论文部分内容阅读
为了克隆人脂联素基因(ACRP30),获得有免疫活性的重组人脂联素,以人皮下脂肪组织为材料,提取总RNA,通过RT-PCR得到ACRP30基因。构建重组质粒pET-CKS-ACRP30,转化表达菌BL21(DE3),以IPTG诱导表达人脂联素重组蛋白,利用镍亲和层析纯化,Western blot鉴定其活性,双抗体夹心法检测其免疫活性。获得了人脂联素基因,表达纯化了人脂联素融合蛋白,融合蛋白分子质量约为50ku,表达量约占菌体总蛋白的30%,纯度达到90%,双抗体夹心法检测重组蛋白具有免疫活性。本研究成功表达了人脂联素重组蛋白且表达产物具有免疫活性,为人脂联素检测技术的建立奠定了基础。
To clone the human adiponectin gene (ACRP30), an immunocompetent recombinant human adiponectin was obtained. Human subcutaneous adipose tissue was used as the material to extract total RNA. The ACRP30 gene was obtained by RT-PCR. The recombinant plasmid pET-CKS-ACRP30 was constructed and transformed into E.coli BL21 (DE3). The recombinant human adiponectin was induced by IPTG. The recombinant protein was purified by nickel affinity chromatography and identified by Western blot. The immunocompetence . The human adiponectin gene was obtained and the human adiponectin fusion protein was expressed and purified. The molecular weight of the fusion protein was about 50ku, the expression level was about 30% of the total bacterial protein and the purity was 90%. The double antibody sandwich method was used to detect the recombinant protein Immune activity. This study successfully expressed human adiponectin recombinant protein and the expression product has immunological activity, which laid the foundation for the establishment of human adiponectin detection technology.