尼罗罗非鱼3种Siglecs like融合蛋白在COS-7细胞中的表达及其结合活性的初步研究

为获得尼罗罗非鱼Siglecs like融合蛋白,开展相关Siglecs like蛋白的功能研究,深入了解罗非鱼与无乳链球菌相互作用机制。本研究利用前期构建的3个含Siglecs like ORF的克隆质粒,PCR扩增获得Siglec-1、Siglec-4b和Siglec-14 like的膜外段序列,插入真核表达载体pc DNA3.1(+)h Ig G1 Fc中,双酶切、测序鉴定后转染COS-7细胞。q PCR、Western-blot对目的蛋白的表达进行检测;亲和层析法纯化融合蛋白,SDS-PAGE电泳检测纯化效率。ELISA检测融合蛋白与GBS的结合活性。测序结果显示,成功构建3个融合蛋白真核表达载体pc DNA3.1(+)h Ig G1 Fc-Siglecs like/Ex。检测结果显示,转染细胞中3个Siglecs/Ex-Fc融合蛋白在mRNA和蛋白水平都有高效表达,且过柱后的融合蛋白具有较高纯度;3个融合蛋白与罗非鱼源GBS的结合活性较对照组均有显著差异。研究表明,利用真核表达系统成功制备了具有较高纯度的尼罗罗非鱼3种Siglecs融合蛋白,且均有与GBS的结合活性。

Expression of three Oreochromis niloticus Siglecs-like fusion proteins in COS-7 cells and characterization of their binding activities with GBS

Siglecs, sialic acid-binding immunoglobulin-like lectins is a class of membrane receptors which could specifically recognize sialic acid polysaccharide of self or some pathogens.In order to prepare Nile tilapia(Oreochromis niloticus)Siglecs-like fusion proteins and study their functions in the interaction of host-pathogen, we prepared O.niloticus Siglecs like fusion proteins using eukaryotic expression system.In this research, the extracellular domain of the three O.niloticus Siglecs like were respectively amplified from the cloned vectors containing the ORF of Siglec-1/-4b/-14 like and then inserted into the expression vector of pcDNA3.1(+)hIgG1 Fc.After double digestion and sequencing, they were transfected into the COS-7 cells in vitro.The expressions of target genes was detected by qPCR and Western-blot analysis.The fusion proteins were purified by affinity chromatography, and their purity was detected by SDS-PAGE electrophoresis.To study the biological activities of these Siglecs like fusion proteins, binding activities of the fusion proteins to Group B Streptococcus(GBS)were detected by ELISA.The results showed that these three recombinant plasmids containing extracellular domains of tilapia Siglecs like with human IgG1 Fc fragment were constructed successfully.These three genes were highly expressed in COS-7 cells both in mRNA and protein levels.Three Siglecs/Ex-Fc fusion proteins were proved to be of high purity.The binding activities between fusion proteins and control were significantly different(P < 0.05).This research showed we successfully obtained purified fusion proteins by COS-7 cells expression system.A preliminary study proved they could bind GBS.