奶牛α-1,3-岩藻糖基转移酶基因的克隆表达

Clone and Expression of Alpha-(1,3)-Fucosyltransferase from Bos Taurus

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。克隆表达Slex合成过程中的关键酶,就可以在体外进行Slex的生物合成,从而进行相关生物制剂的开发。α-1,3-岩藻糖基转移酶(alpha-(1,3)-fucosyltransferase,FT)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FT基因进行了生物信息学的分析,了解了FT的相关理化性质。通过PCR的方法获得了FT基因,构建了重组质粒pMD19-FT,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-FT整合到宿主菌Pichia pastoris GS115基因组上,构建了重组酵母GS115-FT。经诱导表达后,SDS-PAGE检测到了目的蛋白质条带,证明了此基因在P.pastoris GS115中能够可溶性表达。

Abstract:

Sialyl lewis x(Slex) was one of the common glyco ligands of the selectin family. Slex combining with the inflammatory cells competitively could make inflammation reaction restrain efficiently. Clone and expression of the key enzymes in the biosynthesis process of Slex made the biosynthesis process of Slex in vitro possible,and it was also good for some related research of biological agents. Alpha-(1,3)-fucosyltransferase(FT) was the very one of the key enzymes in the biosynthesis process of Slex. In order to understand some related physicochemical properties of the FT gene,its sequence was analyzed by using approaches of bioinformatics online. The FT gene was inserted into a recombinant plasmid pMD19-T,and subcloned to the expression plasmid pPIC9K later. The linear expression plasmid pPIC9K-GT was integrated to the genome of Pichia pastoris GS115 by electrotransformation. After inducible expression,the soluble target protein was detected by SDS-PAGE. The result demonstrated that successfully expressed in P. pastoris GS115.