传染性造血器官坏死病病毒G基因全长cDNA克隆及重组表达研究

发布时间：2018-04-11 23:39

本文选题：传染性造血器官坏死病病毒(IHNV) + 糖蛋白　；参考：《中国兽医杂志》2017年01期

【摘要】：本研究旨在克隆G基因全长并与其他传染性造血器官坏死病病毒(IHNV)株进行分析,为IHNV的预防检测提供依据。通过提取IHNV-G蛋白RNA并进行扩增,将其克隆到p MD-18T并与p Chlamy-4载体相连,重组到莱茵衣藻中并提取总蛋白,结果可得:扩增片段长度为1 500 bp,经SDS-PAGE电泳得到一条大约为58 k Da的蛋白条带,Westen Blot分析结果显示,重组莱茵衣藻所表达的蛋白可以与6×HIS标签抗体特异性结合;并且以重组表达的G蛋白为抗原,鼠抗IHNV血清为一抗,辣根过氧化物酶标记的驴抗鼠IgG为二抗,再次进行Westen Blot检验,其结果显示,经诱导后的G蛋白能与鼠抗IHNV血清反应,并在58 k Da处出现了一条明显的特异性条带,具有良好的反应原性。表明重组莱茵衣藻表达系统构建成功。
[Abstract]:The purpose of this study was to clone the G gene and analyze it with other infectious hematopoietic organ necrosis virus (IHNV) strains in order to provide evidence for the prevention and detection of IHNV.IHNV-G protein RNA was extracted and amplified, then cloned into p MD-18T and ligated with p Chlamy-4 vector. The protein was recombined into Chlamydomonas rhinorrhoeae and the total protein was extracted.The results showed that the amplified fragment length was 1 500 BP, and a protein band of about 58 kDa was obtained by SDS-PAGE electrophoresis. The results showed that the expressed protein of Chlamydomonas rhinella could specifically bind to 6 脳 HIS tag antibody.Using recombinant G protein as antigen, mouse anti IHNV serum as first antibody and horseradish peroxidase labeled donkey anti mouse IgG as second antibody, Westen Blot test was carried out again. The results showed that the induced G protein could react with mouse anti IHNV serum.There was a specific band at 58 kDa, which had good reactivity.The results showed that the recombinant expression system of Chlamydomonas rhinella was successfully constructed.
【作者单位】：吉林农业大学动物科学技术学院;内蒙古乌兰察布市农牧业综合行政执法支队;内蒙古乌兰察布市水产站;吉林省卫生检测检验中心;
【基金】：国家自然科学基金(30972191) 吉林省留学人员创新创业项目(201523) 长春市农业先进实用技术的示范推广项目(20130215) 农业部948项目(2014Z34)
【分类号】：S852.65

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