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目的构建人脂联素的真核表达载体,并检测其融合蛋白在真核细胞COS-7中的表达。方法从人脂肪组织中提取总RNA,通过RT-PCR方法获得人脂联素基因,将其克隆到真核荧光表达载体pEGFP-N1上,构建重组质粒pEGFP-N1-AdipoQ,转染至真核表达细胞COS-7中,并通过SDS-PAGE和Western blot检测融合蛋白在真核细胞中的表达。结果通过RT-PCR方法克隆人脂联素基因744 bp,测序结果显示,1个碱基发生突变,654位:A→T,突变率为0.1%,氨基酸Glu→Asp。通过SDS-PAGE和Western blot检测融合蛋白分子质量约为55×103(绿色荧光蛋白分子质量约为27×103),说明脂联素基因在真核细胞中表达并与绿色荧光蛋白融合。结论成功的构建了真核表达载体,并在真核细胞中表达。
Objective To construct an eukaryotic expression vector of human adiponectin and detect the expression of its fusion protein in eukaryotic cells COS-7. Methods Total RNA was extracted from human adipose tissue. The human adiponectin gene was obtained by RT-PCR and cloned into eukaryotic expression vector pEGFP-N1. The recombinant plasmid pEGFP-N1-AdipoQ was constructed and transfected into eukaryotic Cells were expressed in COS-7 and the expression of the fusion protein in eukaryotic cells was examined by SDS-PAGE and Western blot. Results The human adiponectin gene 744 bp was cloned by RT-PCR and sequenced. One nucleotide mutation was found at position 654: A → T, the mutation rate was 0.1% and the amino acid Glu → Asp. The molecular weight of the fusion protein was about 55 × 103 by SDS-PAGE and Western blot (the molecular mass of green fluorescent protein was about 27 × 103), indicating that adiponectin gene was expressed in eukaryotic cells and fused with green fluorescent protein. Conclusion The eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells.