| Infectious laryngotracheitis (ILT) is one of the important respiratory diseases in chickens, which causes significant economic losses in the poultry industry. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The main clinical signs of this disease include bloody mucus in the trachea with high mortality in chicken, nasal discharge, conjunctivitis, and egg drop. The disease could be found in chicken farms all over the world since it firstly reported in 1925. It was endemic in China. In this study, we expressed and purified recombinant protein His-gJ of ILTV, and developed three monoclonal antibodies (MAbs) to gJ protein. In addition, the ILTV reference antigen and reference antiserum were successfully made with WG strain of ILTV. It will provide effective materials for diagnosis.1. Development of monoclonal antibodies against the gJ Protein of ILTVThe gJ sequence (445-745 amino acids) of ILTV was amplified from the genome of ILTV-WG strain by polymerase chain reaction (PCR). The gJ fragment was inserted into pET-32a and successfully expressed in E.coli BL21 (DE3). SDS-PAGE analysis showed that the fusion protein His-gJ was soluble and its molecular weight was about 55KD. The recombinant protein his-gJ was successfully purified by affinity column. Western-blot analysis indicated that the fusion protein His-gJ could be specifically recognized by anti-ILTV chicken sera. An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to ILTV was established with purified recombinant protein His-gJ as coating antigen.Three MAbs against gJ were developed by fusion between SP2/0 cells and spleen cells from mice immunized with the ILTV, which were named ILTV-gJ-4C6, ILTV-gJ-5B8 and ILTV-gJ-6B6 respectively. Western-blot and indirect immunofluorescence assay (IFA) results indicated that all three MAbs could specifically react with ILTV. Immunoglobin isotype investigations revealed that all the MAbs were IgGl subtype, and the light chains were k type. These MAbs will be useful in ILTV research.2. Development of ILTV reference antigen and antiseraWG strain of ILTV was propagated in 10-to 12-day-old SPF chicken embryos and the chorioallantoic membrane (CAM) was collected at 7 days post infection. The titer of ILTV propagated in SPF chicken embryos was 10-4.9 ELD50/0.2mL. The prepared ILTV were not found contamination with other viruses such as infectious bursal virus (IBDV), infectious bronchitis virus (IBV), gosling pest virus (GPV), Marek’s disease virus (MDV), avian influenza virus (AFV), of Newcastle disease virus (NDV), chicken egg drop syndrome (EDS76), avian leukosis virus (ALV) in hemagglutination test (HA), PCR, and ELISA test. Finally, the reference antigen was characterized by physical properties, sterility, mycoplasma contamination, uniformity, stability, expiration date.For reference sera to ILTV, SPF chickens were immunized with ILTV-WG strain. The titer of antisera was tested as 1:1200 by indirect ELISA using purified recombinant protein His-gJ as coating antigen. The antisera had no cross-reactivity with other avian viruses. The reference antisera was sub-packaged, lyophilized and characterized by physical properties, sterility, mycoplasma contamination, uniformity, stability, expiration date. The positive and weak positive reference antisera were defined by indirect ELISA. The indirect ELISA method and reference antisera were applied to evaluate the reliability of commercial ELISA kits for ILTV antibody detection. The results confirmed the availability of two commercial ELISA kits for detecting clinical samples. |
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