表达H5亚型禽流感同义(Consensus)HA蛋白的重组质粒构建及体外表达

Construction and in vitro expression of consensus-hemagglutinin-based recombinant plasmid of H5 subtype avian influenza virus

为研究禽流感病毒(AIV)H5亚型同义Consensus HA(Cons-HA5)基因的重组表达质粒在体外的表达情况,本研究通过对从NCBI流感数据库中获得的5000条H5亚型AIV的HA蛋白的序列比对、分析获得一条同义HA蛋白,将其相对应的核苷酸序列进行鸡体偏嗜性密码子优化,人工合成Cons-HA5基因并克隆于真核表达载体pCAGGS中构建重组表达质粒pCACons-HA5。将重组质粒转染293-T细胞,在激光共聚焦显微镜下观察转染后不同时间HA蛋白的表达情况,同时在转染后24 h、48 h后分别进行表达蛋白的western blot检测。结果表明,pCACons-HA5转染293-T细胞后,其表达的HA蛋白先分布于细胞质中,而后转移至细胞膜表面;westernblot鉴定结果表明,Cons-HA5重组蛋白可以与AIV多克隆血清反应分子量约为70 ku。该重组质粒的构建将为进一步Cons-HA5核酸免疫对H5亚型不同抗原群AIV的交叉保护免疫效力研究奠定基础。

To expression a consensus HA （Cons-HA5） gene of H5 subtype avian influenza virus （AIV） and localize the recombinant protein in transfected cells, a Cons-HA5 gene was designed by clustering 5,000 HA protein sequence of H5 subtype A1V available in GenBank and synthesized based on chicken-bisaed codon-optimization. The Cons-HA5 gene was cloned into the eukaryotic expression vector of pCAGGS to construct the recombinant plasmid pCACons-HA5. The pCACons-HA5 was transfected into 293-T cells for expression and identification. The result showed that the localization of expressed Cons-HA5 was initially found in the cytoplasm and then transported to the cell surface at 4 hours post transfection. Western blot result indicated that the molecular weight of Cons-HA5 was about 70 ku. These results provided a base for further study of the Cons-HA5 based DNA vaccine for protection against H5 subtype AIV strains in chickens.