酵母表达的戊型肝炎病毒结构区ORF_2蛋白的免疫原性研究

Immunogenicity of recombinant HEV ORF_2 protein expressed in pichia pastoris

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 。

Objective To study to immunogenicity of recombinant HEV ORF 2 protein expressed in %pichia pastoris%.Methods BALB/c mice were immunized with the recombinant HEV ORF 2 protein.The ability of antiserum to bind HEV was tested using affinity-capture reverse transcription polymerase chain reaction (RT-PCR).Moreover,the recombinant protein was used to immunize BALB/c mice by different routes with different adjuvants.Serum conversion rate of anti-HEV antibody and the ELISA titer were detected.Results The antiserum could capture native HEV for RT-PCR.As to the immunization effect,the immune response by intramuscular route was better than that of the intraperitoneal route.The protein with alum and CpG adjuvant could elicits more significant immune responses than using the alum adjuvant alone.The best way was to immunize with the protein with alum and CpG adjuvant by intramuscular route with a boosted injection on the 4th week after the first immunization.The ED 50 was 0^023 μg.This is the first report that the antibody elicited by recombinant HEV ORF 2 protein expressed in %pichia pastoris %recognizes native HEV.High immunogenicity of this kind of ORF 2 was also demonstrated by inducing strong immune response in mice with good ED 50 result.Conclusion The high immunogenicity of this kind of HEV ORF 2 may make a foundation for the development of new type of hepatitis E vaccine.