鲤春病毒血症病毒糖蛋白基因的克隆及其在毕赤酵母中的初步表达

Clone and construction of the expression strain of Pichia pastoris for glycoprotein gene of spring viremia of carp virus

根据SVCV糖蛋白基因序列设计引物,在5’引物和3’引物中分别引入酶切位点。以病毒核酸为模板用RT-PCR方法扩增该段糖蛋白基因,将所得的基因片段(517和521)克隆至穿梭载体pGAPZαA/B之GAP启动子下游位点,构建含α信号肽的重组表达载体。获得的重组质粒pGAPZαA-517和pGAPZαB-521经线性化后,电击法转化毕赤酵母SMD1168菌株,构建分泌型表达SVCV糖蛋白的重组酵母菌株。酵母菌落PCR法检测表明,已成功构建分泌表达SVCV糖蛋白的酵母基因工程菌株,斑点印迹法进一步分析表明有目的蛋白的成功表达。

PCR primers were designed according to glycoprotein nucleotide sequence of SVCV, the enzyme recognizing sites being introduced into 5＇- and 3＇- primers. 2 glycoprotein fragments were obtained by means of RT-PCR with SVCV nucleotide taken as template, and then cloned into plasmid pGAPZαA/B. The recombinant plasmids pGAPZαA-517 and pGAPZαB-521 were linearized and transformed respectively into SMD1168 by electroporation. Checking with yeast colony PCR indicated that the gene-engineering strains which could express SVCV glycoprotein had been constructed successfully. Furthermore, analysis of Western-blot also testified that the objective protein was expressed.