摘要
通过对犬传染性肝炎病毒(CAV-I)基因组的提取,PCR扩增获得了大小为1109bpCAV-I病毒结构IX基因片段,成功地将IX基因克隆至载体pMD-18T中,构建了pIX原核表达载体pGEX-4T1-IX,将重组质粒pGEX-4T1-IX转化至大肠杆菌BL21(DE3)感受态细胞中,并实现了IX基因在该菌中的高效表达。对表达产物进行了Westernblot检测,分析结果表明表达蛋白能与抗CAV-I阳性血清发生特异性的抗原-抗体反应,为CAV-I的血清学诊断和免疫监测提供大量优质的抗原奠定了基础。
The author firstly adopted the method of concentration of the virus and extract its genome.Then referring to the sequence which is published in gene bank,the author designed a pair of primers,product of the PCR amplification,pIX protein,was analyzed by agarose gel electrophoresis and demonstrated to be a specific band of 1099bp.The fragment was cloned into the pMD18-T vector.After cloning it into the T vector,the author sequenced the targeted fragment and the result showed that it was right.Then the author ligated it into the expression vector pGEX-4T-1 with DNA T4 ligase,which was digested by the same two enzymes.Furtherly digesting by EcoRI and SalI for identification,the author finally got the positive expression vector pGEX-4T1-IX.After transforming the recombinant plasmid pGEX-4T1-IX to the host E.coli BL21（DE3）for 2～3 hours,the author induced it by IPTG with the terminal concentration of 1mmol/L.The whole quantity of expression was up to 21% in the host cell.In the western blotting assay,the protein expressed could be detected by CAV-I positive serum,which would contribute greatly to the establishment of CAV-I antibody detecting method.
出处
《中国农学通报》
CSCD
北大核心
2010年第22期12-15,共4页
Chinese Agricultural Science Bulletin
基金
吉林省科技平台建设项目"吉林省试验动物质量检测中心平台建设"(20071138)
国家大学生创新性试验计划"杯状病毒RT-PCR检测方法的建立及初步应用"(2009079)
