Cloning And Expression Of Cervus Nippon Gamma-Interferon Gene And Its Application In Tuberculosis Diagnosis

Cervus nippon is considered as the endangered species by International Union for Conservation of Nature (IUCN) red list and provided the first-level protection in China. The parts from the whole Cervus nippon body such as antler, meat, leather, etc are treasures for medicinal purposes in the East Asia.Mycobacterium bovis (M bovis), is one of the pathogens threatening the survival of wildlife species including Cervus Nippon by causing tuberculosis (TB). M.bovis has an extraordinarily broad mammalian host rang covering humans, domestic livestock, and wildlife. Like TB in other animals and humans, Cervus TB is chronic consumptive infectious disease, mostly occurs in Cervus nippon, reindeers, black deers and red deers. This disease may cause large numbers death, decline the velvet production, and reduce the product quanlity. The spread and prevalence of this disease seriously endangers the Deer industry, simultaneously threatens human health and other animals’security. In order to ensure the deer industry and human health, effective mothds for control and prevent this disease are needed as soon as possible.Tuberculin skin test (TST) is one of the most widely used means in the diagnosis of tuberculosis, although this is the oldest diagnostic test in use. Furthermore, PPD skin test mayl show large individual differences resulting from different batches and production procedures of factories. Besides, the results determined by the increase in skin thickness could be easily subjected to artifical mistakes.Interferon-gamma (IFN-γ) is produced primarily by activated T lymphocytes and natural killer (NK) cells in response to mitogens or antigens and has been referred to as "immune interferon". IFN-y release assay of bovine tuberculosis in vitro is based on following principle:the specific antigens of mycobacterium bovis was used to stimulate peripheral mononouclearcells (PPMC) and the cells from the infected animals thus secrete massively IFN-γwhich is then detected by enzyme-linked immunosorbent assay (ELISA).In this study, we cloned, expressed and purified the Cervus nippon IFN-γgene(CerIFN-γ), prepared IFN-γmonoclonal antibody and polyclonal antibody, established CerIFN-γsandwich ELISA method, and applied it in tuberculosis diagnosis.1. Cloning and expression of the CerIFN-γin Escherichia coli and MDBK cellsTotal RNA was isolated from Cervus Nippon peripheral blood lymphocytes, which were stimulated with Con A. Basing on the published GenBank sequence of CerIFN-γcDNA, two pairs of primers were designed, and CerIFN-γwas amplified.and separately cloned into the prokaryotic expression vector pET32a and the eukaryotic expression vector pCI-neo to construct prokaryotic (eukaryotic) expression recombinant plasmids. The prokaryotic expression recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed soluble protein with the size of about 23KD was purified. Eukaryotic expression recombinant plasmid was transfected into bovine kidney cells (MDBK) using calcium phosphate-mediated method. The stable secreted expression resistant cell lines were obtained by different concentration of G418 selection.2. Cervus Nippon Interferon-gamma (IFN-γ) antiviral activity analysisIn order to identify whether the expressed IFN-γin this study has characteristics of the natural IFN-y, the ability to inhibit the replication of VSV or IBRV in MDBK cell was detected, and CerIFN-γantiviral activity was proved to be approximately 7.25×104 U/mL,4.61×104 U/mL. Its physical and chemical characteristics were also analyzed, showing that the CerIFN-γhad a strong resistance to acid, but sensitive to heat. These findings demonstrated that the expressed recombinant CerINF-γis very similar to the natural INF-γ.3. CerIFN-γmonoclonal antibody preparation and identificationCerIFN-γexpressed by E.coli was purified and used as an immunogen to immunize mice and rabbits respectively. Hybridoma cells were screened with protein expressed by eukaryon cells line remianed above.Three monoclonal antibodies with high titres were obtained and named 4C2, 1F10 and 1G10. Mice were induced with hybridoma cells to obtain ascites. ELISA showed that the three ascites antibodies had titers of 2.56X104, 1.28X104, and 6.4X103. Western-blot analysis showed that three monoclonal antibodies were all able to bind specifically 23KD CerIFN-γ, showing that they were CerIFN-γspecific monoclonal antibodies. In order to further confirm the specificity of McAb, the blocking test was carried out. Result showed that the anti-viral activity of recombinant CerIFN-γcan be effectively blocked by its monoclonal antibody, and the minimum dilution was of 1:1 600 for the complete block of 100 U VSV infection. Non-immune mouse serum was completely unable to block the recombinant CerIFN-γanti-viral activity, while the anti-recombinant bovine INF-γfrom hyperimmunized rabbits could cause 46% blocking rate at the dilution of 1:100.4. Establishment of CerIFN-γin vitro release assayMcAb 1C4, purified polyclonal antibody and horseradish peroxidase (HRP) labeled goat anti-rabbit secondary antibody were used to establish the double-antibody sandwich ELISA method to detect CerIFN-γ. Results showed that the sensitivity of this ELISA reached 24.9 pg/ml, and the specificity was also good since no reaction was displayed with other tested antigens. This ELISA method laid the foundation for clinical application.5. CerIFN-γassay application in clinical Tuberculosis diagnosisThe method was used to detect 120 deer blood samples, which were collected from a deer farm in China. The result showed that the positive rate was 17%,The antibody detection with Immunochromatographic Rapid Assay strips or ELISA in parallel detected the samples, and the positive rates with the Strips was 2% and 11.2% with indirect ELISA. Thus the IFN-γtest has higher sensitivity and would has a broad application in the future.