Duck Plague Virus Ul-6 Gene Antigenic Domain Of The Prokaryotic Expression And Application

This article as to the UL6 gene(GenBank Accession No.EF055890) found by our research team has carried out the folloing series of studies:codon usage bias analysis of the DPV UL6 gene and epitope prediction of the polypeptide;clone,prokaryotic express of major antigen domain and antibody preparation;establish the UL6-M indirect ELISA method by utilizing UL6-M proteinum;prepare subunit vaccine using UL6-M proteinum and carry out experiment of infect protection.Establishment of indirect immunoperoxidase staining & indirect fluorescent antibody technique on account of UL6-M proteinum to the detection for Duck Plague Virus in the paraffin wax tissue splice;The monitorion of DPV UL6 gene products in duck tissues by artificial infecting DPV. The results are as follows:1.Using DNA Star Protean software,the UL6 gene amino acid of the duck plauge virus(DPV) CHv strain(GenBank accession number EF055890) was analyzed on secondary structure and hydrophilicity and antigenic index,with the B cell epitopes to be predicted.The results showed that the B cell epitopes of UL6 were situated in the C-terminal regions Thr507-Gln515,Thr521-Met529,Asp531-Phe539,Gly544-Asn562,Thr568-Gln585,Lys592-Val604,Ala681-Ala778 and Tyr780-Lys790. It was showed that the UL6 polypeptide chain was composed of 61 codes by codon analyzed,and the third codes prefer basic radical A and T.There was great diffrencecs between19 E colis,14 yeasts and 15 the human beings in frequency of code utilization.2.The genomic DNA of DEV CHv was extracted as PCR template,One pair of specific primers was designed by using Oligo6.0 software.A fragment coding for UL6 major antigen epitopes(1483-2385bp) was amplified by PCR technique and was expressed by prokaryotic expression.Western-blot analysis indicated that multiclone anti-serum of DEV had specific reaction with the recombinant protein.The expression product was purified successfully by passing the Ni~+ affinity chromatograph Collurmn using the recombinant protein with a tag of 6×His.The rabbit was immunized with the purified protein,and ELISA showed that the valence was up to 512000.3.Establish indirect ELISA method of detecting anti-DPV specific IgG antibody using the purified UL6M protein as antigen.It was established the best operating conditions by standardized research,The results showed that good sensitivity can be received with the coating concentration of UL6M antigen of 3.125μg each well,It revealed a negative reaction with positive sera of DHBV,DHV,RA,E.coli, SB and a positive reaction with DEV standard positive serum.The variation coefficient were within 10%.The results showed that the UL6M-ELISA method should be advanced by its high sensibility,strong specificity and good repeatability and could be used to identify the serum of DPV infection.4.Utilizing the UL6M protein obtained by genetic engineering technology to prepare subunit vaccine,infect protection experiment shows:after immuning recombinant subunit vaccine,about 7 days,the vaccine can stimulate organism producing antibody.when 14 days,the valence of antibody can reach the top about OD 2.535,the value is higher than the control group.Comparing to the attenuated vaccine group,valence variation of recombinant group similar to it,and sustain high level in 56 days.Recombinant protein group which was 50%(5/10) morbidity and 40%(4/10) mortality respectively,were significantly lower than the control group 100%(10/10),100%(10/10).This manifest UL6M has good immunogenicity,but lower efficient on protection compared to the attenuated vaccine group.5.By detecting DPV UL6 gene products in artificial infected tissues,dynamic state and distribution indicate:after infecting DPV 4 hours,UL6 capsid protein can express in the immune organ such as bursa of Fabricius,thymus et al.Then UL6 capsid protein can lasting express on the target cell of alimentary canal.Afer 3 days each organ tissue can detect the UL6 protein.We can conclude that UL6 gene belong to the first category in Late expression gene,and continually participate in the process of viral DNA replication.