Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.
Gene. 2012 Apr 25;498(1):120-7. doi: 10.1016/j.gene.2012.01.076. Epub 2012 Feb 17.
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.
豚鼠（Cavia porcellus）是最适合模拟人类结核病（TB）的小型动物之一，因为其对低剂量气溶胶感染、肉芽肿形成、肺外传播和疫苗诱导保护的易感性。它也被认为是许多其他传染性和非传染性疾病的金标准；然而，由于缺乏现成的免疫试剂，这种动物模型存在一个主要的缺点。在本研究中，我们使用基因组计划提供的 DNA 序列信息，成功地从近交系 2 号豚鼠中克隆了关键的 Th2 细胞因子白细胞介素-10（IL-10）的 cDNA。完整的开放阅读框（ORF）由 537 个碱基对组成，编码 179 个氨基酸的蛋白质。该 cDNA 序列与人类 IL-10 具有 87%的同源性。令人惊讶的是，它与先前从 C4 缺陷（C4D）豚鼠中发表的 IL-10 序列仅显示 84%的同源性，这促使我们从 Hartley 豚鼠中克隆 IL-10 cDNA。Hartley 株豚鼠的 IL-10 基因与 Strain 2 豚鼠的 IL-10 序列完全同源。为了验证存在于 Genbank 中唯一发表的来自 C4D 豚鼠的 IL-10 序列，从 C4D 豚鼠的组织中分离基因组 DNA。用各种引物进行扩增表明，从 C4D 豚鼠报告的 IL-10 序列包含许多错误。因此，我们报告的 IL-10 序列取代了早期的序列，使我们的 IL-10 序列成为豚鼠的第一个准确序列。随后在原核和真核细胞中表达了重组豚鼠 IL-10 蛋白，并通过 N 端测序进行了确认。使用本研究中表达的重组 IL-10 蛋白在兔子中产生了多克隆抗 IL-10 抗体。综上所述，我们的结果表明，基因组计划提供的 DNA 序列信息可用于直接克隆研究豚鼠结核病所需的 cDNA。新克隆的豚鼠 IL-10 cDNA 和重组蛋白将成为研究豚鼠结核病和其他疾病的宝贵资源。
