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The Cioning, Expression Of The Gene Of Canine Interferon α And Activity Analysis Of This Expression Product

Posted on:2006-09-30Degree:MasterType:Thesis
Country:ChinaCandidate:Y WangFull Text:PDF
GTID:2120360152993861Subject:Prevention of Veterinary Medicine
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During recent years, canine animals have become more and moreimportant in human lives, such as chaperon animals, police dogs, rescue dogs et al. But various virus diseases disturb canine husbandry. One hand, there isn't an efficient medicine to cure it and some viruses become more virulent. On the other hand, the immunized protocol failed because of usage and storage. The repeated occurrence of canine madness and canine parvovirus disease bring human harm and some viruses are the pathogens of zoonoses, which raises the concentration of all the society. As a result, from the view of veterinary medicine and human medicine clinic, control of viral diseases of canine needs medicine with safety, efficiency and less side effect, and canine interferon alpha has high clinic value whose biological products is necessary. Canine interferon alpha has wide anti-virus bioactivity and high research value. So the topic modified the gene of canine interferon alpha, expressed it in various systems, analyzed its bioactivity. This research project was consisted with several sections:1, With the reverse transcription chain reaction (RT-PCR), the gene of Canine's interferon-alpha (CalFN-α) was amplified and cloned, from the total RNA in the lymphocytes stimulated with concanavalin A from the peripheral blood of dog. Then the amplified fragment was inserted into the prokaryotic expressing vector pBV220 and then sequenced. Sequencing result showed that the homology was 100% between the gene we cloned and the one published in GenBank. The recombinant expression vector was transformed into several strains of the E.coli and induced to express by different temperature. The result showed that some strains couldn't express the protein and others could express it. Through analysis, there were not the enough bias codons required by the translating mechanism of the E.coli, which resulted in no expression of the recombinant canine interferon alpha. SDS-PAGE assay showed that the target protein, with the molecular weight of 19kDa, could be expressed in a high level and in the E.coli cell, in the form of inclusion bodies. The target protein was added to MDCK cells, which were subseq-uenttly infected by the 100 TCID50 VSV. The result showed the good cytokine activity. The recombinant CalFN-α activity was up to 5. 11 X 106 U / mg.2, The gene of canine interferon alpha was modified to get the recombinant protein with natural structure. After the first code near 5' terminal were modified to the hobby of Pichia Pastoris yeast, the gene of Canine mature interferon-alpha (CalFN-a) was inserted into Pichia Pastoris yeast expression vector pPICZα-A. The resulted recombinant expression vector was linearized with Sac I and then transformed into Pichia Pastoris X-33 yeast strain by electroporation to integrate into yeast genome and obtained engineering Pichia Pastoris strain X-33/pPICZα-A-CaIFN-α. The transformants with the high copies were selected by Zeocin and were induced to secretive express with methanol added every 24 hours. SDS-PAGE showed that expression product of the gene of Canine's mature interferon-alpha in culture supernatant of X-33/pPICZα-A-CaIFN-α was about 27kDa, which bigger than 19kDa. Because there were two glycosation sites which led to the glycosation of expressed product or there were other protein ornament process. In addition, the target protein was added to MDCK, which were subsequently infected by the 100 TCID50 VSV. The result showed the good cytokine activity. The recombinant CalFN-a activity was up to 1.61-6.51 X 106U / mg.Through this study, the interferon expressed from the E.coli has high production, but the experiment was complex and time-consuming which needs high special technique during the process. The interferon expressed in yeast is similar to natural protein structure which process is easy to maneuver, needs low level of technique and machines and is easy to great scale industrial production.
Keywords/Search Tags:Canine, Interferon, Prokaryotic Expression, Pichia Pastoris Yeast, Secretive Expression, Antiviral Activity
PDF Full Text Request
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