人TALL-1基因在毕赤酵母中的分泌表达与鉴定

目的 研究在毕赤酵母(Pichia Pastoris)系统中人TALL-1基因的分泌表达,获得具有生物活性的重组TALL-1.方法 将人TALL-1基因插入含AOX1启动子和α分泌信号肽序列的酵母表达载体pPIC9K中,用Sac Ⅰ将重组质粒线性化,电转化Pichia Pastoris GS115感受态细胞,筛选阳性整合子并进行甲醇诱导表达,采用MTY对表达产物进行初步的生物活性检测.结果 经过诱导表达条件的优化,人TALL-1在酵母培养基BMMY中实现了分泌表达;免疫印迹和酶联免疫检测结果显示,重组产物可以结合其特异抗体;生物学活性检测证实其可抑制Hela细胞增殖.结论 在毕赤酵母系统中实现了人TALL-1的分泌表达,具有生物学活性的重组TALL-1分子可以用于进一步的结构与功能研究.

Expression and characterization of human TALL-1 in Pichia Pastoris

Objective To study the secretion expression of human TALL-1 cDNA in Pichia Pastoris and to obtain recombinant human TALL-1 of good biological activity. Methods The human TALL-1 gene was cloned into yeast expression vector pPIC9K containing AOXI promoter and the sequences of secreting signal peptides. Recombinant plasmid was linearized by Sac I and transformed into Pichia Pastoris GS115 competent cells. Positive integrated clones were screened out and cultured in flasks, and hTALL-1 was expressed under the induction of 1% methanol. Biological activities were analyzed with ELISA and MTr assay. Results Human TALL-1 gene was expressed in BMMY after optimization of inducing expression condition. The recombinant could combine with specific antibody and the expressed product could inhibit the proliferation of Hela cells. Conclusion The secretion expression of human TALL-1 was successfully achieved in Pichia Pastoris expression system. The recombinant human TALL-1 could be used for further study on its structure and functions.