表达H3N2亚型猪流感病毒HA基因重组腺病毒对小鼠免疫原性的研究

【目的】构建一株表达H3N2亚型猪流感病毒(SIV)HA基因的复制缺陷型重组腺病毒,并测定其对小鼠的免疫效力。【方法】以含有SIV A/Swine/Guangdong/9/2005(H3N2)HA基因的重组质粒pMD18-H3HA为模板,利用带特定酶切位点的引物PCR扩增HA基因,将其亚克隆入质粒pIRES2-EGFP中,再次将含有H3HA及EGFP的基因片段克隆到腺病毒的穿梭质粒pDC315,构建重组穿梭质粒pDC315-H3HA-EGFP。利用脂质体转染方法将穿梭质粒pDC315-H3HA-EGFP和腺病毒骨架质粒pBHGloxΔE1,E3Cre共转染HEK293细胞,基于腺病毒感染后形成的典型细胞病变及EGFP基因在细胞中的表达筛选重组腺病毒rAd-H3HA-EGFP。将重组病毒rAd-H3HA-EGFP以108TCID50两次接种6周龄的Balb/c小鼠,时间间隔为3周,通过检测免疫小鼠的抗体水平及对病毒攻击的保护情况评价该重组病毒的免疫原性。【结果】HA基因已被重组到腺病毒的基因组中,并能够伴随病毒的复制而表达,表达蛋白具有良好的生物性活性。重组腺病毒rAd-H3HA-EGFP经增殖、纯化后其TCID50可达1.58×1010.mL-1,以108TCID50的剂量免疫小鼠后,能够诱导产生高水平的特异性抗体,并对H3亚型SIV的攻击提供有效保护。【结论】构建了一株具有良好免疫原性的复制缺陷型重组腺病毒,为H3亚型SI活载体疫苗的研制奠定了基础。

Immunogenicity of a Recombinant Adenovirus Expressing HA Gene of H3N2 Subtype Swine Influenza Virus in Mice

Objective]A strain of replication deficient recombinant adenovirus encoding HA of H3N2 subtype swine influenza virus (SIV) was generated and the immune efficacy was evaluated in mice.Method]To construct a recombinant adenovirus shuttle plasmid pDC315-H3HA-EGFP,HA gene of A/Swine/Guangdong/9/2005(H3N2) amplified by PCR from the recombinant plasmid pMD18-HA was sub-cloned into plRES2-EGFP.The gene fragment containing HA and EGFP was then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing HA gene (rAd-H3HA-EGFP) was generated by cotransfecting the recombinant shuttle plasmid pDC315-HA-EGFP and the backbone plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was screened by the typical cytopathic effect and expression of EGFP gene in HEK293 cells.The irnmunogenicity of the recombinant adenovirus was evaluated by inoculating 6-week-old BALB/c mice,through detecting specific antibody titer and protection against the virus challenge. Result]HA gene was recombined into the genome of the recombinant adenovirus rAd-H3HA-EGFP,and HA protein could be efficiently expressed in vitro.The TCID_(50) of the rAd-H3HA-EGFP was evaluated as 1.58×10~(10)·mL~(-1) after propagation and purification.Mice inoculated with 10~8 TCID_(50) were protected against the challenge with H3 subtype SIV and accompanied with high titer of antibodies. Conclusion]A strain of replication-defective adenovirus rAd-H3HA-EGFP with good immunogenicity was constructed,which would lay a foundation for the development of the engineered live virus vectored vaccine of H3 subtype of SIV.