Fusion expression of human X-box binding protein 1 and preparation of polyclonal antibody against protein
AIM: To express prokaryotically and purify the unspliced human X-box binding protein1 (XBP1u) recombinant plasmid and prepare polyclonal antibody against protein. METHORDS: We transformed the recombinant plasmid pET32aXBPIu into host bacterium E. coli BL21 (DE3), induced its expression with IPTG, and purified the protein with Ni~(2+) -NTA collumn after identified by SDS-PAGE, then immunized two New Zealand rabbits with the purified protein as antigen after renaturation with dialytic bag, then collected blood from the heart after the last immunization, detected the titer of antiserum by ELISA, detected the antiserum specificity by Western Blot and immunohistochemistry. RESULTS : XBP1u expressed in the inclusion body of E. coli BL21, the titer of antiserum was beyond 1:64 000 after immunize rabbit with purification and renaturation XBP1 u protein. Besides, it is confirmed that the antiserum reacted specifically to the XBPlu protein which is Mr 33 x 10~3 by Western Blot. It is successful to detect the expression in hepatocyte with immunohistochemistry. CONCLUSION: It is successful to induce XBPIu expression and purification. The highly specific and titer polycional antibody have been obtained on basis of this purified protein.
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