Preparation and antigenic epitope mapping of a specific monoclonal antibody against glycoprotein E2 of classical swine fever subgenotype 2.1 field strain
In order to develop the differential monoclonal antibody(MAbs)that specifically reacts with the dominant subgenotype 2.1 field strains of classical swine fever virus(CSFV)in China and to reveal the antigenic difference of E2 protein between the vaccine C-strain and the field strains,this study used subgenotype 2.1 recombinant plasmid pFastBac1-2.1 JL23 E2 by baculovirus expression recombinant E2 protein(rE2).The efficiency of expression and purification were assessed by SDS-PAGE following Ni column purification,and its reactivity was further evaluated using western blot analysis.The findings from both methods confirmed the expression of the subgenotype 2.1 CSFV E2 protein with commendable purification efficiency and reactivity.Monoclonal antibody(MAb)specific to the E2 protein of 2.1b subgenotype CSFV was prepared by hybridoma technique.79 strains of CSFV subgenotypes 1.1,2.1,2.2 and 2.3 were inoculated with PK-15 cells,and the reaction patterns of MAb to different genotypes of CSFV tested by IFA after 72 hours.The reactivity between MAb and 18 E2 proteins derived from CSFV belonging to 10 subgenotypes were verified by western blot.The MAb was subsequently purified through affinity chromatlography,and its concentration was precisely determined using BCA method and detection subclasses of MAb.The immunofluorescence assay(IFA)was used to identify the neutralizing activity of MAb against the diverse genotypes of CSFV.Following IFA identification,the positive nderwent additional subcloning,leading to the acquisition of 16 hybridoma cells that secreted the E2 MAb,The IFA results singled out one particular MAb,TCH061,which exhibited a unique reactivity profile,binding exclusively to cells infected with all variants of subgenotype CSFV 2.1(2.1a,2.1b,2.1c,2.1g,2.1h,2.1i,2.1j,2.1k,2.1l,2.1m,2.1n),while showing no reaction to the vaccine strain or other subgenotype CSFV.Western blot results showed that the TCH061 could specifically recognize the E2 protein subgenotype of CSFV 2.1(2.1a,2.1b,2.1c,2.1g,2.1h,2.1i,2.1j),with a specific band at 90ku,while it remained unreactive to E2 proteins from other genotype CSFV.The results indicated that TCH061 was a specific MAb of CSFV E2 protein of gene subgenotype 2.1.The purification of TCH061 yielded distinct bands at 50ku and 25ku,with the MAb concentration quantified at 1.70μg/μL.The MAb TCH061 heavy chain was IgG2a,and the light chain was kappa chain.The IFA results of MAb neutralizing activity showed that TCH061 had certain neutralizing activity against JL23 strain(2.1b)and GDLF1 strain(2.1c),but could not neutralize C strain.Further exploration into the antigenic epitopes of the E2 protein was conducted using truncated proteins from four different regions of the GD53 strain as antigens,with the reactivity being preliminarily mapped by western blot.Utilizing CLC Sequence Viewer and SWISS-MODEL,we compared the amino acid sequences of the CSFV E2 protein reactive to the MAb,predicting key amino acid sites for antigen recognition.Through a series of point mutations in the E2 protein of the CSFV JL23 strain expressed by baculovirus,the reactivity of the mutated E2 protein with MAb TCH061 was identified by western blot.The results showed the epitope recognized by TCH061 within the first 90 amino acids of the E2 protein.Amino acid sequence alignment within this region showed that TCH061 recognized P20,but not recognized L20 in E2 protein of CSFV,and I18,G19,P20,L21,G22,A23 and E24 were spatially similar predicted,and MAb TCH061 reacted with the I18M mutant E2 protein of JL23 strain,and did not react with the E2 protein mutated in G19K,P20L and L21P,and was weakly reactive with the E2 protein with the G22K mutation,but did not react with the E2 protein with the HCLV L20P mutation.It was confirmed that the amino acid sequence of TCH061 to recognize the epitope of E2 protein was 19GPLG22,which was determined as a spatial conformational epitope by PyMOL structural simulation.Except for aa20,other sites were very conserved in each genotype CSFV,indicating that P20 was a key amino acid for MAb TCH061 to recognize the epitope of E2 protein of subtype 2.1 CSFV field strains.In summary,this study obtained a MAb TCH061 for the first time to distinguish between CSFV vaccine strain and CSFV subtype 2.1 field strain,revealing the difference in antigenic amino acid sites of E2 protein between vaccine strain and field strain,and providing an important MAb resource for the development of serologic identification detection methods for swine fever vaccine and CSFV field strain infection.
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