梅花鹿胸腺素β4基因真核表达载体的构建及鉴定

Construction and identification of recombinant eukaryotic plasmid of sika deer thymosin beta4

为了构建梅花鹿胸腺素β4(Tβ4)基因真核表达载体,使用TRIzol法提取梅花鹿鹿茸总RNA,RT-PCR方法特异性扩增梅花鹿Tβ4基因,目的基因插入真核表达载体VR1012构建表达质粒,将质粒瞬时转染到293T人胚肾细胞系,使用Western blot和免疫荧光方法检测目的基因的表达。结果显示:克隆得到的梅花鹿Tβ4基因长度为135 bp,编码44个氨基酸,成功构建真核表达载体VR1012-Tβ4-HA,转染后梅花鹿Tβ4在293T细胞中表达,而且主要表达在细胞浆中。本试验为进一步研究Tβ4在鹿茸生长中的作用奠定了基础。

The present study was conducted to construct the eukaryotic expression vector of thymosin beta4 gene（ Tβ4）. The total RNA was extracted from sika deer antler by TRIzol regent and the coding region of the Tβ4 gene was amplified by RT-PCR,and then the gene fragment was inserted into the recombinant eukaryotic expression vector VR1012,and the eukaryotic expression plasmid was transfected into 293T cells by Fugene6 agent. The expressed products were detected by Western blot and immunofluorescence. The ORF of Tβ4 cDNA was 135 bp,coding for a protein of 44 amino acids. Sika deer Tβ4 was cloned and constructed into VR1012. The expression of sika deer Tβ4 was detected in 293T cells by Western blot. Immunofluorescence detected the expressed fusion protein of the Tβ4-HA,which located in the cytoplasm in 293T cells. This finding lays experimental basis for study of Tβ4 roles in the antler growth.