水牛CD14 cDNA的克隆、表达及鉴定  

Cloning,Expression and Identification of CD14 cDNA of Buffalo

作　　者：陈品林[1] 杜丽[1] 雷明[1] 成鹰[1] 陈兴生 莫蘼 刘涛[1] 张冬琳[1] 崔森[1] 王凤阳[1] 

机构地区：[1]海南大学农学院海南省热带动物繁育与疫病研究重点实验室(筹)海口市动物基因工程重点实验室,海南海口570228 [2]海南省定安县畜牧兽医局,海南定安571200

出　　处：《生物技术》2011年第1期28-32,共5页Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2009ZX08007-009B)资助

摘　　要：目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定。方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入Ecoli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定。结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带。结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础。Objective：This study was aimed to clone and express bCD14（buffalo cluster of differentiation antigen 14）,then carry out Western Blot for it.Method：RT-PCR method were used to clone CDS area of cluster of bCD14,then,the PCR products of bCD14 gene was cloned into the expression vector PET-28a,and transformed into the host strain E.coli BL21.After the inducible expression by IPTG,the target protein was analyzed under denaturing condition,Western blot were used to identify the fusion protein.Result：The results showed that bCD14 gene contain a 1122 bp ORF encoding 373 amino acids,compared with Indian buffalo,Norway rat and human,the homology of cDNA were 97.95%,68.78% and 78.60%;the homology of amino acid were 96.78%,61.27% and 72.34%,then the 46kD target protein was confirmed by Western blot.Conclusion：The CDS area of bCD14 was successfully cloned,expressed,which laid a solid foundation for the in-depth function study of bCD14.

关 键 词：水牛 CD14 克隆 表达 纯化 

分 类 号：Q786[生物学—分子生物学]