Construction and expression of anti-CD62P/anti-GPIIb-IIIa diabody

OBJECTIVE: To construct 3 expression plasmids for the targeted therapy of thrombosis: single chain variable fragment (scFv) of monoclonal antibody (mAb) 7E3 that can identify and bind platelet glycoprotein GPIIb-IIIa complex, scFv of mAb WAPS12.2 that can identify and bind P selectin (CD62P), a diabody that can identify and bind GPIIb-IIIa and CD62P imultaneously, and to investigate whether the vectors can express correctly.

METHODS: This study was carried out at the Laboratory of Hunan Yuantai Biological Technology Co. Ltd, Hubei, China from September 2007 to May 2008. Total RNA of mAb 7E3 cells and WAPS12.2 cells were obtained. Reverse transcriptase polymerase chain reaction (PCR) was carried out to obtain the genes of variable regions of light and heavy chains of 7E3 and WAPS12.2. Target genes were named 7E3VL, 7E3VH, CD62PVL, and CD62PVH. The 7E3VL-7E3VH and CD62PVL-CD62PVH were obtained by PCR and connected with pET-22b(+). Products were named pET-scFv7E3 and pET-scFvCD62P. The 7E3VL-CD62PVH and CD62PVL-7E3VH were obtained by using overlap PCR and were ligated to pET-22b(+). The products were named pET-ED1 and pET-ED2. The PCR was performed by taking pET-ED2 as a template to obtain the complete operon gene and was ligated to pET-ED1. The product was named pET-7ECD.

RESULTS: The identification by restriction endonuclease cleavage and DNA sequencing confirmed that the construction of these expression plasmids was successful. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed that these plasmids expressed correctly.

CONCLUSION: The expression plasmids pET-scFv7E3, pET-scFvCD62P, and pET-7ECD were constructed and expressed successfully, and laid a good foundation for further research on target-oriented thrombolytic agents.