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目的构建分泌人干扰素α-2a(IFNα-2a)的重组卡介苗(rBCG)。方法采用聚合酶链反应(PCR)技术从卡介苗基因组中扩增出 Ag85B 的信号肽基因序列,从 pBIFNα-2a 质粒中扩增出IFNα-2acDNA 全长序列。利用 DNA 重组技术将以上两个片段插入质粒 pMV261结核杆菌 HSP60启动子下游,构建一个分泌型的卡介苗穿梭表达质粒 pMSIFNα-2a。利用电穿孔技术将质粒 pMSIFNα-2a 转入卡介苗得到 rBCG,分别用 PCR 扩增和 Western 印迹检测 rBCG 菌体和培养上清中 IFNα-2a 的DNA 和蛋白表达,用酶联免疫吸附法(ELISA)检测 rBCG 培养上清中 IFNα-2a 的含量。结果酶切鉴定、PCR 扩增和 DNA 测序分析结果均表明,所克隆的信号肽 DNA 片段和 IFNα-2a 片段与文献结果完全一致,pMSIFNα-2a 的连接方向正确,阅读框与预期完全一致。以 rBCG 质粒 DNA 为模板,通过PCR 扩增得到 IFNα-2a 的基因片段,Western 印迹证实 rBCG 分泌的蛋白能与人 IFNα-2a 的抗体特异性结合,ELISA 法检测到 rBCG 培养上清中高含量的 IFNα-2a(324.57 pg/ml)。结论构建的 rBCG 能够分泌具有功能活性的细胞因子 IFNα-2a,有望成为膀胱肿瘤免疫治疗的一种有效方法。
Objective To construct a recombinant BCG secreting human interferon α-2a (IFNα-2a). Methods The signal peptide gene sequence of Ag85B was amplified from BCG genome by polymerase chain reaction (PCR), and the full-length IFNα-2 cDNA was amplified from plasmid pBIFNα-2a. The above two fragments were inserted into the downstream of the promoter of Mycobacterium tuberculosis HSP60 by using DNA recombination technology to construct a secreting BCG shuttle plasmid pMSIFNα-2a. The plasmid pMSIFNα-2a was transferred into BCG by electroporation to obtain rBCG. The DNA and protein expression of IFNα-2a in rBCG and culture supernatant were detected by PCR amplification and Western blot respectively. The expression of IFNα-2a was detected by enzyme-linked immunosorbent assay (ELISA) The content of IFNα-2a in rBCG culture supernatant was detected. Results The results of restriction endonuclease digestion, PCR amplification and DNA sequencing showed that the cloned signal peptide DNA fragment and IFNα-2a fragment were completely consistent with the results in the literature. The orientation of pMSIFNα-2a was correct and the reading frame was exactly the same as expected. The gene fragment of IFNα-2a was amplified by PCR using rBCG plasmid DNA as a template. Western blotting confirmed that the protein secreted by rBCG specifically binds to the human IFNα-2a antibody. The high level of IFNα in the rBCG culture supernatant was detected by ELISA -2a (324.57 pg / ml). Conclusion The constructed rBCG can secrete IFNα-2a, a functional cytokine, which is expected to be an effective immunotherapy for bladder cancer.