人防御素6基因的克隆、亚克隆及在酵母中的分泌表达

Clone,Sub-Clone and Secretion Expression of Human Defensin 6 Gene in Pichia pastoris

探讨人防御素6(HD-6)在毕赤酵母中表达的可行性,为进一步研究HD6的功能提供理论依据和实验基础。采用PCR方法,设计引物从cDNA文库中扩增出人α防御素6基因片段,并将其插入到克隆载体pMD-18T中,再与毕赤酵母表达载体pPICZαA重组,以得到重组的HD-6酵母表达载体pPICZαA/HD-6,并进行琼脂糖电泳和测序鉴定。再将构建好的毕赤酵母重组表达质粒pPICZαA/HD-6经SacⅠ线性化后,应用LiCl法转化毕赤酵母菌株GS115感受态中,Zeocin平板筛选,PCR鉴定转化子。经摇瓶发酵和甲醇诱导,SDSPAGE分析重组HD-6的表达。从cDNA文库中扩增出的HD-6基因片断大小正确;电泳和测序结果均证明已将此片段克隆到酵母表达载体pPICZαA内;线性化的重组质粒pPICZαA/HD-6成功转化进入毕赤酵母感受态中,PCR鉴定结果与预期相符;蛋白电泳证实重组HD-6在酵母中获得分泌表达。提示重组HD-6可以在毕赤酵母中实现分泌表达。

The feasibility of expression of human defensin 6 （HD-6） in Pichia pastoris was investigated in order to provide theoretical foundation and experiment bases for further study on HD 6 function. A primer was designed, from cDNA library a fragment of α HD6 gene was amplified with PCR method. Then inserted it into HD-6 segment into a clone vector pMD 18T. Then recombine the α HD-6 fragment with expression vector pPICZαA to obtain a recombinant expression vector of pPICZαA/HD-6 and then identified by electrophoresis and sequence analysis. After the constructed P. pastoris recombinant plasmid pPICZαA/HD-6 was linearized by Sac Ⅰ, then was transformed into competence P. pastoris strains GS115, screened on Zeocin plate, then identified the transformant with PCR. Induced through shaker flask fermentation and alcohol, the expression of recombinant HD-6 was analyzed by SDS-PAGE. The PCR results of HD-6 gene from cDNA library had the correct size and sequences. The results of electrophoresis and sequencing had all proved that the fragment had been cloned into the expression vector plasmid pPICZαA. The linearized recombinant plasmid pPICZαA/HD-6 had successfully transformed into competence P. pastoris. The PCR identified results accorded with the one expected. Protein electrophoresis had proved that recombinant HD 6 had obtained secretion expression in P. pastoris. Suggested that the HD 6 could be realized the secretion expression in P. pastoris.