Bioinformatics Analysis And Monoclonal Antibody Preparation Of Porcine Acute Diarrhea Syndrome Coronavirus M Protei

A new swine enteric coronavirus has emerged referred to as swine acute diarrhea syndrome coronavirus(SADS-CoV),which can cause swine acute diarrhea syndrome(SADS).The clinical manifestation is acute gastrointestinal symptoms of newborn piglets,and the mortality is more than 90%.Like other coronaviruses,the virus has four main structural proteins: spike(S),envelope(E),membrane(M),and nucleoprotein(N),and the M protein is a that performs a wide range of biological functions.It’s an important structural protein.However,few reports on the biological characteristics and related research of SADS-CoV-M protein.Based on this,in order to clarify the biological function of the SADS-CoV-M protein,In this study the SADS-CoV-GDLS-2017 M protein was systematically analyzed using bioinformatics methods.The results showed that SADS-CoV-M protein amino acid mutation is less,highly conserved,and stable hydrophilic,containing 3 potential N-glycosylation sites and long transmembrane regions,with an average antigen index of up to 0.6268 and 7 epitopes.The proportion of β-turn and irregular curly structure conducive to antibody chimerism reached44.73% in the secondary structure.A total of 8 linear and 5 discontinuous epitopes were generated in the mapping of tertiary structural protrusion epitopes,with scores above 0.8 and stable binding of TLR-3.The results showed that M protein was a good candidate protein for SADS-CoV vaccine development,antiviral drug design and detection method establishment.In order to further study the SADS-CoV-M protein,In this study,the SADS-COV-M protein was prokaryotic expressed,and the recombinant M protein was analyzed by SDS-PAGE,gel cutting purification,concentration determination and Western blot identification.The results showed that the recombinant M protein was mainly expressed in the form of inclusion bodies,and the stripe was single after the purification of the cut gum,and the concentration and purity were high,Moreover,it could react specifically with goat anti-mouse primary antibody labeled with His and the target band appeared at 26 KDa.The results showed that the recombinant SADS-CoV-M protein was successfully expressed in this experiment.In this study,two monoclonal antibodies with specific and stable secretion against SADS-CoV-M protein were prepared by hybridoma technique using purified recombinant SADS-CoV-M protein as immunogen,named 62F7 and 62E8 respectively.Antibody chromosome,titer and subclass identification results showed,The number of chromosomes is 103 and 104,the potency is higher than 1:12800,the heavy chain subtype is Ig G2 b,and the light chain is κ chain.The Western blot identification results showed that none of them reacted with PEDV,but only with the recombinant SADS-CoV-M protein.The IFA identification results showed that both 62F7 and 62E8 had specific reactions with SADS-CoV strains..In conclusion,In this study,the SADS-CoV-M protein was successfully bioinformatics analysis and prokaryotic expression.Two monoclonal antibodies with good immunogenicity and specificity were successfully prepared by hybridoma technique,It provides a material basis for the development of SADS-CoV vaccines,the design and detection of antiviral drugs..