title: Molecular cloning and expression of the IL-10 gene from guinea pigs.
abstract: The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.
authors.affiliation: Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA. dirisala@medicine.tamhsc.edu
authors.forename: Vijaya R
authors.initials: VR
authors.lastname: Dirisala
authors.forename: Amminikutty
authors.initials: A
authors.lastname: Jeevan
authors.forename: Gregory
authors.initials: G
authors.lastname: Bix
authors.forename: Teizo
authors.initials: T
authors.lastname: Yoshimura
authors.forename: David N
authors.initials: DN
authors.lastname: McMurray
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006168:Guinea Pigs / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D013045:Species Specificity
chemical_list: D011994:Recombinant Proteins; D016753:Interleukin-10; D004247:DNA