人TALL-1可溶性功能片段在毕赤酵母中的分泌表达

Secretory expression of human soluble TALL-1 in Pichia pastoris

目的在毕赤酵母系统中分泌表达具有生物活性的人TALL-1基因胞外区片段(sTALL-1)。方法构建酵母重组表达质粒pPIC9K-sTALL-1,电转化法将SacI线性化的重组质粒转导入毕赤酵母菌GS115中,经G418筛选获得高拷贝转化子及表型鉴定后,用含1%甲醇的培养基诱导其分泌表达。结果成功构建酵母重组表达质粒pPIC9K-sTALL-1;甲醇诱导表达后,sTALL-1在酵母培养基中得到分泌表达;Westernblot和ELISA检测证实重组产物可以结合其特异抗体;MTT检测证实其对Hela细胞具有细胞毒效应。结论在毕赤酵母系统中实现了人sTALL-1的分泌表达。

Objective To study the secretory expression of human soluble TALL-1 in Pichia pastoris. Methods The human sTALL-1 cDNA was cloned into yeast expression vector pPIC9K containing AOXI promoter and the sequences of secreting signal peptides. Recombinant plasmid was lined by Sac Ⅰ and transformed into Pichia Pastoris GS115. Positive integrated clones were screened and cultured in flasks, and sTALL-1 was expressed under the induction of 1% methanol. Results Recombinant expression plasmid pPIC9K-sTALL-1 was successfully constructed. The human sTALL-1 was expressed in BMMY after optimization of inducing expression condition. Western blot and ELISA assay revealed recombinant products could combine with specific antibody. The expressed product could inhibit the proliferation of Hela cells by MTT assay. Conclusions The secretory expression of human sTALL-1 had been successfully achieved in Pichia pastoris expression system.