[Preparation and characterization of monoclonal antibody against N-terminal domain of polycystin 1].

AIM

To prepare and identify monoclonal antibody (mAb) against N-terminal domain of polycystin 1.

METHODS

Total RNA was extracted from kidney tissue of a healthy man. Gene sequence encoding polycystin 1 N-terminal domain was amplified by one-step RT-PCR. The target gene was inserted into prokaryotic expression vector pQE30 and transformed into competent cells E. coli M15. The fusion protein was expressed under IPTG induction and purified by affinity chromatography. The purified fusion protein was then used to immunize BALB/c mice. The splenocytes from immunized mice were fused with myeloma cells Sp2/0 by PEG 4000 mediator method and the hybridomas were selected in HAT medium. The hybridoma clones secreting mAb against polycystin 1 amino-terminal domain were detected by ELISA and cloned by limiting dilution. The specificity of mAb against polycystin 1 N-terminal domain was verified by ELISA and Western blot.

RESULTS

cDNA encoding polycystin 1 extracellular region was obtained. Fusion protein of polycystin 1 N-terminal domain were expressed in pQE30 expression system. The relative molecular masses (Mr) of the two fusion proteins were 19,800 and 18,900, respectively. One hybridoma cell 7B1 secreting specific mAb was obtained. Western blot analysis showed that the mAb reacted strongly and specifically to polycystin 1 N-terminal domain.

CONCLUSION

polycystin 1 N-terminal fusion proteins have been expressed in E.coli M15. Anti-fusion protein mAb with antigen-binding activity has been prepared successfully.