Construction and expression of anti IL-4R single antibody of prokaryotic expression vector
10.11958/20170284
- VernacularTitle:抗IL-4R单链抗体原核表达载体的构建与表达
- Author:
Guangyong YANG
;
Qianming LIU
;
Lili LIU
;
Wenjia WANG
;
Guangzhi HE
- Keywords:
receptors;
interleukin-4;
recombinant proteins;
electrophoresis;
polyacrylamide gel;
scFv;
prokaryotic expression
- From:
Tianjin Medical Journal
2017;45(9):897-901
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct anti-IL-4R murine anti-human single-chain variable fragment (scFvs) antibodies through BL21 (DE3) prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 (DE3) prokaryotic expression bacteria to express the pET-32a-scFv recombinant protein in E.coli BL21 (DE3). The purification and renaturation were researched, and SDS-PAGE analysis was studied. The molecular weight of ScFv against IL-4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6 × His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.
