| Duck virus hepatitis(DVH)is an acute,highly contagious and fatal disease in young ducklings,which mainly infects duckings under 3 weeks and has been widely popular in many areas of the country.It has been a severe disease threaten to duck industry.Duck tembusu disease is a rapidly spreading acute infectious disease that can lead to nerve symptoms and severe egg drop syndrome.Since 2010 the outbreak of it has affected major regions in China and has caused a serious economic loss.Currently,the antibody detection of DHAV(Duck hepatitis A virus)and DTMUV(Duck tembusu virus)mainly depend on neutralization test and enzyme-linked immunosorbent assay(ELISA)test which took more time and efforts to detect samples in batches.Therefore,it is urgent to develop an efficient and rapid detection method for the antibody detection of multi-pathogen.VP1 and E genes are the primary antigen genes of DHAV and DTMUV,respectively.This experiment mainly studied the expression of the VP1 gene of DHAV-1 and the E gene of DTMUV in BL21 of Escherichia coli,respectively.We proposed to develop a liquid-phase chip detection method with bacterially expressed recombinant viral protein as antigen that can simultaneously detect antibodies against DHAV-1 and DTMUV,respectively,to develop a liquid-phase chip detection method.This research mainly includes the following three contents: 1.Isolation and identification of duck hepatitis A virus & sequence analysis of VP1 geneA total of 8 liver tissues were collected from ducks infected with duck hepatitis A virus in Shandong,Henan and other areas in 2016,and were inoculated with duck embryo after grinding,freezing and thawing,and diagnosis by neutralization test.The result showed 2 strains of isolated virus could be neutralized by the positive serum of duck hepatitis A virus type 1,6 strains of isolated virus could be neutralized by the positive serum of duck hepatitis A virus type 3.So there were 2 strains of virus isolated from duck hepatitis virus type 1,and 6 isolates of virus isolated from duck hepatitis virus type 3 by preliminary diagnosis.The DHAV-1 and DHAV-3 were amplified by RT-PCR respectively with the designed specific primers.The purified VP1 type 1 and VP1 type 3 genes were cloned into pMD18-T vector,respectively,to construct the recombinant plasmid of pMD18-T-1VP1 and pMD18-T-3VP1.Using program MegAlign of DNA star software,sequence alignment analysis of 1VP1 gene and 3VP1 gene of DHAVwas carried out.We found that the homology of duck hepatitis A virus type 1 isolated in 2016 1-JSDH/16 strain and 1-HZYC/16 strain with classical strain 1-R85952/55 were 93.8% and 93.4% and varies from the lowest 92%~ maximum 94.5% with other isolates;the homology of 3-GD/16 strain,3-HNSQ/16 strain,3-SDGT/16 strain,3-SDXD/16,3-SC1/16 and 3-SC2/16 strain with classical strain AP04114/03 were 92.1%,92.5%,92.4%,93.5,92.4 and 92.4% and varies from the lowest 90.1%~ highest 99.7% with other isolates;two phylogenetic trees indicated that 1-JSDH/16 strain and 1-HZYC/16 strain were in the same branch,and were closely related to the epidemic 1-CS/14 strain in recent years,and were relatively far with the classical strain 1-R85952/55;3-GD/16 strain,3-SC1/16 strain and 3-SC2/16 strain had the closest relationship in the same small branc;3-HNSQ/16 strain and 3-SDGT/16 strain were in the same branch,the 3-SDXD/16 strain was isolated in a lonely branch,and was far away from the classical strain 3-AP04023/04.2.Prokaryotic expression of pCold-1VP1 and pET-28a-EClone the purified 1VP1 gene into the prokaryotic expression vector of pCold-III,and obtained pCold-1VP1 with authentic sequence and orientations,and the recombinant plasmid containing the 1VP1 gene was inserted into the correct reading frame,and the recombinant plasmid pCold-1VP1 was obtained.Nde I and Hind III were used for double enzyme digestion,and two bands were detected by agarose gel electrophoresis,which were about 4,377 bp and 714 bp respectively,which was in line with the expected size.Extracting the RNA of duck Tembusu virus(DTMUV)with specific primers for RT-PCR amplification,we can obtain the E gene with 1503 bp.The purified E gene was cloned into pMD18-T vector to construct the recombinant plasmid of pMD18-T-E,and then inserted into the prokaryotic expression vector of pET-28a(+)to screen the positive recombinant plasmid pET-28a-E.EcoR I and Xho I were used for double enzyme digestion,and two bands were detected by agarose gel electrophoresis,which were about 5,285 bp and 1,503 bp respectively,which was in line with the expected size.The pCold-1VP1 and pET-28a-E recombinant expression vector were transformed into BL21(DE3).DHAV-1VP1 and DTMUV-E fusion protein expressed steadily and in a high yield following induction by IPTG.SDS-PAGE analysis of extracts of pCold-1VP1 and pET-28a-E transformed E.coli revealed that the recombinant protein with a mass of 26.5kDa and 54.8kDa approximatly.Western blot,whose first antibodies were positive serum(DHAV-1)and positive serum(DTMUV),showed that the expressed proteins were very antigenic and reactive to DHAV-1 and DTMUV positive sera respectively,showing good immunogenicity.3.Establishment of a liquid-phase chip detection method for antibodies against DHAV-1 and DTMUVAccording to the principle of liquid chip technology,the recombinant proteins were conjugated to microspheres with different ID to get conjugated complexes as capture vectors to establish a liquid-phase chip for detecting DHAV-1 and DTMUV antibodies.The results showed that the method of liquid phase chip detection is specific,sensitive and reproducible and provided a high-throughput,reproducible,specific detection system for detecting DHAV-1 and DTMUV antibodies.It is conducive to rapid detection of duck hepatitis A virus disease and duck tembusu virus disease,to improve production life and reduce economic losses. |
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