结合利用pGAPZαA和pPICZαA构建目的蛋白的多拷贝载体并在毕赤酵母中表达  

Combination Utilization of pGAPZαA and pPICZαA to Construct Multi-Copies Expression Plasmid of the Interested Protein and Express the Protein in Pichia Pastoris

作　　者：冯菲[1] 王雪妍[2] 陈晓飞[1] 宁萌[2] 周伏忠[1,2] 刁文涛[1,2] Feng Fei;Wang Xueyan;Chen Xiaofei;Ning Meng;Zhou Fuzhong;Diao Wentao(Institute of Biology Co. Ltd.,Henan Academy of Sciences,Zhengzhou 450008;Key Laboratory of Microbial Engineering of Henan Province,Zhengzhou 450008)

机构地区：[1]河南省科学院生物研究所有限责任公司,郑州450008 [2]河南省微生物工程重点实验室,郑州450008

出　　处：《河南科学》2017年第1期83-87,共5页Henan Science

基　　金：河南省省级重点实验室专项(122300413214);河南省重点攻关计划项目(142102210087;152102210167);河南省科学院2016年基本科研费项目;河南省科技开放合作项目(152106000052)

摘　　要：结合利用载体pGAPZαA和pPICZαA构建目的蛋白的可线性化多拷贝表达载体,并转化毕赤酵母表达目的蛋白.首先将目的蛋白基因构建到组成型表达载体pGAPZαA中,然后依据载体自身特性,用同尾酶BglⅡ和Bam HⅠ切取其表达盒并连接到甲醇诱导型载体pPICZαA中的Bam HⅠ位点处,经多次重复后获得目的蛋白多拷贝表达载体,线性化后电转化毕赤酵母表达目的蛋白.灵芝免疫调节蛋白LZ8是一个已成功在毕赤酵母中表达的蛋白,作为检验该方法的样本其基因被首先构建到载体pGAPZαA中得到质粒pGAPZαA-LZ8,切取其表达盒构建到载体pPICZαA中,经过4次重复得到了多拷贝表达载体pPICZαA-[GAPZαA-LZ8]4,然后利用pPICZαA特有的限制性内切酶位点SacⅠ进行线性化,最终电转化到毕赤酵母GS115中进行组成型表达蛋白LZ8,其表达量达到pGAPZαA-LZ8转化菌株表达量的2~3倍.经检验,结合利用载体pGAPZαA和pPICZαA可以构建目的蛋白的多拷贝表达载体,并且可以对载体进行线性化而不影响转化酵母时的转化效率.Vectors pGAPZαA and pPICZαA were utilized to construct the multi-copies expression plasmid that can be linearized to maintain the transformation rate at a high level and have a higher yield of interesting protein than the single-copy plasmid. The gene of the interesting protein was originally constructed in the constitutiveexpression vector pGAPZαA,then the expression cassette is digested out with the isocaudamers BglⅡand BamHⅠand constructed in the methanol inducible expression vector pPICZαA at BamHⅠsite. The multi-copies expression plasmid was constructed after several times repeating. The LZ8 which is a fungal immunomodulatory protein from Lingzhi is used as an example. Its multi-copies expression plasmid pPICZαA-[GAPZαA-LZ8]4 is constructed after repeating 4 times. Then the plasmid is linearized at the unique Sac Ⅰsite of pPICZαA and transformed in GS115. The yield of LZ8 expressed in the clones is transformed with pPICZαA-[GAPZαA-LZ8]4 reaches 2 or 3 times of that in the clones which is transformed with pGAPZαA-LZ8. We find the linearizable multi-copies expression plasmid bases on combination utilization of pGAPZαA and pPICZαA can express the interesting protein in GS115 at a higher level than the single-copy plasmid.

关 键 词：pGAPZαA pPICZαA 多拷贝 毕赤酵母 

分 类 号：Q785[生物学—分子生物学]