摘要
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（ACRP30） cDNA and obtain the recombinant adiponectin with immunological activity,fresh human fat tissue was used for extracting total RNA and amplificating the adiponectin cDNA through RT-PCR.The recombinant plasmid pET-CKS-ACRP30 was constructed and confirmed,and it was transduced into BL21（DE3）.Then the transformation was used for induced expression.The recombinant protein was identified by Western blotting and purified by Ni affinity chromatography.Finally,the sandwich ELISA method was developed for detecting the immunological activity of human adiponectin.Human adiponectin was expressed and purified,and the target protein is about 30% of the total bacterial protein,and the purity is around 90%.It is domenstrated that the aim protein has immunological activity with the sandwich ELISA.Adiponectin with immunological activity has been obtained from prokaryotic expression,which lays a foundation for establishing the detection method of human adiponectin.
出处
《动物医学进展》
CSCD
北大核心
2011年第10期36-39,共4页
Progress In Veterinary Medicine
基金
河南省科技创新团队和郑州市科技创新团队资助项目
关键词
人脂联素
原核表达
免疫活性
human adiponectin
prokaryotic expression
immunological activity
