黄颡鱼自噬相关基因 LC3B和Beclin1的原核表达及多克隆抗体制备

为深入研究黄颡鱼Pelteobagrus fulvidraco自噬的发生过程和机制, 从黄颡鱼cDNA中克隆获得372和1344 bp的黄颡鱼LC3B(PfLC3B)和Beclin1(PfBeclin1)基因编码序列, 并成功构建了pET32a(+)-PfLC3B和pET32a(+)-PfBeclin1重组表达载体, 分别采用亲和层析和包涵体纯化的方法得到了纯度较高的重组PfLC3B和PfBeclin1蛋白; 以重组PfLC3B和PfBeclin1蛋白免疫Balb/C小鼠, 制备多克隆抗体。通过Western blot鉴定了PfLC3B和PfBeclin1抗血清可以分别特异性识别重组PfLC3B和PfBeclin1蛋白; PfLC3B抗血清可以特异性识别黄颡鱼内源性LC3-Ⅰ和LC3-Ⅱ蛋白, PfBeclin1抗血清可以特异性识别黄颡鱼内源性Beclin1蛋白。蛋白水平的组织分布显示, 黄颡鱼LC3和Beclin1蛋白在肝脏中表达水平均较高, 在肾脏和脾脏中的表达水平较低。综上所述, 研究成功制备了黄颡鱼LC3B和Beclin1的多克隆抗体, 为深入研究黄颡鱼自噬机制提供了有力工具。

PROKARYOTIC EXPRESSION AND POLYCLONAL ANTIBODY PREPARATION OF AUTOPHAGY-RELATED GENES LC3B AND BECLIN1 OF YELLOW CATFISH PELTEOBAGRUS FULVIDRACO

In order to investigate the mechanism of autophagy in yellow catfish Pelteobagrus fulvidraco, we cloned P. fulvidraco LC3B (PfLC3B) and Beclin1 (PfBeclin1) genes that were 372 bp and 1344 bp, respectively, and constructed the plasmid pET32a(+)-PfLC3B and pET32a(+)-PfBeclin1 encoding recombinant PfLC3B and PfBeclin1 protein, respectively. The recombinant PfLC3B was highly purified by affinity chromatography, while the recombinant PfBeclin1 protein was highly purified under inclusion body conditions. The polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies were generated by immunizing Balb/C mouse with recombinant PfLC3B and PfBeclin1 protein, respectively. The results of western blot showed that the polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies could recognize recombinant and endogenous PfLC3B and PfBeclin1 protein, respectively. PfLC3 and PfBeclin1 were predominantly expressed in the liver, but not in the kidney and spleen. In conclusion, we generated polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies, which provided powerful tools to investigate the mechanism of autophagy in yellow catfish.