基因组学与应用生物学
基因組學與應用生物學
기인조학여응용생물학
Genomics and Applied Biology
2011年
2期
190-196
,共7页
张晓茹%匡文华%成鹰%杜丽%张冬琳%郝永昌%雷明%焦寒伟%刘涛%祁超%王凤阳
張曉茹%劻文華%成鷹%杜麗%張鼕琳%郝永昌%雷明%焦寒偉%劉濤%祁超%王鳳暘
장효여%광문화%성응%두려%장동림%학영창%뢰명%초한위%류도%기초%왕봉양
MyD88%水牛%基因克隆%原核表达
MyD88%水牛%基因剋隆%原覈錶達
MyD88%수우%기인극륭%원핵표체
MyD88%Buffalo%Cloning%Prokaryotic expression
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。
採用RT-PCR方法從水牛外週血白細胞總RNA中擴增齣髓樣分化因子88（mydoid differentiation factor88,MyD88）cDNA序列,PCR產物分離純化後,與pMD20-T載體連接,重組質粒經PCR、酶切鑒定後測序,併進行生物信息學分析;構建pET28a-MyD88錶達載體,併將其轉化至E.coli BL21（DE3）,經IPTG誘導錶達後,進行SDS-PAGE、鎳柱親和層析純化和Western blotting分析。結果顯示,剋隆到的水牛MyD88cDNA全長為1189bp,含有1箇891bp的開放閱讀框,編碼296箇氨基痠,理論等電點為5.65。經IPTG誘導錶達後,得到一箇帶His·Tag的約39kD的重組融閤蛋白。用抗His單剋隆抗體進行Western blotting,得到1條約39kD特異性抗體結閤帶,錶明水牛MyD88原覈錶達載體成功構建併錶達。本研究為進一步開展水牛MyD88的結構功能分析奠定瞭基礎。
채용RT-PCR방법종수우외주혈백세포총RNA중확증출수양분화인자88（mydoid differentiation factor88,MyD88）cDNA서렬,PCR산물분리순화후,여pMD20-T재체련접,중조질립경PCR、매절감정후측서,병진행생물신식학분석;구건pET28a-MyD88표체재체,병장기전화지E.coli BL21（DE3）,경IPTG유도표체후,진행SDS-PAGE、얼주친화층석순화화Western blotting분석。결과현시,극륭도적수우MyD88cDNA전장위1189bp,함유1개891bp적개방열독광,편마296개안기산,이론등전점위5.65。경IPTG유도표체후,득도일개대His·Tag적약39kD적중조융합단백。용항His단극륭항체진행Western blotting,득도1조약39kD특이성항체결합대,표명수우MyD88원핵표체재체성공구건병표체。본연구위진일보개전수우MyD88적결구공능분석전정료기출。
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.
