摘要
构建表达甲型H1N1流感病毒血凝素(hemagglutinin,HA)的重组腺病毒,探讨其经滴鼻和肌肉注射免疫小鼠后诱导机体产生特异性免疫应答的能力。通过人工合成HA基因,克隆其至穿梭质粒pShuttle-CMV中,经同源重组获得重组腺病毒质粒,转染Ad-293细胞,包装携带H1N1HA基因的重组腺病毒Ad-HA。RT-PCR和免疫荧光检测HA基因在Vero细胞中成功的转录和表达。CsCl密度梯度离心纯化重组腺病毒,通过鼻腔免疫和肌肉注射免疫小鼠,ELISA法检测免疫小鼠血清中抗HA抗体滴度。结果显示Ad-HA通过鼻腔免疫和肌肉注射两种途径均能刺激小鼠产生抗HA的抗体,鼻腔免疫能在初次免疫后两周刺激机体产生抗体,最高抗体效价可达1∶103.4,而肌肉注射初次免疫两周后未出现明显的免疫应答,加强免疫后抗体水平出现明显的上升,最高抗体效价可达1∶104。结果表明表达甲型H1N1流感病毒HA蛋白的重组腺病毒通过肌肉注射和滴鼻免疫两种途径均能刺激小鼠产生针对HA的IgG抗体。
To construct a recombinant adenovirus expressing the hemagglutinin（HA） antigen of the influenza A（H1N1） virus, and evaluate its immunization effect in ICR mice by intramuscular and intranasal administration, synthetic HA gene was insert- ed into the shuttle plasmid-pShttle-CMV, which was then transformed into bacteria for homologous recombination with the ad- enovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombination virus Ad- HA. The recombinant adenovirus were constructed successfully, and the transcription and expressing of HA were determined by RT-PCR and immunofluorescence assay. The Ad-HA was amplified and then purified by CsC1. ICR mice were inoculated through intramuscular and intranasal routes. The specific antibody against HA in serum was determined by ELISA. The results showed that inoculation of the recombinant adenovirus by any of the two routes elicited HA specific serum antibodies. Antibody stimulated by nasal immunization occurred two weeks after the initial immunization, and the highest antibody titers reached up to 1：10 000. There is no apparent immune response after the first immunization through the intramuscular route, however, antibody levels after the booster immunization was apparently increased with the highest titers up to 1 ： 1 000. Our results of the study indicate that the recombinant adenovirus expressing the HA antigen of the H1N1 virus can stimulate IgG （anti-HA） anitbodies in the host through both of intramuscular and intranasal immunization.
出处
《现代免疫学》
CAS
CSCD
北大核心
2012年第4期308-314,共7页
Current Immunology
基金
中国医学科学院医学生物学研究所基金资助项目(IMB2009YB02)
