| Interferon can be a natural antiseptic and adjuvant because of its antivirus, antitumor andimmune regulation. Given that there is still no related report about interferon-gamma(IFN-γ) ofLushi chicken which is the local fine varieties, the mature IFN-γ gene was cloned, analyzed forgenetic variation and expressed in prokaryotic in this study. To provide the theoretical basic forthe future development and application of Lushi chicken IFN-γ, the prokaryotic expressionproducts were collected by ultrasonic method, a combination of ultrasonic andlysozyme, high-pressure slurry method and a combination of high-pressure slurry andlysozyme, and the effects of their extraction were studied. Mainly including as follows:1. Cloning, identification and expression for the mature gene of chickeninterferon-gammaAt first, the complete sequence of Lushi chicken IFN-γ gene was cloned from thechicken embryos spleen by RT-PCR, and the location of signal peptide was predictedby biologic software to design the primers of the mature Lushi chicken IFN-γ sequence.The mature Lushi chicken IFN-γ gene without N-signal peptide was firstly amplified byPCR, then cloned into the plasmid PET-28a. Enzyme digestion, PCR, sequencing andSDS-PAGE were done for identification after the recombined expression plasmid PET28a-IFN-γwas transformed into BL21.The results showed the mature Lushi chicken IFN-γ gene wassuccessfully cloned and expressed, which were respectively about438bp and22KDa.2. The homologous analysis for IFN-γ genes of Lushi chicken and the other poultryThe IFN-γ gene homologies of Lushi chicken and the other poultry were analysed, thephylogenetic tree was also designed. The nucleotide sequence homologies of IFN-γ genes were all99.5%when Lushi chicken was compared with Gallus gallus breed white leghorns, Gallusgallus murghi and Gallus gallus, respectively96.6%,95.2%,95.0%. When it was compared with Meleagris gallopavo, Phasianus colchicus and Coturnix coturnixjaponica.It were respectively92.5%,70.8%,31.5%compared to Numida meleagris,different kinds of Numida meleagris and Columba livia.3. The analysis results of measuring endotoxin and pyrogen of different extractionways for IFN-γAll the pyrogen limitation of chicken IFN-γ extracted in four different ways meet thecorrelated national standards through animal heat test. Compared with other methods,the temperature change was minimal by the combining lysozyme and ultrasonic, whichshowed the pyrogen content of recombinant protein extracted by this way was thelowest.Endotoxin levels in different extraction ways did not exceed the limits（10EU/ml）and were consistent with the national standards through gel method.4. The effects of four different extraction ways on poultry growth and immuneorgan indexCompared with control group, the effects of the combination of ultrasonic andlysozyme, high-pressure slurry method and the combination of high-pressure slurry andlysozyme, were insignificant on the parts of bursal index and spleen index in0~21daysby oral or injection. Compared with control group, the combination of ultrasonic andlysozyme had significant difference in thymus index after21days by oral or injection,and the averages of other methods were higher than the control group. The resultsrevealed chicken IFN-γ had no influence on bursal and spleen. However, it couldpromote the development of thymus.By observing the growth of the experimental animals, it showed mental status, diet,feces, necropsy and weight of the test animals in oral and injection groups had nosignificant difference with the control group.5. The analysis results of measuring the affect for antibody levels of the Newcastledisease Ⅳ vaccinationThe recombinant protein in different extraction ways were used in combinationwith Newcastle disease Ⅳ vaccination by oral and injection, the results showed theantibody titers in oral and injection groups were higher than control group after7d,which indicated that the recombinant chicken in the form of inclusion body couldimprove the immune level of Newcastle disease Ⅳ vaccination. |
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