人类肥胖相关新基因LYRM1真核表达载体的构建及稳定转染3T3-L1细胞系的建立

Construction of eukaryotic expression vector of LYRM1,a novel gene related with human obesity,and establishment of its stable transfected 3T3-L1 cell line

目的:构建人LYRM1基因的真核表达载体,转染3T3-L1前体脂肪细胞,建立稳定过表达人LYRM1基因的3T3-L1前体脂肪细胞系。方法:运用RT-PCR技术从人网膜脂肪组织中分离LYRM1基因的完整编码框,将其亚克隆到真核表达载体pcDNATM3.1/myc-His B,脂质体转染3T3-L1前体脂肪细胞,通过G418筛选,建立稳定转染的3T3-L1前体脂肪细胞系,并利用Westernblot方法鉴定其表达。结果:PCR、酶切鉴定及测序结果表明重组质粒构建正确。建立了稳定转染LYRM1的3T3-L1前体脂肪细胞,成功地表达目的基因。结论:LYRM1真核表达载体的成功构建及稳定转染3T3-L1细胞系的建立为进一步研究其功能奠定了良好的实验基础。

Objective- To construct the eukaryotic expression vector of LYRM1 and to transfect it to preadipocytes cell line 3T3-L1. Methods： The complete coding sequence of LYRM1 gene was amplified by RT-PCR from human omental adipose tissue and was subcloned into eukaryotic expression vector pcDNA^TM3. 1/myc-His B. The recombinant plasmid was then transfected into 3T3-L1 preadipocytes by liposome. After screening culture by G418,stable transfected 3T3-L1 cell line was established. The expression of LYRM1 protein was identified by Western blot. Results.. The recombinant plasmid was confirmed by PCR, restriction enzyme digestion and sequencing. The stable transfected 3T3-L1 cell line was established and the LYRM1 protein was expressed successfully. Conclusion： The eukaryotic expression vector of LYRM1 has been successfully established and the stably transfected 3T3-L1 cell line also established.