Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.
Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.
复制标题
在大肠杆菌中质粒定向合成真正的腺病毒 2 早期区 1A 和 1B 蛋白。
DOI：
10.1128/mcb.4.8.1427-1439.1984
复制
发表时间：
1984
影响因子：
5.3
通讯作者：
Harter,ML
中科院分区：
文献类型：
--
作者：
Ko,JL;Harter,ML
人腺病毒2的转化区位于病毒基因组的左侧11.2%，由两个不同的遗传单位组成，称为E1 A和E1 B。含有与这些遗传单位互补的成熟E1 A 13 S和E1 B 22 S mRNA的完整核苷酸序列的cDNA已被引入到大肠杆菌Iac启动子下游短距离处的细菌质粒中。转化为适当的E。大肠杆菌宿主中，其中一个质粒pKHAO指导45千道尔顿（kd）蛋白质的合成，另一个质粒pKHBO指导54.9kd蛋白质的合成。这两种质粒编码的蛋白质均占总细胞蛋白质的0.1 - 0.3%，并且通过凝胶电泳、免疫沉淀和胰蛋白酶指纹分析确定，与真正的腺病毒2 E1 A 42- 50-kd和E1 B 53- 58-kd肿瘤抗原（T抗原）几乎相同。通过使用我们的pKHBO表达质粒，我们还能够证明E1 B 22 S mRNA中出现的第二个AUG序列对应于编码大腺病毒2 T抗原的基因的起始。这证实了基于DNA测序分析的理论推断，即大T抗原的翻译在内部ATG而不是在5 '近端ATG启动翻译。
The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from theEscherichia coli lacpromoter. Upon transformation into appropriateE. colihosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5′-proximal AUG.