表达鸡补体因子C3d基因重组杆状病毒的构建及初步应用  

Preliminary application and construction of recombinant baculovirus expressing chicken complement component 3d gene

作　　者：孙盈[1,2] 闫敏鑫[1] 黄秀芬[1] 孔瑞丽 黄金海[2] 李永清[1] 

机构地区：[1]畜禽疫病防控技术北京市重点实验室北京市农林科学院畜牧兽医研究所,北京100097 [2]天津大学化工学院,天津300072 [3]北京市延庆县农业局,北京102100

出　　处：《中国兽医学报》2015年第8期1254-1259,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31372420);北京市自然科学基金资助项目(6122018)

摘　　要：为研制鸡体内补体因子C3d抗体的检测方法,采用RT-PCR方法从鸡肝脏组织细胞的总RNA中扩增出1 002bp的鸡补体因子C3d基因全长,然后将其克隆到杆状病毒载体pFB-LIC-Bse中获得重组质粒pFB-C3d。将pFB-C3d转化到带有杆状病毒基因组的DH10Bac感受态细胞中,制备重组杆粒rBacmid-C3d。再将该重组杆粒转染到昆虫细胞sf9中,通过病毒拯救技术构建携带C3d基因的重组杆状病毒。Western blot试验结果表明,重组杆状病毒能在昆虫细胞sf9中表达出大小约为35 000的C3d-his融合蛋白。以该融合蛋白作为抗原建立的Western blot和间接免疫荧光试验(IFA)显示,经C3d-M2-GST融合蛋白免疫的鸡血清能够与杆状病毒表达的C3d结合,说明本研究构建的重组杆状病毒可以用来检测C3d作为分子佐剂及其融合蛋白免疫鸡后产生针对C3d自身的抗体,为今后建立体内C3d水平监测的指示系统奠定基础。To develop a technology to detect antibody against C3 din chicken.Full length of chicken complement component 3dgene（cC3d）of（cC3d）with 1 002 bp was amplified from total RNA extracted from chicken liver by RT-PCR with specific primers in this study.Then the full length of cC3 dgene was cloned into a novel baculovirus vector pFB-LIC-Bse and recombinant transfer vector pFB-C3 dwas constructed.The recombinant bacmid rBacmid-C3 dwas constructed after pFB-C3 d was transformed into DH10 Bac competent cells.The recombinant baculovirus expressing C3 dgene was constructed and rescued by transfection with rBacmid-C3 dDNA into Sf9 cells.Western blot analysis showed that C3d-his fusion protein with about 35 000 molecular weight was expressed in sf9 cells by the recombinant baculovirus.Indirect immunoflourescence（IFA）and further Western blot analysis based on this fusion protein as antigen indicated that C3 dprotein expressed by recombinant baculovirus reacted to chicken serum against C3d-M2-GST fusion protein.These results suggested that the recombinant baculovirus could be applied to detect anti-C3 dantibodies when C3 dwas employed to immunize chicken as molecular adjuvant with fusion.This is a basic work for establishing a indicator system to monitor C3 dlevel in vivo.

关 键 词：补体因子C3d 重组杆状病毒 pFB-LIC-Bse 蛋白表达 

分 类 号：S852.4[农业科学—基础兽医学]