梅花鹿胸腺素beta10基因真核表达载体的构建及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2016-1000

生物技术通报 ›› 2017, Vol. 33 ›› Issue (6): 149-154.doi: 10.13560/j.cnki.biotech.bull.1985.2016-1000

梅花鹿胸腺素beta10基因真核表达载体的构建及鉴定

王佳雯, 高云鹏, 孙天霞, 刘美辰, 赵雨, 王思明

长春中医药大学中医药与生物工程研究开发中心,长春130117

收稿日期:2016-11-02 出版日期:2017-06-26 发布日期:2017-06-19
作者简介:王佳雯,女,助理研究员,研究方向：中药生物技术;E-mail：wangjiawen1229@163.com
基金资助:
吉林省科技发展计划（20140622003JC）

Construction and Identification of Recombinant Eukaryotic Plasmid of Sika Deer Thymosin Beta10

WANG Jia-wen, GAO Yun-peng, SUN Tian-xia, LIU Mei-chen, ZHAO Yu, WANG Si-ming

Traditional Chinese Medicine and Biotechnology Research and Development Center,Changchun University of Traditional Chinese Medicine,Changchun 130117

Received:2016-11-02 Published:2017-06-26 Online:2017-06-19

摘要/Abstract

摘要： 转录组测序发现Tβ10在鹿茸中高表达,为了研究Tβ10与鹿茸生长发育间的关系,构建Tβ10真核表达载体。使用Trizol法提取梅花鹿鹿茸总RNA,PCR技术特异性扩增梅花鹿Tβ10基因,利用酶切位点将目的片段插入真核表达载体VR1012构建表达质粒,通过Fugene^®6将质粒瞬时转染到293T细胞中,使用Western blotting和免疫荧光方法检测目的基因的表达。结果发现克隆得到的梅花鹿Tβ10基因长度为129bp,编码42个氨基酸,成功构建真核表达载体VR1012- Tβ10-HA,转染后使用Western blotting方法检测到梅花鹿Tβ10在293T细胞中表达,免疫荧光方法证明梅花鹿Tβ10主要定位在细胞浆中。

关键词: 梅花鹿, 胸腺素beta10, 质粒构建, 转染, 真核表达

Abstract: Tβ10 was discovered to be highly expressed in sika antler in transcriptome sequencing. In order to study the relationship between Tβ10 and the growth and development of antler,the Tβ10 eukaryotic expression vector was constructed. Extracting the total RNA from sika deer antler by Trizol regent,the coding region of Tβ10 gene was amplified by RT-PCR,and then the gene fragment was inserted into the recombinant eukaryotic expression vector VR1012. The eukaryotic expression plasmids were transfected into 293T cells by Fugene^®6 agent. The ORF of Tβ10 cDNA was 129 bp,encoding for a protein of 42 amino acids. The eukaryotic expression vector VR1012- Tβ10-HA was constructed successfully. The expression of sika deer Tβ10 was detected in 293T cells by Western blotting. Immunofluorescence demonstrated that the expressed fusion protein of the Tβ10 was located in the cytoplasm in 293T cells.

Key words: sika deer, thymosin beta10, plasmid construction, transfection, eukaryotic expression

王佳雯, 高云鹏, 孙天霞, 刘美辰, 赵雨, 王思明. 梅花鹿胸腺素beta10基因真核表达载体的构建及鉴定[J]. 生物技术通报, 2017, 33(6): 149-154.

WANG Jia-wen, GAO Yun-peng, SUN Tian-xia, LIU Mei-chen, ZHAO Yu, WANG Si-ming. Construction and Identification of Recombinant Eukaryotic Plasmid of Sika Deer Thymosin Beta10[J]. Biotechnology Bulletin, 2017, 33(6): 149-154.

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梅花鹿胸腺素beta10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 Beta10

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