牛抵抗素克隆及高效可溶性表达研究

Bovine Resistin Cloning and Its High Level of Soluble Expression

【目的】抵抗素(resistin,RETN)在机体脂糖代谢和促炎过程中都发挥着重要作用。获得有活性、高效表达的可溶性牛抵抗素并鉴定其正确性,以期为后续其相关疾病的研究和防治奠定基础。【方法】以肉牛大网膜脂肪组织为原料提取总RNA,经RT-PCR获得目的基因,克隆至原核表达载体PET-21a中,构建重组表达质粒PET-Resistin。经双酶切及测序鉴定正确后,对宿主菌、IPTG浓度、诱导温度及时间进行优化,用抗His抗体鉴定融合蛋白表达正确。【结果】在宿主菌Rosetta,IPTG终浓度1.0 mmol/L,20℃诱导14 h时,牛抵抗素的表达量最高;在上述条件下,牛抵抗素存在于上清液中,为可溶性表达。【结论】成功克隆表达出仅含His标签的可溶性牛抵抗素,为进一步研究牛抵抗素参与相关疾病的调控途径和防治奠定了基础。

【Objective】Resistin plays an important role in the process of proinflammatory and metabolism of lipid and sugar. Our study aims to obtain soluble expression of bovine resistin with high activity and high expression and identify it for further study and prevention of related diseases.【Method】Total RNA was extracted from bovine omental adipose tissue,the cDNA sequence obtained by PCR was cloned into the prokaryotic expression vector PET-21a to recombinant cloning vector PET-RETN. After identifying with double-enzyme digestion and sequence analysis,we optimizated host bacteria,IPTG concentration,induction temperature and time and then analyzed by Western blotting.【Result】When the host bacteria was Rosetta,the final concentration of IPTG was 1.0 mmol/L,the induction temperature was 20 ℃,the induction time was 14 h and the expression of bovine resistin was the highest. Under the above-mentioned condition,the bovine resistin was soluble expression which presented in the super natant.【Conclusion】Through the PET-21a vector,the bovine resistin only with His-tag was successfully cloned and expressed,which laid a foundation for further study of bovine resistin regulation and control of related diseases.