论文部分内容阅读
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 18cDNA。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 18cDNA克隆入pIRES1neo中 ,构建cIL 18真核表达载体。以 2 0 0 μg∶2 0 0 μg的比例 ,与狂犬病病毒糖蛋白表达载体pIGneo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 .6kb核酸带 ,测序证明长度为 5 82bp ,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pIG neo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 5 82bp,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
Objective: To obtain canine interleukin 18 (cIL 18) gene and explore its immune effect as a gene vaccine adjuvant. Methods: Leukocytes were isolated from peripheral blood of dogs and stimulated with ConA for 5-12 hours. Total canine leukocyte RNA was extracted as a template and cIL 18 cDNA was amplified by RT PCR. It was cloned into vector pMD18 T and sequenced and compared with published sequences. The cIL 18 cDNA was cloned into pIRES1neo to construct cIL 18 eukaryotic expression vector. The dogs were immunized with the pIGneo of rabies virus glycoprotein expression vector at the rate of 200 μg / 200 μg and compared with the immune response of dogs immunized with glycoprotein alone. RESULTS: A single 0.6 kb band of nucleic acid was obtained by PCR. The length of the DNA was 5 82 bp, encoding a protein of 193 amino acids. The sequence of cIL 18 gene amplified from canine pulmonary macrophages was completely identical. Compared with pIG neo immunization, the level of anti-rabies virus-specific antibody of the constructed expression vector pIIL18 and pIGneo was significantly lower than that of the pIG neo immunization alone. However, the mixed immunization group of peripheral blood lymphocytes against specific and non-specific antigens Stimulation reactivity was significantly higher than glycoprotein alone immunization group. CONCLUSION: The total length of cIL 18 is 5 82bp. The eukaryotic expression vector has the ability of enhancing cellular immune response, and can significantly inhibit the humoral immune response. This study laid the foundation for the study of cIL 18 as a gene vaccine adjuvant