Preparation And Biological Activity Study Of Fully Humanized Antibody Against Porcine Lysi

Streptococcus suis（S.suis）is an important zoonotic pathogen.The sporadic cases have been documented since 1968,patients mainly manifesting as meningitis.However,streptoccal toxin shock-like syndrome（STSLS）was found in Sichuan epidemic,China,causing 38 death out of 200 patients in 2005.The research have proved that the outbreak strain is ST7 S.suis,and the patients with STSLS were observed higher level of pro-inflammatory cytokine,such as IL-6,TNF-αthan those of meningitis patients.Previous findings have revealed that suilysin（SLY）activation of NLRP3inflammasomes leading to a cytokine storm may be the main pathogenic mechanism for STSLS occurrence.Antitoxins played an important role in the treatment of bacterial infections in pro-antibiotics era,saving countless lives.The antitoxin products against tetanus,botulinum,and anthrax are still available in clinic so far.Owing to the nature of STSLS patients as acute onset,short hospitalization time and high mortality（up to 81.3%）.It is necessary to develop some specific drug to block cytokine storm caused by in S.suis.The goal of this study was to raise fully human antibody by immunizing the transgenic mouse CAMouse with r SLY^P353L,a non-toxic mutant of SLY.And its physical and chemical characteristics,epitope recognition,neutralization and mechanism of action were studied.The main research methods and results are as follows:1.Preparation of fully human m Ab and their physicochemical characteristicsPurified with Ni-NAT agarose media column the proteins r SLY^P353Land r SLY.With r SLY^P353Limmunotransgenic mouse CAMouse,splenocytes and myeloma cells SP2/0electrofusion after electrofusion on HAT selective semi-solid medium,126double-positive hybridoma cells were screened by indirect ELISA and hemolytic suppression tests.The type of antibody is identified by the double antibody sandwich method,and the light chain belongs to the human kappa chain.The results of heavy chain detection showed that 102 strains were murine Ig M,5 strains of murine source Ig G,5 strains of human Ig G.The numbers of hybridoma cells producing fully human monoclonal antibodies are:1-2C,1-10F,2-2G,4-2G,and 5-4G,respectively.Indirect ELISA determined the subtypes of 1-2C is the Ig G1 subtype,and the remaining four subtypes are Ig G3.The hybridoma cells were cultured by serum-free medium Hyber TM-B100.The m Ab 1-2C was purified with Prism A affinity chromatography column,and the remaining four m Abs were purified with Protein L.The apparent dissociation constant（K_d）values of the indirect ELISA determination of m Abs 1-2C,1-10F,2-2G,4-2G,and 5-4G were 0.87 nmol/L,0.11 nmol/L,1.19 nmol/L,2.32 nmol/L,and 53.25nmol/L,respectively,and the dissociation equilibrium constant（K_D）values of the m Ab5-4G and r SLY by biofilm interference technique were 1.25×10^-8mol/L,and the remaining m Abs were K_D<1.0×10^-12mol/L,the results show that m Abs have high affinity.2.Monoclonal antibody sequences and the predicted recognition domainThe Fab segment sequences of m Abs 1-10F,2-2G,4-2G,5-4G were amplified by RACE technology,the V_H+C_H1 amplification sequences were about 500 bp,the V_L+C_Lamplification sequences were about 550 bp,and the measured nucleotide sequence was compared by IMGT/V-QUEST to prove that the heavy chain and light chain of several m Abs were in line with the characteristics of human Ig G,and the length of the V_Lsequence was 333 bp and 333 bp,333 bp,306 bp,V_Hsequence lengths of 360 bp,306bp,306 bp,372 bp,respectively.MAbs 1-10F,2-2G,4-2G V_Land V_HCDR1,CDR2,CDR3 have the same number of amino acid residues,[11.3.8]and[10.9.10].Comparing the V_Land V_Hamino acid sequences of several m Abs with Clustalx software,in addition to the m Ab 5-4G V_L,the V_Land V_Hsequences of 1-10F,2-2G,4-2G are more than 95%.The reactivity of m Abs 1-10F,2-2G,4-2G,and 5-4G with r SLY and r SLY D4was detected by indirect ELISA method and Western-Blot,and the results showed that5-4G recognized the domain 4,and the remaining three antibodies recognize the domains 1-3.3.The neutralizing effect and mechanism of fully human genetically engineered antibodiesThe antibody expression plasmid was constructed with pc DNA3.4,and purified to genetically engineered antibodies（GEAb）1-10F,2-2G,4-2G after co-transfection of HEK293F cells,and the K_dvalues of three GEAbs and r SLY were measured by indirect ELISA method to be 0.18 nmol/L,0.61 nmol/L,and 7.78 nmol/L;biofilm interference technology determined the binding kinetics of antibodies to different concentrations of r SLY,when the r SLY concentration was greater than 125 nmol/L,respectively.K_D<1.0×10^-12mol/L,all with high affinity.The neutralization of antibodies was explored at the level of erythrocytes and HEK293T cells,and the results at the erythrocyte level showed that when the concentration of r SLY is 1 nmol/L and the concentration of 3 strains of GEAbs is 5.3μmol/L,more than 60%of erythrocyte lysis can be blocked.At the HEK293T level,when the concentration of r SLY is 3.26μmol/L and the concentration of antibody is 6μmol/L,1-10F is capable of neutralizing 50%of HEK293T cell lysis,but 2-2G,4-2G behave as weak neutralizing activity.SDS-AGE detection showed that no hyperpolymer bands appeared on the erythrocyte membrane after binding to the antibody,but rather a dimer and oligomer.In summary,this study screened five strains of anti-suilysin fully human m Abs by immunizing the humanized antibody transgenic mouse CAMouse with r SLY^P353L,of which five strains had high affinity.Three antibodies recognize suilysin domains 1-3and 1 strain recognizes suilysin domains 4.There are differences in the sequence of the variable region of several strains of antibodies,and the three strains of fully human GEAbs constructed from antibody variable region genes also have a neutralizing effect and affect the formation of prepore complex on the surface of the red blood cell membrane by suilysin.