绵羊黏膜趋化因子CCL28基因的克隆、序列分析与原核表达  

Molecular Cloning,Sequence Analysis and Prokaryotic Expression of Ovine Mucosal Chemokine CCL28

作　　者：乔新安[1] 李宏基[1] 王月影[1] 朱彦彩[2] 韩立强[1] 杨国宇[1] 王艳玲[1] 

机构地区：[1]河南农业大学动物生理生化实验室,郑州450002 [2]河南科技学院细胞工程重点实验室,新乡453003

出　　处：《畜牧兽医学报》2008年第6期842-847,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：河南省重点科技攻关项目(0523010500)

摘　　要：利用同源序列克隆原理设计1对克隆引物,从绵羊结肠黏膜组织提取mRNA,通过RT-PCR获得CCL28基因,将PCR产物克隆、测序,并进行序列分析;再根据克隆的序列设计1对表达引物,用PCR法从重组克隆载体中扩增出含BamHⅠ/XhoⅠ酶切位点的CCL28片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-CCL28,转化宿主菌E.coliBL21(DE3),经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊CCL28基因包含完整的开放阅读框,克隆片段长444 bp,ORF为387 bp,编码129个氨基酸,与人、小鼠、猪、牛该基因的同源性分别为76.4%、61.4%、84.3%和92.9%,推测的氨基酸序列与人、小鼠、猪、牛的同源性分别为76%、63%、84%和93%,表达的融合蛋白分子量约为39 ku,并以包涵体形式存在。为下一步研究绵羊CCL28的生物学功能奠定基础。A pair of cloning primers were designed according to the cloning principle of homologous sequence CCL28. CCL28 mRNA extracted from mucosal tissue of ovine colon was amplified by RT-PCR, then the PCR product was cloned, sequenced and analyzed. Then, a pair of expres- sion primers were designed according to the cloned sequence. A fragment of ovine CCL28 cDNA containing BamH Ⅰ/Xho Ⅰ were amplified from recombinant vector by PCR. The fragment cleaved by BamH Ⅰ/Xho Ⅰ was subcloned into pGEX-4T-1 vector to construct a recombinant expression vector, pGEX-CCL28. Subsequently, E. coli BL21 competent cells were transformed by the recombinant vector. The fusion protein, which was induced by IPTG was analyzed by SDSPAGE. The cloned sequence containing the open reading frame （ORF） of ovine CCL28 consists of 444 bp, the ORF is 387 bp and encodes 129 amino acids. Analysis showed that the ovine CCL28 nucleotide sequence shared 76.4%, 61.4%, 84.3% and 92.9% homology with that of human, mouse, pig and cattle, the predicted amino acid shared 76%, 63%, 84% and 93% homology with that of human, mouse, pig and cattle. The fusion protein expressed in E. coli BL21 （DE3） was about 39 ku and mainly existed as inclusion bodies. Our research provides the experiment basis for further researching biological function of CCL28.

关 键 词：绵羊 黏膜相关上皮趋化因子 分子克隆 序列分析 原核表达 

分 类 号：Q786[生物学—分子生物学] S852.4[农业科学—基础兽医学]