Preparation of monoclonal antibodies with blocking activity against cat TNF-α
10.16303/j.cnki.1005-4545.2024.08.12
- VernacularTitle:猫TNF-α蛋白的原核表达及阻断活性单克隆抗体的制备
- Author:
Yue WANG
1
,
2
;
Jiakang LI
;
Qiuyan LI
;
Shengbo CAO
;
Jing YE
;
Longlong CAO
;
Dengyuan ZHOU
Author Information
1. 华中农业大学动物科学技术学院与动物医学院,湖北武汉 430070
2. 农业微生物资源发掘与利用全国重点实验室,湖北武汉 430070
- Keywords:
tumor necrosis factor-α;
monoclonal antibody;
blocking activity;
recombinant protein ex-pression;
cat
- From:
Chinese Journal of Veterinary Science
2024;44(8):1667-1673
- CountryChina
- Language:Chinese
-
Abstract:
In order to prepare monoclonal antibodies with blocking activity against feline TNF-α,this study successfully constructed,expressed and purified the recombinant plasmid pET-28a-sTNFα based on the soluble feline TNF-α(sTNFα)gene,and further investigated the induced ex-pression.The conditions were explored and optimized to identify its biological activity;secondly,the feline TNF-α recombinant protein was used as an immunogen for mouse immunization,after cell fusion,screening of blocking active hybridoma cells and ascites preparation,the obtained mon-oclonal antibodies were tested.The results showed that the pET-28a-sTNFα plasmid was success-fully constructed and the bioactive feline TNF-α recombinant protein was expressed in E.coli sys-tem.The molecular weight was 34 kDa and the 50％inhibitory concentration was 1.22 pg/L.Three monoclonal antibodies(A6-B7-9,H5-E2-94 and C8-A10-100)with blocking activity were success-fully screened out.The results of Western blot showed that all the three mAbs could specifically bind to TNF-α with a titer of 1:512 000.When the concentration of the three mAbs was 100 mg/L,the inhibitory effect on TNF-α was the strongest.In this study,we screened antibodies that can block the activity of cat TNF-α,in order to provide novel,safe and effective candidate drugs for the treatment of TNF-α mediated diseases in cats.
