猪清道夫受体SRA/CD204多克隆抗体的制备以及该受体介导的细菌吞噬功能分析  

GENERATION OF POLYCLONAL ANTIBODIES AGAINST PIG SCAVENGER RECEPTOR SRA/CD204 AND FUNCTIONAL ANALYSIS OF BACTERIAL PHAGOCYTOSIS MEDIATED BY PIG SRA/CD204

作　　者：黎倩倩[1,2] 张彦兵[1,2] 相笑 魏建超[2] 齐鹏飞[2] 石元元[2] 陆莹梅 夏鹏[2] 刘珂[2] 邵东华[2] 李蓓蓓[2] 马志永[2] 孙延鸣[1] 邱亚峰[2] 

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2015年第6期42-47,共6页Chinese Journal of Animal Infectious Diseases

基　　金：中国农业科学院上海兽医研究所中央级公益性科研院所基本科研业务费专项资金项目(2014JB06)

摘　　要：针对猪SRA/CD204胞外区对应的基因片段（614～1324 bp）进行引物设计,PCR扩增获得目的片段并克隆至原核表达载体p ET-28a中,构建重组表达质粒p ET-SRA-c。利用原核表达系统获得高效表达于包涵体中的重组蛋白r SRA-c（约32 k Da）,利用亲和层析技术获得纯化的r SRA-c。利用纯化的r SRA-c为免疫原,免疫Balb/c小鼠（100μg/小鼠）,制备多抗血清。经Western blot结果显示,该多抗血清不仅可以识别r SRA-c,而且可以识别真核表达的全长的猪SRA/CD204（约55 k Da）。免疫荧光分析表明该多抗血清可以清楚地识别表达于CHO细胞膜上的SRA/CD204。最后,利用表达猪SRA/CD204的CHO细胞,对其介导的细菌吞噬功能进行了研究,结果表明猪SRA/CD204可以有效地介导大肠杆菌和金黄色葡萄球菌的吞噬。本研究为进一步研究猪SRA/CD204在病原微生物感染中的作用奠定了基础。To gain insights on biological functions of pig scavenger receptor SRA/CD204,we first sought to generate polyclonal antibodies against pig SRA/CD204.Firstly,a C-terminal gene fragment（614-1324bp） of pig SRA/CD204 gene was amplified in PCR from pMD18-SRAwt and cloned into pET-28 a to construct the recombinant plasmid pET-rSRA-c.The recombinant protein rSRA-c（about32kDa） was mainly expressed as inclusion body and purified by His-Bind affinity chromatography.Furthermore,Balb/c mice were immunized with purified rSRA-c for generation of polyclonal antibodies.The results showed that the polyclonal antibodies recognized rSRA-c and full-length pig SRA/CD204 expressed in CHO cell line（about 55 kDa） in Western blot.Furthermore,the polyclonal antibodies strongly reacted with pig SRA/CD204 in indirect immunofluorescence assay,which mainly located in membranes and cytosol of CHO cells. Finally, we used the CHO cell line for over-expressing pig SRA/CD204 via transient transfection. The results showed that pig SRA/CD204 mediated phagocytosis of bacteria like E.coli and S. aureus.

关 键 词：清道夫受体 重组蛋白 多克隆抗体 细菌 吞噬 

分 类 号：S852.61[农业科学—基础兽医学]