汉滩病毒包膜糖蛋白G1和G2重组杆状病毒表达载体的构建与表达及其免疫原性

BACULOVIRUS EXPRESSION OF M GENOME SEGMENT OF HANTAAN VIRUS AND IMMUNOGENIC STUDIES OF THE EXPRESSED GLYCOPROTEINS

将汉滩病毒（ＨＴＮＶ）Ｍ基因插入杆状病毒转移质粒ｐＡｃＹＭＩＢ多角体启动子下游附近，与Ｂｓｕ３６１酶切线性化的杆状病毒（ＡｃＶＥＰＡ）ＤＮＡ共同转染Ｓｆ９细胞，经空斑筛选获得了表达包膜糖蛋白（Ｇ１、Ｇ２）的重组杆状病毒（ＡｃｖＨａｎＭ）。经纯化ＡｃＶＨａｎＭＤＮＡ的Ｓｏｕｔｈｅｍｂｌｏｔ证实，Ｍ基因正确插入了杆状病毒基因组中。用抗糖蛋白混合单克隆抗体和病人血清做免疫荧光染色，观察到细小的特异性荧光颗粒，呈典型的核周分布。免疫荧光检测还证实，重组糖蛋白与９株抗糖蛋白单抗均起反应，提示重组糖蛋白的抗原位点分布与病毒毒粒糖蛋白相同或相似，用放射免疫沉淀（ＲＩＰ）分析仅显示Ｇ２带，且表达的Ｇ２分子量略小于病毒Ｇ２，这可能与两者的糖基化程度不同有关。研究还表明，重组糖蛋白具有细胞融合活性。

he recombinant baculovirus ,AcVHanM ,which contains the M genome segment of Hantaan virus ,was generated by cotransfection of Sf9 cells with the recombinant plasmid AcVHanM)and Bsu36-linearized AcVEPA DNA. The restriction enzyme analysis and Southern blot of purified AcVHanM DNA showed that the M gene was correctly inserted intothe recombinant baculovirus DNA adjacent to the downstream of the polyhedrin promotorgene.By immunoflurescent stain ing with anti-glycoprotein McAbs and HFRS patients sera,fineand perinuclear fluorescent granules were demonstrated in the infected cells.Only glycoprotein G2was immuno-precipitated from the radiolabeled AcVHanM infected Sf9 cell lysate and HTNVinfected vero E6 cell lysate.The reactivity of the expressed glycoproteins with two anti-G1and seven anti -G2 "McAbs indicated that the epitopes of the expressed glycoproteins and the authentic viral glycoproteins are similar.Under Low pH condition (pH5.5),cell fusion wasdemonstrated,in Sf9 cell infected with the recombinant virus which is generally taken as a biological characteristics of the envelope glycoproteins of Hantavirus