犬IL-18 cDNA的克隆及其免疫学特性

目的：获得犬白介素-18(cIL-18)基因并探讨其作为基因疫苗佐剂的免疫效果。方法：从犬的外周血中分离白细胞，经ConA刺激培养5～12h。提取犬白细胞总RNA作为模板，通过RT-PCR技术扩增cIL-18 cDNA。将其克隆到载体pMD18-T中测序，并与已发表的序列进行比较。将cIL-18 cDNA克隆入pIRES1 neo中，构建cIL-18真核表达载体。以200txg：200txg的比例，与狂犬病病毒糖蛋白表达载体plGneo混合免疫犬，并与糖蛋白单独免疫犬的免疫应答进行比较。结果：扩增获得单一长约0．6kh核酸带，测序证明长度为582bp，编码193个氨基酸，与从犬肺巨噬细胞中扩增的cIL-18基因的序列完全一致。以构建的表达载体pIIL18与pIGneo混合免疫与用pIGneo单独免疫相比较，前者抗狂犬病病毒特异性抗体的水平显著低于后者；但混合免疫组犬外周血淋巴细胞对特异性和非特异性抗原刺激的反应性显著高于糖蛋白单独免疫组。结论：cIL-18全长为582bp，其真核表达载体具有增强细胞免疫应答的能力，同时可显著抑制体液免疫应答。本研究为cIL-18作为基因疫苗佐剂的研究奠定了基础。

Cloning and immunological characteristics of canine IL-18 cDNA

AIM: To clone the canine IL-18 cDNA and to explore the immunological effectiveness of canine IL-18 as an adjuvant of genetic vaccine. METHODS: Canine leukocytes were separated from peripheral blood stimulated with ConA for 5-12 h. The full length cDNA of canine IL-18 was amplified by RT-PCR using leukocyte mRNA as a template. IL-18 cDNA was cloned into pMD18-T. Sequencing result showed that the full length cDNA was 582 bp, encoding 193 amino acids, which was identical with that published. The eukaryotic expression vector pIIL18 was constructed. Dogs were inoculated in mixture form of pIGneo and pIIL18. RESULTS: Canine IL-18 cDNA was successfully cloned and eukaryotic expression vector pIIL18 was constructed. The immunological assay result showed that the anti-rabies virus-specific antibody level of the group immunized with mixture of pIIL18 and pIGneo was obviously lower than that of the group immunized with pIGneo alone, but the level of cellular immunity of the former was higher than the latter. CONCLUSION: Canine IL-18 can enhance cellular immunity but at the same time suppress humoral immunity, which lays the foundation for IL-18 as an adjuvant of genetic vaccine.