Prokaryotic Expression, Purification And Anti-NDV Activity Detection Of Chicken Interferon-α And Interferon-γ

Interferon (IFN) is secreted mainly by the spleen cells or liver cells to produce a kind of multifunctional glycoprotein or cytokines, and with broad-spectrum anti-viral, anti-tumor and immune function. Because of its important biological activity, including antivirus and immune regulation, which makes chicken interferon extensively attracted, and it has become a hot spot of research in recent years. Currently, more researches focus on the interferon of human and mammalian, while the study of chicken interferon was often neglected. Some studies have shown that antiviral activity of chicken recombinant interferon- (IFN- ) and chicken recombinant interferon-γ(IFN-γ) were produced in vitro prokaryotic expression system. However, the studies of chicken interferon were just limited to the expression and activity of a single type interferon, and the anti-viral combination synergies of IFN- and IFN-γhave not been reported. On the other hand, in normal chicken body, with less the secretion of interferon, this largely restricts its application for antivirus. Therefore, establishing a kind of high efficient and stable expression system, and key research for alpha andγinterferon synergy has the vital significance. In this study, using molecular biology techniques for prokaryotic expression of the chicken ,γinterferon, and their vitro and vivo antiviral activity were studied systematicly. The whole experiment was divided into the following two parts:First, According to GenBank, designing the specific primers for chicken ,γinterferon gene sequences, from Shanghai normal SanHuang chicken spleen total RNA extraction, the establishment of nested PCR amplification method for chicken ,γinterferon gene. The results showed that amplification of the chicken ,γinterferon gene fragments were 447bp and 438bp.Recombinant expression plasmid was constructed by pET-30a. After identified by restrict enzyme and sequencing, Recombinant expression plasmid was transformed into the receptive cells of E. coli BL21(DE3) for induced , and then recombinant protein was purified by Ni-NTA chromatography column, thus to provide a candidate protein for further study on anti-NDV activity.Second, anti-NDV activity of the recombinant expression product was tested by cytopathogenic effect inhibition assay in BHK-21 cell, The results showed that chicken recombinant single interferon had a certain antiviral activity, and the inhibitory activity against Newcastle diseasevirus replication showed the highest value in the group that received the combination of recombinant interferon (IFN- : IFN-γ= 125:1).The antiviral activity of chicken recombinant interferon was detected by using the chicken embryos-infected. The results of antiviral activity on chicken embryos showed that the HA titers of IFN- , IFN-γand combination group were significant lower than that of the virus control group. In terms of the average death time of chicken embryos, antiviral effect of combination group was better than that of each IFN group alone. This proved that chicken IFN- , IFN-γand combination of interferon could inhibit the replication of Newcastle disease virus significantly.