Expression and analysis of ScFv antibody of mcAb 50A anti-RBC blood group A substance

Aim To construct and express ScFv fusion protein against human blood group A substance. Methods The restriction enzyme sites were added to the previously cloned VH and VL genes fo 50A by add on PCR. The 50A ScFv genes constructed by the splicing overlap extension (SOE) PCR using a (Gly4Ser)3 linker were sequenced by Sanger's method and then inserted into fusion expression vector pET 28a. The recombinant proteins were examined by immunoblot. Results The nucleotide sequencing indicated that the cloned genes coded the light and heavy chain variable domains in mouse antibody. The 50A ScFv gene consisted of 747 bp, encoding 249 amino acids. After induction, a new protein band with relative molecular mass(Mr) of 29 000 appeared on SDS PAGE gel and amounted to 16.2% of total bacteria protein in a form of inclusion body. Immuno blot analysis identified that the recombinant protein retained the specific affinity of the intact 50A mAb. Conclusion The 50A ScFv gene has been constructed successfully and expressed in E.coli BL 21(DE3). This provided a foundation for studying engineering antibody as blood grouping reagent.