McGarry T J, al-Ahdal M N
Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.
Biomed Chromatogr. 1989 Sep;3(5):221-5. doi: 10.1002/bmc.1130030510.
Rabbit kidney (RK-13) and human jejunum and ileum (I-407) cells infected with herpes simplex virus type 1, strain F, were radiolabelled with [14C]glucosamine or [35S]methionine for 24 h. The cells were extracted with 1% Triton X-100 and the extracts were separated by gel filtration high performance liquid chromatography. Monoclonal antibody immunoprecipitation of the fractions collected from the column revealed a monomeric glycoprotein D (gD) of 52 - 56,000 molecular weight from RK-13 cells and two monomeric forms of gD, 54,000 and 58,000 molecular weight, from I-407 cells. Densitometry scanning of the autoradiograms from SDS-PAGE showed gD from the RK-13 host cells to be 98.7% pure with the [35S]methionine label and 97.0% pure with the [14C]glucosamine. On the other hand, gD from the I-407 host cells was only 78.6% with the [35S]methionine label and 96% pure with the [14C]glucosamine. This method could provide a means for the isolation of native gD for structural and immunological studies.
用1型单纯疱疹病毒F株感染的兔肾（RK - 13）细胞以及人空肠和回肠（I - 407）细胞，用[14C]葡萄糖胺或[35S]甲硫氨酸进行放射性标记24小时。用1% Triton X - 100提取细胞，提取物通过凝胶过滤高效液相色谱进行分离。对从柱上收集的各组分进行单克隆抗体免疫沉淀，结果显示RK - 13细胞中有分子量为52 - 56,000的单体糖蛋白D（gD），而I - 407细胞中有两种单体形式的gD，分子量分别为54,000和58,000。SDS - PAGE放射自显影片的光密度扫描显示，RK - 13宿主细胞的gD用[35S]甲硫氨酸标记时纯度为98.7%，用[14C]葡萄糖胺标记时纯度为97.0%。另一方面，I - 407宿主细胞的gD用[35S]甲硫氨酸标记时纯度仅为78.6%，用[14C]葡萄糖胺标记时纯度为96%。该方法可为分离天然gD用于结构和免疫学研究提供一种手段。
