Establishment Of Dot Blot Hybridization Detection Method For Duck Tembusu Virus And Prokaryotic Expression Of E Protein

Duck Tembusu virus disease is a kind of infectious disease that uses Duck Tembusu virus(DTMUV)as a pathogen.It has been reported that DTMUV is associated with slow development,decreased egg production,paralysis and other clinical symptoms.TMUV belongs to the Flaviviridae,Flavivirus,Entaia virus population and it poses a potential health threat to mammals including humans.Establishing a rapid,specific and sensitive diagnostic method for TMUV and developing a new vaccine with high protection efficacy are the most effective measures for preventing and controlling the epidemic of this disease.In this study,firstly we established a nucleic acid dot blot hybridization method,which has a character of the unique specificity and sensitivity,based on the TMUV NS5 gene.Subsequently,this method was used to detect the suspected tissue samples,and a TMUV strain SD1601 was isolated from the positive tissue samples.Finally,the TMUV E protein prokaryotic expression plasmid was constructed and the GST-E fusion protein was obtained through the E.coli expression system.1.Establishment of TMUV nucleic acid dot blot hybridization methodIn this study,according to the character of the gene sequence of the virus NS5,the digoxin was used to prepare the DNA probes,and a nucleic acid hybridization detection method for TMUV virus nucleic acid was established.Primers 5.0 software was used to design the primers and the digoxin labeling kit was used to prepare NS5 nucleic acid probes by PCR amplification.The results showed that the NS5-based nucleic acid dot blot hybridization method established in this study has good specificity and sensitivity.The method can specifically detect TMUV nucleic acid,and is negative for common viral nucleic acid samples such as DPV,AIV,NDV,etc.The minimum detectable amount is 10 pg,which is higher than conventional RT-PCR method.Subsequently,a total of 40 tissue samples were collected from the duck farms in Shandong Province were detected by the nucleic acid dotblots in this study.Four positive samples were detected.The results were consistent with classical RT-PCR methods.The nucleic acid dot blot hybridization method established in this research has laid the foundation for the prevention and epidemiological investigation of duck Tembusu virus disease.2.Isolation and identification of TMUV SD1601 strain and analysis of its whole genome sequenceThe spots that were positive for spot hybridization were collected,ground,filtered,and inoculated into the BHK-21 cells.One strain of TMUV field virus was isolated and identified which was named SD1601 strain.By amplifying and analyzing the whole genome sequence of SD1601 strain,the results showed that the genome of the SD1601 strain virus contained an open reading frame,and the encoded polyprotein was composed of 3426 amino acids.The nucleotide sequence homology and the phylogenetic tree of the TMUV genome of SD1601 strain with the reference strains were compared and analyzed.The phylogenetic tree showes that the strain SD1601 is belonged to Ia branch,The complete genome sequences of the SD1601 shares 96.9%-99.4% nucleotide identity with the reference strain in this study.and it shows the highest nucleotide identity of the strains JS804 and BYD-1,which is 99.4%.The homology with HZ3-2015 was the lowest,only 96.9%.E protein homology comparison results showed that SD1601 strain protein and SDHS,SDMS,TA2010 three strains E protein gene homologous highest homology,but shows the lower identity with the classical strains which were isolated in the early.The isolation and identification of SD1601 strain provides a theoretical basis for further understanding of the genetic evolution of TMUV.3.Prokaryotic expression of E protein of TMUV SD1601 strainThe full-length E gene of TMUV SD1601 was amplified and cloned into the prokaryotic expression vector pGEX-6P-1.After correct identification,it was transformed into competent cells of the recipient strain E.coli BL21(DE3),induced by IPTG and expressed.The soluble proteins were analyzed by SDS-PAGE electrophoresis.To determine the optimal induction conditions by varying the induction time,the induction temperature,and the IPTG concentration,we usually perform purification analyses on soluble proteins with the help ofglutathione gel resin and validated by western-blot.Results showed the E protein of SD1601 was successfully expressed.The temperature of the induction was 30°C,the concentration of the IPTG was 0.8 mM,and the time of the induction was 4 hours.The recombinant protein was present both in the inclusion body and the supernatant.We purified the soluble GST-E protein by the method described above.This protein is specifically recognized by anti-TMUV positive antibodies.The expression of TMUV E protein laid the foundation for the development of ELISA assays and vaccines.