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GM-CSF与BZLF1融合基因修饰的重组BCG的构建与表达

, PP. 1035-1039

Keywords: 融合基因GCBF,基因重组,重组卡介苗

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Abstract:

目的将人粒细胞-巨噬细胞集落刺激因子基因(GM-CSF)与EB病毒即刻早期基因(BZLF1)重组为融合基因GCBF,并转化入卡介苗(BCG)中进行表达。方法用随机引物Oligo(dT)15进行RT-PCR,分别获得人GM-CSF和BZLF1编码序列的cDNA。将纯化GM-CSF和BZLF1PCR扩增产物插入分泌表达载体pMV261,并转化入感受态细胞EscherichiacoliDH5α(E.coliDH5α),在LB培养基上进行卡那霉素抗性选择,测序正确的序列进行剪接式重叠延伸,将目的基因GM-CSF和BZLF1编码经多肽接头(Gly4Ser)3序列连接,构建融合基因GCBF,将GCBF克隆至pMV261,转化感受态细胞E.coliDH5α,在LB培养基平板上进行卡那霉素抗性选择,阳性克隆提取质粒后转化感受态BCG,western-blot检测GCBF在rBCG中的表达。结果目的基因GM-CSF和BZLF1RT-PCR产物大小分别为461bp和788bp,与预期值一致。构建的重组质粒经双酶切、扩增及测序鉴定证实,融合基因GCBF(1209bp)正确插入载体,成功转化入BCG感受态细胞,且被正确表达。结论融合基因GCBF修饰的rBCG构建及表达成功,为进一步探讨rBCG杀伤EB病毒阳性肿瘤的免疫活性研究奠定了实验基础。

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