人白细胞介素12在昆虫细胞中的表达

Expression of Human Interleukin 12(hIL 12) in Insect Cells

人白细胞介素１２（ｈＩＬ１２）是一种异源二聚体细胞因子，由ｐ４０和ｐ３５两个亚基通过二硫键连接而成。首先构建了两个表达载体ｐＶＬ１３９２ｈｐ４０和ｐＶＬ１３９３ｈｐ３５，并分别与线性化多角体病毒基因组ＤＮＡ共转染昆虫细胞株Ｓｆ９，用目视法筛选出重组病毒ＡｃＮＰＶｈｐ４０和ＡｃＮＰＶｈｐ３５。利用ＰＨＡ激活的人淋巴细胞增殖试验，在条件培液中检测到重组ｈＩＬ１２的生物活性，表达量约为１５～２μｇ／１０６细胞。经实时ＢＩＡ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析，ｈｐ３５和ｈｐ４０两亚基在昆虫细胞中获得表达。非还原条件下的Ｗｅｓｔｅｒｎｂｌｏｔ结果显示，重组ｈＩＬ１２的表观分子量为７６ｋＤａ，ｈｐ４０同源二聚体的表观分子量为９２ｋＤａ。重组ｐｈ４０具有明显抑制ｈＩＬ１２生物活性的作用。

Human interleukin 12(hIL 12) is a heterodimer cytokine,which consists of two disulfide linked subunits,p40 and p35.This paper reports the expression of hIL 12 using Baculovirus Expression System in insect cells.We firstly constructed two expression vectors pVL1392 hp40 and pVL1393 hp35,and then they were used to co transfect the insect cells(Sf9) separately with linearized polyhedrosis virus genomic DNA.Two kinds of recombinant viruses Ac NPV hp40 and AcNPV hp35 were visually screened out.Biological activity of the recombinant hIL 12(rhIL 12) was detected in the conditioned medium using proliferation assay of PHA activated human lymphocytes and the expression level was about 1 5～2μg/10 6cells.The results of real time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the subunits of rhIL 12,hp35 and hp40 were expressed successfully in the insect cells.The apparent molecular weights of rhIL 12 and hp40 homodimer were 76kDa and 92kDa under non reducing conditions of Western blot,respectively.The recombinant hp40 can significantly inhibit the biological activity of hIL 12.