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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) of the influenza A (H1N1) virus and investigate its ability to induce the body to produce a specific immune response after intranasal and intramuscular immunization. The HA gene was synthesized and cloned into the shuttle plasmid pShuttle-CMV. The recombinant adenovirus plasmid was obtained by homologous recombination and transfected into Ad-293 cells to package the recombinant adenovirus Ad-HA carrying H1N1 HA gene. RT-PCR and immunofluorescence were used to detect the successful transcription and expression of HA gene in Vero cells. The recombinant adenovirus was purified by CsCl density gradient centrifugation and immunized by nasal and intramuscular injection. The titer of anti-HA antibody in the serum of immunized mice was detected by ELISA. The results showed that nasal immunization and intramuscular injection of Ad-HA could both stimulate the production of anti-HA antibodies in mice. Nasal immunity stimulated the body to produce antibodies two weeks after the first immunization. The highest antibody titers reached 1:103.4. Two weeks after the first immunization, there was no obvious immune response, and the antibody level increased significantly after immunization. The highest antibody titer reached 1:104. The results showed that recombinant adenovirus expressing HA protein of influenza A (H1N1) virus can stimulate mice to produce IgG antibodies against HA by both intramuscular injection and intranasal immunization.