唾液酸化路易斯-X合成关键酶基因的克隆表达  

Cloning and Expression of the Key Enzyme Gene in Biosynthesis of Sialyl Lewis X

作　　者：姚晶[1,2] 任婧[2] 吴正钧[2] 孙克杰[2] 郭本恒[1,2] 

机构地区：[1]上海海洋大学食品学院,上海201306 [2]乳业生物技术国家重点实验室光明乳业股份有限公司技术中心,上海200436

出　　处：《中国生物工程杂志》2011年第12期51-56,共6页China Biotechnology

基　　金：国家"973"计划(2010CB735705);上海市科委课题(09DZ2251400)资助项目

摘　　要：唾液酸化路易斯-X(sialyl lewis X,Slex)是选择素家族的一个共同糖配体,通过与选择素竞争性地结合炎性细胞,可以抑制炎症反应。通过克隆表达Slex合成过程中的关键酶,在体外进行Slex的生物合成,用于相关乳腺炎防治药物的研发。β-1,4-半乳糖基转移酶(β-1,4-galactosyltransferase,GT)就是参与Slex生物合成过程的关键酶之一。利用相关软件对牛的GT基因进行了生物信息学的分析,了解了GT的相关理化性质。通过人工合成的方法获得了GT基因的CDS,构建了重组质粒pMD18-GT,并亚克隆至表达载体pPIC9K。通过电转化将线性化的表达质粒pPIC9K-GT整合到宿主菌P.pastoris GS115基因组上,构建了重组酵母GS115-GT。经诱导表达后,SDS-PAGE检测到了目的蛋白条带,证明了此基因在P.pastoris GS115中能够可溶性表达;并用苯酚红法测定了粗酶液的活性,其比酶活为16.4 U/ml,这为其进一步研究奠定了基础。Sialyl lewis X （Slex）, one of the common glyco ligands of the selectin family, restrains inflammation reaction by combining with the inflammatory cells competitively. Clone and expression of the key enzymes in the biosynthesis process of Slex could make its biosynthesis in vitro, and some related mastitis therapy research possible. Beta 1, 4 galactosyhransferase （GT） was the very one of the key enzymes in the biosynthesis process. In order to understand some related physicochemical property of GT gene, the gene sequence was analyzed by using approaches of bioinformaties online. The synthetic CDS of this gene was inserted into a recombinant plasmid pMD18-T, and subcloned to the expression plasmid pPIC9K later. The linear expression plasmid pPIC9K-GT was integrated to the genome of P. pastoris GS115 by electrotransformation. After inducible expression, the soluble target protein was detected by SDS-PAGE. It was proved that this gene could be expressed successfully in P. pastoris GS115. The phenol red method was used to determine the activity of the unpurified enzyme, and the specific activity was 16.4U/ml. This might be the foundation work for the further study.

关 键 词：乳腺炎 唾液酸化路易斯-X 生物合成 牛 β-1 4-半乳糖基转移酶克隆表达 

分 类 号：Q786[生物学—分子生物学]