Expression and functional detection of recombinant immunotoxins anti-B7-H4-scFv/PE38KDEL
10.3969/j.issn.1000-484X.2018.03.008
- VernacularTitle:抗B7-H4-scFv/PE38KDEL重组免疫毒素的表达和功能性检测
- Author:
Hong-Shuai JI
1
;
Jin-Rui GUO
;
Ying YANG
;
Wei-Ping MAO
Author Information
1. 江苏省分子医学重点实验室
- Keywords:
B7-H4;
Single chain antibody;
Pseudomonas aeruginosa exotoxin PE38KDEL;
Recombinant immunotoxin;
Targeted therapy
- From:
Chinese Journal of Immunology
2018;34(3):354-361,366
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody (scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography (Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease di-gestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.
