汉滩病毒膜蛋白重组腺病毒的构建表达及免疫效果研究

Recombinant adenovirus construction of Hantaan virus M genome segment and immunogenic studies of the expressed glycoproteins

探讨利用腺病毒载体作为汉滩病毒基因工程疫苗载体的可行性。通过PCR扩增,得到完整的汉滩病毒76-118株M片段编码区,将该片段克隆入质粒pAdTrackCMV的CMV启动子下游,得到阳性克隆pAdTrackCMV-M。PmeI线性化的阳性克隆与腺病毒骨架载体pAdEasy-1共转化大肠杆菌BJ5183,经同源重组后得到重组病毒pAdEasy-M。pAdEasy-M经PacI线性化后,脂质体介导转染293细胞,经Western-blot检测表明,G1、G2基因在293细胞中得到表达。重组病毒免疫BALB/c小鼠,产生了具有一定中和活性的抗体。该研究为进一步研制以腺病毒为活载体的工程疫苗奠定了基础。

In order to study the immune responses of the recombinant adenovirus which contain the Hantaan virus M seg-ment.The76-118M genome segment,which codes for the two envelope glycoproteins G1and G2,was cloned to the plasmid pAdTrackCMV.Then the positive clone pAdTrackCMV-M was proved by restriction enzyme digestion.The lin-earized positive clone was cotransformed the E.coli BJ5183cells with the adenoviral plasmid pAdEasy-1.After homologous recombination,the recombinant adenoviral plasmid pAdeasy-M was constructed.The pAdEasy-M was linearized with PacI digestion,then transfected to293cell by lipofection.The SDS-PAGE and Western-blot proved the expression of G1and G2in293cells.The neutralizing antibodies were obtained by immune the BALB/c mice with pAdEasy-M.This result make the base of the future study.