Purification And Comparative Study On Anti-tumor Effects Of Recombinant MUC1Peptides And MUC1-MBP Fusion Protein

MUC1is a tumor-associated antigen, glycoprotein.of high molecular weight.MUC1-based vaccine has been rapidly applied to human clinical trials, anddemonstrates a good efficacy. Studies have shown that MUC1peptides can notinduce the host to produce antibodies, CTL or DTH; MUC1peptides gearing KLHand GST can induce the host to produce antibodies and DTH, but no CTL; however,MUC1connecting oxidized mannan can induce strong CTL responses andanti-tumor effects. E. coli maltose binding protein (MBP) can regulate the activity ofDCs. Our research team find that MBP can promote cellular immune responses.Therefore, our research group used pMAL-c2, a prokaryotic vector, as a carrier toinsert a repeating sequence of MUC1gene into the downstream of MBPgene—malE gene and established a vector that could successfully expressMUC1-MBP recombinant gene and obtained engineered bacteria which couldexpress MUC1-MBP fusion protein steadily and efficiently. Thus we prepared theMUC1-MBP fusion protein of higher purity and carried out a preliminary study ontheir anti-tumor effects. The purpose of this subject, is to conduct a comparativestudy on the anti-tumor effects of recombinant MUC1peptides and MUC1-MBPfusion protein on the basis of our previous research work.Methods and Results of the Research:1. Preparation for the protein We cultured a large number of pMAL-MUC1/DH5α, pMAL-c2/DH5α and pGEX-MUC1/BL21and used IPTG for theexpression of MUC1-MBP, MBP and MUC1-GST protein. Bacterial specimenswere lysis by sonication and the supernatant were obtained. MUC1-MBP fusionprotein and MBP protein of high purity were prepared by affinity chromatography of Amylose resin. We purified MUC1-GST through Glutathione-Sepherose4B. Weused thrombin to cleavage the MUC1-GST protein in order to get the recombinantMUC1peptides. The purified protein were identified by Western blotting.2. The establishment of the subcutaneous B16-MUC1melanoma model In theexistence of G418we cultured B16melanoma cells stably transfected with humanfull-length MUC1. The expression of MUC1protein was identified by flowcytometry.2*106cells were injeced into each C57BL/6mouse subcutaneouslyand the subcutaneous B16-MUC1melanoma model was successfully established.3. Comparative analysis on the protein’s anti-tumor effects C57BL/6micewere immunized with recombinant MUC1peptides, MUC1-MBP protein ofdifferent doses and MBP protein and the above-mentioned protein joint BCGsubcutaneously every seven days and immune totally three times. The weightchanges of the mice were monitored and analyzed by statistical software. After5-7days of the third immunization,2*106mouse melanoma cells stably transfectedwith the MUC1gene (B16-MUC1) were subcutaneously injected into each mouse.We monitored tumor growth carefully. The mice were killed9-13days after theinjection of tumor cells. We weighed the mice, stripped subcutaneous melanomaxenograft tumor completely and weighed the tumor. Subcutaneous melanomaxenograft tumor inhibitory rates of each group were calculated. Separated andweighed the spleen. Combining with the body weights of the mice, spleen indexes ofall groups were culculated. The results showed that:1) MUC1peptides immunized alone slightly inhibited the growth of subcutaneoustransplanted melanoma tumor; BCG immunized alone and MUC1peptidesimmunized with BCG had no tumor inhibition effect.2) It was not obvious of tumor inhibition when MBP protein was immunizedalone; on the contrary, immunizingMBP protein combined with BCG distinctlyprohibited the growth of subcutaneous melanoma with the rate of55.5%.3) Immunizing MUC1-MBP protein alone slightly reduced the growth ofsubcutaneous transplanted melanoma and the inhibitory rate was higher when the doses of MUC1-MBP increased. Inhibitory rates of subcutaneous transplantedmelanoma clearly increased when mice were immunized with MUC1-MBP proteincombined with BCG. Importantly, the inhibitory rate increased to a maximum of98%with the administered dose increased to50μg.4) With regard to the mice spleen indexes, the MUC1peptides group,MUC1-MBP fusion protein group and MBP group had no significant differencescompared with the control group (P>0.05). Furthermore, the MUC1peptidescombined with BCG group, MUC1-MBP fusion protein combined with BCG groupand MBP combined with BCG group had distinct differences compared with thecontrol group (P<0.05). So spleen index can be used as an indicator of immuneactivity of the MUC1-MBP vaccine or BCG.5) The body weights of the mice immunized with recombinant MUC1peptides,MUC1-MBP fusion protein and MBP had no significant differences compared withthe control group (P>0.05). When joint BCG, the body weights of mice declinedslightly but didn’t significantly compared with the control group (P>0.05). Theresults indicate that BCG prompts a slight toxic effect in miceIn conclusion, MUC1-MBP protein combined BCG has significant anti-tumoreffect on transplant malignant melanoma while MUC1peptides combined BCGhas no evident anti-tumor effect. The results further suggest the valuation ofMUC1-MBP combined with BCG as a anti-tumor vaccine.