论文部分内容阅读
目的 研究巴氏毕赤酵母表达系统表达的戊型肝炎病毒 (HepatitisEvirus ,HEV)结构区ORF2 蛋白的免疫原性。方法 将重组蛋白免疫BALB c小鼠 ,首先通过亲和捕获反转录PCR鉴定免疫小鼠抗血清是否能结合感染HEV恒河猴粪便及胆汁中的病毒颗粒。其次将重组蛋白分别以不同免疫途径、不同佐剂免疫小鼠 ,通过检测小鼠血清中抗 HEVORF2 抗体阳转率及抗体滴度 ,观察其免疫原性。结果 亲和捕获反转录PCR阳性 ,表明免疫鼠抗血清可以捕获感染恒河猴粪便及胆汁样品中的HEV。在小鼠免疫实验中 ,肌内免疫优于腹腔免疫 ,抗原联合铝佐剂及CpG佐剂优于抗原联合铝佐剂。以抗原联合铝佐剂和CpG佐剂经股四头肌免疫小鼠 ,4周后加强一次的免疫效果最佳 ,ED50为 0 0 2 3μg。实验表明 ,巴氏毕赤酵母表达的重组HEVORF2 蛋白刺激小鼠产生的抗体 ,不仅可以特异性结合天然HEV ,而且该蛋白在小鼠体内可以有效地诱发体液免疫反应。结论 巴氏毕赤酵母表达系统表达的HEVORF2 蛋白具有良好的免疫原性 ,这为新型戊型肝炎疫苗的研制奠定了基础
Objective To study the immunogenicity of ORF2 protein in the structural region of Hepatitis Virus (HEV) expressed by Pichia pastoris expression system. Methods The recombinant protein was immunized with BALBc mice. The antiserum of the immunized mice was identified by affinity capture reverse transcription polymerase chain reaction (PCR). Secondly, the recombinant protein was immunized by different immunization routes and different adjuvants respectively. The immunogenicity of the recombinant protein was tested by detecting the anti-HEVORF2 antibody positive rate and antibody titer in the serum of mice. Results The result of affinity capture reverse transcription PCR showed that the immunized murine antiserum could capture HEV in rhesus monkey feces and bile samples. In mouse immunization experiments, intramuscular immunization was superior to intraperitoneal immunization. Antigen combined with aluminum adjuvant and CpG adjuvant was superior to antigen combined with aluminum adjuvant. The mice immunized with quadriceps muscle antigen combined with aluminum adjuvant and CpG adjuvant had the best immunization effect after 4 weeks, with an ED50 of 0 0 2 3 μg. Experiments show that recombinant HEVORF2 protein expressed by Pichia pastoris stimulates the antibody produced by mice, which not only can specifically bind to natural HEV, but also can effectively induce humoral immune response in mice. Conclusion HEVORF2 protein expressed in Pichia pastoris expression system has good immunogenicity, which lays a foundation for the development of a new hepatitis E vaccine