pPICZα-synBPIm600酵母表达载体的构建和鉴定

首都医科大学学报 ›› 2004, Vol. 25 ›› Issue (1): 35-39.

pPICZα-synBPI_m600酵母表达载体的构建和鉴定

丛敏¹, 靖学芳², 刘振龙³, 孙明洁³, 安云庆³

1. 首都医科大学附属北京友谊医院肝病中心;2. 吉林大学免疫学系;3. 首都医科大学免疫学教研室

收稿日期:2003-10-14 修回日期:1900-01-01 出版日期:2004-01-15 发布日期:2004-01-15

Construction and Identification of pPICZα-synBPI_m600 Recombinant Expression Vector

Cong Min¹, Jing Xuefang², Liu Zhenlong³, Sun Mingjie³, An Yunqing³

1. Liver Center, Beijing Friendship Hospital, Affiliate of Capital University of Medical Sciences;2. Docter of Immunology, Jilin University;3. Department of Immunology, Capital University of Medical Sciences

Received:2003-10-14 Revised:1900-01-01 Online:2004-01-15 Published:2004-01-15

摘要/Abstract

摘要： 为使rBPI_m23重组抗菌蛋白在毕赤酵母表达系统中分泌表达,拟构建synBPI_m600酵母表达载体。以pUC18synBPI₄₁₄质粒为模板扩增synBPI₂₀₀基因片段,经XhoI/EcoRI双酶切获得synBPI_185bp基因片段;EcoRI/SalI双酶切PBV220BPI_m600质粒获得BPI_m420基因片段;将上述2个基因片段连接后定向克隆到pPICZα酵母表达载体上。连接产物转化E.coliTOP10F′,在含Zeocin的低盐LB固体培养基上筛选阳性转化子。阳性克隆菌经菌落PCR法鉴定、酶切分析和测序鉴定,结果与预期相符。提示:成功构建了pPICZαsynBPI_m600酵母表达载体.

关键词: 杀菌/渗透增强蛋白, rBPI_m23重组抗菌蛋白, 表达载体, 巴斯德毕赤酵母

Abstract: In order to express rBPI_m23 recombinant bactericidal protein in Pichia pa storis, synBPI_m600 expression vectors were constructed. synBPI₂₀₀ bp fragment was amplified by PCR from pUC18-synBPI₄₁₄ vector, then the product was diges ted by XhoI and EcoRI to obtain synBPI_{185 bp} fragment; PBV220-BPI_m600 recombinant expressing vector was digested by EcoRI and SalI to obtain BPI_{m420 bp} fragment; the above two fragments were linked and cloned in pPICZα e xpression vector to form intact pPICZα-synBPI_m600 recombinant expression vector. After E.coli TOP 10 F' was transformated by pPICZα-synBPI_m600 vector, positive cloning strains were selected by low salt LB medium with Zeocin and id entificated by PCR amplication, restriction analysis by XhoI/EcoRI/SalI and DNA sequencing. The results show that intact pPICZα-synBPI_m600 recombinant expression vectors are constructed successfully.

Key words: bactericidal/permeability-incr easing protein(BPI), rBPI_m23 recombinant bactericidal protein, ex pression vector, Pichia pastoris

中图分类号:

Q782

丛敏;靖学芳;刘振龙;孙明洁;安云庆. pPICZα-synBPI_m600酵母表达载体的构建和鉴定[J]. 首都医科大学学报, 2004, 25(1): 35-39.

Cong Min;Jing Xuefang;Liu Zhenlong;Sun Mingjie;An Yunqing. Construction and Identification of pPICZα-synBPI_m600 Recombinant Expression Vector[J]. Journal of Capital Medical University, 2004, 25(1): 35-39.

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