猪细小病毒7型cap蛋白的原核表达及多克隆抗体的制备  

Prokaryoticexpression and polyclonal antibody preparation of cap protein of porcine parvovirus Type 7

作　　者：吕莫然 袁枫 李子璇[2] 全荣[2] 朱珊珊[2] 刘爵[2] LV Moran;YUAN Feng;LI Zixuan;QUAN Rong;ZHU Shanshan;LIU Jue(College of Animal Science and Technology,Beijing University of Agricultural,Beijing 102206,China;Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京农林科学院,北京100097

出　　处：《北京农学院学报》2020年第4期77-81,共5页Journal of Beijing University of Agriculture

基　　金：北京市农林科学院创新能力建设项目(KJCX201914)。

摘　　要：【目的】为后续研发最新型PPV疫苗,控制猪细小病毒传染性,以及建立ELISA检测方法鉴定基础。【结果】对已发表的37株完整猪细小病毒7型cap蛋白的核苷酸序列进行比对,将得到的保守序列根据大肠杆菌的偏好进行密码子优化后合成基因,得到全长为1422 bp的基因,并与原核表达载体pET-32a(+)连接,获得重组质粒pET-32a-cap,经PCR扩增测序鉴定后,将其转化到大肠杆菌BL21(DE3)感受态细胞中,进行IPTG诱导表达及Western blotting鉴定。对获得的重组融合蛋白进行可溶性分析、纯化,免疫BALB/c小鼠和豚鼠制备多克隆抗体。【结论】成功制备了具有免疫原性的cap蛋白及其鼠源多克隆抗体,为猪细小病毒7型cap蛋白生物学功能及相关致病机制研究提供了基础材料。The aim of the experiment was to expressand purify the cap protein of porcine parvovirus type 7 in prokaryotic system and prepare polyclonal antibodies of guinea pig and mouse. 37 published nucleotide sequences of complete porcine parvovirus type 7 were compared. The obtained conserved sequences were synthesized after codon optimization according to the preference of E. coli, a gene of 1 422 bp was obtained and connected with the prokaryotic expression vector pET-32 a(+) to obtain recombinant plasmid pET-32 a-PPV7-cap. After PCR and sequencing analysis, the plasmidwas transformed into E. coli BL21(DE3) competent cells, expressed by IPTG and identified by western-blotting. The obtained recombinant fusion protein was soluble analyzed, purified and used to immunize BALB/c mice and Guinea pigs to obtain polyclonal antibodies. The immunogenic cap protein and its mouse source-derived polyclonal antibodies were successfully obtained in this experiment, which provided basic materials for the study on the biological function of cap proteinof porcine parvovirus type 7 and the pathogenic mechanism of related diseases.

关 键 词：猪细小蛋白 CAP蛋白 克隆 原核表达 多克隆抗体 

分 类 号：S851[农业科学—预防兽医学]