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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(d T)15进行RT-PCR,分别获得人GM-CSF和BZLF1编码序列的c DNA。将纯化GM-CSF和BZLF1 PCR扩增产物插入分泌表达载体p MV261,并转化入感受态细胞Escherichia coli DH5α(E.coli DH5α),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p MV261,转化感受态细胞E.coli DH5α,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r BCG中的表达。结果目的基因GM-CSF和BZLF1 RT-PCR产物大小分别为461 bp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r BCG构建及表达成功,为进一步探讨r BCG杀伤EB病毒阳性肿瘤的免疫活性研究奠定了实验基础。
Objective To recombine human granulocyte-macrophage colony-stimulating factor gene (GM-CSF) and Epstein-Barr virus immediate early gene (BZLF1) into fusion gene GCBF and transform into BCG for expression. Methods RT-PCR was performed with a random primer Oligo (d T) 15 to obtain c DNA of human GM-CSF and BZLF1 coding sequences, respectively. The purified GM-CSF and BZLF1 PCR products were inserted into the secretion expression vector pMV261 and transformed into competent cells Escherichia coli DH5α (E.coli DH5α). Kanamycin resistance was selected on LB medium and sequenced correctly (Gly4Ser) 3 sequence of target gene GM-CSF and BZLF1 to construct a fusion gene GCBF. The GCBF was cloned into pMV261 and transformed into competent cells E. coli DH5α. The kanamycin resistance selection was carried out on the medium plate. The positive clones were transformed into competent BCG after the plasmid was extracted, and the expression of GCBF in r BCG was detected by western-blot. Results The size of the target gene GM-CSF and BZLF1 RT-PCR products were 461 bp and 788 bp, respectively, consistent with the expected values. The constructed recombinant plasmid was confirmed by double enzyme digestion, amplification and sequencing. The fusion gene GCBF (1209bp) was correctly inserted into the vector and successfully transformed into BCG competent cells and correctly expressed. Conclusion The construction and expression of fusion gene BCBG modified by GCBF is successful, which lays the foundation for further research on the immunological activity of rBCG-killing EBV-positive tumors.