利用同源重组构建BMP6基因真核表达载体及在成纤维细胞中表达量的研究  

Study on Construction of Eukaryotic Expression Vector for BMP6 Gene by Homo Iogous Recombination and Its Expression in Fibroblasts

作　　者：张梦瑶 杨峰 刘开东 刘积凤[1] 柳楠[1] 贺建宁[1] ZHANG Meng-yao;YANG Feng;LIU Kai-dong;LIU Ji-feng;LIU Nan;HE Jian-ning(College of Animal Science and Technology Qingdao Agricultural University,Shandong Qingdao 266109,China;College of Animal Science and Technology,Inner Mongolia Agricultural University,Inner Mongolia Huhhot 010018,China;Qingdao Institute of Animal Husbangdry and Vetrrinary,Shandong Qingdao 266121,China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]内蒙古农业大学动物科技学院,内蒙古呼和浩特010018 [3]青岛畜牧兽医研究所,山东青岛266121

出　　处：《中国畜牧杂志》2018年第8期101-106,共6页Chinese Journal of Animal Science

基　　金：国家自然科学基金(31402047);国家绒毛用羊产业技术体系专项资金(CARS-39-05);青岛农业大学高层次人才科研基金(631410)

摘　　要：本研究旨在利用同源重组技术构建敖汉细毛羊BMP6基因表达载体及转染成纤维细胞后基因的表达量变化。实验以敖汉细毛羊为研究对象,采集40日龄的胎羊。首先,提取RNA并反转录成c DNA,参照Gen Bank中BMP6基因序列信息及pc DNA3.1载体序列设计1对含有同源臂的引物,通过PCR扩增获得BMP6基因片段、利用So So试剂盒将目的片段连接到pc DNA3.1真核表达载体上。鉴定pc DNA3.1-BMP6表达载体构建成功后,转化大肠杆菌(E.coli)DH5α感受态细胞;其次对敖汉细毛羊的成纤维细胞进行分离培养,并将构建的pc DNA3.1-BMP6表达载体转染成纤维细胞,利用实时荧光定量PCR技术和Western Blot技术检测BMP6基因在成纤维细胞中表达量的变化。结果表明:经酶切、测序鉴定pc DNA3.1-BMP6表达载体构建成功,并且转染成纤维细胞后BMP6基因的m RNA、蛋白表达量均明显升高,且转染组的表达量极显著高于对照组(P<0.01)。在本实验条件下,利用同源重组技术成功构建了敖汉细毛羊BMP6基因表达载体,并且成功转染成纤维细胞,基因在细胞中过表达,为进一步研究其功能奠定基础。The study aimed to construction plasmids of BMP6 genes by Homologous Recombination and the expression of fi broblast gene. 40 days old fetal Aohan Fine Wool Sheep were selected. Firstly, A pair of primers were designed by RNA extraction and referring to BMP6 gene sequence information of Aohan Fine Wool Sheep in Gen Bank and pc DNA3.1 sequence, and the BMP6 gene fragment was amplifi ed by PCR method and use So So kit to connect the target fragment to pc DNA3.1 eukaryotic expression vector. Identification of pc DNA3.1-BMP6 recombinant plasmids after successful construction, and transformed into E.coli DH5α competent cells. Secondly, fi broblasts of Aohan fi ne wool sheep were isolated and cultured,and the constructed plasmids pc DNA3.1-BMP6 were individually and co-transfected into fi broblasts, and BMP6 was detected by fl uorescence quantitative PCR and Western Blot techniques. As the results showed that, pc DNA3.1-BMP6 plasmid constructed successfully and identifi ed by enzyme and sequencing. The m RNA and protein expression level of BMP6 gene in fi broblasts was higher than in the control group（P0.01）.Under this experimental condition, Construction of BMP6 gene expression vector of Aohan fi ne wool sheep by homologous recombination technology and transfected into fi broblasts successfully. These results would lay a foundation for the further research..

关 键 词：同源重组 成纤维细胞 BMP6基因转染 RT-PCR WESTERN BLOT 

分 类 号：Q786[生物学—分子生物学]