Cloning And Prokaryocyte Expression Of Duck Interferon- Ⅰ Gene

Interferon-I was a giycoprotein produced by PBMC or fibroblasts which is induced by viruses. Interferon-I was notable for its functions of anti-virus. It can interfere virus in synthesis and reproduction. It is the first defending system of antiviral infection.Duck interferon (DuIFN) gene was first cloned by Schultz in 1995, and was expressed in E.coli and COS7 cells. Recombinant DuIFN had a strong antiviral effect toward several RNA viruses and was a potent inhibitor of Duck hepatitis B virus (DHBV) in primary duck hepatocytes.According to the complete nucleotide sequence of DuIFN published in GenBank, a pair of primers were designed with Oligo 4.1. By reverse transcription-polymerase chain reaction (RT-PCR), a DNA fragment of about 600bp was amplified. And then, the fragment was cloned into pMD18-T vector. The result of sequencing shows that the open reading frame (ORF) of Duck interferon-I (DuIFN-I) gene was consist of 576bp, and encodes a protein of 191 amino acids. The 30 hydrophobic amino acids at the N-terminus may serve as signal peptide. Mature polypeptide of DuIFN-I seems to consist of 161 amino acids. Sequence comparison with the published DuIFN gene in GenBank revealed 99.65% identity at the nucleotide level. Identity at the amino acid level was 98.95%. But it only shared 31.73% identity in nucleotide level and 7.58% identity in amino acid level with DuIFN-II gene. By comparing of the homology, DuIFN-I gene share 71.82%, 42.86%, 39.21% identities with Chicken, Canine and human IFN gene in nucleotide respectively, and shares 46.88%, 16.36%, 16.50% identities in amino acid respectively.Then, the DuIFN-I gene was cloned specially into pGEX-6p vector between the BamH I and Sal I cleavage site, the recombinant plasmid was transformed into E.coli TGI and BL21(DE3)PlysS, and was induced with 1.0mmol/L IPTG for DuIFN-I gene expression. The expressed product was identified by SDS-PAGE, a fusion protein about 47ku as we expected was found.The secondary structure and disulfide bonds of DuIFN-I and DuIFN were predicted and compared by Antheprot5.0 software. The result suggested that there was no difference between DuIFN-I and DuIFN. It demonstrated that DuIFN-I had biological ability as soon as DuIFN published in GenBank.This study provided the basis evidence for the further research about DuIFN molecular biological properties and nucleotide sequence evolution relationship between domestic and exterior countries. It also provided a new reagent for resisting duck viral disease.Candidate: Xu LinaMajor: Preventive Veterinary ScienceSupervisor: Prof. Wang Junwei...