Tianjin Med J ›› 2017, Vol. 45 ›› Issue (9): 897-901.doi: 10.11958/20170284

• Cell and Molecular Biology •     Next Articles

Construction and expression of anti IL-4R single antibody of prokaryotic expression vector

YANG Guang-yong, LIU Qian-ming, LIU Li-li, WANG Wen-jia, HE Guang-zhi   

  • Received:2017-03-07 Revised:2017-05-12 Published:2017-09-15 Online:2017-09-25

Abstract: 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.

Key words: receptors, interleukin- 4, recombinant proteins, electrophoresis, polyacrylamide gel, scFv, prokaryotic expression