Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris

LI Zhi-hua; XU Wei-ming; JI Qiu-yan; LONG Hai-ting; PEN Zheng-hua; LI Jian-feng

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February 2006

LI Zhi-hua, XU Wei-ming, JI Qiu-yan, LONG Hai-ting, PEN Zheng-hua and LI Jian-feng. Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris[J]. Virologica Sinica, 2006, 21(1): 6-10.

Citation: LI Zhi-hua, XU Wei-ming, JI Qiu-yan, LONG Hai-ting, PEN Zheng-hua, LI Jian-feng. Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris .VIROLOGICA SINICA, 2006, 21(1) : 6-10.

单纯疱疹病毒I型糖蛋白D在酵母中的表达

1.
中国医学科学院/中国协和医科大学医学生物学研究所

摘要

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。
- 单纯疱疹病毒
- , 包膜糖蛋白D
- , 毕赤酵母表达
- , 纯化

Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris

1.
Chinese Academy of Medical Sciences /Peking Union Medical college

Abstract

The extracellular portion of glycoprotein D gene(gDt),encoding 1-314 amino acids,was amplified from extracted HSV-1 DNA and cloned into expression vector pPIC9K.After electroporation transformation of Pichia pastoris GS115 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.
- Herpes simplex virus (HSV)
- , Glycoprotein D (gD)
- , Pichia pastoris
- , Purification

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Expression of Herpes Simplex Virus 1 Glycoprotein D in Pichia pastoris

1. Chinese Academy of Medical Sciences /Peking Union Medical college

Abstract: The extracellular portion of glycoprotein D gene(gDt),encoding 1-314 amino acids,was amplified from extracted HSV-1 DNA and cloned into expression vector pPIC9K.After electroporation transformation of Pichia pastoris GS115 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.