Legrain M, Portetelle D, Dumont J, Burny A, Hilger F
Unit of Microbiology, Faculty of Agronomy, Gembloux, Belgium.
Gene. 1989 Jul 15;79(2):227-37. doi: 10.1016/0378-1119(89)90205-9.
The nucleotide sequence coding for bovine leukemia virus (BLV) envelope glycoprotein gp51 was inserted into a yeast-Escherichia coli shuttle vector carrying the promoter and secretion signal sequence of PHO5 (the yeast gene coding for repressible acid phosphatase) and the CYC1 transcriptional terminator. Yeast cells transformed by this construction synthesized gp51 after PHO5 induction by inorganic phosphate deprivation. The yeast-expressed gp51 was partially glycosylated into heterodisperse protein molecules ranging from 40 to 48 kDa. No gp51 was excreted in the culture medium. The amount of protein accumulated in yeast cells was estimated to reach 0.06% of soluble proteins. This modest level of expression seemed to be due to the toxicity of gp51 to the yeast cell. The yeast-expressed gp51 products were used in enzyme-linked immunosorbent assays for the detection of antibodies in sera from BLV-infected animals; they were also screened for the presence of well-defined biological epitopes. In both studies poor reactivity was observed. Rabbits immunized with the recombinant gp51 showed high antibody titers to native BLV gp51. However, these antibodies did not neutralize BLV in vitro.
将编码牛白血病病毒（BLV）包膜糖蛋白gp51的核苷酸序列插入到一个酵母-大肠杆菌穿梭载体中，该载体带有PHO5（编码可阻遏酸性磷酸酶的酵母基因）的启动子和分泌信号序列以及CYC1转录终止子。通过这种构建体转化的酵母细胞在无机磷酸盐缺乏诱导PHO5后合成了gp51。酵母表达的gp51被部分糖基化，形成了分子量在40至48 kDa之间的异质分散蛋白分子。没有gp51分泌到培养基中。估计酵母细胞中积累的蛋白量达到可溶性蛋白的0.06%。这种适度的表达水平似乎是由于gp51对酵母细胞有毒性。酵母表达的gp51产物用于酶联免疫吸附测定，以检测BLV感染动物血清中的抗体；还对其进行筛选，以确定是否存在明确的生物学表位。在这两项研究中，均观察到反应性较差。用重组gp51免疫的兔子对天然BLV gp51显示出高抗体滴度。然而，这些抗体在体外不能中和BLV。
