在用³⁵S-甲硫氨酸标记的细胞中检测单纯疱疹病毒胸苷激酶多肽。

Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine.

作者信息

Kit S, Jorgensen G N, Dubbs D R, Trkula D, Zaslavsky V

出版信息

Intervirology. 1978;9(3):162-72. doi: 10.1159/000148933.

PMID:201586

Abstract

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.

摘要

为了研究单纯疱疹病毒（HSV）胸苷激酶（TK）多肽的大小，已设计出从用35S-甲硫氨酸标记的感染细胞中纯化该酶的方法，具体如下：（i）在琼脂糖-5'-氨基-5'-脱氧胸苷上进行亲和层析；（ii）制备性等电聚焦或制备性聚丙烯酰胺凝胶电泳；（iii）甘油梯度离心。在每个纯化步骤中，酶组分的一部分也用免疫吸附剂——琼脂糖-抗HSV-1 TK免疫球蛋白（IgG）处理。从免疫吸附剂上洗脱的标记多肽通过SDS平板凝胶电泳和放射自显影进行分析。结果表明，HSV TK多肽的分子量约为40,000。TK阴性的HSV-1突变体B2006未能诱导出40千道尔顿的多肽。