摘要
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 bacillus Calmette-Gu6rin vaccine （BCG） secreting human interferon α-2a （IFNα-2a）, Methods BCG Ag85B signal sequence was amplified from the genome of BCG by using polymerase chain reaction （PCR） and cloned in E. coli-BCG shuttle-vector pMV261 to get pMS. The cDNA fragment encoding human IFNα-2a was amplified from the plasmid pBIFNct- 2a by using PCR and inserted into the shuttle expression vector pMV261. The recombinant plamid pMSIFNα-2a was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing, pMSIFNα-2a was electroporated into BCG to get rBCG. The DNA and protein expressions of IFNα-2a gene in rBCG were determined by PCR and Western blotting respectively. IFNα-2a in the culture superuatant of rBCG was detected by enzyme-linked immunosorbent assay （ELISA）. Results The recombinant plamid pMSIFNα-2a was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and nucleotide sequencing analysis, pMSIFNα-2a was successfully transformed into BCG by eletroporation and were capable of synthesizing and secreting cytokine IFNα-2a. Western blotting revealed that the secretive proteins could specially combine with antibody against human IFNα-2a. the level of IFNα-2a （324.57 pg/ml）in the culture supernatant of rBCG was higher than control group by ELISA assay. Conclusion The constructed recombinant BCG strain produces and secretes human IFNα-2a and it will be used in the treatment of superficial bladder cancer.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第34期2417-2420,共4页
National Medical Journal of China
基金
上海市科委重点基金(034119849)
