Aim: To express human HMGB1 B box protein and obtain monoclonal antibodies (mAbs) against HMGB1 B box for further study of the function of human HMGB1 protein.
Methods: pET28-HMGB1 B box plasmid transfected the DH5α, then expressed. And the extracted protein was purified by protein purification system. BALB/c mice were immunized with recombinant human HMGB1 B box protein. Hybridoma cell lines secreting mAb against human HMGB1 B box protein were screened by ELISA and subcloning approach. The characteristics of these mAbs were identified by ELISA and Western blot.
Results: Two hybridoma cell lines (1D2F4E3 and 2D4E3A2) stable secreting specific mAbs were successfully obtained.Western blot exhitited the two mAbs binded specifically to human HMGB1 B box protein. The immunoglobulin (Ig) class of two mAbs belonged to IgG, their titers were 1×10(6);, and the A(450); of mAb1D2F4E3, 2D4E3A2 were 0.324±0.093, 0.296±0.085, respectively.
Conclusion: Two of high specificity mAbs against human HMGB1 B box protein have been successfully prepared, which laid the foundation for further study of biological function of human HMGB1 protein.