| Purposes:On February 11,2020,pathogen of global pneumonia was named as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)by International Committee on Taxonomy of Viruses,and the resulting disease was named as coronavirus disease-19(COVID-19)by the World Health Organization(WHO).COVID-19 is highly contagious,with symptoms such as fever,cough,and even difficulty breathing.So far,there is no drug that can completely treat the disease.So the only way to get herd immunity is to get an effective vaccine.SARS-CoV-2 belongs to beta coronavirus,in which the nucleocapsid(N)protein is one of its structural proteins,which is expressed in large quantities after infecting cells.Studies have shown that the current research on the new coronavirus protein vaccine mainly focuses on the S protein,but the S protein is easily mutated,and the cross-protection between different types is poor.Compared with the S protein,N protein is more conservative.The N protein contains the specific antigenic epitopes of B cells,CTL cells and Th1 cells can stimulate the body to produce high-level specific immune responses.Therefore,in view of the highly mutated characteristics of SARS-CoV-2,the development of vaccines using N protein as an antigen is expected to reduce the incidence of different mutant strains probability of severe infection.The use of immune adjuvants can greatly improve the immune effect of vaccines,and the immune enhancement effect of traditional Chinese medicine extracts has been continuously proved.Lentinan(LNT)is a high-molecular-weight polysaccharide extracted and isolated from the fruiting bodies of Lentinus edodes fungus.Lentinan can enhance the body’s non-specific immune function by activating macrophages,activating T cells,regulating cell metabolism,and has the advantages of low toxicity and no residue.Lentinan was used as the research object,and the N protein was expressed by the prokaryotic expression system,and the optimal expression and purification conditions were explored;the purified N protein was immunized BALB/c mice by subcutaneous injection.It was administered by oral or subcutaneous co-injection,and the auxiliary effects of different administration methods on recombinant N protein were analyzed to provide theoretical support for the development of Chinese medicine adjuvant in the development of new coronary pneumonia N protein vaccine.Methods:1.Construction of SARS-CoV-2 N protein expression vector: The p UC19-N plasmid was used as a template,and the N gene was obtained by amplification,which was inserted into the p EASY-Blunt vector.After the sequencing was correct,N was recovered by double-enzyme digestion.The gene was connected to the p ET-28 a prokaryotic expression vector.After preliminary identification by bacterial liquid PCR and double digestion,the plasmid with correct sequencing was named p ET-28a-N.2.Expression and purification of SARS-CoV-2 N protein: transform p ET-28a-N into E.coli BL21(DE3)for induction expression,explore the induction conditions(time and IPTG concentration),use Ni-TED 6FF agar The N protein was purified by sugar purification resin,and its specificity was identified by Western blot.3.Mouse immunization experiment.The antigenicity of the SARS-CoV-2N protein prepared in this study was analyzed.We compared the immune-enhancing effects of lentinan administered by oral or subcutaneous co-injection on the N protein vaccine.4.Establishment of indirect ELISA detection method for SARS-CoV-2 N protein antibody.The detection method was optimized in terms of antigen coating concentration,coating time,serum dilution,blocking conditions,antibody incubation time,substrate action time,and repeatability,and its accuracy and repeatability were verified.5.Monitoring of serum antibody titers in mice.The mouse serum was collected at 7 days,35 days and 49 days after the primary immunization.After Western blot analysis of serum antibody specificity,the SARS-CoV-2 N protein antibody indirect ELISA method established in this study was used to detect the primary immunization and secondary immunization.And the N protein antibody titer was monitored after three immunizations.6.Analysis of mouse serum antibody Ig G type.The Ig G1,Ig G2 a and Ig G2 b antibody levels in the serum of immunized mice were detected by ELISA,and the tendency of lentinan to activate cellular and humoral immunity through different administration routes was determined.7.Analysis of immune cell proliferation by flow cytometry.49 days after primary immunization,the mice were euthanized and their spleens were used to prepare single cell suspension.The numbers of Tfh cells were detected by flow cytometry.Results:1.The recombinant SARS-CoV-2 N protein was successfully expressed and purified by using the E.coli prokaryotic expression system.We determined that the recombinant strain could express N protein at a high level after being induced under the condition of IPTG 0.8 m M for 12 h,and the N protein with higher purity could be obtained by using Ni-TED 6FF agarose purification resin.2.An indirect ELISA detection method for SARS-CoV-2 N protein antibody was established.The recombinant N protein prepared in this study was used as the coating antigen,the coating amount was 8 μg/m L,and the serum to be tested was diluted to 1:12000 and incubated for 60 min better.3.Antigenicity of SARS-CoV-2 recombinant N protein and lentinan enhance humoral immune response.The commercial SARS-CoV-2 N protein antibody was used to determine the reactogenicity of the recombinant N protein by Western Blot.The recombinant N protein was used as an antigen to immunize BALB/c mice.The titers of the antibodies can reach 1:25000,1:1000000 and 1:1400000 respectively.It is determined that using lentinan as an adjuvant,both oral and subcutaneous injection can improve the body’s immune response to SARS-CoV-2 recombinant N protein,and oral administration of 20 mg/kg lentinan can significantly improve the antibody levels of the N protein vaccine.The results of antibody type analysis showed that the immune-enhancing effect of lentinan on the body tended to be Th2 type,indicating that the immune-enhancing effect of lentinan on the body was more inclined to humoral immunity.4.Subcutaneous injection and oral administration of 20 mg/kg lentinan can promote the proliferation of Tfh cells in mice,strengthen the specific immune response of mice against N protein,and the immune enhancement effect of oral high-dose group was significantly higher than that of low-dose group,subcutaneous injection group and no lentinan group.Conclusion:The high-purity SARS-CoV-2 recombinant N protein was successfully expressed and purified by using the E.coli prokaryotic expression system,and an indirect ELISA method for the N protein was established,the N protein vaccine constructed in this experiment has better immunogenicity and immunoreactivity;lentinan was administered orally and subcutaneously to assist the N protein vaccine and they can improve the specific immune response of the body.Therefore,the recombinant N protein vaccine is expected to become a candidate vaccine for the COVID-19;when inoculating the COVID-19 vaccine,injection or oral administration of lentinan has a better immune enhancement effect,and lentinan is expected to become complementary drug for COVID-19 or other viral vaccines. |
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