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人IL-12的作用具有种属特异性,无法对鼠淋巴细胞起作用。因此,为了在啮齿动物模型上研究该细胞因子的免疫学效应,并评价其临床应用前景,就很有必要获得重组鼠IL-12(mIL-12)。为此,我们首先构建了两个表达载体pVL1393-mp40和pVL1393-mp35,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-mp40和AcNPV-mp35,然后共感染昆虫细胞,使mp40和mp35在昆虫细胞中共表达。经实时BIA和Northern blot分析,表明重组mIL-12在昆虫细胞中表达成功。经还原条件下的SDS-PAGE分析,重组mp40和mp35的分子量分别为40KDa和22KDa。非还原条件下的Western blot结果显示,重组mIL-12的表观分子量为80KDa。用抗体捕获法在条件培液中测到了重组产物的生物活性,经与参照标准相比照,重组mIL-12的表达量约为10-15μg/10~6细胞。
The role of human IL-12 is species-specific and can not act on murine lymphocytes. Therefore, in order to study the immunological effects of this cytokine in rodent models and to evaluate its clinical application prospects, it is necessary to obtain recombinant murine IL-12 (mIL-12). To this end, we first constructed two expression vectors, pVL1393-mp40 and pVL1393-mp35, and co-transfected the insect cell line Sf9 with the linearized polyhedrosis virus genomic DNA respectively to screen the recombinant viruses AcNPV-mp40 and AcNPV -mp35 and then co-infected with insect cells to co-express mp40 and mp35 in insect cells. Real-time BIA and Northern blot analysis showed that recombinant mIL-12 was successfully expressed in insect cells. After SDS-PAGE analysis under reducing conditions, the molecular weights of recombinant mp40 and mp35 were 40 kDa and 22 kDa, respectively. Western blot under non-reducing conditions showed that the apparent molecular weight of recombinant mIL-12 was 80 kDa. The biological activity of the recombinant product was measured by antibody capture in conditioned medium. The expression level of recombinant mIL-12 was about 10-15 μg / 10-6 compared to the reference standard.