Enhanced protective efficacy of avian influenza DNA vaccine with expressive vector pCAGGS

将A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]的HA基因插入鸡β-actin启动子高效真核表达载体pCAGGS，构建了DNA疫苗质粒pCAGGHA5，以提高H5亚型禽流感DNA疫苗的表达水平和免疫保护效果。将pCAGGHA5和表达GD/1/96(H5N1)HA基因的质粒pCIHA5通过间接免疫荧光法和Western-blot分析检测转染293T细胞后瞬时表达的HA抗原蛋白，随之将pCAGGHA5及pCIHA5分别以100 andmicro;g和10 andmicro;g剂量一次免疫3周龄SPF鸡，4周后用100 LD50的HPAIV GD/1/96(H5N1)鼻腔途径进行攻击。间接免疫荧光法和Western-blot分析表明2种表达质粒均可正确表达H5亚型HA抗原蛋白，载体pCAGGS表达水平显著高于载体pCI；免疫SPF鸡后，100andmicro;g pCAGGHA5可形成5/5的免疫保护，100andmicro;g pCIHA5可形成2/4的免疫保护，10andmicro;g pCAGGHA5可形成对免疫鸡5/5的免疫保护，而10andmicro;g pCIHA5 则基本不能形成免疫保护，pCAGGHA5诱导的HI抗体水平远远高于pCIHA5。鸡β-actin启动子表达载体pCAGGS可显著提高HA基因体外表达水平和H5亚型禽流感DNA疫苗诱导的保护性抗体免疫反应水平，增强免疫保护效果。 To improve the protective efficacy of H5 subtype avian influenza DNA vaccine, the HA gene of A/Goose/GuangDong/1/96(H5N1) [GD/1/96(H5N1)] was inserted into pCAGGS, which contains chickenβ-actin promoter, and was designated as pCAGGHA5. The monolayer 293T cells were transfected by pCAGGHA5 and pCIHA5, which contained HA gene of GD/1/96(H5N1). The HA protein expression was detected by indirect i