水牛MyD88 cDNA的克隆与原核表达  

Gene Cloning and Prokaryotic Expression of MyD88 cDNA from Buffalo

作　　者：张晓茹[1,2] 匡文华[1,2] 成鹰[2] 杜丽[2] 张冬琳[2] 郝永昌[2] 雷明[2] 焦寒伟[2] 刘涛[2] 祁超[1] 王凤阳[2] 

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]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,海口570228

出　　处：《基因组学与应用生物学》2011年第2期190-196,共7页Genomics and Applied Biology

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

摘　　要：采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88(mydoid differentiation factor88,MyD88)cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21(DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析。结果显示,克隆到的水牛MyD88cDNA全长为1189bp,含有1个891bp的开放阅读框,编码296个氨基酸,理论等电点为5.65。经IPTG诱导表达后,得到一个带His·Tag的约39kD的重组融合蛋白。用抗His单克隆抗体进行Western blotting,得到1条约39kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达。本研究为进一步开展水牛MyD88的结构功能分析奠定了基础。Mydoid differentiation factor 88 （MyD88） cDNA was amplified from total RNA of peripheral blood leukocyte in buffalo by RT-PCR,and cloned into pMD20-T vector after purified.The recombinant plasmid was confirmed by PCR,endonuclease digestion,sequencing and bioinformatics analysis.The PCR product was then subcloned into pET28a vector and transformed into E.coli BL21 （DE3）.Protein expression was induced by IPTG,purified by Ni-NTA superflow cartridge and analyzed by SDS-PAGE and Western blotting.The results showed that the MyD88 cDNA was 1 189 bp in length and contained a 891 bp ORF,encoding 296 amino acids.The theoretical IP of the encoded protein was 5.65.A 39 kD recombine fusion protein with a His tag was induced by IPTG and confirmed by Western blotting using anti-his tag monoclonal antibody,a special antibody binding band was obtained,which indicated that prokaryotic expression vector of MyD88 from buffalo was constructed and expressed successfully.This work laid a foundation for the further investigation of the molecular constitution and function of MyD88 in buffalo.

关 键 词：MYD88 水牛 基因克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]