猪带绦虫未知基因SLC10在毕赤酵母中的表达

An Unknown Taenia Solium' Gene of SLC10 Expressed in Pichia pastoris

SLC10是以反式剪切引导序列为基础,通过RT-PCR从猪囊尾蚴中克隆到的一个未知基因。将猪囊尾蚴SLC10基因从重组克隆载体pGEM-SLC10扩增并酶切后,与相应酶切处理的毕赤酵母分泌性表达载体pPIC9K相连接,构建重组表达载体pPIC9K-SLC10,转化大肠杆菌JM109,经PCR、酶切和测序鉴定,基因序列完全正确。纯化的重组质粒pPIC9K-SLC10用内切酶SacⅠ线性化,电转化毕赤酵母,使重组表达载体与酵母染色体发生同源整合;采用G418抗性梯度法筛选多拷贝重组菌株,用甲醇进行诱导表达;通过SDS-PAGE和Western-blot分析表达产物,结果表明目的蛋白得到了表达,但不具有免疫反应性。

An unknown gene, SLC10, was cloned by RT-PCR from Cysticercus cellulosae on the base of trans-spliced leader sequence. The SLC10 gene was cloned from vector of pGEM-SLC10 and inserted into pPIC9k to construct a recombinant secretory vector named pPIC9K-SLC10. Then the pPIC9k-SLC10 was transformed into E. coli JM109. The recombinant vector was identified by PCR, enzymatic analysis and sequencing. The positive plasmid was linearized with Sac I and transformed into GS115 by electroporation and the insert was integrated into genomic DNA of Pichia pastoris by means of homologous recombination. The recombinants with multiple integrated copies of SLC10 were screened using gradient concentration G418 and induced with methanol. The results of SDS-PAGE and Western-blot showed that the expressed protein was not of immunoreaction.