汉滩病毒膜蛋白重组腺病毒的构建表达及免疫效果研究

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。Pmel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。

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

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.