摘要
为了制备猪IL-10的单克隆抗体(MAb),本研究根据GenBank登录的猪白细胞介素10(PIL-10)基因序列设计两对特异性引物,利用套式PCR扩增PIL-10成熟蛋白编码基因(490bp),构建原核表达载体pET28a-PIL-10,并转化至大肠杆菌BL21,经IPTG诱导,SDS-PAGE和western blot分析鉴定,证明PIL-10重组蛋白获得表达。提取纯化该重组蛋白包涵体,免疫BALB/c小鼠,经过3次细胞融合,间接ELISA方法筛选,获得一株能稳定分泌抗猪IL-10MAb的杂交瘤细胞,将其命名为2F4。Western blot结果证明该MAb能够与重组蛋白发生特异性反应。间接ELISA测定细胞上清抗体效价为1∶1024,腹水效价为1∶128000,该MAb为IgG3亚型,轻链为λ型。杂交瘤细胞连续传代20代,分泌抗体的效价基本一致,从而为进一步建立猪IL-10的检测方法奠定了物质基础。
The cDNA of porcine interleukin-10（PIL-10） was amplified with nested PCR and cloned to construct recombinant plasmid pET-28a-PIL-10,then transformed into E.coli BL21. Recombinant bacteria was induced by IPTG and expression of PIL-10 was confirmed by SDS-PAGE and western blot. Monoclonal antibody（MAb） specific to PIL-10 protein was prepared by fusion spleen cells of BALB/c mice immunized with purified PIL-10 inclusion body. Positive hybridoma cell line was screened by indirect ELISA and confirmed by western blot,One MAb 2F4 cell line,which had an antibody titers of 1∶1 024 in cell culture medium,and 1∶128 000 in asicites,was obtained. The isotype of 2F4 belongs to IgG3/λ. The titer of the antibody was stable during 20 passages in vitro.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第4期281-284,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30871868)
国家科技支撑计划(2007BAD86B02-3
2006BAD06A04)
江苏省科技攻关项目(BE2007342)
教育部博士点基金项目(20060307007)
