Expression Of Monkey B Virus Glycoprotein D Gene And The Establishment Of Indirect ELISA

Monkey B virus is a member of the a-herpesvirus sub-family and is a common pathogen of macaque monkeys.The infection rate of B virus in its natural host is greater than60%,but infection in macaque monkeys is frequently asymptomatic.However,the infection is usually fatal in human beings(and other unnatural host),the case fatality rate in untreated human BV infection has been greater than80%, survive will leave aphasia,paralysis and other serious neurological sequelae.Epidemiological investigations revealed that most cases of human B-virus infection have involved direct contact with macaques,such as a bite,scratch.Human-to-human transmission has been documented in one case. Once has clinical signs in primates and monkeys indicates that the virus has invaded the nervous system,treatment will lose its significance.It is important to establish rapid diagnosis method for early diagnosis and therapy of monkey B virus infection.Monkey B virus is similar to the herpes simplex viruses (HSV-1and HSV-2) of humans,the extreme cross-reactivity of primate alphaherpesviruses has required the development of diagnostic methods that can differentiate between HSV and B-virus infection.By the impact of HSV and monkey B virus infection characteristics and other factors,the monkey B virus detection methods reach the purpose of quick and specific.Commonly using of baboon herpes virus (Herpesvirus papio2, HVP-2) and simian agent8(SA8) and HSV and so on other viral proteins as a diagnostic antigen detection of monkey B virus in clinical medicine, but its sensitivity is low.Researcher developed the monkey B virus inactivated vaccine,but the vaccine protection rate is less than50%.Recently,with the deepening of the structural studies of monkey B virus,especially the completion of the whole-genome sequencing,the monkey B virus glycoprotein is becoming the main target of the monkey B virus diagnosis and prevention research.Glycoprotein D and B are the important target of the immune response to alpha-herpes virus infection in primates,gD and gB antibodies first appeared in the serum of acute or recurrent infected human HSV and reached very high titers,and can be maintained for at least2years. gB is a main reasons that lead to antigenic cross-reactivity among of primate herpes virus,the amino acid sequence of the gD is relatively conservative,and inferred monkey B virus gD can stimulate its body to produce specific antibodies.View of the monkey B virus glycoprotein the important role in the differential diagnosis process,we started works as following:(1)Compared the homology of monkey B virus and HSV)-1and HSV-2glycoproteinprotein of B, C and D,analysising of monkey B virus gD gene sequence;(2)Cloning gD extracellular region gene which from the infected tissues of the rhesus monkey B virus in China source,and analysis its sequence features;(3)The prokaryotic expression of Monkey B virus gD gene;(4)Using purified gD recombinant protein as coating antigen, establishment of Indirect ELISA for detectig of monkey B virus.1. Bioinformatic analysis of Monkey B virus glycoproteinUsing DNAMAN to compare the homology of monkey B virus glycoprotein(gB, gC and gD)with HSV-1and HSV-2,the homology of monkey B virus gB and HSV-1and HSV-2are78%,77%,gC and HSV-1and HSV-2are42%,48%,gD and HSV-1and HSV-2are56%,58%. Studies have shown that gB is a main reasons that lead to antigenic cross-reactivity among of primate herpes virus,amino acid sequence of the gD is more conservative.The researcher of our lab had get the recombinant glycoprotein C and establish of gC-ELISA,the sensitivity and specificity were94.1%and100%.Therefore B virus glycoprotein D as the object of the present study.Using bioinformatics analysis the structure of monkey B virus gD gene,hydrophilic,flexible,structure regions,antigenicindex and B-cell epitope,the signal peptide of the protein is located in the N-terminal1-24amino acids,25-341amino acids in the N-terminal extracellular region,transmembrane region is located in the N-terminal342-364amino acids,the intracellular domain is located in the N-terminal365-394amino acids.Flexible regions of the protein molecules evenly distributed,the hydrophilic is well and the surface probilitity plot is higher.The average antigen index of section24to44,49to76,107to118,141to150,164to174,206to214,287to308,322to339and365to382are0.983,1.854,0.901,1.763,1.62,1.1,2.028,0.979,0.676and1.148, these data imply the possibility of the existence of B-cell epitope in these section.Finally,we choice the monkey B virus gD extracellular region as a candidate fragment.2. Expression of Monkey B virus glycoprotein D gene and its characterization analysisAccording to the NCBI published standards strain of monkey B virus E2490genome sequence,monkey B virus glycoprotein D gene extracellular domain as the purpose fragments,analyzing the sequence of restriction sites,found that the sequence exists203common enzymes, including SmaⅠ, SacⅡ, NdeⅠ, and the HindⅢ58enzyme in the sequence has not exesit,we choice NdeⅠ and the HindⅢ as restriction sites,design specific primers, PCR amplification the target gene and cloned into the pGM-T vector,and sent for sequencing after PCR and restriction enzyme digestion.The PCR amplification of the target gene about1000bp,further analysis of the target sequence found that the homology of amplified fragment and B virus standard strain E2490is97.21%,GC content up to75%and showed regional concentrated,there were20rare codons in the sequence,accounting for5.99%.The comparison of Monkey B virus gD with other species,revealed that gD of Monkey B virus was most similar to BV-MC1gD gene with98.6%identity,and shared lowest homology61.7%with HSV-1gD gene. By analysis of phylogenetic trees, we found the evolutional relation of gD of Monkey B virus and BV-MC1gD gene was very close, while it had a relatively low evolutional relation with Felis catus. HSV-2333gD gene.3. Recombinant expression of Monkey B virus glycoprotein D geneThe extracellular domain gene of Monkey B virus glycoprotein D was amplified by PCR and cloned into the prokaryotic expressive vector pET-28b(+) after PCR,restriction digestion and DNA sequencing.Recombinant plasmid transfected into E.coli BL21(DE3) and protein expression was induced by IPTG.The recombinant protein had a Mr of48ku,accounted for35%total proteins,and expressed in form of inclusion body.The recombinant protein was purified by using AKTA protein purification system,after dialysis to remove urea and imidazole.The purified recombinant proteins by SDS-PAGE electrophoresis,using BIO-RAD semi-dry transfer system to transfer membrane (PVDF).Western blot showed that the recombinant protein could react specifically to the monkey B virus positive sera and anti-histidine monoclonal antibody.It indicate that recombinant glycoprotein D from prokaryotic expression system had perfect reactionogenicity,which would can be used as the candidate antigen of monkey B virus serological testing.4-The establishment of gD-ELISA for detecting antibodies of monkey B virus infectionUsing recombinant gD protein as the coated antigen,establishment of indirect ELISA,which for detecting antibodies of monkey B virus infection.The ELISA the optimizated conditions are antigen concentration2μg/mL,serum dilution of1:8, the blocking solution of PBS containing8%rabbit serum,37℃dark color for12min,rabbit anti-monkey IgG-HRP diluted1:25000;positive critical value of the OD450nm≥0.166. Established gD-ELISA specificity is well,can effectively distinguish between BV and of HSV.Using established monkey B virus gD-ELISA and gC-ELISA established by Wang daiping in our lab,and the "gold standard"—dot immuno-binding method(DIA, American the VRL Laboratory) detected166(90Beijing, Sichuan76) monkey serum samples,the positive rates were12.65%,13.25%and12.05%,respectively.Among the positive rate of90monkey serum samples from Beijing were13.33%(12/90),13.33%(12/90) and12.22%(11/90),respectively,the positive detection rate of76samples from Sichuan were11.84%(9/76),13.16%(10/76)and11.84%(9/76),respectively.Taking gD-ELISA and the "gold standard" DIA as screening test evaluation,the authenticity of assessment results of the screening test gD-ELISA sensitivity and specificity were95%and97.26%,Youden index was0.9226,indicating that the established monkey B virus gD-ELISA and Monkey B virus "gold standard" DIA were in line with a higher degree. Reliability evaluation of screening test results is observed consistent with rate Po was98.19%,opportunities consistent with rate Pe was78.35%and Kappa value was0.92,indicating well consistency of monkey B virus "gold standard" DIA and established gD-ELISA.