| Objective The COVID-19 pandemic caused by severe acute respiratory syndrome(SARS-COV-2)and its impact on global population structure and economic destruction marks the third highly pathogenic coronavirus to threaten humanity after severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East Respiratory Syndrome(MERS-CoV).While previous epidemics of SARS-COV and MERS-CoV have increased awareness of clinical treatment or prevention interventions.to date,no effective prevention is available.The number of cases caused by SARS-COV-2 has overwhelmingly surpassed those caused by SARS and MERS in terms of the number of people infected and the spatial scale of the affected areas.SARS-COV-2 has the characteristics of high infectivity,extensive tissue tropism and easy mutation,and its spread has posed a great threat to the global public health system.To date,countries have relied on isolation,quarantine and clinical care for affected individuals to control the rapid spread of the disease.In the face of the rampant virus,SARS-CoV-2 vaccine development and vaccination is particularly important to prevent SARS-COV-2 infection and reduce the disease.However,an important issue in vaccine development is antibody dependent enhancement(ADE),in which non-neutralizing or subneutralizing antibodies bind to viral surface proteins and facilitate the entry of the virus into the host cell.The abundant and relatively stable major structural proteins are generally thought to be antigens that induce antibody production.Among the four main structural proteins of SARS-COV-2,S and N proteins have obvious immunogenicity.For M protein,there is little research attention.The aim of this study was to investigate the immunological characteristics of membrane protein(M),the most abundant structural protein in SARS-COV-2 virus,and in particular the possibility of inducing ADE effects.Methods In this study,a recombinant plasmid pET-32a-M,which can express SARS-CoV-2 full-length Membrane(M)protein,was constructed by prokaryotic expression system.The full-length M protein with His label was purified by lysozyme treatment,ultrasonic crushing,urea washing,gel cutting,dialysis and concentration after induced expression by IPTG in BL21 cells.The purified recombinant protein(50μg/mouse)was mixed with Freund’s adjuvant and immunized normal BALB/c mice subcutaneously for three times(day 1,14 and 21).The immune serum was collected and isolated before immunization,for one time,for two times and for three times,respectively.The specificity of serum antibodies was identified by Western blotting,and the titer of serum antibodies was detected by ELISA and neutralization test.Then dendritic cells(DC),natural killer cells(NK),T-lymphocyte and B-lymphocyte were separated from the spleen of normal BALB/C mice.Different concentrations of anti-M protein serum were added to the cells and sarS-COV-2 was subsequently infected.In vitro culture techniques were used to detect virus replication and proliferation in these immune cells at 12,24,and 48h after infection,as well as induced cytokine expression changes related to natural immune activation.Results We constructed the prokaryotic expression vector pET-32a-M,and induced the expression system of E.coli to express the full-length M protein by 1mM IPTG induction.SDS-PAGE confirmed that escherichia coli expression system can successfully express M protein,and the prokaryotic expression of M protein mainly concentrated in inclusion body.Subsequently,4M urea was used to wash the inclusion body to remove miscellaneous proteins,and the target protein was purified by gel cutting,and relatively pure M protein was obtained through dialysis.Mice were subcutaneously immunized with the purified and concentrated M protein at the dose of 50μg/mouse with Fredrin’s adjuvant for 3 times.The serum of mice was obtained by tail vein blood sampling before enhanced immunization,and the serum of mice was obtained by eyeball blood sampling on the 28th day after primary immunization(7 days after the last immunization).The antibody titer of BALB/c mice immunized with purified SARS-CoV-2 full-length M protein was about 1:200 by ELISA.Western Blot was used to test the specificity of the obtained immune serum,and it was confirmed that the immune serum could specifically recognize M protein in SARS-CoV-2.In addition,neutralization test was used to detect the neutralization capability of the immune serum in novel coronavirus,and it was found that the neutralization titer of the prepared anti-M protein antibody was<1:4(negative).Furthermore,DC,NK,T and B cells were isolated from the spleen of blank BALB/c mice by in vitro culture technique.In the presence of different concentrations of anti-M protein serum,DC,NK,T and B cells were infected with SARS-COV-2(MOI=0.01),respectively.PCR was used to detect virus proliferation at 12,24 and 48h after infection.The results showed that in the presence of antiserum at different dilutions,viral load was similar to that of the control group(without antiserum)in B cells,DC cells,NK cells and T cells,and there was no significant trend of increase within 12-48h of infection.At the same time,the viral load of Vero cells lacking Fc receptor(FcR)was similar to that of the control group(without antiserum)in the presence of different diluents,and began to show a slight increase trend at 24h after infection.In addition,we also detected the expression levels of cytokines associated with natural immunity after virus infection in these cells.The results show that the expression levels of various factors in the experimental group infected with novel coronavirus cells of different immune cells(in the presence of antisera with different dilutions)are similar or lower than those in the control group infected with viruses of the same cells(without antisera).Conclusions Full-length M protein was successfully prepared by prokaryotic expression system,and mice were immunized with this protein to prepare anti-M protein antiserum.This serum can specifically identify SARS-CoV-2 M protein with antibody titer of about 1:200,but it is a nonneutralizing antibody with no neutralizing ability.This non-neutralizing antibody induced by M protein cannot assist the virus in infecting various types of immune cells with Fc receptor(FcR)and proliferating or inducing abnormal immune responses,that is,it cannot induce ADE effects through the FcR pathway. |
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