Homology Modeling Of Variable Regions Of Monoclonal Antibody 2E4 Against ORFV And Gene Editing Experiments On B2L In Vitro

Contagious ecthyma(CE)known as Orf is a viral infectious diseases caused by orf virus(ORFV)belonging to Parapoxvirus genus of Poxviridae family.The ORFV has a world-wide distribution and causes contagious skin diseases affecting sheep,goats and other small ruminant animal,sometimes even including humans.Of late,there have been an increasing number of reports on ORFV and its pathogenic factors customized has appeared to become hotspots because of the potential threat to animal husbandry and the risk of zoonotic infetion.B2 L gene is an irreplaceable key gene and always attracts more and more researchers which pay close attention to it.The B2 L gene is located at the central conserved region of ORFV viral genome and acts as major protective antigen gene of Orf.The gene encoding envelope proteins of ORFV have been identified.Cloning and expression of the ORFV gene has shown that both antibody and T-cell responses are directed.It's essential for spread and infection of the virus,even specific for the extracellular enveloped form of the virus.The B2 L gene has been used for detection of orf virus by PCR.PCR based detection of the B2 L gene has been the most widely used approach to diagnosis of Orf and this method has shown good sensitivity and specificity.Nevertheless,investigation of the role of B2 L in nucleic acid structure and function has been rarely reported.Currently,we attempt to verify it by using a novel methods for gene editing in vitro and in vivo.Firstly,we analyzed and determined complementarity determining regions(CDRs)of heavy chain variable region and light chain variable region of monoclonal antibody 2E4 against B2 L protein with a conformational epitope respectively.In the molecular level,accouplement and specificity between B2 L epitope and CDRs were investigated by homology modeling method and amino acid substitution.Briefly,after amplification and sequencing in vitro,the amino acid structure of the CDRs and the framework regions(FRs)in the variable region of 2E4 antibody were provided by Ig BLAST software followed by molecular modelling for exhibition of the three dimensional spatial configuration of antibody light and heavy chain using available SWISS-MODEL homology modeling method,and the possible binding site(Pocket)of B2 L antigen epitope polypeptide was simulated.The importance of B2 L antigen binding to 2E4 was verified subsequently by amino acid mutation in CDRs regions.Secondly,CRISPR/Cas9 system was used for editting the B2 L gene in vitro and in vivo.In order to verify the feasibility of the g RNA sequence in our constructed system,several specific targeting g RNA sequences were designed and transcripted into single stranded m RNA(sg RNA)in vitro.B2L-p ET-32 a recombinant plasmid was cleaved expectedly in an in vitro CRISPR digestion assay.Then the plasmids of PMJ920 and g RNA1-p GEMT-easy were co-transfected into HEK293 cells,and finally,the proliferated virus variants were isolated by plaque purification technology.Sequencing analysis showed that the 5' terminal near to the PAM sequence(CGG)in which the fifth bases cytosine(C)was mutated into guanine(G)in one of the mutant strains,but the serine(S)was stabilized.Contrastly,four-base deletion was found in another strains(named OV_HLJ-?B2L),leading to the subsequent disruptive expression of the amino acid sequence such as appearance of the terminator in OV_HLJ-?B2L.It was confirmed that OV_HLJ-?B2L could not be recognized by 2E4 monoclonal antibody during the indirect immunofluorescence.When ORFV of the OV_HLJ-?B2L strain was inoculated into the sheep experimentally,the lesions of Orf were found alleviative significantly compared with that of the wild-type virus.In summary,we exhibited the three dimensional spatial configuration of the heavy chain variable region and light chain variable region of monoclonal antibody 2E4 by using available SWISS-MODEL homology modeling method,and simulated the possible binding site(Pocket)of B2 L antigen epitope polypeptide,then identified the correspondence and specificity between B2 L epitope and the CDRs.More importantly,in this study,except for the emergence of escaping viruses,we have operated on the B2 L gene by using CRISPR/Cas9 technology and isolated the OV_HLJ-?B2L strains with the disturbed B2 L gene.It should lay the foundation for the application of B2 L gene and the study of other functional genes of ORFV in the future.