Analysis of IL-1β- and IL-8-encoding gene sequences and their recombinant protein structure and function of avian species

• Main Authors: Yung-Fu Wu, 吳永富
• Other Authors: Long-Huw Lee
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/79604435274769913242

博士 === 國立中興大學 === 獸醫微生物學研究所 === 96 === Interleukin (IL)-1β-encoding regions of chicken, duck, goose, turkey, and pigeon were cloned and sequenced. Each IL-1β-encoding region of chicken, duck, goose, and turkey is 804 nucleotides long and encodes IL-1β protein that is 268 amino acids. Pigeon IL-1β-encoding region is 810 nucleotides long and encodes IL-1β protein that is 270 amino acids. Two one-nucleotide and one four-nucleotide insertions of pigeon IL-1β-encoding region sequence were found, resulting in two amino acid insertions in pigeon IL-1β. Pairwise sequence analysis showed that the sequence identities of IL-1β-encoding genes ranged from 77 to 99% which were also found for IL-1β protein sequence identities, with an average level of both sequence identities of 89%. Phylogenetic analysis indicated that IL-1β-encoding regions and the encoded proteins of chicken, duck, goose, and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from the binding reaction of antiserum against each recombinant IL-1β (rIL-1β) protein to homologous or heterologous rIL-1β, the enhancement levels of K60 mRNA expression in rIL-1β-treated DF-1 cells, or the reduction levels of K60 mRNA expression in DF-1 cells treated with rIL-1β that was preincubated with homologous or heterologous antiserum showed that all five rIL-1β were functional active and shared significantly structural and functional homology. Inhibitors of viral disassembly or RNA and protein synthesis, viral disassembly intermediates (infectious subviral particles, ISVP), binary ethylenimine-inactivated virions, and viral particles lacking genomic double-stranded RNA (empty particles) used to assess the expression of interleuin-1β (IL-1β) mRNA in chicken macrophages in response to avian reovirus. The results demonstrated that two distinct expression patterns of chIL-1β mRNA mediated by different steps in viral replication were found. Viral disassembly was required for induction of a rapid, transient expression of chIL-1β mRNA that was rapidly induced at 30 min, with maximal levels reached at 2 h, and fell to a low level within 6 h post-inoculation; while viral RNA synthesis rather than protein translation, which were subsequent to membrane penetration, was required to induce a stable, sustained expression of chIL-1β mRNA that occurred at and after 6 h post-inoculation. In addition, the induction of chIL-1β mRNA expression by the empty particles and ISVP was exceedingly weak, compared to the active double-stranded (ds) RNA+ virions or binary ethylenimine-inactivated virions, suggesting that the presence of dsRNA, even if transcriptionally inactive, may be an important factor in this response. Interleukin (IL)-8-encoding regions of five avian species were cloned, sequenced and characterized. Each IL-8-encoding region is 312 nucleotides long and encodes IL-8 which is 103 amino acids. Pairwise sequence analysis showed that sequence identities of IL-8-encoding regions ranged from 87% to 100%. The IL-8 protein identities varied from 84% to 100%. Phylogenetic analysis indicated that IL-8-encoding regions and encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from binding reactivities of antiserum against each recombinant IL-8 (rIL-8) protein to homologous or heterologous rIL-8 proteins, chemotactic activities of each rIL-8 protein or reduction levels of the chemotactic activity of rIL-8 protein which was pretreated with homologous or heterlogous antiserum have suggested that all five IL-8 proteins were functionally active, and shared structural and functional identity with each other.