Expression, purification and activity identification of human single-chain antibody against esophageal cancer cells
QIAO Yuanyuan
XIONG Ming
QU Xiuhua
WANG Yuxiao
ZHOU Lijun
ZHANG Dajin
Abstract：Objective To study the function of single chain antibody against esophageal cancer cells from phage antibody library and purify the ScFv. Methods Two single chain antibody genes were screened from phage antibody library and respectively connected to pET-28a and pET-SUMO prokaryotic expression vectors. The recombinant vectors were constructed respectively into E. coli BL21 (DE3). The induction conditions of IPTG concentration, time and temperature were optimized. The antibodies against surface antigen of esophageal cancer cells were renaturation by guanidine hydrochloride dilution titration and identified by SDS-PAGE and mass spectrometry. Results The recombinant vectors were successfully constructed. The recombinant strains were identified, the induction conditions were 0.5 mmol/L IPTG, 3 h, 18 t, and the recombinant protein was expressed in the form of inclusion body. The recombinant protein with relative molecular weight of 30 KD and 40 KD. Cell ELISA was used to identify the biological activities of two proteins and esophageal cancer cells. Conclusion The single chain antibody NFC20b and NFC70a can be successfully constructed in the bacteriophage and express the inclusion body and the protein activity can be obtained after purification. It can be used for the research and development of subsequent experiments and testing reagents.
Keywords：Protein expressionProtein purificationActivity identification
Publication Date：2018-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 31-34,38 )