去N端信号肽和C端跨膜区猪卵透明带-3β蛋白在原核系统表达的研究  

Study on the Expression of Pig Zona Pellucida-3βExcluding N-terminus Signal Peptide and C-terminus Transmembrance-like Domain in Escherichia coli

作　　者：徐万祥[1] 邱德义[1] 熊艳[1] 申庆祥[1] 顾少华[2] 曹根涛[3] 应康[2] 谢毅[2] 

机构地区：[1]上海市计划生育科学研究所,上海200032 [2]复旦大学生命科学院遗传所遗传工程国家重点实验室,上海200433 [3]中国科学院上海细胞生物学研究所,上海200031

出　　处：《生物工程学报》2001年第1期16-19,共4页Chinese Journal of Biotechnology

基　　金：国家计划生育药具重点实验室和上海市计划生育委员会局管基金资助项目(97JG05022)

摘　　要：猪卵透明带 (Zonapellucida ,ZP)由生物化学和免疫学性质截然不同的三种糖蛋白 (pZP1、pZP3α和pZP3β)组成。由于抗 pZP3β抗体能在体外与人ZP发生交叉反应 ,因此 pZP3β一直被认为是研制抗受精避孕疫苗的潜在抗原之一。为了能在原核系统中获得无卵巢因子污染的 pZP3β蛋白 ,本文采用PCR方法删除了原cDNA中编码5′ 和 3′ 端信号肽和跨膜区的DNA顺序。扩增的 pZP3β基因核心片段 (pZP3β″)经DNA测序验证后 ,通过引入其两端的EcoRI和SalI酶切位点被定向插入 pBV2 2 1质粒PRPL 启动子下游的多克隆区。SDS PAGE分析表明 :工程菌在热诱导后能特异地表达pZP3β蛋白 ,并能在蛋白印迹实验中被兔抗 pZPIgGs识别。Pig oocytes are surrounded by an acellular translucent envelope zona pellucida(pZP),which is comprised of three biochemically and immunologically distinct glycoproteins(pZP1,pZP3α and pZP3β).Due to the cross reactivity of anti pZP3β antibodies with human ZP in vitro, pZP3β has been considered as potential one of immunogens for developing human contraceptive vaccine.In order to express pZP3β directly in E.coli, we amplified the core fragment of pZP3β cDNA by PCR that was deleted 5′ and 3′ terminal sequences of coding for signal peptide and transmembrane like domain.The DNA sequenced Eco RI and Sal I restriced core fragment was cloned in a frame downstream of P RP L promoter in the pBV221 vector.SDS PAGE analysis showed that pZP3β protein was expressed especially in E.coli after thermal induction,which the expression band displayed the molecular weight of about 38 kD matching with its deduced molecular weight(36 5 kD).In addition,the specially expressed protein band on SDS PAGE gel showed specific immunological reaction with anti pig ZP IgGs of rabbit in Western blot.

关 键 词：pZP3β蛋白 基因表达 免疫印迹 

分 类 号：Q78[生物学—分子生物学]