中华鼢鼠卵透明带3基因(mZP3)真核表达载体的构建及其在中国仓鼠卵巢(CHO)细胞中的表达  

Construction of Eukaryotic Expression Vector for Myospalax fontanierii Zona Pellucida 3 Gene(mZP3) and Its Expression in Chinese Hamster Ovary(CHO) Cell

作　　者：张冬辉[1] 郑雪莉[1] 李昊[1] 周智敏[1] 吴景龙[1] 隋丹丹[1] 韩崇选[1] 

机构地区：[1]西北农林科技大学鼠害治理研究中心,杨凌712100

出　　处：《农业生物技术学报》2015年第3期408-413,共6页Journal of Agricultural Biotechnology

基　　金：国家林业公益性行业科研专项(No.201404405)

摘　　要：卵透明带3(zona pellucida 3,ZP3)蛋白是精卵结合最重要的受体。为了构建中华鼢鼠(Myospalax fontanierii)卵透明带3(mZP3)基因真核表达载体,本研究利用PCR方法得到加入酶切位点的mZP3片段,并将其克隆到真核表达载体pEGFP-N1上,体外转染中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO cell)和尾静脉注射昆明小鼠(Mus musculus)体内,利用RT-PCR和Western bolt技术检测其表达情况。双酶切和基因测序鉴定结果表明,所构建的表达载体是pEGFP-mZP3;倒置显微镜观察到发绿色荧光的成功转染的细胞;RT-PCR和Western bolt检测结果表明,目的基因mZP3在CHO细胞中成功表达,并且小鼠肝脏内也检测到目的基因。研究结果表明,卵透明带3基因能够在CHO真核细胞内表达,为后期基因疫苗防治中华鼢鼠提供依据。Zona pellucida 3 （ZP3） is the most important acceptor in the sperm-egg binding. In order to construct Myospalax fontanierii mZP3 eukaryotic expression vector pEGFP-mZP3 and detect the expression, mZP3 fragment was obtained by PCR and inserted into the eukaryotic expression vector pEGFP-N1, pEGFP- mZP3 was transfected to Chinese hamster ovary （CHO） cell and injected into Kunming mice （Mus musculus）, its expression was detected using RT-PCR and Weston bolt. Identification result showed that expression vector pEGFP-mZP3 was constructed successfully, the transfected cells emitted green fluorescence. RT-PCR and Western bolt test results showed that mZP3 expressed in CHO cells, and the target genes were also detected in the liver of mice. These results suggested that expression vector pEGFP-mZP3 successfully expressed in CHO cells, and provide the basis for the control of Myospalaxfontanierii by immunization approach.

关 键 词：中华鼢鼠 卵透明带3(ZP3) 真核表达 免疫不育 蛋白免疫印迹 

分 类 号：Q279[生物学—细胞生物学]