Toll样受体4胞外区在毕赤酵母系统的初步表达及鉴定  

Primary expression and identification of TLR4 extracellular fragment in Pichia pastoris

作　　者：杨清武[1] 牟玲[2] 朱佩芳[3] 王正国[3] 蒋建新[3] 王景周[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所神经内科,重庆400042 [2]第三军医大学预防医学院毒理学教研室,重庆400038 [3]第三军医大学大坪医院野战外科研究所第四研究室,重庆400042

出　　处：《第三军医大学学报》2005年第1期20-23,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划资助项目 ("973"项目 ) (G19990 54 2 0 3);国家自然科学基金资助项目 ( 30 170 96 8);全军医药卫生科研基金资助项目 ( 0 1Q10 5)~~

摘　　要：目的　利用毕赤酵母系统对Toll样受体 4(Toll likereceptor 4,TLR4)胞外区进行初步表达及鉴定 ,为相关研究奠定基础。方法　用PCR获得扩增TLR4胞外区DNA ,经序列分析后 ,插入含AOX1启动子和α分泌信号肽序列的毕赤酵母表达载体中 ,构建pPIC9K/TLR4胞外区的重组质粒 ;转化酵母宿主菌GS115后 ,G418筛选多拷贝转化子 ;菌落PCR鉴定后 ,摇菌培养 ,1%甲醇诱导表达。SDS PAGE分析表达产物 ,并利用Westernblot法鉴定。结果　PCR扩增的TLR4胞外区基因序列与GenBank数据库中的序列一致 ,构建的重组质粒经酶切及测序证实 ,成功构建了pPIC9K/TLR4胞外区的重组质粒。经过G418筛选后 ,共获得 2 0 0个高拷贝转化子 ,菌落PCR鉴定其中 5个克隆 ,能扩增出特异的TLR4胞外区基因片段 ,表明TLR4胞外区基因完全整合到毕赤酵母中 ;SDS PAGE分析显示 ,表达产物以可溶性分子形式存在于上清中 ,薄层凝胶扫描分析显示诱导表达 4d的表达量达上清的 5 0 3 5 % ,Westernblot分析表明表达蛋白能与TLR4单抗结合。结论　利用毕赤酵母系统成功地对TLR4胞外区进行了表达及鉴定 ,为进一步筛选高表达菌株、蛋白纯化、功能研究奠定基础。Objective To express and identify of Toll-like receptor 4 (TLR4) extracellular fragment in Pichia pastoris and to provide basis for the related researches. Methods The human TLR4 extracellular cDNA fragment was amplified by PCR and after confirming by DNA sequence analysis, was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of α factor gene to form a pPIC9K/TLR4 extracellular recombinant expression plasmid. The recombinant plasmid was transformed into GS115 and high copies transformants were screened with G418. After being identified by colony PCR, transformants were cultured in flasks and induced by 1% methanol. The expression products were analyzed by SDS-PAGE and were identified by Western blot analysis. Results The sequencing showed that TLR4 extracellular gene was identical to that in Genbank. The analysis for the recombinant plasmid DNA digested by enzyme demonstrated that the recombinant expression plasmids of pPIC9K/TLR4 extracellular were constructed successfully. After screening by G418, 200 high-copy-number of transformants were acquired. The special TLR4 extracellular gene segment was obtained by identification with colony PCR, which showed that TLR4 extracellular gene of 5 clones were inserted into Pichia pastoris. After methanol induction for 4 d, the expression proteins came up to 50.35% of total proteins in medium supernatant as shown by SDS-PAGE. Western blot analysis proved that the expression protein could bind to TLR4 monoclonal antibody specifically. Conclusion TLR4 extracellular protein is expressed and identified successfully by Pichia pastoris system, which can provide basis for the researches of further screening of high expression colony, protein purification, and functional research.

关 键 词：TOLL样受体4 脓毒症 内毒素 毕赤酵母 

分 类 号：R392.13[医药卫生—免疫学] R394-33[医药卫生—基础医学]