含PD-1 B细胞表位的乙型肝炎病毒核心蛋白病毒样颗粒的制备及其抗结肠癌活性OA

Objective:To establish a recombinant particle protein using hepatitis B core protein(HBc)virus-like particles(VLPs)as the scaffold,with chimeric incorporation of PD-1 B cell epitopes,and to investigate its inhibitory effect on colorectal cancer in mice.Methods:A PD-1 HBc VLPs plasmid was designed and introduced into Escherichia coli BL21(DE3)to obtain recombinant PD-1 HBc VLPs-expressing strains.Soluble protein expression was achieved by optimizing the culture temperature.Subsequently,high-purity PD-1 HBc VLPs were obtained via a series of purification steps,including ammonium sulfate precipitation,size-exclusion chromatography,and anion-exchange chromatography.The expression level,protein purity,particle formation efficiency,morphological characteristics,and antigen-binding activity of the obtained PD-1 HBc VLPs were systematically characterized by electrophoresis,WB,high-performance liquid chromatography(HPLC),and transmission electron microscopy.Immunogenicity was evaluated by ELISA to determine serum antibody titers in immunized mice.CT26 colorectal carcinoma cell transplant tumor model was constructed in mice,and the anti-tumor activity of PD-1 HBc VLPs was further assessed in vivo by comparing tumor growth,body weight and tumor volume changes.Results:At 28 ℃,the soluble target protein accounted for 10.27％of total protein.The target protein was purified using Sepharose 4 Fast Flow molecular sieve and DEAE anion resin chromatography,with a purity of(91.77±0.62)％and a molecular weight of 23 000.The particle size measured by electron microscopy was(31.90±1.88)nm,while particle size analyzer showed a particle size of(30.66±0.62)nm,and the polydispersity index(PDI)was(0.18±0.05).WB analysis demonstrated that the recombinant PD-1 HBc VLPs could bind to anti-HBc VLPs serum and anti-PD-1 antibodies,respectively.ELISA results showed that on day 14 after the third immunization with PD-1 HBc VLPs,the total antibody titer in mouse serum reached 1∶640 000,with an anti-PD-1 antibody titer of 1∶40 000.Anti-tumor experiments showed that PD-1 HBc VLPs achieved a tumor inhibition rate of(27.99±2.98)％in mice with colorectal carcinoma.All mice in the control group died by day 45,whereas the median survival time(MST)in the PD-1 HBc VLPs group was prolonged by 20 days.Conclusion:PD-1 HBc VLPs containing PD-1 B cell epitopes were successfully prepared and demonstrated therapeutic efficacy against colorectal cancer in mice.