在昆虫细胞中产生的腺病毒5型和12型E1A蛋白的表达、纯化及功能特性分析

Expression, purification, and functional characterization of adenovirus 5 and 12 E1A proteins produced in insect cells.

作者信息

Peeper D S, Zantema A, Dowdy S F, van der Eb A J

机构信息

Department of Molecular Carcinogenesis, Sylvius Laboratory, University of Leiden, The Netherlands.

出版信息

Virology. 1992 Oct;190(2):733-45. doi: 10.1016/0042-6822(92)90911-8.

DOI:10.1016/0042-6822(92)90911-8

PMID:1387752

Abstract

The 12 S and 13 S E1A cDNAs from both the Adenovirus (Ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. Upon infection of Spodoptera frugiperda cells, the production of E1A proteins reached a level of about 15 micrograms/10(6) cells. The E1A proteins are highly soluble and apparently are processed authentically. They are readily recognized by various antibodies and display phosphorylation patterns similar to those of E1A proteins synthesized in mammalian cells. Single-step immunoaffinity chromatography was used to purify the Ad5 E1A proteins to near homogeneity under nondenaturing conditions. The Ad5 and Ad12 E1A proteins are able to form complexes with the retinoblastoma susceptibility gene product (Rb) and other cellular proteins. Interestingly, the presence of a cellular extract seems to be a prerequisite for association between highly purified E1A and Rb polypeptides.

摘要

来自腺病毒（Ad）5型非致癌株和12型致癌株的12S和13S E1A cDNA在昆虫细胞/杆状病毒系统中过表达。感染草地贪夜蛾细胞后，E1A蛋白的产量达到约15微克/10⁶个细胞的水平。E1A蛋白高度可溶，显然经过了正确的加工。它们很容易被各种抗体识别，并且显示出与在哺乳动物细胞中合成的E1A蛋白相似的磷酸化模式。在非变性条件下，采用单步免疫亲和层析法将Ad5 E1A蛋白纯化至接近均一。Ad5和Ad12 E1A蛋白能够与视网膜母细胞瘤易感基因产物（Rb）及其他细胞蛋白形成复合物。有趣的是，细胞提取物的存在似乎是高度纯化的E1A与Rb多肽之间发生结合的前提条件。