摘要
为了研发非洲猪瘟病毒(African swine fever virus, ASFV)的免疫学诊断试剂,试验将ASFV B646L基因克隆至pET-30a(+)载体中构建表达载体pET-30a(+)-p72,利用大肠杆菌表达系统表达重组蛋白,采用SDS-PAGE分析诱导表达的大肠杆菌,以镍柱对重组蛋白进行纯化,通过SDS-PAGE分析和Western-blot鉴定纯化的重组蛋白。用纯化后的重组蛋白免疫Balb/c小鼠,通过细胞融合方法制备杂交瘤细胞。采用间接ELISA方法筛选并检测单克隆抗体,以Western-blot及IFA方法鉴定单克隆抗体的特异性,并通过Mouse Monoclonal Antibody Isotyping ELISA Kit鉴定单克隆抗体的亚型。结果表明:PCR扩增得到大小约为1 941 bp的B646L基因,并成功构建了表达载体pET-30a(+)-p72。诱导表达的大肠杆菌在72 ku处出现目的条带,纯化后的重组蛋白p72在72 ku处出现目的条带。筛选出4种单克隆抗体2C7D8、2E6D12、3B10E3、4G5E6,其效价在1∶100 000~1∶500 000之间,且4种单克隆抗体均能特异性识别真核及原核表达的p72蛋白。4种单克隆抗体的重链亚型均为IgG2b,单克隆抗体2C7D8和4G5E6的轻链亚型为Lambda, 2E6D12和3B10E3的轻链亚型为Kappa。说明利用大肠杆菌表达系统原核表达ASFV重组蛋白p72及制备相应的单克隆抗体是可行的。
In order to develop immunological diagnostic reagents for African swine fever virus(ASFV), ASFV B646L gene was cloned into pET-30a(+) vector, and the prokaryotic expression of pET-30a(+)-p72 was constructed and identified. The recombinant protein was expressed in E. coli expression system, and the induced expression of E. coli was detected by SDS-PAGE. The recombinant protein was purified by Ni column, and identified by SDS-PAGE and Western-blot methods. Balb/c mice were immunized with purified recombinant protein, and hybridoma cell lines were prepared by cell fusion. The monoclonal antibody was screened and detected by indirect ELISA method, and the specificity of monoclonal antibodies was identified by Western-blot and IFA method, and the subtype of monoclonal antibodies were identified by Mouse Monoclonal Antibody Isotyping Elisa Kit. The results showed that the B646L gene with a size of about 1 941 bp was amplified by PCR, and the pET-30a(+)-p72 expression vector was successfully constructed. The target band appeared at 72 ku in the induced E. coli, and the target band appeared at 72 ku in the purified recombinant protein p72. Four monoclonal antibodies(2C7D8, 2E6D12, 3B10E3, 4G5E6) were screened, and their titers ranged from 1∶100 000 to 1∶500 000. The four monoclonal antibodies could specifically recognize p72 protein expressed in eukaryotes and prokaryotes. The heavy chain subtypes were identified as IgG2b, the light chain subtypes of 2C7D8 and 4G5E6 monoclonal antibodies were Lambda, and the light chain subtypes of 2E6D12 and 3B10E3 monoclonal antibodies were Kappa.The results indicated that the ASFV recombinant p72 protein could be expressed by the E. coli prokaryotic expression system and the corresponding monoclonal antibody could be prepared.
作者
吴宏举
李潮
郑南南
侯浩宇
陈寅龙
靳家鑫
孙爱军
杜永坤
张改平
WU Hongju;LI Chao;ZHENG Nannan;HOU Haoyu;CHEN Yinlong;JIN Jiaxin;SUN Aijun;DU Yongkun;ZHANG Gaiping(National Joint Research Center for Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2023年第1期1-6,13,122,共8页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金重点专项(31941001)
河南省高等学校重点科研项目(21A230010)。
关键词
非洲猪瘟病毒
p72蛋白
单克隆抗体
原核表达
细胞融合
African swine fever virus
p72 protein
monoclonal antibody
prokaryotic expression
cell fusion
