脂联素球状结构域真核表达载体pcDNA3.0-gAd的构建

目的构建脂联素球状结构域（globular adiponectin,gAd）的真核表达载体,为研究gAd对糖尿病、动脉粥样硬化等疾病的治疗奠定基础。方法从健康人全血中提取基因组DNA,PCR法获得脂联素球状结构域基因,经EcoRⅠ和XhoⅠ酶切后与pcDNA3.0真核表达载体重组得到pcDNA3.0-gAd,分别用PCR法和DNA测序法鉴定pcDNA3.0-gAd。结果以从健康人全血中提取出的高纯度基因组DNA为模板,PCR扩增出gAd基因,真核载体pcDNA3.0经EcoRⅠ和XhoⅠ双酶切后与gAd基因进行体外重组,PCR法筛选出阳性克隆,DNA测序显示重组质粒pcDNA3.0-gAd的gAd基因与基因库中报道的脂联素基因序列（Gen-Bank：EU420013.1）中gAd序列完全一致,说明成功构建了pcDNA3.0-gAd,且序列正确。结论本研究为进一步从分子水平探究gAd基因和蛋白质的功能、深入探讨其生物作用机制创造了条件。

Construction of Eukaryotic Expression Plasmid PcDNA3.0-gAd Containing the Globular Adiponectin

Objective To construct the eukaryotic expression plasmid pcDNA3.0-gAd containing the globular adiponectin.Methods Genomic DNA was extracted from healthy human whole blood and the globular adiponectin gene was acquired by PCR,The gAd and pcDNA3.0 vector were enzymed by EcoRⅠand XhoⅠ.The two segments were linked in vitro.The positive clones were identified with PCR and DNA sequencing.Results The high-purity genomic DNA was extracted and the gAd gene was amplified by PCR.The gAd gene and pcDNA3.0 were enzymed by EcoRⅠand XhoⅠ,then were linked in vitro.The positive clones were identified with PCR.DNA sequencing showed that the target segment gAd was obtained successfully.The homology of the nucleotide in this segment gAd between the sequences from genbank was 100%.The recombinant plasmid of pcDNA3.0-gAd was successfully constructed.Conclusion This study established foundation for the further exploring the function of gAd genes and gAd proteins at the molecular level.