| Hepatitis C is a viral hepatitis caused by hepatitis C virus(HCV),which is a global epidemic and costs more than $100 billion annually for hepatitis C treatment worldwide.Most patients with hepatitis C virus infection are persistently infected,which in turn causes chronic liver inflammation or necrosis,cirrhosis and even some patients can transform into liver cancer.Existing therapeutic drugs are only effective for some hepatitis C virus infected patients,and have long treatment time,high cost,and side effects.Vaccines are an economical,simple,and effective means of preventing viral diseases.Hepatitis C virus is a single-stranded positive-sense RNA virus with significant heterogeneity and high variability,and there is no vaccine against hepatitis C virus.Therefore,it is of great scientific value to develop genetically engineered vaccines for hepatitis C virus.In this study,based on the amino acid sequence of hepatitis C virus genotype I(Beijing 1b)in China,the genes of six conserved sequences Bp(E1313-327 aa,E2396-424 aa,E2438-447 aa,E2523-540 aa,E2610-627 aa,E2631-648aa),mimotope R9 peptide,and immune adjuvant thermosensitive enterotoxin B unit(LTB)in hepatitis C virus envelope protein E1/E2 were synthesized to construct the expression vectors p ET28a-LTB-R9-Bp and p ET28a-R9-Bp expressing these multi-epitope genes.The recombinant plasmid expressing the multi-epitope vaccine gene was successfully obtained by sequencing identification.The conditions for efficient and stable expression of LTB-R9-Bp and R9-Bp proteins were explored in an E.coli system,and a large number of expressed proteins were purified using nickel columns,and we finally successfully obtained the purified target proteins.The purified LTB-R9-Bp and R9-Bp proteins were immunized to Kunming mice by three immunization methods: IP,IM,and Oral,and antisera were collected to detect the induced humoral immune response.The results of ELISA showed that the titer of LTB-R9-Bp antibody expressed by fusion reached1:341000 after intraperitoneal injection,while the highest dilution of R9-Bp protein antiserum was 1:85000,which had significant difference,indicating that LTB had immune enhancement effect;in the intramuscular injection group,both recombinant LTB-R9-Bp and R9-Bp protein immunized mice caused humoral immune response in mice,but the induced antibody level was equivalent;LTB was a good oral immune adjuvant,and oral immunization of mice with LTB-R9-Bp protein was attempted,and no good immune effect was detected.Real-time PCR was used to detect the expression levels of IL-4 and IFN-γcytokines in splenocytes of immunized mice,and it was found that the cytokine contents in the experimental groups immunized with LTB-R9-Bp and R9-Bp proteins were higher than those in the control group,indicating that LTB-R9-Bp and R9-Bp proteins could cause cellular immunity in mice.The reaction of LTB-R9-Bp and R9-Bp antisera with HCV viral proteins was detected by Western blot,and the results showed that the self-made antisera could specifically recognize HCV viral antigens E1 and E2.In order to study whether the antisera obtained by immunization with LTB-R9-Bp and R9-Bp could inhibit HCV infection to a certain extent,virus neutralization experiments were designed to infect Huh7 cells after incubating different dilutions of sera with the virus,and the results revealed that the antisera from immunization with LTB-R9-Bp and R9-Bp could partially protect cells from hepatitis C virus infection in an antibody-dose dependent manner.The above results indicate that hepatitis C virus multi-epitope antigens LTB-R9-Bp and R9-Bp proteins have good immunogenicity and immune specificity,further deepening our potential understanding of HCV envelope proteins E1 and E2 as vaccine development.And the serum antibody produced by mice against hepatitis C virus multi-epitope protein has a certain neutralizing effect on the virus.It lays a preliminary experimental foundation for the development of hepatitis C virus multi-epitope genetic engineering vaccine. |
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