Accurate Identification Of The Linear Epitopes In VP1Proteins Of Duck Hepatitis A Virus Type1and Type3

Duck viral hepatitis is an acute and fatal disease of ducklings, characterized by livernecrosis, hemorrhage and high mortality. The disease is caused by at least three different typesof duck hepatitis virus (DHV) and no antigenic relationships have been found among theDHVs. According to the Virus Taxonomy ninth Report of the International Committee onTaxonomy of Viruses (ICTV), the three types of DHV are all RNA viruses, namly duckhepatitis A virus (DHAV), duck astrovirus type1(DAstV-1) and duck astrovirus type2(DAstV-2) respectively. Among the DHVs, DHAV-1is the most common, most virulent andworldwide serotype.Present study on DHAV is undergoing a stage from basic genetic analysis to functionalresearch. After obtaining the mAb4F8and learning its one general epitope position in VP1protein of DHAV-1in preliminary work of our lab, we decide to make an accurateidentification of this epitope. Additionally, VP1proteins of DHAV-2and DHAV-3wereexpressed in prokaryotic expression system, and were used to find whether there is anotherepitope of mAb4F8in them. The main contents are summarized as follows:1. Determination of the neutralizing activity for mAb4F8against DHAV-1In this study, mAb4F8against DHAV-1was prepared by intraperitoneal injection ofhybridoma to six-week-old female BALB/c mice. Neutralizing activities of mAb4F8with2.056mg/mL protein content against DHAV-1and DHAV-3were determined usingquantitative virus respectively. Using0.1mL mAb4F8(2.056mg/mL) to neutralize100ELD50of DHAV-1and DHAV-3, with protection for more than50%of duck embryo from death, theantibody dilution titers were5.0to DHAV-1and9.8to DHAV-3. The results indicated thatmAb4F8had weak neutralizing activities to both types of DHAV, and although mAb4F8wasmade by DHAV-1immunization, it could neutralize DHAV-3better.2. Accurate identification of the linear epitope of mAb4F8in DHAV-1VP1proteinIn our early research, the linear epitope domain of mAb4F8in DHAV-1VP1protein hasbeen located to74SGEIILTIV82. To identify the accurate location of the epitope against4F8, amino acids of SGEIILTIV were reduced one by one from both ends. Six recombinantplasmids were constructed and transformed into Rosetta(DE3) PLys cells. Expression of thefusion proteins were induced by IPTG and identified by SDS-PAGE. The results of westernblot analysis with mAb4F8indicated that the accurate epitope against4F8was75GEIILT80inVP1protein of DHAV-1. Compared with other serotypes of DHAV in NCBI, this epitope washighly conservative in VP1protein of DHAV-1and mutations existed in same sites ofDHAV-2and DHAV-3.3. Research of epitope of mAb4F8in DHAV-2VP1proteinAs a new serotype, DHAV-2has only been isolated from Taiwan. In this research, VP1gene of DHAV-2was directly synthesized according to the DNA sequence released in NCBI.Two recombinant plasmids were constructed and transformed into Rosetta(DE3) PLys cells.Expression of the fusion proteins were induced by IPTG and identified by SDS-PAGE. Theresults of western blot analysis with mAb4F8showed that there was no epitope against4F8in VP1protein of DHAV-2.4. Research of epitope of mAb4F8in DHAV-3VP1proteinCompared with amino acid sequence of DHAV-1VP1protein, there was the onlymutation in75~80aa domain, the Val of site77, in VP1of DHAV-3. According to this result,four recombinant plasmids were constructed and transformed into Rosetta(DE3) PLys cells.Expression of the fusion proteins were induced by IPTG and identified by SDS-PAGE. Theresults of western blot analysis with mAb4F8indicated that75GEVILT80conservatively sitingin VP1protein of DHAV-3was also an epitope against mAb4F8.