论文部分内容阅读
目的 :探讨 HBV核酸免疫的可行性。方法 :采用 PCR技术 ,扩增得到编码 HBV HBs Ag的 S基因片段 ,将其插入到真核表达载体 pc DNA3,酶切鉴定并测序确证 ,得到质粒 pc DNA3- S,转染至 COS- 7细胞表达。将所构建的核酸疫苗免疫小鼠 ,观察其诱导产生的特异性体液免疫和细胞免疫功能。结果 :HBV S基因序列与文献报道一致 ,转染真核细胞后能表达目的蛋白。免疫小鼠后实验组和阳性对照组抗 - HBs阳性率分别为 70 % (7/ 10 )和 80 %(8/ 10 ) ,抗体水平分别为 (32 .14± 13.79) m IU/ ml和 (2 8.5 0± 11.87) m IU/ ml,两者比较无显著性差异 (P>0 .0 5 )。对照组和空白组抗 - HBs均为阴性 ,小于 10 m IU/ ml。实验组和阳性对照组的特异性 CTL杀伤率在效∶靶比为 2 0∶ 1时分别为 (35 .4 0± 4 .85 ) %和 (38.2 0± 7.6 9) % ,效∶靶比为 10∶ 1时则分别为 (2 3.95± 3.98) %和 (2 4 .5 5±3.5 9) % ,两者比较无显著性差异 (P>0 .0 5 )。对照组和空白组的 CTL杀伤率在效∶靶比为 2 0∶ 1和 10∶ 1时均小于 5 %。结论 :pc DNA3- S直接经肌肉免疫小鼠可诱导产生特异性体液和细胞免疫反应
Objective: To investigate the feasibility of HBV nucleic acid immunization. Methods: S gene fragment encoding HBV HBsAg was amplified by PCR and inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid was identified by restriction enzyme digestion and confirmed by sequencing. The plasmid pcDNA3-S was obtained and transfected into COS-7 cells expression. The constructed nucleic acid vaccine was immunized to observe the specific humoral and cellular immune functions induced by it. Results: The sequence of HBV S gene was consistent with that reported in the literature. After transfected with eukaryotic cells, the target protein could be expressed. The positive rates of anti - HBs in experimental group and positive control group after immunization were 70% (7/10) and 80% (8/10) respectively, and the antibody levels were (32.14 ± 13.79) m IU / ml and 2 8.5 0 ± 11.87) m IU / ml, no significant difference between the two (P> 0.05). The anti - HBs of the control group and blank group were all negative, less than 10 m IU / ml. The specific killing rates of CTL in experimental group and positive control group were (35.4 ± 4.85)% and (38.2 ± 7.69)%, respectively, at effect: target ratio of 20: 1 (2 3.95 ± 3.98)% and (24.55 ± 3.59)% respectively at 10: 1. There was no significant difference between the two groups (P> 0.05). The killing rates of CTLs in the control group and the blank group were less than 5% at the effect: target ratios of 20: 1 and 10: 1. Conclusion: pcDNA3-S can induce specific humoral and cellular immune responses directly via muscle immunization