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Abstract 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 cDNA clone of bovine (Bos taurus) MEN1 gene (bMEN1), and analyze its expression levels of mRNA and target encoded protein menin in different cell lines. According to the deposited sequence of bMEN1 in GenBank, primers attached with EcoRⅠand HindⅢ restriction enzymes were designed and amplified by using qRT-PCR method. The digested PCR product was further ligated into eukaryotic vector pcDNA3.1-myshis-(A). Under in vitro transfection, the expected mRNA 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 qRT-PCR and Western blot. Double restriction enzymes digestion and sequencing results indicated that the eukaryotic expression vector of bMEN1 gene was successively cloned, named as pcDNA3.1-myc-his-bMEN1. Under the constructed transfection system, both mRNA 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, bMEN1 mRNA 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.
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Received: 28 September 2015
Published: 05 February 2016
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