Prokaryotic Expression Of Chicken Interferon-α And Its Antiviral Effect On Newcastle Disease Virus

The interferon is a glycoprotein produced by host cells under the action of special inducers, which is able to initiate the antiviral mechanisms of cell, and play an important role in immunomodulation. The interferon-α (IFN-α) belongs to interferon-I and involves in the in vivo antiviral process. Although IFN-α does not directly act on the virus, it can stimulate the the host cells to produce antiviral protein rapidly. Recently, there are more and more studies on the anivirus effect of poultry IFN-α, and recombinant chicken IFN-α (ChIFN-α) produced by genetic engineering technique has entered the market. Newcastle disease virus (NDV) is one of the most important pathogen that threatening the poultry industry, and it is highly contagious and pathogenic. The outbreaks of NDV can be put down by the emergency vaccination, but the effect is limited. Therefore, the development of therapeutic drugs is still important for prevention and control of NDV.1. The cloning and analysis of ChIFN-α geneThe full length of ChIFN-α gene was amplified by the polymerase chain reaction (PCR) from total liver genome of Tibet Chicken (582bp).The amplified fragment was then ligated with pMD19-T vector and transformed into the E.coli DH5a competent cells. Confirmation of clones containing recombinant plasmid was achieved by PCR and restriction enzyme digestion. The plasmid was then sent to Shanghai Sang-gong Biological Engineering Technology&Services Co., Ltd (Shanghai, China) for sequencing..The results showed the ChIFN-α ORF sequence was582bp long, and the homology of this sequence and other ChIFN-a gene sequence in GenBank was99%.2. The prokaryotic expression of ChlFN-a maturation proteinA pair of primers with restriction sites EcoRl and HindⅢ was designed and synthesized to amplify the mature protein gene by using the plasmid containing the ChIFN-α ORF sequence as the template.Then the amplified fragment was subcloned into pET-32a and transformed in E.coli BL21competent cells. The recombination protein was induced by IPTG and analyzed by SDS-PAGE.. Optimization of expression conditions such as tempreture, the concentration of IPTG and induction time was also conducted..The sequencing result showed no genetic mutation was observed in the subcloned mature protein gene. The recombinant protein was expressed mainly in the form of inclusion body, and its molecular weight was about32KD as detected by SDS-PAGE. For the optimal expression condition, the optimal IPTG concentration of was0.6mM, the optimal induction time was4h, and the optimal temperature was37℃. E.Coli BL21was better than Rosetta to express the protein. The optimal condition was chosen to express the protein, and purified protein was achieved by inclusion body washing and renaturation. The concentration of purified protein reached1.56mg/ml as detected by protein measuring instrument.3. The anti-NDV effect of recombinant ChIFN-α.3.1The anti-NDV effect of recombinant ChIFN-α on CEFThe protein was diluted with PBS from10-1to10-9and added into the96well plate with CEF for12h, each dilution was inoculated into8wells. Then100TCID50NDV F48E9was added into each well and the growth of cells was observed and recorded every8h.The result showed that the recombinant ChIFN-α can inhibit the replication of NDV virus in CEF, and the antiviral activity of ChIFN-α reached3.16×105IU/mL.3.2The anti-NDV effect of recombinant ChIFN-a on non-immunized chicken embryos30μg of recombinant ChIFN-α was inoculated into10-days-old non-immunized chicken embryos infected by100EID50NDV F48E9. the addition of the recombinant ChIFN-α into the chicken embryos was performed at two time points, one was the same time as the inoculation of NDV, the other is24hours after the inoculation of NDV. The result showed that the average time of death can be prolonged by17and6hours. It can be concluded that recombinant ChIFN-α could inhibited the replication of NDV in chicken embryo, but it can not inhibit the replication of NDV completely.3.3The anti-NDV effect of recombinant ChIFN-α on chickenThirty3-days-old non-immunized chickens were divided into six groups. Group1,2,3and4were the test groups injected with100EID50F48E9virus in left side breast muscle, and injected with10μg,20μg,40μg and80μg of recombinant ChIFN-a in right side breast muscle intramuscularly, respectively. Group5was the control group injected with PBS. Group6was the positive control group injected with100EID50F48E9. Clinical signs as well as mortalities were recorded for a period of seven days after injectionChicks of group6appeared depression, loss of appetite, downer and other clinical symptoms within24h, and began to die after24h. Chicks in group1, group2and group3showed clinical symptoms, and began to die after48h. Group4showed clinical symptoms after38h, and began to die after72h. The average time of death of chickens was prolonged with the increase of the dose of recombinant ChIFN-α, suggesting that the recombinant ChIFN-α can inhibit the replication of virus in vivo. However, all the chickens in test groups died7days post challenge, indicating that recombinant ChIFN-α can not inhibit the infection and pathogenesis of NDV in chickens completely.