Study On Therapeutic Vaccine For Colorectal Cancer With HBcAg As Carrier

Colorectal cancer is one of the malignant tumors that seriously threatens human health.At present,the clinical treatment for CRC is still limited to surgical resection,radiotherapy and chemotherapy.In recent years,immunotherapy,especially those targeting neoantigen,has attracted much attention in cancer treatment.Neoantigen is a tumor-specific antigen,which is produced by genic mutation of tumor cell and not expressed in normal cells.Unlike autoantigens,neoantigens are recognized as a "non-self" antigen by the immune system,which in turn stimulates the immune system to produce a specific,efficient,and safe immune response.Many studies have shown that cytotoxic T lymphocytes(CTLs)targeting neoantigen are the main T cell populations that kill tumor cells.However,under normal circumstances,due to the low expression level of neoantigens in cancer cells or the low efficiency of antigen presentation,the number of specific CTLs targeting neoantigens in patients is very small.Therefore,it is necessary to enhance the body’s immune response to neoantigens by designing cancer vaccines targeting neoantigens.In this study,the gene encoding neoantigen Adpgk was inserted into position between amino acids 78 and 79 of the truncated hepatitis B virus core antigen(HBcAg)(1-149aa)by fusion PCR and the fusion gene was cloned into the prokaryotic expression vector pET-22b(+).The fusion protein was expressed by E.coli expression system.After induced expression by IPTG,the expression of fusion protein was detected by SDSPAGE and Western-blot.SDS-PAGE results showed that after the introduction of the RD sequence at the C-terminus of the insertion site,the soluble fusion protein HBcRD-Adpgk could be expressed;the fusion protein HBc-Adpgk without the RD sequence was insoluble.It shows that RD sequence can change the physical and chemical properties of HBc-Adpgk and improve its solubility.The soluble target protein after expression was purified by affinity chromatography and ultrafiltration.Transmission electron microscope observation results show that HBc-RD-Adpgk protein can be correctly assembled into virus-like particles with a diameter of about 30 nm,indicating that the insertion of the neoantigen Adpgk and RD sequence into HBcAg did not change the self-assembly characteristic of HBcAg.In this study,the recombinant HBcAg VLP was successfully prepared,which laid the foundation for the research of recombinant VLP cancer vaccine targeting neoantigen.In order to increase the number of neoantigens within the recombinant HBcAg to increase the therapeutic target of the vaccine,the coding genes of the neoantigens Adpgk,Reps1 and Dpagt1 were inserted into position between amino acids 78 and 79 of the truncated HBcAg and in both sides of the insertion site are supplemented with flexible amino acid sequences.After codon optimization of the full-length gene sequence,the gene is synthesized and called HBc-neoantigens and then cloned into the prokaryotic expression vector pET-22b(+).The fusion protein was expressed by E.coli expression system.The induced expression conditions were optimized and the expression of fusion protein was detected by SDS-PAGE.The results showed that the protein of interest was expressed inclusion body under the induction conditions of 0.1m M IPTG at 37℃,30℃ and 16℃.This study laid the foundation for the research of recombinant muti-epitope VLP cancer vaccine.