单纯疱疹病毒I型糖蛋白D在酵母中的表达

Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。

The extracellular portion of glycoprotein D gene （gDt）, encoding 1-314 amino aods, was amplified from extracted HSV-1 DNA and cloned into expression vector pPIC9K. After electroporation transformation of Pichia pastoris GS 115 and selection with G418, a strain of P. pastoris with high yield of secreted recombinant gD was obtained. The yield of recombinant gD was 250mg/liter in shake flask cultures. The recombinant protein could be identified with specific monoclonal antibody 1-I-9 using ELISA and Western blotting assays. Recombinant gD was purified to almost homogeneity by Q-Sepharose ion exchange, Chelating Sepharose immobilized metal ion affinity and Sephacryl S-200 gel filtration chromatographies. Purified gD expressed in P. pastoris elicited high level of specific antibodies in BALB/c mice, indicating recombinant gD were characteristic high immunogenicity and capable of inducing significant titers of specific antibodies.