Purification Of Recombinant MUC1-MBP Fusion Protein And Study On Its Security And Stability

MUC1, a transmembrane glycoprotein of the mucin family. In normal cells, MUC1 is heavily glycosylated and its distribution is limited to the apical surface of ductal cells. In tumor cells, underglycosylated MUC1 is markedly upregulated and distributed along the entire cell surface. Hence MUC1 has been considered as an attractable target for tumor immunotherapy. Currently, many MUC1-based cancer vaccines have shown effective anti-tumor by generating cellular immune response and some of that have already entered phaseⅢclinical trials. Since 1999, we have studied MUC1-based fusion protein vaccine and produced recombinant MUC1-MBP fusion protein. We have found that MUC1-MBP can induce not only humoral immune response but also effective cellular immune response, resulting in expect effective anti-tumor. Even though MUC1-MBP tumor vaccine has also made encouraging progress, purification method of the protein has not been immatured, studies of MUC1-MBP on security and stability have not been carried out. Therefore, in this study, to further purify MUC1-MBP protein we prepared specific anti-MBP antibody affinity chromatography; to study MUC1-MBP tumor vaccine safety, we did allergy test and acute toxicity test; to determine the stability of MUC1-MBP protein stability, we did classical isothermal accelerated test and the stabilizers which protect the protein were investigated.1. A large of pMAL/DH5αinduced by IPTG were broken and removed supernatant containing fusion protein. MBP protein was purified by Amylose affinity chromatography and obtained higher purity of MBP.2. The rabbit was immunized by MBP protein, we obtained the MBP anti-serum with the titer of 320 000.3. We Purified the MBP anti-serum by a saturated ammonium sulfate and protein A affinity chromatography in turn, we obtained high purity of anti-MBP antibody.4. To prepare anti-MBP antibody affinity chromatography, anti-MBP antibody and CNBr-Sepharose 4B were mixed and incubated at room temperature for 4h. The result showed that we successfully produced the anti-MBP antibody affinity chromatography with the coupling ratio of 90%.5. E.coli DH5αwere transformed by pMAL-MUC1, pMAL-MUC1/DH5αexpressing MUC1-MBP protein was obtained. We found that it can stably express MUC1-MBP till seventh generations.6. A large of pMAL-MUC1 / DH5αcultured were inducted by IPTG and broken by sonication to obtain the whole cell supernatant. MUC1- MBP fusion protein from the supernatant was purified by Amylose and further by anti-MBP antibody affinity chromatography. We analyzed the purified MUC1- MBP by native PAGE and GIS.The result showed the protein with purity of 92.75% .7. In systemic allergy in guinea pig experiments, we use the purified MUC1-MBP fusion protein of the clinical equivalent doses to sensitize subcutaneously by three times and attack by quadruple dose, and the results showed that there is no systemic allergy exceeding second level. It indicates recombinant MUC1-MBP fusion protein is qualified on guinea pig allergic.8. In acute toxicity tests, three dose groups including clinical human dose of 32 times, 64 times and 125 times, 10 mice per group were designed, injected subcutaneous once, observed mortality for ten days.There were no one dead.As we could not measure the LD50, so to do the maximum tolerance test done instead. The dose of clinical human dose of 125 times, and the number of mice of 20 were designed, injected subcutaneous once, observed weight,food consumption and symptom for one week. As a result, there was nothing anormal.After a weak, we did anatomy to detect no viscera anormal.The result demontrates that MUC1-MBP maximum tolerated dose is the clinical human doses of 125 times, the vaccine is nonpoisonous.9. In classical isothermal accelerated test, utilizing the relation of the degradation amount of MUC1-MBP fusion protein and time in four temperatures of 37℃, 45℃, 50℃, 55℃, we obtained their degradation rate constants (K), and then deduced relation between K and temperature: lgK = - 5.8012 (1 / T)×103 +17.009. Eventually we worked out expiry period of MUC1-MBP in room temperature 25℃and 4℃are respectively 1 month and 2.5 years.10. Adding sucrose, mannitol, glycerol, gelatin as a single stabilizing agents to MUC1-MBP protein solution to test their protective effect on MUC1-MBP protein. The results showed that only glycerol has played a weak protective effect.In summary, we have optimized the purification process of MUC1-MBP fusion protein and obtained MUC1-MBP fusion protein with purity of 92.75%; detected the security and the stability of MUC1-MBP fusion protein, and found it is secure and stability. This study provided the basis for the further research and development of MUC1-MBP vaccine.