黄颡鱼细胞因子信号传导抑制因子SOCS1的原核表达及多克隆抗体制备

Prokaryotic protein expression of suppressor of cytokine signaling 1 of yellow catfish (Pelteobagrus fulvidraco) and preparation of their polyclonal antibodies

摘要:

细胞因子信号传导抑制因子(suppressor of cytokine signaling, SOCS)是一类由细胞产生的胞内蛋白，能够反馈性阻断细胞因子信号传导过程的负性调控因子。为深入研究黄颡鱼细胞因子信号传导的发生过程和机制，实验从黄颡鱼cDNA中克隆获得561 bp的黄颡鱼SOCS1(PfSOCS1)基因编码序列，成功构建了重组质粒pET32a(+)-PfSOCS1，并转化至大肠杆菌BL21(DE3)感受态细胞中，在不同温度、IPTG浓度、诱导时间等条件下进行表达，确定最佳表达条件。本实验采用包涵体纯化的方法得到纯度较高的重组SOCS1蛋白；以重组PfSOCS1蛋白免疫Balb/C小鼠，制备多克隆抗体。通过Western blot鉴定了PfSOCS1抗血清可以特异性识别重组PfSOCS1蛋白。蛋白水平的组织分布显示，黄颡鱼SOCS1蛋白在肝脏中表达水平较高。

Abstract:

Suppressor of cytokine signaling (SOCS) is a class of intracellular proteins that inhibited cytokine signaling. We cloned the sequence of SOCS1 gene from yellow catfish Pelteobagrus fulvidraco, whose length was 561 bp. We constructed the recombinant expression plasmid [pET32a (+)-SOCS1] carrying PfSOCS1 protein. The recombinant PfSOCS1 protein was optimally expressed by transformation into Escherichia coli BL21 (DE3) and then highly purified under inclusion body conditions. The polyclonal anti-PfSOCS1 antibodies were prepared by immunizing Balb/C mouse with recombinant PfSOCS1 protein, which could be used to recognize recombinant PfSOCS1 protein by Western blotting. The protein expression of PfSOCS1 was detected in kidney, intestine, liver and ovary, with relatively predominant level in the liver in P. fulvidraco.