摘要
目的为进一步研究脂联素(ADPN)的功能提供实验基础,构建含重组人脂联素(hADPN)真核表达载体的3T3-L1细胞株。方法酶切载有人脂联素cDNA的重组克隆质粒pMD18-T和真核表达质粒pcDNA3.1+,回收目的基因片段与线性化的真核表达质粒,经连接、转化大肠杆菌JM109,筛选阳性克隆,经酶切和核苷酸序列测定鉴定。脂质体法转染3T3-L1细胞,G418筛选阳性细胞克隆扩增,通过RT-PCR的方法逆转录合成脂联素基因片段。结果重组真核表达质粒酶切后获得5.4kb和800bp两个片段,大小与理论值一致。并经核苷酸序列测定证实。RT-PCR产物经凝胶电泳后于紫外分析仪下可见在250bp前有一清晰条带,和扩增目的基因片段长度234bp一致。结论成功地构建了人重组脂联素真核表达质粒并在3T3-L1细胞中稳定表达。
[Objective] To provide experimental basis for further investigating on adiponectin （ADPN） function, whose eukaryotic recombinant was constructed and expression in 3T3-L1 preadipocytes. [Methods] The recombi- nant plasmid pMD18-T/hADPN and eukaryotic expression vector pcDNA3.1 ＋ were digested by two restrictive endonuelease and hADPN and linear pcDNA3.1＋ were obtained, then, they were linked and translated into JM109, and at last, the recombinant pcDNA3.1＋/ADPN was obtained and was identified by digest of restrictive endonuclease and nucleotide sequencing. The 3T3-L1 preadipocytes were transfected using SuperFect Transfection Reagent （QIAGEN）. The positive clones screened out by G418, and the positive clones were cloned again. The adiponectin cDNA was synthesized by a reverse transcription by using the total RNA as a template, and then complete adiponectin was synthesized by PCR by the cDNA as a template. [Results] 800 bp fragment and 5.4 kb fragment were consistant with theoretic values after eukaryotic recombinant was digested by Hind Ⅲ and EcoRⅠ , [Conclusion] The eukaryotic expressing recombinant with complete adiponectin cDNA was constructed.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2007年第16期1927-1930,共4页
China Journal of Modern Medicine
基金
安徽省自然科学基金资助(03043719)
