人IL-2与EB病毒LMP1融合基因重组BCG的构建与表达

Construction and expression of rBCG with a fusion gene consisting of human IL-2 and Epstein-Barr virus LMP1

目的构建并鉴定人IL-2与EB病毒LMP1融合基因重组BCG(卡介苗)。方法利用RT-PCR从EB病毒阳性的B95-8细胞中获得去除致癌部分的LMP1基因,与扩增的IL-2通过overlap-PCR扩增获得融合基因。将融合基因连接到穿梭表达质粒pMV261,进行酶切、PCR及测序鉴定。重组质粒经电转导入BCG,通过Western blot鉴定目标抗原在rBCG中的表达。结果 PCR扩增获得453bp IL-2和1 225bp LMP1,利用overlap-PCR获得1 623bp的融合基因,通过电转仪将重组质粒导入BCG感受态细胞,构建的重组质粒能够在BCG中表达相对分子质量为5.7×104的目的蛋白,Western blot显示该蛋白能被兔IL-2单抗及鼠LMP1单抗识别。结论构建的rBCG能稳定表达融合蛋白,为rBCG的抗肿瘤作用和抗肿瘤疫苗的研发奠定了基础。

Objectives To clone the latent membrane protein 1(LMP1)gene of the Epstein-Barr virus(EBV)with its carcinogenic portions removed and the human IL-2gene,to ligate the target fragments to the shuttle expression vector pMV261,and to transform the recombinant plasmid into BCG in order to obtain recombinant BCG that could stably express LMP1 and IL-2. Methods The LMP1 gene was obtained from EBV-positive B95-8cells,and its carcinogenic portions were removed using RT-PCR.The LMP1 and IL-2genes were amplified to obtain a fusion gene using overlapping PCR.The fusion gene was transformed into the shuttle expression plasmid pMV261 and the recombinant plasmid was identified using enzymatic digestion,PCR,and sequencing.The constructed recombinant plasmid was electrotransposed into BCG,and expression of the recombinant plasmid was identified using Western blotting. Results IL-2with 453 bp and LMP1 with 1,225 bp were obtained via amplification with PCR,and a fusion gene with 1,623 bp was obtained using overlapping PCR.The recombinant plasmid was ligated into BCG competent cells using electroporation.The target protein with a relative molecular mass of 5.7×104 was expressed in BCG,and it was recognized by a rabbit anti-IL-2monoclonal antibody and a mouse anti-LMP1 monoclonal antibody according to Western blotting. Conclusion The constructed rBCG stably expressed the fusion protein,thus laying the foundation for the further study of the antitumor action of rBCG and the development of antitumor vaccines.