禽流感病毒HA基因真核表达质粒的构建与表达

血凝素蛋白（HA）基因是禽流感病毒（AIV）重要的保护性抗原基因.为了研究 HA基因疫苗，用PCR扩增H5亚型AIV HA基因，将其克隆到质粒pcDNA4/HisMax和pRc/CMV上得到真核表达质粒pC4H5和pCMVH5.采用Tfx^TM-20、Superfect转染试剂和电转染法转染HeLa细胞，转染后的HeLa细胞经蛋白质印迹和血凝试验检测HA蛋白及其活性.结果表明，Superfect转染和电转染均能正确表达HA蛋白并具有生物学活性，蛋白质印迹检测到HA和HA裂解的HA1和HA2，与AIV 的HA、HA1、HA2蛋白的分子质量一致.从血凝试验结果看，Superfect和电转染表达的HA均具有血凝活性，而经Superfect转染的pC4H5的表达量是pCMVH5的8倍，表明pC4H5是一高效的真核表达质粒.

Construction and Expression of Avian Influenza Virus HA Gene Eukaryotic Vectors

Hemagglutinin (HA) gene is an important gene of protective antigen of Avian Influenza Virus (AIV).In order to study HA gene vaccine, the H5 gene of AIV, amplified by PCR, was subcloned into eukaryotic expressing vectors pcDNA4/HisMax and pRc/CMV. The recombinant plasmids were transfected into HeLa cell by Tfx^TM-20 transfection reagent, Superfect transfection reagent and electroporation respectively. The expressed HA was examined and identified by Western blotting and hemagglutination assay. The results showed that HA was expressed precisely in HeLa cells and had bioactivity. Three special bands (HA, HA1 and HA2) were found by Western blotting, which were identical to those of AIV. The quantity of expressed HA from pC4H5,transfected by superfect transfection reagent, was approximately 8 times as much as that from pCMVH5. These results suggest that pC4H5 is a highly efficient eukaryotic expressing vector.