重组猪IL-7在真核细胞的分泌表达及生物活性分析

Expression of recombinant porcine interleukin-7 in eukaryotic cells and analysis of its biological activity

为了解猪IL-7(pIL-7)在防治猪免疫抑制性传染病及疫苗生物佐剂中的作用,通过RT-PCR方法从猪脾脏中扩增出含有自身信号肽序列的pIL-7 cDNA基因,将其克隆到pcDNA3.1A表达载体上,构建出羧基端与Myc/His融合的pIL-7基因真核表达载体pcDNA3.1A-pIL-7/MH。由磷酸钙介导将pcDNA3.1A-pIL-7/MH质粒转染人类胚胎肾细胞293T(HEK293T)使其进行瞬时表达,以Western-blot检测构建的pIL-7基因表达载体是否可介导重组pIL-7在真核细胞中进行分泌型表达。然后利用Ni-NTA琼脂糖亲和层析纯化表达的重组pIL-7,并通过淋巴细胞增殖试验检测其的生物活性。结果表明,扩增的pIL-7基因序列与GenBank序列完全一致;构建的表达载体可介导重组pIL-7在真核细胞HEK293T中进行分泌型表达。表达的重组pIL-7对猪脾淋巴细胞有明显的促增殖效应。由此可见,利用真核细胞表达的重组pIL-7具有生物活性。

To investigate the effect of porcine IL-7 （plL-7） on prevention and treatment of por- cine immunosuppressive diseases and adjuvant functions of animal vaccine, plL-7 gene contai- ning the signal peptide sequence was amplified from porcine spleen lymphocytes by RT-PCR, and then was inserted into the eukaryotic expression vector pcDNA3.1A to construct the Myc/ His-tag-fused pIL-7 gene eukaryotic secretory expression vector （pcDNA3. 1A-pIL-7/MH）. To determine whether the expression vector could mediate pIL-7 gene expression in eukaryotic cells, the pcDNA3.1A-pIL-7/MH plasmid was transfected into HEK293T cells using calcium phosphate transfection method for transient expression. The expressed recombinant plL-7 was purified using Ni-NTA agarose affinity chromatography. The biological activity of the recombi- nant pIL-7 was detected using lymphocyte proliferation assay. The results showed that the se- quence of pIL-7 gene amplified in this study was consistent with that in GenBank. Western blot showed that the recombinant pIL-7 could be expressed and secreted in HEK293T cells. Lymphocyte proliferation assay showed that the recombinant plL-7 significantly promoted por- cine spleen lymphocyte proliferation, which indicates that the recombinant plL-7 prepared in this study possesses the biological activity.