含有鸡体偏嗜性密码子的H5亚型禽流感病毒HA基因的优化构建及其DNA疫苗体外瞬时表达研究

不同物种间细胞在蛋白翻译过程中具有密码子使用的偏嗜性,鸡与A型流感病毒的密码子使用频率存在着明显差异.为此,利用寡核苷酸合成、重叠延伸PCR及限制酶切法构建了密码子优化的表达A/Goose/GuangDong/1/96(H5N1)GD/1/96(H5N1)]HA蛋白的基因optiHA5,并将其插入CMV启动子表达载体pCI构建了H5亚型DNA疫苗质粒pCIoptiHA5,将含有GD/1/96(H5N1)野生型HA基因的质粒pCIHA5和pCIoptiHA5分别转染293T细胞,间接免疫荧光检测转染后24～48h293T细胞中瞬时表达的HA抗原蛋白;Westemblot证实上述2种表达质粒均可正确表达H5亚型HA抗原蛋白,结果表明密码子优化的基因optiHA5体外瞬时表达水平显著高于野生型HA基因.这一结果为进一步开展SPF鸡免疫保护效果的比较研究奠定了基础.

Construction of codon optimized H5 avian influenza HA gene with chicken bias codons and in vitro expression of its DNA vaccine

Codon usage optimization of the immunogenic genes is considered effective strategy for improving the protection efficacy of DNA vaccine.It is well known that different species have different bias in using the codons for protein translation.The codons in the influenza viruses are quite different from the codons in chickens.Therefore,using synthesized oligos and over-lapping extension PCR techniques,we constructed a codon optimized H5 avian influenza HA gene by replacing the codons of avian influenza virus with the codons of chickens.The optimized HA gene was inserted into pCI,and was designated as pCIoptiHA5.The monolayer 293T cells were transfected by pCIoptiHA5 and pCIHA5,which contained wild type HA gene of A/Goose/GuangDong/1/96(H5N1)GD/1/96(H5N1)].The HA protein expression was detected by indirect immunofluorescence assay after transfection 24?h to 48?h,and was confirmed by western-blotting analysis.The results indicated the optimized HA gene was better expressed than the wild type HA gene in the same vector,and they were the basis for valuing the protection efficacy in SPF chickens.