Expression Of Infectious Laryngotracheitis Virus Glycoprotein D,E And J In E.Coli And The Development Of Indirect ELISA

Avian Infectious laryngotracheitis (ILT) is an important worldwide occurring acute respiratory disease of poultry characterized by gasping, expectoration of bloody mucus and increased tracheal mucosal thickness. The disease might lead to a severe economic loss due to a high mortality rate or reduced egg production. The causative agent designated as ILTV or gallid herpesvirus 1. ILTV field strains and conventionally attenuated live vaccine can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. In this study, three important glycoproteins of ILTV were expressed in E.coli and indirect ELISA were developed, in order to choose an ELISA antigen for serum antibody detection of ILTV field strain infected chickens.In this study, the gene coding glycoprotein J of ILTV WG strain was amplified and sequenced based on the published sequence of ILTV USDA challenge strain. The cloned gJ gene contains a complete open reading frame of 2988bp, 30bp more than ILTV USDA challenge strain which may coding a new epitope in glycoprotein J. Later, fragments coding major antigenicity portion of ILTV envelope glycoprotein D, E and J were amplified and inserted into the expression vector pET32a, recombinant proteins, designated r-gD, r-gE, and r-gJ were expressed in E. coli after IPTG induction. A significant portion of r-gD, r-gE, and r-gJ was found in the soluble fraction. Western blot with His monoclonal antibody showed that r-gD, r-gE, and r-gJ contain His tag which promotes the purification of r-gD, r-gE, and r-gJ by nickel chromatography under native conditions. Prepared rabbit antiserum by immunization with r-gD, r-gE, r-gJ and Western blot with anti-ILTV serum showed that r-gD, r-gE, and r-gJ all retain at least part of the antigenicity.The purified r-gD, r-gE, and r-gJ were then used as antigens to develop r-gD, r-gE, and r-gJ indirect ELISA. Different antigen amounts, primary serum dilutions, second antibody dilutions and blocking medium were tested to obtain the highest P/N ratio. The cutoff values of r-gD, r-gE, and r-gJ indirect ELISA were established by the mean plus 3 standard errors of 36 SPF sera. Cross-reaction test, competitive-supression test were carried out; and tallying test with 139 sera showed 92.8%, 77.0% and 77.0% coincidence rate between r-gD, r-gE, r-gJ indirect ELISA and ILTV whole virus indirect ELISA, respectibely. In detecting ILTV-specific antibodies of ILTV WG strain experimently infected chicekens, r-gD, r-gE, and r-gJ indirect ELISA were showed significant antibody change curves after virus challenge. Among them, r-gD indirect ELISA reflects the humoral immunity of chickens after ILTV infection in a more reasonable way. Based on our results, we chose r-gD as ELISA antigen and r-gD indirect ELISA was developed.In this study, we chose r-gD from r-gD, r-gE, r-gJ as ELISA antigen and developed r-gD indirect ELISA, which may serve as an alternative ELISA for detecting ILTV infected or attenuated live-vius vaccine immunized chicken serum antibodies.