猪重组RBP4蛋白可溶性原核表达纯化及生物学活性鉴定

Soluble expression,purification and biological activity identification of recombinant porcine retinol binding protein 4

视黄醇结合蛋白4(retinol binding protein,RBP4)是一种主要在肝脏细胞和脂肪细胞中合成和分泌的细胞因子。RBP4在机体的脂肪代谢、慢性炎症、胰岛素抵抗等疾病过程中发挥重要作用,最新的研究也发现其与病毒感染密切相关。为了获得可溶性猪重组RBP4蛋白,本研究克隆了猪rbp4(prbp4)基因,并成功构建了原核表达载体pET32a-pRBP4。将该原核表达载体转化至BL21(DE3)感受态细胞中,分别对诱导剂浓度、诱导时间、诱导温度及超声功率进行条件优化,结果显示重组pRBP4以0.5 mmol/L IPTG在16℃条件下诱导12 h能够获得最大量的可溶性蛋白。进一步研究发现,以60 mmol/L咪唑纯化重组蛋白可获得最高产量。利用纯化的重组pRBP4蛋白处理猪肺泡巨噬细胞,qPCR检测炎症因子表达情况,发现重组pRBP4可显著诱导IL-1β、TNFα及IL-6的表达,表明获得的重组蛋白具有良好的生物学活性。以上结果为深入研究pRBP4的生物学功能提供了有利的生物学候选工具。

Retinol binding protein(RBP4)is a cytokine synthesized and secreted mainly in hepatocytes and adipocytes,and plays important roles in lipometabolism,chronic inflammation,insulin resistance and virus infection.In order to obtain soluble recombinant porcine RBP4 fusion protein,porcine rbp4(prbp4)gene was amplified by specific primers and the pET32a-pRBP4 prokaryotic expression vector was successfully constructed.The expression plasmid was then transformed into BL21(DE3)competent cells,and the recombinant pRBP4 expression conditions including IPTG concentration,induction time,induction temperature were optimized,respectively.The results showed that the expression of recombinant pRBP4 reached to the maximum amount of soluble protein when it was induced by 0.5 mmol/L IPTG at 16℃ for 12 h.Further study revealed that the highest yield of crude recombinant pRBP4 could be purified with 60 mmol/L imidazole.Moreover,the expression of inflammatory mediators such as IL-1p,TNFα and IL-6 was significantly increased in porcine alveolar macrophages upon treatment with purified recombinant pRBP4,indicating that the soluble recombinant protein had good biological activities.Hence,this study provides a useful tool for further study the biological functions of pRBP4.