摘要
目的构建高效表达人β3-肾上腺素受体(hβ3-AR)的真核细胞表达载体,为进一步研究hβ3-AR的功能以及与过氧化物酶体增殖体激活受体-γ2(PPAR-γ2)调控基因之间的相互影响奠定基础。方法从人肾旁脂肪组织提取总RNA,应用逆转录-聚合酶链式反应(RT-PCR)方法扩增hPPAR-γ2、hβ3-AR cDNA全长序列。将EcoRⅠ和XhoⅠ双酶切后的hβ3-AR cDNA与同样进行EcoRⅠ和XhoⅠ双酶切的pcDNA3·1(+)载体连接形成重组载体pcDNA3·1(+)/hβ3-AR。将XbaⅠ和AflⅡ双酶切后的hPPAR-γ2cDNA与同样进行XbaⅠ和AflⅡ双酶切的pcDNA3·1(+)载体连接形成重组载体pcDNA3·1(+)/hPPAR-γ2。通过限制性内切酶酶切鉴定后对DNA序列进行测序,鉴定重组质粒中hβ3-AR和hPPAR-γ2基因的完整性和可靠性。运用定点诱变方法,对野生型hPPAR-γ2和hβ3-AR质粒进行定点诱变,构建Pro12Ala突变的hPPAR-γ2和Trp64Arg突变的hβ3-AR的pcDNA3·1(+)载体,电穿孔法将人hβ3-AR重组表达载体pcDNA3·1(+)/hβ3-AR(突变型和野生型)转染到SH-SY5Y细胞中,用G418筛选获取稳定转染细胞株,并用RT-PCR、Western blot方法进行鉴定。结果酶切和测序分析显示重组质粒构建正确,并在转染的SH-SY5Y细胞中获得hβ3-AR的高效表达。结论成功克隆了人PPAR-γ2、β3-AR基因全长cDNA,并成功构建了人野生型和突变型的hPPAR-γ2和hβ3-AR的真核表达重组体〔pcDNA3·1(+)/hPPAR-γ2和pcDNA3·1(+)/hβ3-AR〕。成功获得了稳定表达hβ3-AR基因的SH-SY5Y细胞株。
Aim A high efficient eukaryotic expression vector carrying human β3-adrenoceptor （AR） genes was constructed to investigate the function of human β3-AR and interaction between β3-AR and PPAR-γ2 gene. Methods The paranephric fat tissue from a patient with kidney stones was disrupted and homogenized, total RNA was finally extracted by Trizol kit.Human PPARγ2 （ hPPARγ2 ） gene and human β3-AR （hβ3-AR） gene cDNA were amplified by using RTPCR and then subcloned into pcDNA 3.1 empty vector. The pcDNA 3.1 （ ＋ ）/hβ3-AR（ Mutant and Wild Type）was transfected into SH-SYSY cell by electroporation, G418 （300 mg · L^-1） was added into the media after 48 h transfection, and medium was changed every 3 - 5 days. The expression level of β3-AR protein was detected by RT-PCR and Western blot. Results The sequencing results of amplified target genes showed that the sequence of human PPARγ2 gene and human β3-AR gene were the same as that of hPPARγ2 gene and hβ3-AR in Genebank. The mutants of pcDNA 3. 1 （ ＋ ）/hPPAR,/2 （Pro12Ala） and pcDNA 3. 1 （ ＋ ）/ hβ3-AR plasmid （Trp64Arg）were successfully con- structed. Anti-G418 cell clones were formed after 2 -4 weeks of G418 selection. RT-PCR results and Western blot analysis showed that anti-G418 SH-SYSY cells transfected with PeDNA 3. 1 （ ＋ ） / hβ3-AR had the DNA fragment of hβ3-AR gene. Conclusions The plasmids for encoding hPPARγ2 and hβ3-AR cDNAs were successfully cloned and the eukaryotic expression vectors [ PeDNA 3. 1 （ ＋ ）/h PPARγ2 and PeDNA 3. 1 （ ＋ ）/hβ3-AR] of hPPARγ2 and hβ3-AR wild type and mutant genes were also successfully constructed.SH-SY5Y cell lines which stably expressed β3-AR genes were achieved successfully.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第4期461-468,共8页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No30572230)
教育部新世纪优秀人才支持计划资助项目(No教技函[2005]35号)
