牙鲆NPY的体外表达及体内检测

Expression of recombinant flounder NPY protein in vitro and detection of endogenous NPY in flounder

神经肽Y(Neuropeptide Y,NPY)被普遍认为是一种重要的促食因子,在调节鱼类的摄食行为方面起着至关重要的作用。为进一步研究牙鲆NPY蛋白的生物学功能,作者克隆了牙鲆NPY成熟肽序列(m-NPY)及含有信号肽的全长序列(f-NPY),利用原核表达系统分别进行体外重组表达,筛选出诱导剂IPTG最佳诱导浓度为0.8 mmol/L及最佳诱导时间3 h;此外,根据牙鲆NPY第49-64位氨基酸序列制备了多克隆抗体并通过Western blot验证该多抗能够有效检验重组NPY及牙鲆体内NPY的表达。研究结果为研究重组牙鲆NPY蛋白在牙鲆水产养殖产业中的应用及检测提供了依据。

Neuropeptide Y （NPY） plays a key role in regulation of food intake in fish, which is essential for aqua-culture. In order to investigate the effects of NPY on olive flounder （Paralichthys olivaceus）, two types of flounder NPY gene, full length NPY （f-NPY） with signal peptide and mature peptide NPY （m-NPY） without the signal, were cloned into expression vector pPROEXTM HTa and expressed in E. coli BL21 （DE3） respectively. The recombinant NPY proteins were induced by IPTG. The optimal concentration of IPTG was 0.8 mmol/L and the optimal induction time was 3 h. In addition, the NPY polyclonal antibody was prepared according to its amino acid sequence. Western blot analysis confirmed that both the recombinant NPY and the flounder endogenous NPY protein could be detected by the polyclonal antibody. In summary, our study will provide basis for further study on the application of recom-binant olive flounder NPY in aquaculture.