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| Cloning, Sequence Analysis of Feline IL-18 Gene And Its Expression in E.coli |
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Abstract According to feline IL-18 gene sequence reported in GenBank, feline IL-18 gene was amplified by RT-PCR from the PBMCf stimulated by ConA. The PCR product was purified and ligatured with pMD18-T. The recombinant plasmid was used for sequencing. The full length of feline IL-18 gene was 579 bp , which encoded 192 amino acids. There were no signal peptide and N-glycosylation sites, while had 4 conservative Cys amino acid in the deduced amino acid sequence. Compared with IL-18 genes of other different species, the nucleotide sequence of feline pIL-18 gene shares higher identities with IL-18 genes of canine, ovine, bovine and porcine, but displays significant differences with mouse and chicken IL-18 gene sequence which published in GenBank. Meanwhile, the feline IL-18 gene was subcloned into the prokaryotic expressing vector pET28a and recombinant vector further transformed into host E.coli strain BL21 (DE3) for expression under the induction of IPTG. The results of SDS-PAGE revealed that it had a molecular weight of 27.5 kDa, which amounts to 13.6% in the total protein of the induced bacteria by the assaying of gel scanning.
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Received: 10 April 2006
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