The Anti-melanoma Research Of A New Recombinant Vaccine PNb

IntroductionMalignant tumors rank first in the cause of death in China,followed by heart disease and cerebrovascular disease.Malignant tumors significantly reduce the patient's survival time and bring a huge economic burden to the patient.People attach great importance to it.There are many therapeutic strategies for malignant tumors,of which vaccine therapy is a research hotspot.The most important advantage of vaccine treatment over traditional treatments(surgical treatment,radiotherapy,and chemotherapy)is that the immune response it stimulates plays a role in the body for a long time to minimize tumor recurrence.As one of many tumor vaccine targets,NY-ESO-1 has become a promising tumor vaccine target because of its high specific expression and expression in a variety of tumor tissues.NY-ESO-1 combined with various adjuvants has a large number of basic studies for the preparation of tumor vaccines.In animal experiments,the vaccine has shown excellent anti-tumor effects.In addition,NY-ESO-1 target vaccines have seen numerous clinical studies on the clinicaltrials.gov website.Completed Phase I clinical trials of the NY-ESO-1 vaccine in combination with ipilimumab for the treatment of melanoma,and the NY-ESO-1 vaccine with resiquimod adjuvant for phase I clinical trials of tumours.The results of these clinical trials showed that the NY-ESO-1 vaccine is effective for cancer patients.In addition,another anti-tumor strategy is to inhibit tumor angiogenesis,because it has been recognized in earlier studies that the importance of angiogenesis for tumors.In the absence of angiogenesis,the tumor may not even grow to a diameter of 2 mm.Fibroblast growth factor(bFGF)as the first identified angiogenic growth factor plays an important role in tumor angiogenesis and tumor growth.Although animal experiments with the bFGF vaccine showed that the vaccine significantly inhibited tumor angiogenesis and inhibited tumor growth,no clinical trials were found for antitumor drugs targeting the bFGF target.P4 is a T-cell epitope peptide that activates T cells and increases the immunogenicity of various vaccines.P4 is often used as an intramolecular adjuvant for various vaccines,and the results of animal experiments show that the vaccine containing the p4 adjuvant has a better anti-tumor effect than the vaccine without the p4 adjuvant.It is now recognized that the T cell immune response is extremely important for the entire anti-tumor immunity,and p4 has thus become a popular vaccine intramolecular adjuvant.Considering the above-mentioned aspects,this research project was designed to select NY-ESO-1 and bFGF bi-target vaccines for cancer vaccines.In addition,NY-ESO-1 and bFGF single-target vaccines were used as controls.All three vaccines contained the intramolecular adjuvant T cell epitope peptide p4.Purpose(1)Construction of recombinant plasmids and expression of three recombinant vaccine molecules.(2)To study the anti-tumor effect of dual target vaccine molecules.Methods(1)Using the pET-32 a plasmid as a vector to synthesize the desired sequences therein,three kinds of recombinant plasmids are obtained,which are pNb,pN and pb.pNb contains p4,NY-ESO-1,and bFGF(10aa-150aa).pN contains p4 and NY-ESO-1.Pb contains p4 and bFGF.The three plasmids were respectively transferred into BL21(DE3),and IPTG induced and optimized the expression conditions so that the target protein was well expressed.(2)The bacterial cells after the expression of the protein were collected by centrifugation,resuspended and sonicated,and the supernatant and the inclusion body were again centrifuged.Total protein,protein total protein,supernatant protein and inclus ion body protein before protein expression were subjected to SDS-PAGE electrophoresis,followed by staining and decolorization.Based on the above results,the next purification method was determined.The purification methods used were urea conversion,nickel column purification,and ion column purification.(3)To establish a melanoma model,C57BL/6 mice were selected as experimental animals and B16-F10 was used as experimental tumor cell line.In order to stably express NY-ESO-1 in B16-F10,the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro and the lentivirus packaging plasmid psPAX2,pMD2.G were used.In the experiment,NY-ESO-1 was constructed into pCDH-CMV-MCS-EF1-Puro,and the recombinant plasmid was then transfected into HEK293 T cells together with the two lentivirus packaging plasmids.The supernatant of HEK293 T cells was filtered to obtain virus particles,and the virus particles were transfected into B10-F10 cells.(4)Animal experiments C57BL/6 females were divided into 6 groups,pNb group,pN group,pb group and AL(OH)3 group were immunized groups,and Tumor group and Normal group were also included.Animal vaccines were immunized three times.After completion,tumor cells were implanted subcutaneously on the right back of the mice.(5)Mouse serum antibodies were tested for titers using ELISA.Mouse tumors were measured using vernier calipers and their size was calculated.Histological experiments were performed on the tumor tissues after sacrifice.Results(1)Successfully constructed three recombinant plasmids containing the gene of interest.(2)Successful expression of recombinant molecules in E.coli BL21(DE3)and completion of protein purification for immunized animals.(3)All three vaccine molecules have an anti-melanoma effect,and dual-target vaccines may have better anti-melanoma effects.ConclusionsSuccessfully expressed recombinant proteins pN,pb,and pNb immunized mice with a positive anti-melanoma effect.Among them,pNb may have an anti-melanoma effect better than pN and pb.