梅山猪β干扰素基因的克隆、原核表达及生物活性  

Cloning,Expression and Biological Activity of Meishan Porcine Interferon Beta Gene

作　　者：彭贵青[1] 陈焕春[1] 钱平[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2005年第1期40-42,共3页Chinese Journal of Veterinary Science

摘　　要：利用 PCR技术从中国梅山猪白细胞总 DNA中扩增出猪β干扰素基因 (MS- IFNβ)。将 PCR产物克隆至 p MD18-T载体 ,利用双脱氧末端终止法测定了全基因的核苷酸序列。序列分析表明 ,β干扰素基因全长 5 6 1bp,编码 186个氨基酸 ,与 Gen Bank中 S4 1178的序列同源性达 99% ,在 12 8、177位核苷酸处发生了点变异 ,由 G变为 A,T变为 C,但仅导致 4 3位氨基酸由 G变为 E。在此基础上 ,合成了 1对引物 ,通过 PCR方法将编码成熟蛋白基因亚克隆到 p GEX-KG表达载体 ,转化宿主菌 BL2 1 (DE3) ,经 IPTG诱导后 ,得到高效表达 ,所表达蛋白质的大小约为 4 70 0 0 ,占总蛋白的2 3%。表达产物主要为不溶性的包涵体 ,包涵体经提纯后 ,能够抑制口蹄疫病毒 (FMDV)The completed interferon beta gene(MS-IFNβ) was amplied from leucocyte genome DNA of Meishan porcine by PCR.The PCR products were cloned into pMD-IFNβ and sequenced.The squence result indicated that the MS-IFNβ gene was consisted of about 561 bp and en coded 186 amino acids.The homology analysis of the gene with S41178 in GenBank showed that it was highly conserved,only 128 and 177 sites nucleotide to be different.Then the interferon beta gene en coded mature protein was cloned by using of another pair of primers.The PCR products were inserted into PGEX-KG and transformed BL_(21)(DE_3) bacteria.The expression of IFN-β was induced with IPTG and confirmed by SDS-PAGE,the recombinant protein had a molecular weight of (47 000) and could inhibit FMDV replication.

关 键 词：基因 Β干扰素 克隆 原核表达 IFN 包涵体 白细胞 梅山猪 FMDV 总DNA 

分 类 号：R512[医药卫生—内科学] S828[医药卫生—临床医学]