黄颡鱼长型肽聚糖识别蛋白（pfPGRP－L）原核表达和多克隆抗体的制备

摘要

In order to study the important roles of peptidoglycan recognition proteins (PGRPs) in the in⁃nate immunity,primers were designed to amplify the conding segment,then,recombinant expression vectors containing yellow catfish PGRP⁃L (pfPGRP⁃L) were constructed and transformed into E. coli BL21(DE3). The recombinant fusion protein was induced with IPTG and isolated by SDS⁃PAGE.The isolated fusion protein was used to produce the polyclonal antibody of pfPGRP⁃L by immunizing rabbit.Then,the antibody was tested by ELISA and western blotting,and the results showed that the antibody had fine sensitivity and specificity.Furthermore,distribution of pfPGRP⁃L protein in different tissues was examined by western blotting analysis, and pfPGRP⁃L had a significant expression in all examined tissues,with a higher expression in intestine and spleen.This study provides some useful information for studying the antibacterial activity and other functions of pfPGRP⁃L.%为进一步研究肽聚糖识别蛋白（peptidoglycan recognition proteins，PGRPs）在先天性免疫应答中的生物学功能，在前期克隆黄颡鱼长型肽聚糖识别蛋白（pfPGRP－L） cDNA 全长的基础上，根据 pfPGRP－L 基因中高亲水性区域序列设计特异性引物扩增出编码序列，将该基因片段克隆到 pGEX－4T－1质粒，构建重组表达载体。SDS－PAGE 电泳检测该目的蛋白大小约为51 ku，与理论值大小符合，回收蛋白并纯化，与弗氏佐剂等量混合后注射新西兰大白兔制备多克隆抗体，用 ELISA 和 Western blotting 分别检测该抗体，结果显示：抗体效价可达1∶256000，且有特异性条带，表明 pfPGRP－L 的多克隆抗体制备成功。此外，Western blotting 检测黄颡鱼不同组织 pfPGRP－L 蛋白的表达，发现在鳃、胸腺、肝脏、肾脏、头肾、脾脏、血液和肠道等不同组织中都有目的条带，而在脾脏和肠道中表达更强。为 pfPGRP－L 的抗菌作用和功能研究奠定基础。