摘要
目的构建一种具有生物学活性的血管活性肠肽(VIP)的真核表达质粒。方法利用RT-PCR从小鼠胸腺淋巴细胞中克隆有信号肽序列的VIPcDNA,插入真核表达载体pcDNA3.1,构建含VIP的重组质粒pcDNA3.1-VIP,转染COS-7细胞,用ELISA和Westernblot鉴定表达的蛋白,细胞因子释放抑制法测定生物学活性。结果经酶切和基因测序证实,克隆的基因片段为带有信号肽序列的VIP基因;经转染的COS-7细胞表达VIP蛋白,并能有效地抑制巨噬细胞的TNF-α释放。结论成功构建了能够表达具有生物学活性的真核表达质粒pcDNA3.1-VIP。
Objective: To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities. Methods: VIP cDNA including the sequences of signal peptide was cloned from mouse thymus by RT-PCR,and then inserted into the mammalian expression vector pcDNA3.1 between Hind III and EcoR I restriction sites.COS-7 cells were transfected with pcDNA3.1-VIP using liposome,the expression of VIP was identified by Western blot and ELISA.Supernatant of transfected cell culture was added to LPS-stimulated macrophages and the TNF-αproduction in cell medium was observed by ELISA. Results: The cloned VIP cDNA was confirmed by enzyme digestion and DNA sequencing.The expression of VIP was detected in the pcDNA3.1-VIP transfected COS-7 cells by Western blot and ELISA.The VIP in culture supernatant potently inhibited TNF-αproduction by LPS-induced Macrophages in vitro. Conclusion: The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully. [
出处
《浙江大学学报（医学版）》
CAS
CSCD
2005年第2期148-151,共4页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金项目(30170874)
浙江省教育厅科研基金项目(20040232)
关键词
血管活性肠肽
质粒
克隆
分子
基因表达
Vasoactive intestinal peptide
Plasmids
Cloning,molecular
Gene expression
