| Herpes B virus is a zoonotic disease caused by double-stranded enveloped DNA virus taken cercopithecidae as its natural host.Generally,the clinical symptoms in macaques post BV infection are mild or inapparent.Sometimes,symptoms were presented as oral herpes lesions such as oral ulcers,conjunctivitis or gingivitis,and then developed into chronic infection.The virus was discharged primarily in saliva or genital tract secretions,transmitted to human being through bitten or scratched by infected monkeys.The mortality rate of infected human could upto more than 70%with the fatal encephalitis and encephalomyelitis.Upto now,there is no any effective treatment for BV infection.Due to the high death rate of BV infected person and non-macaque primate,BV is a potential harzard for animal breeder or related investigators.Among the various proteins encoded by monkey B virus,gD,a conserved structural protein,is population and type-specific.During the evolution of the virus,mutation rate of it is relatively low,at the same time,it carries the group-specific antigenic sites,and can induce the production of group-specific antibodies against the virus,which harbors the important application value for the serological diagnosis of frequent variations of the monkey B virus.Group-specific gD of BV was expressed in E.coli by a recombinant vector in this study.Five McAbs against group-specific antigen gD of BV were prepared.This study paves the way for effective and quick diagnosis reagen research.The main contents and results are as follows:1.Expression and antigentic characterization of the major envelope protein gD of Herpes B virusWe synthesize published BV strain E2490 complete sequence with DNA synthesizer of Suzhou Jin Weizhi Biological Technology Co.Ltd and analysis the gene sequence which encode gD protein,preference codon optimized according to E.coli codon bias.pET-32a was selected as the prokaryotic expression vector to express the synthesized fragment.By PCR,resistance screening,restriction endonucleases and sequencing confirmation,the positive clones with correct open reading frame were selected.The recombinant expression vectors were transformed into E.coli Rosetta(DE3)and induced by IPTG.And the higher expression vector pET-32a/gD was screened.The His-tagged recombinant protein was purified using a His-tag affinity chromatography column on Ni2+-nitrilotriacetate(NTA)resin.The results of SDS-polyarcylamide gel electrophoresis(SDS-PAGE)and Western-blotting revealed that the recombinant fusion protein was expressed with a molecular mass of approximately 53kDa.The indirect enzyme-linked immunosorbent assay(ELISA)indicated that the expressed BV gD was highly immunogenic.2.Preparation of Monoclonal Antibodies against serogroup-specific antigen gD of Herpes B virusBy the prokaryotic expression of Herpes B virus envelope protein(gD)as immunogens,were purified and used to subcutaneously immunize BALB/c mice in this study.Then splenocytes from the immunized mice were fused with SP2/0 myeloma cells.The recombinant gD were coated as the detection origin and indirect ELISA was used to screen positive hybridoma clones.Limited dilution method was performed to subclone the positive clones.After three cycles of subcloning,five McAbs against the gD of BV were obtained,and designated as 4E3,3E7,3F8,1H3 and 4B6.The ELISA titer of culture supernatant were1:640,and ascites were 1:2×106,1:2×105,1:2×105,1:2×103 and 1:2×102,respectively.Subtype identification results showed that monoclonal antibody 1H3 and 4E3 were IgG2b subtype,monoclonal antibody 3E7,3F8 and 4B6 were IgGl subtype,light chains of them were all kappa chain.SDS-PAGE analysis showed that five strains of monoclonal antibody could have two distinct bands after purification.The indirect immunofluorescence analysis showed that five McAbs could react specifically with the gD homologous protein in Vero cells infected with HSV-2 and the Western-blotting analysis showed that 3E7,3F8 and 4E3 McAbs could react specifically with the gD homologous protein in Vero cells infected with HSV-2 but 4B6 and 1H3 can not.Which indicating that 3E7,3F8 and 4E3 mAbs could bind with the natural BV gD protein specificly,while 4B6 and 1H3 may only recognize gD protein conformational epitopes or recognize as BV gD protein of HSV-2 differs form the segment of the virus that has a strong specificity to BV.Our research prepares abundant recombinant BV-gD protein antigen,and the ELISA experiments show that there is a specific reaction with an infected monkey serum BV,the total coincidence rate is 75%,and this has significant reference on the research of BV detection kit.The prepared monoclonal antibody can provide basis and a powerful tool for further research of the characteristics and function of envelope protein and its antibody. |
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