辽宁绒山羊DLX3毛囊表达载体的构建及其转染成纤维细胞的研究

›› 2014, Vol. 41 ›› Issue (11): 29-34.

辽宁绒山羊DLX3毛囊表达载体的构建及其转染成纤维细胞的研究

舒国涛¹, 姚娜¹, 董坤哲¹, 康晔^1,2, 浦亚斌¹, 赵倩君¹, 何晓红¹, 马月辉¹

1. 中国农业科学院北京畜牧兽医研究所, 农业部畜禽遗传资源与种质创新重点实验室, 北京 100193;
2. 云南农业大学, 云南昆明 650201

收稿日期:2014-04-08 出版日期:2014-11-20 发布日期:2014-12-06
通讯作者: 马月辉 E-mail:yuehui.ma@263.net
作者简介:舒国涛(1987-),男,四川人,硕士,研究方向:动物种质资源。
基金资助:
国家绒毛用羊产业技术体系(nycytx-40-5)。

Construction of Hair Follicle Expression Vector of DLX3 and its Transfection into Liaoning Cashmere Goat Fibroblasts Cell

SHU Guo-tao¹, YAO Na¹, DONG Kun-zhe¹, KANG Ye^1,2, PU Ya-bin¹, ZHAO Qian-jun¹, HE Xiao-hong¹, MA Yue-hui¹

1. Key Laboratory of Farm Animal Genetic Resources and Utilization of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Yunnan Agricultural University, Kunming 650201, China

Received:2014-04-08 Online:2014-11-20 Published:2014-12-06

摘要/Abstract

摘要： 根据GenBank中已发表的DLX3基因(登录号:XM_005694267.1)序列设计引物,以辽宁绒山羊毛囊生长期皮肤组织cDNA为模板,采用RT-PCR方法,扩增出DLX3基因的CDS区,连接平末端载体并验证后与真核表达载体pIRES2-EGFP连接。真核表达载体经Xho Ⅰ和BamHⅠ双酶切鉴定后,通过脂质体法转染绒山羊耳源成纤维细胞,在荧光显微镜下观察细胞中增强型绿色荧光蛋白(EGFP)的表达,并通过RT-PCR和Western blotting技术检测目的基因转录、蛋白质表达情况。结果表明,成功克隆了绒山羊DLX3基因,并构建了真核表达载体pIRES2-EGFP-DLX3。重组质粒转染绒山羊成纤维细胞24 h后在荧光显微镜下观察到绿色荧光,通过RT-PCR扩增909 bp的转录产物,并利用Western blotting检测到32.87 ku目的蛋白DLX3的表达。本试验结果为研究DLX3基因在绒山羊毛囊生长周期内的功能以及调控毛囊生长发育的机制奠定了基础。

关键词: DLX3; 绒山羊; 成纤维细胞; 真核表达载体

Abstract: The complete CDS of DLX3 gene was amplified from skin during Liaoning cashmere goat hair follicle growth phase by RT-PCR using one pair of primer which was designed and synthesized according to the DLX3 gene sequence in GenBank(No:XM_005694267.1),inserted into blunt-vector after DNA sequencing verification,it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP.After the restriction enzyme (XhoⅠ/BamHⅠ) digestion of the eukaryotic expression vector and sequencing,the plasmid had been transfected into the goat fibroblasts by Lipofectamine 2000.We also observed the fluorescent under the microscope,examined the transcription vector pIRES2-DLX3-EGFP by RT-PCR and Western blotting.The results showed that we had successfully cloned goat DLX3 gene and constructed eukaryotic expression vector pIRES2-DLX3-EGFP,we could observe the green fluorescent after 24 h transfection of the plasmid.Then, we amplified the transcription product of 909 bp by RT-PCR.The targeted protein 32.87 ku was also detected by Western blotting.This result has paved the way for investigating the influence of DLX3 on hair follicle development and cycling as well as the research of the regulation mechanism of hair follicle growth and development.

Key words: DLX3; cashmere goat; fibroblasts cell; eukaryotic expression vector

中图分类号:

S826

舒国涛, 姚娜, 董坤哲, 康晔, 浦亚斌, 赵倩君, 何晓红, 马月辉. 辽宁绒山羊DLX3毛囊表达载体的构建及其转染成纤维细胞的研究[J]. , 2014, 41(11): 29-34.

SHU Guo-tao, YAO Na, DONG Kun-zhe, KANG Ye, PU Ya-bin, ZHAO Qian-jun, HE Xiao-hong, MA Yue-hui. Construction of Hair Follicle Expression Vector of DLX3 and its Transfection into Liaoning Cashmere Goat Fibroblasts Cell[J]. , 2014, 41(11): 29-34.

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