HIV-1 型 gag 前体蛋白片段在大肠杆菌中的表达、纯化及鉴定

Expression and purification of gag fragments of human immunodeficiency virus type 1 in E.coli

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Ｔ７噬菌体基因１０（ｇ１０）的核糖体结合位点（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阳性血清发生特异反应，可以用于ＨＩＶ－１抗体检测。

A novel expression plasmid, which utilizes λ PR promoter and T7 g 10 RBS, was used to overproduce gag fragments of human immunodeficiency virus type 1 (HIV 1). One of the structures which was named PG1 encodes 13 amino acid residues of carboxyl terminal of p17, the entire p24 and 74 amino acids of the amino terminal of p15. Another clone was similar to PG1 except that it contained no p17 sequences and deleted 77 amino acid residues of amino terminal residues of p24. Both recombinant proteins were expressed to take more than 20% of the cell proteins, and existed as inclusion bodies. Following solubilizing with 8 mol/L urea, both could be purified to 90% purity after a simple ion exchange chromatography. PG1 could be 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.