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Expression of Chicken Interleukin-2 in Pichia Methanolica and Analysis of Biological Activity

Author: DuMingHua
Tutor: CaoMinJie
School: Jimei University
Course: Of Food Science
Keywords: Chicken Interleukin -2 (IL-2) Pichia pastoris Recombinant expression Ferment Immunostimulants
CLC: S852.5
Type: Master's thesis
Year: 2008
Downloads: 99
Quote: 1
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Poultry infectious disease is the biggest threat in the poultry industry, causing serious economic losses to the aquaculture industry, the immune prevention is important strategy to control the disease. But in actual production, not all diseases have a vaccine is available, not all vaccines can be completely immune to the body to produce protection. Especially some of inactivated vaccine and gene engineering vaccine due to the low antigenicity cause immune effect is often not ideal, often causing immune failure. The chicken interleukin -2 (Chicken Interleukin-2, ChIL-2) in the poultry industry and can be used as immune adjuvants to improve the immune system of the chicken body resistance pathogen, has been since its discovery in 1982, is the hot spot in the study of cytokines one is expected to be used in conjunction with the vaccine has become an important cellular immune adjuvant to improve the body's resistance to infectious enhanced inactivated vaccine or immune effects of genetic engineering. In this paper, the expression of Pichia yeast (Pichia Pastoris) of reorganization ChIL-2 and improve the physiological activity of the chicken immune function was studied. First ChIL-2 was cloned into the the Pichia yeast shuttle plasmid pPIC9K construct the recombinant plasmid pPIC9K/ChIL-2 linearized by Sal I digestion, electroporation integrated into the chromosome of Pichia pastoris GS115 (His Mut), the use of MM, MD screened on His Mut positive clones with high concentration of G418-YPD plate screening high-copy transformants. Five positive clones were screened using colony PCR further screening, plant height copy strains. Screening to high-copy strains BMGY / BMMY shake flask cultures induced expression of the reorganization ChIL-2. By SDS-PAGE and Western blot analysis confirmed the ChIL-2 gene was successfully expressed in recombinant GS115 approximately 16 kDa and 14 kDa two specific bands, with the the natural ChIL-molecular weight of the same size, suggesting that natural protein, restructuring ChIL-2 may also protein glycosylation. The recombinant yeast strain for fermentation production, its expression level compared to shake flask culture was improved about 20 times, and up to about 100 mg / L, the recombinant target protein mass fraction of more than 50% of the total protein. Very different target protein hybrid protein with size, high purity target protein through the membrane filter interception, and simple to operate. In this study, for the industrial production of chicken interleukin-2 and laid the foundation. Purified ChIL-2 with vaccine combined immune chickens to study the effect of its avian influenza virus (avian influenza virus as immunostimulants AIV) vaccine and Newcastle disease virus (Newcastle disease virus NDV) vaccine enhancement using hemagglutination inhibition the AI ??antibody serum test for detection of chicken body and ND antibody levels. The experimental results show that: ChIL-2 with avian influenza or Newcastle disease vaccine vaccine immune enhancing effect, can effectively improve the antibody titers of chicken body, extend the time of antibody titers and improve survival. ChIL-2 use the dose should be 8.35μg / chicken around. In the process, ChIL-2 did not find any toxic side effects, proved to be a safe and effective immune enhancer.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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