人肝再生增强因子在毕赤酵母中的表达

Expression of human augmenter of liver regeneration in Pichia pastoris

目的从人胎肝cDNA文库中克隆出人肝再生增强因子(human augmenter of liver,regeneration,hALR),并实现hALR在毕赤酵母中的表达方法利用PCR从胎肝cDNA文库中扩增出与文献报道序列一致的ALR cDNA,亚克隆到pPIC9K的α因子下游并置于AOX1启动子控制下,构建成含有ALR基因的酵母表达载体转化酵母细胞后,挑选出能在浓度为4.0g·L^(-1)的G418平板上生长的His^+的重组酵母,利用PCR鉴定是否插入了hALR基因.重组酵母的表达在甲醇培养中进行,并利用SDS-PAGE分析hALR的表达.结果 PCR从胎肝cDNA文库中扩增出与文献报道序列一致的ALR cDNA,实现了其在酵母中的整和表达结论 hALR在毕赤酵母中的表达是可行的.

AIM To clone the human augmenter of liver regeneration (hALR) cDNA from the human fetal liver cDNA panel, and to express hALR in Pichia pastoris. METHODS hALR cDNA was amplified by PCR, and then was sequenced. The expression vector was constructed after in subcloning hALR cDNA into the downstream of the α-factor secretion signal under the control of AOX1 promoter of the pP1C9K vector. His recombinant clones that can resist 4.0 g.L^(-1) G418 and were selected, and hALR cDNA insertion was analyzed by PCR. The expression of recombinant Pichia pastoris strains were conducted under the inducer of methanol and analyzed by SDS-PAGE. RESULTS hALR cDNA whose sequence is the same as reported was amplified by PCR. hALR cDNA insertion was confirmed, and expression of hALR could be detected by SDS-PAGE. CONCLUSION The cloning and expression of hALR in Pichia pastoris is successful.