嗜鞣管囊酵母adh2基因克隆及其原核表达

Cloning and Prokaryotic Expression of Pachysolen tannophilus adh2 Gene

通过对不同酵母乙醇脱氢酶Ⅱ基因的多重同源性分析比对,克隆出嗜鞣管囊酵母P-01(Pachysolen tan-nophilus)乙醇脱氢酶Ⅱ基因的一段长为340 bp的片段(EU570211).利用RACE技术,扩增出嗜鞣管囊酵母P-01adh2基因的全长ORF(1 056 bp).将基因与pMD18-T载体连接,验证后测序.经酶切、酶连到表达载体pET-20b并成功构建了基因的原核表达载体pET-20b-adh2,在E.coliBL21(DE3)中诱导表达出adh2重组蛋白,主要以包涵体的形式存在,预期大小约为35 kD.

A 340 bp length cDNA of adh2 gene（EU570211） is cloned with primers designed based on the conserved domain sequences of yeast gene families.Using the RACE method,a full length cDNA of adh2（1 056 bp） gene from the cDNA library of yeasts is cloned,and then the adh2 ORF is cloned into the vector pMD18-T.PCR amplifying and sequencing confirm that the construction is correct and has no base mutant.The recombinant prokaryotic expression vector pET-20b-adh2 is constructed using vector pET-20b,the prokaryotic expression analysis is carried out through transformation of E.coli（BL21）.The SDS-PAGE electrophoresis analyses show that about a 35 kD recombinant protein is produced in the form of inclusion body.