of relevant nucleic acid and amino acid sequence were compared and analyzed with that of AEV 1143 strain and others of small RNA virus family.Recombinant expression vectors PET30a-VP0 were constructed, by connecting VP0 gene sequences with prokaryotic Expression Vector PET30a, and transduced into BL21 by IPTG and heatshock. To collect inclusion bodies, bacteria with vector were collected and broken up, and then inclusion bodies were analyzed with SDS-PAGE and Western-blot electrophoresis. Results showed that recombinant plasmids could be expressed in Escherichia coli, which were in the form of inclusion bodies; Molecular weight of PET-VP0 expression protein was 52Ku by Western-blot electrophoresis, and relative protein content was 26.3% by TLCS. In addition, specific reaction of these expression products with AEV serum was positive.更多还原