摘要
目的确定小鼠肝脏SDF2L1基因在不同生理和病理条件下的表达情况,并构建SDF2L表达质粒,为进一步研究SDF2L1基因的功能奠定基础。方法 Real-time PCR确定SDF2L1基因在糖尿病条件下(db/db和ob/ob小鼠分别为糖尿病和肥胖模型小鼠)或不同营养状态的表达情况;设计并合成SDF2L1基因的PCR引物,以野生型C57BL/6J小鼠肝脏c DNA为模板,扩增SDF2L1编码区,PCR产物测序正确后连接到pc DNA4/myc-His表达载体,鉴定插入片段序列正确后,将构建的质粒转染293A细胞,Western blot检测SDF2L1的表达。结果糖尿病条件下或饥饿状态下小鼠肝脏中SDF2L1基因表达明显下调(P<0.01);纯化质粒的相对分子质量为5.8 kb,酶切鉴定结果符合目的条带(666 bp)大小,插入的寡核苷酸序列与野生型SDF2L1基因序列完全相符,表达的融合蛋白大小为26ku。结论 SDF2L1基因可能参与机体糖脂代谢;成功构建SDF2L表达质粒。
Objective To determine the expression of SDF2L1 in mouse liver under different physiological and pathophysiological conditions and to perform SDF2L1 overexpression by using eukaryotic expression system for fur- ther function research. Methods The expression of SDF2L1 in mouse liver under different conditions were deter- mined by Real-time PCR. Primers were designed commercially. Total RNA was isolated from the wild type C57BL/ 6J mouse liver and reverse transcribed to eDNA. The coding sequence of SDF2L1 was amplified by PCR. The prod- uct of PCR was purified and inserted into pcDNA4/myc-His vector and then transformed into E. coli competent cells. Expression of recombinant was transfected into 293A cells and identified by SDS-PAGE. Results The fasting or pathological condition lead to decreased expression levels of SDF2L1. The molecular weight of purified plasmid was 5.8 kb. The product was 666 bp and its sequence was identical to that in wide type SDF2L1. Therecombinant protein was 26 ku. Conclusions SDF2L1 may play a significant role in the regulation of hepatic glu- cose and lipid metabolism.
出处
《基础医学与临床》
CSCD
2015年第12期1601-1605,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(81471049)
