重组人A20蛋白在巴斯德毕赤酵母GS115中的表达  

Expression of recombinant human A20 in Pichia pastoris GS115

作　　者：吴丽娟[1] 蒋建新[1] 朱佩芳[1] 康格非[2] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室全军交通医学研究所全军交通医学实验室创伤烧伤与复合伤国家重点实验室,重庆400042 [2]重庆医科大学检验系,重庆40016

出　　处：《第三军医大学学报》2006年第3期201-204,共4页Journal of Third Military Medical University

基　　金：国家科技部创伤;烧伤与复合伤国家重点实验室开放基金资助项目(2003)~~

摘　　要：目的利用酵母表达技术制备重组人A20蛋白(rhA20),为进一步研究其在脓毒血症中的治疗作用奠定基础。方法利用基因工程技术构建rhA20毕赤酵母表达载体yevCFP-hA20,电转化巴斯德毕赤酵母菌株GS115,筛选高拷贝阳性菌株,甲醇诱导表达rhA20。产物用SDS-PAGE和W estern B lot鉴定。同时对诱导表达培养基的pH值和配方,以及培养环境温度等条件进行了优化研究。结果表达产物分子量约93×103,表达条件的优化研究提示:降低诱导培养基pH值、添加低剂量EDTA、蛋白酶水解物和降低培养温度,可以使全长表达产物提高11倍,占总蛋白的18.24%,达到(254.10±24.52)μg/m l水平。结论我们成功在巴斯德毕赤酵母GS115中表达了rhA20,通过对诱导培养基成分及诱导温度的优化,使全长rhA20表达得到明显提高,探索了人A20的生物合成途径,也为巴斯德毕赤酵母表达外源蛋白遭遇降解时的处理提供了新的方法。Objective Human A20 is one of the endogenetic regulating proteins of inflammation in vivo and play important roles in preventing uncontrolled inflammatory response as well as endogenetic cellular protection. Our aim is to prepare a recombinant protein of human A20 （rhA20） in Pichia pastoris and then research its roles in sepsis therapy. Methods rhA20 yeast expression vector yevCFP-hA20 was constructed by genetic engineering. Pichia pastoris with linearized yevCFP-hA20 was electroporated. The high copy clones were screened with MD/His^ - medium and YPD/G418 gradient medium. The number of hA20 in yeast genome was detected by quantitative PCR. The rhA20 was expressed by methanol induction and identified by SDS-PAGE and Western blotting. The optimum conditions （including pH and ingredient of induced medium as well as the temperature of induced culture） were investigated. Results The molecular weight of rhA20 is about 93 × 10^3. After expression condition＇ s optimization by decreasing the pH of BMMY medium, adding a low dosage of EDTA and the substrate of protease, as well as decreasing the induced temperature, the full expressing products was up to 11 times, rose to 18. 24% in total protein and achieved to 254. 1 024. 52μg/ml. Conclusion we have successfully expressed rhA20 in Pichia pastoris GS115. The expression amount of full rhA20 increases clearly by progressing the ingredient and pH of BMMY and using a lower inducing temperature. This starts the biosynthesis of hA20 in vitro. A new solving regime is provided to other biologist when they meet product＇s degradation by using Pichia pastoris GS115.

关 键 词：A20 基因表达 巴斯德毕赤酵母 基因工程 

分 类 号：R341[医药卫生—基础医学] R394-33