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Objective To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP)and to demonstrate the effect of ultrafiltration on endotoxin removal.Methods CM Sepharose FF weak cation exchange (CM)(CM group), CM combined with Phenyl Sepharose 6 FF exchange (C6)(CM+C6 group),CM combined with ultrafiltration (CM+ultrafiltration group), and CM combined with C6 and ultrafiltration (CM+C6+ultrafiltration group)were used to purify the MUC1-MBP from E.coli. and remove endotoxin;the expression level of endotoxin was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference between two groups (P>0.05 );the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group, and there was significant difference (P0.05 ). Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor, especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and ultrafiltration plays an important role in endotoxin removal.
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Objective: To study the anti-tumor effect of recombinant human MUC1-MBP. Methods: The C57BL/6 mice were in oculated with MUC1-MBP by subcutaneous. MUC1 specific CTL activity of spleen were determined by MTT; The effects on prevention and treatment of tumor were observed by establishing lewis lung cancer-carrying mice. Results: The cytotoxicity of CTL from immunized mice to the MCF7 and Lewis lung cancer cells respectively was (47.7?4.3) % and (67.5 ?6.5) %; 5?10 5 lewis lung cancer cells following immunization were injected iv into C57BL/6 mice, after three weeks, the number of lung and tail tumor colonies was 51 and 5 for PBS and MUC1-MBP groups respectively and the suvival time was significantly delayed in immunized mice. The average volume of tumors in mice with MUC1-MBP was 386 mm 3 wherea control group was 4 000 mm 3 at tumor treating experiment. Conclusions: Recombinant human MUC1-MBP have significantly effects on prevention, treatment and inhibiting metastases of tumor. Our results suggested that the recombinant MUC1-MBP might be used to develop protein vaccine against human carcinoma.