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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。
The HIV-1 gag gene fragment was highly expressed in E. coli using a novel prokaryotic expression vector pDOG. The expression vector effectively initiates the translation of the expressed gene using the lambda PR promoter and the RBS of T7g-10. The expression fragment PG1 includes 13 amino acids at p17C-terminal, 74 amino acids at the entire p24 and p15N-terminal. Compared to PG1, the PG2 fragment does not contain the p17 sequence and has a deletion of 77 amino acids at the p24 N-terminus. The expression of both the total bacterial protein accounted for more than 20%. The recombinant protein is in the form of inclusion body. After extracting the inclusion body, it can be purified to over 90% purity by one-step ion-column chromatography. PG1 was specifically recognized by an anti-p24 mAb, while PG2 did not. The purified recombinant protein can react strongly with HIV-1 positive serum and can be used in HIV-1 antibody detection.