鸡γ-干扰素基因的克隆及原核表达  

Cloning and Expression of Chicken IFN-γ gene

作　　者：史耀旭[1] 关贵全[2] 高金亮[2] 党志胜[2] 刘志杰[2] 刘爱红[2] 马米玲[2] 任巧云[2] 罗建勋[2] 殷宏[2] 曾巧英[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽药杂志》2006年第7期12-16,共5页Chinese Journal of Veterinary Drug

基　　金：国家高技术研究发展计划(863)项目(2003AA241110);国家自然科学基金资助项目(30270992)

摘　　要：参照Digby发表的鸡γ-干扰素的核苷酸序列,设计一对引物,应用RT-PCR技术,从ConA诱导过的鸡脾淋巴细胞中特异性地扩增出大小约为500bp的基因片段。将该扩增基因进行克隆测序,结果表明其序列与Digby报道的CHIFN-γ基因序列的同源性达100%。将该基因插入原核表达载体pGEX-4T-1中构建成重组表达载体pGEX-CHIFN-γ,并将其导入大肠杆菌BL21中,诱导表达的重组蛋白经SDS-PAGE分析,分子量约为43ku,表明所克隆的CHIFN-γ基因在原核细胞中得到了表达。The total RNA was abstracted from the concanavalin A（ Con A） -activated spleen lymphocytes. A pair of specific primers was designed by Oligo software based on the sequence of Chicken interferon-gamma submitted by Digby in GenBank （accession No. U27465）. A 500 bp DNA fragment was amplified by reverse transcriptionpolymerase chain reaction （RT-PCR） and then ligated into T-easy vector for sequencing. The result shows that the fragment contained the complete open reading frame of CHIFN-γ gene. In comparison with GenBank data, the homology of the nucleotide sequence is 100% . The sequence was inserted into pGEX-4T-1 and the recombined expressing vector was constructed, then transformed into Escherichia coli BL21. which were further induced by IPTG and cultured, and a fusion protein was obtained with about 43 000 of molecular weight in SDS-PAGE. This result showed that the cloned CHIFN-γ gene was expressed in prokaryotic cells.

关 键 词：Γ-干扰素 鸡 克隆 表达 

分 类 号：Q78[生物学—分子生物学]