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 Bgl Ⅱ and BamH Ⅰ and constructed in the methanol inducible expression vector pPICZαA at BamH Ⅰ site.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 Ⅰ site 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.%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Bgl Ⅱ和BamH Ⅰ切取其表达盒并连接到甲醇诱导型载体pPICZαA中的BamH Ⅰ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.
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