表达流行性感冒病毒HA基因非复制型重组腺病毒的构建及免疫效果的研究

通过RT-PCR扩增流行性感冒(流感)病毒HA基因，克隆至腺病毒穿梭载体pAd Track-MV，该重组质粒与腺病毒DNA共转化E．coli BJ5183，通过细菌内同源重组获得重组腺病毒DNA，将其转染293细胞获得重组腺病毒。PCR证实HA基因已整合至腺病毒基因组中，Western blot结果检测到重组病毒感染293细胞中HA的表达。重组病毒经滴鼻和灌胃两种途径免疫小鼠，结果2次免疫后滴鼻组和灌胃组均产生明显的免疫应答，血清IgG抗体滴度分别为1：10000和1：1000。除血清IgG外，还在肺灌洗液中检测到分泌型IgA。滴鼻组的免疫效果强于灌胃组。经小剂量攻毒实验显示，重组腺病毒保护率为100％。该文成功构建了表达流感病毒HA基因的非复制型重组腺病毒，重组病毒免疫小鼠可产生较好的免疫效果。

Construction of Replication-defective Recombinant Adenoviruses Expressing Influenza Virus Hemagglutinin and Study on its Immune Responses

HA gene of influenza virus A/FM/1/47 was amplified by RT-PCR and cloned into adenovirus shutter vector pAdTrack-CMV.The recombinant plasmid and adenovirus DNA was cotransformed into E.coli BJ5183 and the recombinant adenovirus genomic DNA was obtained through homologous recombination.The recombinant adenovirus was gained after transfecting 293 cell line with the genomic DNA.HA gene was confirmed to be integrated into the genome of recombinant adenovirus by PCR.The expression of HA was detected in the 293 cells infected with recombinant adenovirus.ELISA results showed that systemic immune responses to influenza virus could be induced effectively through both intranasal and oral routes as well as SIgA.The immunization efficacy of intranasal route was superior to that of oral route.Mice immunized with recombinant adenovirus showed complete protection against a challenge with 10 LD_(50) of influenza virus A/FM/1/47 whereas all control mice died.The replication-defective recombinant adenovirus expressing influenza virus hemagglutinin had been consturcted and could produce favorable immune responses.