Fusion Expression and Affinity Identification of Two scFvs Against CEA or CA19-9 in HEK293 Cells

Objective To express two scFv-Fc fusion proteins of anti-CEA scFv-Fc and anti-CA19-9 scFv-Fc in HEK293 cells efficiently, and to determinate the affinity of two single chain antibodies.

Methods Two scFv gene sequences obtained from GenBank were optimized and synthesized. They and pET32a(+) formed double prokaryotic expression vectors, anti-CEA scFv/pET32a(+) and anti-CA19-9 scFv/pET32a(+), respectively, which were translated into E.coli BL21 (DE3) and induced expression with IPTG. Two scFv genes (deleted terminators) were amplified by PCR; in addition, through overlapping region amplification method, we added IL-2 signal peptide into the upstream of anti-CEA scFv gene, getting three scFv genes, and connected these genes respectively with pTT5 containing human IgG1 Fc gene (Fc/pTT5) to constitute three eukaryotic expression vectors (anti-CEA scFv(-t)-Fc/pTT5, IL2-anti-CEA scFv(-t)-Fc/pTT5 and anti-CA19-9 scFv(-t)-Fc/pTT5). Three recombinant plasmids of scFv were transferred into HEK293 cells by Lipofectamine. We purified and identified expressing products by rProtein-A FF affinity chromatography, competitive cellular Elisa and Western blot.

Results Western blot analysis showed that three scFv-Fc fusion proteins were expressed in HEK293 cells. Competitive binding test showed that antibody concentrations of anti-CEA scFv-Fc and anti-CA19-9 scFv-Fc were 8.2 nmol/L and 6.0 nmol/L, respectively.

Conclusion anti-CEA scFv and anti-CA19-9 scFv could express in HEK293 cells, and their purified products could recognize tumor cell surface antigens CEA and CA19-9, although their expression efficiency is relatively low.