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Optimizing Expression of the Fused Chicken IL-18 and HN Genes of ND Virus in Pichia Pastoris and Its Immune Effect Evaluation

Author: WangXueQiang
Tutor: ChengXiangChao
School: Henan University of Science and Technology
Course: Basic Veterinary Science
Keywords: chickenIL-18-HN Optimization of yeast expression activity detection detection of immune effect
CLC: S858.31
Type: Master's thesis
Year: 2010
Downloads: 59
Quote: 0
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Abstract


Interleukin-18,found in recent years, is an important cytokine which induce IFN-γand promote T cell proliferation and differentiation. IL-18 as a natural immunomodulator and vaccine adjuvant can overcome the high cost, side effects of traditional medicines, and is a new alternative to traditional therapy, so it has great application prospect in the poultry industry. Currently the study of IL-18 was focused on mammals such as human and mouse, Few study on chicken IL-18 were reported. In order to determine the efficacy of chicken IL-18 in the vaccination of animal diseases, chicken IL-18-HN fused protein were sucessfully expressed with the yeast expression system. Then immune responses of chickens inoculated with recombinant IL-18 and/or HN were analyzed in the study, this study provide an important data for further study of the biological functions of IL-18 and its application in vaccine prevention. The genetic stability of recombinant strain chIL-18-HN was analyzed. The optimal conditions for inducible expression in Pichia pastoris was optimized, then recombinant chIL-18-HN was expressed efficiently, and purified, meanwhile, the concentration of target protein was detected. Finally Biological activity of rchIL-18-HN was analyzed through Western bloting and MTT methods. The results showed that recombinant bacterial strain can stably express chicken IL-18-HN for 60 generations.The eukaryotic IL-18-HN yeast expression system was able to express efficiently the chIL-18-HN fused protein. The optimal conditions for inducible expreesion were pH6.5 in culture media, add 1.5% methanol 24 hours after culture to induce for 156h at 30℃, and the largest protein concentration was 400mg/L. Purified recombinant chIL-18-HN can effectively stimulate lymphocyte proliferation in vitro activity tests, the result of Western blotting showed that the recombinant chIL-18-HN has better immunoreactity. Meanwhile, The target fragment of HN-IL18 was cloned from pSK- HN-IL18 plasmid, and subcloned into the eukaryotic expression vector pcDNA3.1 to construct the final vector pcDNA- HN-IL18.On the basis of the above work, Immunological efficacy of Recombinant chIL-18-HN in chickens was observed. A total of 300 chickens (1-day-old) were randomly divided into 6 groups of 50 chickens, the 1st group: chIL-18-HN fused protein (50μg/bird), the 2nd group: recombinant HN protein (50μg/bird), the 3rd group: recombinant IL-18 (50μg/bird), the 4th group: recombinant HN + chIL-18 protein (50μg/bird), the 5th group: pcDNA- HN-IL18 plasmid(20μg/bird) and the blank control group. Primary inoculation was at the age of 8 days old, and boost at 12-day-old, and then 20 chickens from each group were challenged two weeks post secondary inoculation to determine the immunity to the virulent NDV F48E9 strain. Serum antibody titer were detected using HI assay at 4d, 7d, 14d, 21d, 28d post primary inoculation. The proliferation activity of T-lymphocytes, NO levels of macrophages in peripheral blood and immune organ index was measured. In additon, the number of T lymphocytes and IgG antibody-producing cells in immune organs were axamined. The results showed that vaccination with HN protein in combination with IL-18 can provide stronger immune response against ND virus than single HN. Distinct increase in the number of T lymphocyte transformation and NO level of macrophage secretion were seen in the chickens vaccinated with HN and IL-18 together, spleen and other immune organs developed and matures faster than other group. At the same time, the number of IgG-producing B cells in immune organs rised significantly relative to other chickens.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Chicken
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