牛MEN1基因真核表达载体的构建及其表达分析  

Construction of Eukaryotic Expression Vector for Bos taurus MEN1 Gene and Its Expression Analysis in Different Cell Lines

作　　者：刘学[1] 李洪辉[1] 王中华[1] 林雪彦[1] 闫振贵[1] 侯秋玲[1] 王云[1] 胡志勇[1] 师科荣[1] 

机构地区：[1]山东农业大学动物科技学院/山东省动物生物工程与疾病防治重点实验室,泰安271018

出　　处：《农业生物技术学报》2016年第3期366-372,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31402054);山东省自然科学基金(No.ZR2013CM013);山东省奶牛良种工程重大课题(No.2012LZ15);山东省泰山学者种业创新人才团队计划培养项目(No.2014LZ07-04);国家奶业产业技术体系资助项目(No.CARS-37);山东省牛产业技术体系资助项目(No.SDAIT-12-011-06)

摘　　要：多发性内分泌腺瘤致病因子1(multiple endocrine neoplasiaⅠ,MEN1)参与乳腺发育与泌乳行为的调控。本研究克隆了牛(Bos taurus)MEN1基因(b MEN1)的全长c DNA,并在不同细胞中检测b MEN1m RNA及其编码蛋白menin的表达情况。根据Gen Bank中b MEN1基因序列,设计特异性引物,用q RTPCR方法得到附带Eco RⅠ和HindⅢ酶切位点的b MEN1片段,并将其克隆到真核表达载体pc DNA3.1-myc-his(A)中。体外转染物种同源性细胞牛乳腺上皮细胞(bovine mammary gland epithelial cell,MAC-T)和异源性细胞中国仓鼠(Cricetulus barabensis)卵巢细胞(Chinese hamster ovary cells,CHO)、小鼠(Mus musculus)成肌细胞(mouse myoblast cells,C2C12),利用q RT-PCR和Western blot技术检测转染前后b MEN1 m RNA和蛋白menin的表达。双酶切和基因测序结果表明,成功构建了b MEN1基因的真核表达载体pc DNA3.1-myc-his-b MEN1。所建立的转染体系可以使目的基因b MEN1在3种不同细胞中成功表达m RNA和目的蛋白,转染后24 h都达到最高表达量,并达到极显著水平(P<0.01),之后逐渐降低。其中,CHO细胞中的转染24 h后b MEN1 m RNA和menin蛋白的表达量分别是对照的28 415倍和5.65倍。本研究建立了b MEN1基因的真核表达载体及其转染体系,能够在不同细胞中成功表达,为体外研究MEN1基因对于乳腺的调节功能及其对于机体代谢的调节机制提供了一种工具和技术体系。Multiple endocrine neoplasia Ⅰ（MEN1） participates in regulatory role in the development of mammary gland and lactation behavior cycling. The objective of this study was to obtain full- length c DNA clone of bovine（Bos taurus） MEN1 gene（b MEN1）, and analyze its expression levels of m RNA and target encoded protein menin indifferent cell lines. According to the deposited sequence of bM EN1 in GenB ank, primers attached with EcoR Ⅰand HindⅢ restriction enzymes were designed and amplified by using qR T-PCR method. The digested PCR product was further ligated into eukaryotic vector pcD NA3.1- myshis-（A）. Under in vitro transfection, the expected mR NA and target encoded protein menin were successively detected in homologous cell line bovine mammary epithelial cells（MAC- T） and heterologous cell lines Chinese hamster（Cricetidae） ovary cells（CHO） and mouse（Mus musculus）myoblast cells（C2C12）, respectively by using qR T-PCR and Western blot. Double restriction enzymes digestion and sequencing results indicated that the eukaryotic expression vector of bM EN1 gene was successively cloned, named as pcD NA3.1-myc-his-bM EN1. Under the constructed transfection system, both mR NA and protein could expectedly express in all three different cell lines, with significantly highest expression levels after 24 h post transfection（P〈0.01） afterwards gradually decreased. Especially in CHO cells, bM EN1 mR NA expression level was 28 415-fold of negative control after 24 h post transfection, and 5.65-fold for encoded menin protein. This study can supply research tool and technical system for further functional or mechanism investigation of bovine MEN1 gene in mammary gland development and body metabolism.

关 键 词：牛多发性内分泌腺瘤致病因子1基因(b MEN1) 真核表达 奶牛乳腺上皮细胞(MAC-T) 中国仓鼠卵巢细胞(CHO) 小鼠成肌细胞(C2C12) 

分 类 号：S823[农业科学—畜牧学]