人类免疫缺陷病毒Ⅰ型Gag前体蛋白片段在大肠杆菌中的表达、纯化及鉴定

利用新型原核表达载体pDOG,在大肠杆菌中高效表达了人类免疫缺陷病毒1型(HIV-1)gag基因片段。表达载体利用λPR启动子以及T7g-10的RBS来有效起始表达基因的翻译。表达片段PG1包括p17C-端13个氨基酸、整个p24以及p15N-端74个氨基酸。与PG1相比,PG2片段不含有p17序列,并缺失了p24N-端77个氨基酸。两者的表达量均占总菌体蛋白的20％以上。重组蛋白以包涵体形式存在,在提取包涵体后,经过一步离子柱层析,可以纯化到90％以上的纯度。PG1可被一株抗p24的单抗特异识别,而PG2则不能。纯化的重组蛋白能与HIV-1阳性血清发生很强的特异反应,可以用于HIV-1抗体检测中。

Expression. Purification and Characterization of Fragments of Gag Protein of Human Immunodeficiency Virus Type 1 (HIV-1) in Escherichia coli

A novel expression plasmid, which utilizes X PR promoter and T7 g-10RBS, was used to overproduce gag fragments of human immunodeficiency virus type 1 ( HIV-1 ) . fine of the constructs encodes 17 ammo amd residues of carboxyl terminal of p!7. the entire p24 and 74 ammo acids of the ammo terminal of p15. Another clone was similar to ,PGI except that it contained no p17 sequences and deleted 77 ammo acid residues of ammo terminal residues of p21. Both recombinanl proteins were expressed to take more than 20K of the cell proteins, and existed as inclusion bodies. Following solubilizmg with 8mol/I, urea, both could be purified to 90% purity after a simple ion exchange chromtography. PG1 could he recognized by one monoclonal antibody directed against HIV-1 p24. but PG2 could not. Both of the purified proteins could be used as highly specific reagents for HIV-1 diagnostic purposes.