Prepartion And B Cell Epitop's Preliminary Identification Of Two Monoclonal Antibodies Against Pseudorabies Virus

Pseudorabies(PR)is an important infectious disease which caused by pseudorabies virus(PRV).Pseudorabies caused great economic losses in China as its outbreak recently.pseudorabies virus(PRV)belongs to the herpes simplex virus.The gB,gC and gE proteins are related to the virulence and immune protection closely.This study expressed PRV gB1,gB2,gC1,gC2 and gE recombinant proteins by prokaryotic expression system;two monoclonal antibodies were prepared successfully,which 2B3 aimed to gC protein,and another one 5C10 aimed to gE protein;B cell epitope of two monoclonal antibodies against pseudorabies virus were preliminary identificatd.1.The cloning and prokaryotic expression of pseudorabies virus envelop glycoprotein gB,gC and gEAccording to the gene sequences of PRV-ZJ01 gB,gC and gE,five pairs of primers were designed to amplify the gB1,gB2,gC1,gC2 and gE gene fragments by polymerase chain reaction.And the size of five gene fragments were 514bp,916bp,874bp,614bp,625bp separately.Then gB1,gB2 and gE genes were cloned into pET-32aM,gC1 and gC2 genes were cloned into pET-28a,resulting in the construction of recombinant plasmid pET 32aM-gB1,pET 32aM-gB2,pET28a-gC1,pET 28a-gC2 and pET 32aM-gE.Through the induction of these recombinant plasmids by IPTG,the recombinant proteins highly expressed in E.coli BL21(DE3).The result of SDS-PAGE showed that gB1,gB2 and gE were in the form of soluble supernatant,being approximately 40 kDa,40 kDa and 50 kDa individually.while gC1 and gC2 recombinant proteins were mainly in the form of inclusion bodies,about being 35 kDa and 37 kDa separately.Western blot results showed that gB1,gB2,gC1,gC2 and gE recombinant proteins could react with pig positive serum,which could be used as antigen of PRV antibody detection because of high reactivity.2.Prepartion and characterization of two monoclonal antibodies against pseudorabies virusThe purified virion of pseudorabies virus(PRV)ZJ01 strain were used to inoculate into Balb/c mice and two hybridoma lines 2B3 and 5C10 that can steadily secret monoclonal antibodies(McAb)against PRV were obtained by cells fusion and ELISA.Among them,2B3 McAb belons to IgG2a subtype,and 5C10 belong IgGl subtype,which light chains are ? type.IFA showed that they can react with PRV in BHK-21 cells.Western blot results showed that 2B3 is specific to PRV gC protein,and 5C10 is specific to PRV gE protein.The titer of ELISA antibodies were 1:8192 and 1:1:163840 respectively,and the cell stability were still fine after 15 passages.It is helpful to develop immunological method for rapid detection of PRV infections.3.B cell epitop's preliminary identification of two monoclonal antibodies against pseudorabies virusSeven gC gene segments and five gE genes segments were cloned into pET-3 2aM by polymerase chain reaction(PCR),His fusion protein used as a tag protein meanwhile.the recombinant proteins highly expressed in E.coli BL21(DE3)by IPTG induction.After identification of whether the Seven truncated gC protein and five truncated gE protein were successfully expressed by SDS-PAGE and Western blot,we used two monoclonal antibodies to determine epitops of PRV gC,gE protein by Western blot.Results indicated that 2B3 McAbs recognized the epitope which lay to 340aa-366aa,5C10 McAbs specifically recognized the epitope which lay to 195aa-207aa.These results provided a good foundation for study of gC and gE protein function.