期刊论文

Construction of GnIH Overexpression Vector and Its Effect on Mouse Leydig Cells

中文

畜牧兽医学报

0366-6964

2021

52

10

2969-2977

10.11843/j.issn.0366-6964.2021.010.028

湖南省教育厅重点项目（18A095）；

本校为第一机构

本试验通过构建GnIH过表达载体,探究其对小鼠睾丸间质细胞（TM3细胞系）的凋亡效应及睾酮合成的调节作用。从6～8周雄性小鼠睾丸组织中提取RNA,反转录获得cDNA样本,利用PCR扩增并纯化GnIH基因,应用T4连接酶将其连入PLVX-IRES-ZsGreen1载体,进一步转化到感受态菌中,通过PCR、双酶切及测序鉴定后提取质粒。随后将构建好的重组质粒转染TM3细胞系72 h,分为空质粒组和GnIH过表达组,通过显微镜观察细胞荧光、实时荧光定量PCR （qRT-PCR）法、ELISA法和流式细胞术检测GnIH表达水平、细胞凋亡率及相关凋亡基因表达变化、睾酮分泌水平和睾酮合成酶基因表达水平。结果显示,GnIH扩增片段序列与参考序列一致...

In this present study,in order to investigate the effect of GnIH on the apoptosis and testosterone synthesis of mouse Leydig cells,its overexpression vector was constructed.RNA was extracted from the testis tissues of male mice at 6-8weeks,reverse transcribed into cDNA. The GnIH gene was amplified and purified by PCR,and connected to the PLVX-IRES-ZsGreen1 vector through T4ligase.Then,the recombinant plasmid was transformed into the competent cells,and extracted the plasmid after identified by PCR,double enzyme digestion and sequencing. Subsequ...