| China is the biggest country of feeding fowl, however, the diseases caused by virus have puzzled the industry for a long time and leaded to huge economic losses. At present, Number of poultry viral diseases is still a lack of effective treatment,the use of chemical drugs cause drug resistance and easily lead to drug residues in animals and affect human health,vaccines have to consider the variation and diversity of the virus. So it is urgently hoped that safe and high effective antiviral therapeutics and alternative forms of vaccine adjuvants could be developed.The protein interferon,produced by animal cells when they are invaded by viruses,is released into the bloodstream or intercellular fluid to induce healthy cells to manufacture enzymes that counters the infection. Interferon has been considered a very exciting group of proteins for many years during which they were initially shown to have antiviral and non-specific effects and lately, immunomodulatory and immunoregulatory effects. While beneficial aspects of interferon therapy have been shown in a variety of experiments in animals, the great potential for interferon treatment has yet to be achieved.Interferon in vitro can be applied to both diseases, but also can be combined with the vaccine.Enhance and improve the immune effect of the vaccine. Interferon-alpha is especially well antiviral applied to animals.In recent years,the antiviral function and vaccine adjuvant activity of chicken interferon were found and conformed in many studies and trials. The aim of the present study is to develop recombinant chicken alpha interferon with self-governed knowledge property right applying the technique of gene engineering and protein engineering and to study the antiviral activity of the obtained recombinant chicken interferon in cultured cells and chicken. The study proved the way for engineering production of the recombinant chicken interferon and its wide usage in chicken industry.This study includes:1 Chicken IFN-a is a multigene family, with more than 20 kinds of different subtypes, the different subtypes have differences in biological activity. After analysising gene sequences of subtypes with DNA Star Software,screened high homology gene.According to the partial tropism of Pichia pastoris, designed a new gene sequences encoding chicken interferona,using the preferential condon of P. pastoris,the chicken interferon a gene 507 bp in length was designed and synthesized.The modified antibacterial peptide gene was cloned into the pPICZa-A vector to construct the recombinant expression vector pPICZa-A-ChIFN-α.The Sac I linearized plasmid pPICZa-A-ChIFN-αwas transformed into P. pastoris X-33 by electroporation. The identified transformants were isolated and assayed for the expression of pPICZa-A-ChIFN-α. The expression of pPICZa-A-ChIFN-αwas induced with methanol.The antivirus activity was about 1.76×106U·mL-1 and the content of the secreted pPICZa-A-ChIFN-αwas about 0.143mg·mL-1.The specific activity of chicken interferon a produced by the Pichia pastoris was approximately 1.23×107U·mg-1.2 Based on the related researches, by contrasting chicken interferon to human interferon protein sequences, found that C-side high homologous. Transformed chicken interferon-a according to the means of mutation in HuIFN-α, constructed five mutants:Mutl:V141I, R142S, A145Y; mutant Mut2:V141L, R142S, A145Y; mutant Mut3:W138H, V141I, R142S,, A145Y, W148S, L150S; mutant Mut4:L114S; mutant Mut5:L114S, W138H, V141I, R142S, A145Y, W148S, L150S and expressed successfully, measured their antiviral activity through the VSV-CEF system, Mut1 and Mut2 have high antiviral activity, improved 28 times and 15 times; antiviral activity of Mut4 is no more than before and Mut3, Mut5 lower 32times and 23 times respectively. Analysis proven and predictable structure, we found that:chicken interferon-a receptor-binding site located at amino acid position 29-35 and 123-140 amino acid sites, C-end can effect the antiviral activity of IFN,mutation of amino acid may increase its affinity and biological activity.3 Two trials were carried out to assess the antiviral activity of recombinant chicken interferon on avian influenza virus (AIV),Newcastle disease virus (NDV).The results of the first trial showed that:recombinant chicken IFN-a can reduce the AIV in chicken embryo fibroblast cells proliferation titer.Titers of AIV in the 1000U/mL and 100U/mL rChIFN-αpretreated cell were respectively 2.76 LoglO TCID50 and 1.93 Log10 TCID50.Lower than in the control CEFs, the high concentration is more obvious. In the second trial,it was found that the inhibition effect of chicken interferon alpha on NDV is more obvious. Titers of NDV in the 1000U/mL and 100U/mL rChIFN-a pretreated cell were respectively 1.73Log10 TCID50 and 0.89 Log1O TCID50.Lower than in the control CEFs.What is more, the replication of NDV was completely inhibited in CEFs pretreated with 1000U/mL of recombinant chicken interferon alpha. 4 The recombinant Chicken Interferon alaph as adjuvant was used to enhance the immune efficacy of inactivated vaccine of avian influenza H9N2 type in the trial. Hens the United States Hailan were raised in cages in pathogen free surroundings and divided into 6 groupes. At 20day age,chickens in vaccine control group were immunized with injecting 0.3mL inactivated vaccine of AI H9N2 type into chest muscle,and the same dosage of AI H9 inactivated vaccine and different dosages of recombinant Chicken Interferon alaph were respectively injected into the experimental group animals. The HA and HI assays were applied to detect the titers of antibody (Ab) after the immunization every 7 days. The result s showed that the recombinant chicken interferon protein of H9 subtype of avian influenza inactivated vaccines to enhance immune function. The rise and fall of antibody titer of integrated features and indicators of cellular immunity,the use of 100,000 U/head of the chickenα-interferon as adjuvant AIV H9N2 inactivated vaccine can be made more satisfactory results. |
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