Brantl S, Eldarov M A, Rössler H, Drescher B, Lang H, Rosenthal S, Skryabin K G
Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of the GDR, DDR, Jena.
Yeast. 1988 Mar;4(1):47-59. doi: 10.1002/yea.320040106.
DNA sequences of the envelope (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast Saccharomyces cerevisiae. Two yeast promoters, the repressible PHO5 promoter and the constitutive PGK promoter, were used to construct four expression plasmids comprising either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence. The expressed heterologous gene products were characterized by Western blot analysis and competitive radioimmunoassay. By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed. The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence--a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus--as well as the PHO5 promoter including PHO5 signal sequence and the PHO5 terminator. The recombinant gp51 was partially glycosylated but the PHO5 signal peptide did not seem to be cleaved off. No immunoreactive material could be found in the periplasm or in the culture medium. By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all four sequential antigenic determinants were detected in the AH 216 (YEpSG 94) expression product.
牛白血病病毒（BLV）包膜（env）基因的DNA序列在酿酒酵母中得以表达。使用了两个酵母启动子，即可阻遏的PHO5启动子和组成型PGK启动子，构建了四个表达质粒，这些质粒包含表面抗原gp51的序列或（gp51 + gp30）序列。通过蛋白质免疫印迹分析和竞争性放射免疫测定对表达的异源基因产物进行了表征。借助Northern印迹分析对env特异性mRNA的稳态水平进行了分析。从包含gp51序列（一个630碱基对的片段，含70%的gp51但缺少N端）以及包括PHO5信号序列和PHO5终止子的PHO5启动子的重组质粒YEpSG 94中获得了最高表达率。重组gp51部分糖基化，但PHO5信号肽似乎未被切割掉。在周质或培养基中未发现免疫反应性物质。借助针对病毒gp51八个不同表位的单克隆抗体，在AH 216（YEpSG 94）表达产物中检测到了所有四个连续的抗原决定簇。
