【作者】 刘洪波； 吴灿权； 谢颖； 梁洪； 蔡昌学；

【Author】 LIU Hong-bo~△,WU Can-quan,XIE Yin,LIANG Hong,CAI Chang-xue.(△Zhongshan Center for Disease Control and Prevention,Zhongshan 528403,China)

【机构】 华中科技大学同济医学院； 中山市疾病预防控制中心；

【摘要】 目的构建分泌表达鼠IFN-γ(Mouse interferon-gamma,mIFN-γ)的重组卡介苗(Bacillus Calmette-Guerin,BCG)。方法PCR扩增BCG抗原85B的信号肽序列,插入到E.coli-BCG穿梭载体pBCG3000的启动子下游,构建分泌性重组穿梭质粒S-paCG3000:以PHA刺激小鼠脾细胞产生的RNA为模板,RT-PCR扩增mIFN-γ成熟肽的编码cDNA,插入到S-pBCG3000质粒的信号肽序列下游,构建mIFN-γ的表达质粒S-pBCG3000-γ;通过电穿孔技术将表达质粒转入BCG,以卡那霉素和PCR筛选重组BCG;ELISA法检测重组BCG培养滤液中的mIFN-γ含量,细胞病变抑制法鉴定mIFN-γ的生物活性。结果经测序、PCR扩增和酶切鉴定分析,Ag85B信号肽和mIFN-γ成熟肽的编码序列均成功克隆并插入到pBCG-3000的相应位点,表达质粒转化BCG后获得的重组BCG分泌mIFN-γ的含量高达1234.1 pg/ml,在L929细胞中的抗VSV活性为64 U/ml。结论所构建的重组BCG可分泌具有功能活性的mIFN-γ,为进一步研究其免疫效应奠定了基础。更多还原

【Abstract】 Objective To construct a recombinant BCG secreting mouse interferon gamma(mIFN-γ).Methods The signal peptide sequence of BCG antigen 85B was obtained by PCR amplification and cloned into downstream to the promoter of E.coli-BCG shuttle vector pBCG3000,yielding the secretory recombinant shuttle vector S-pBCG3000.The cDNA encoding mature peptide of mIFN-γwas amplified by RT-PCR from PHA-activated splenocyte RNA and inserted downstream to the signal sequence of S-pBCG3000.The constructed recombinant plasmid S-pBCG3000 was transformed into BCG by electroporation, and recombinant BCG was screened with kanamycin and PCR.The mIFN-γcontent in culture filtrate of recombinant BCG was determined by ELISA,and the biological activity of mIFN-γby cytopathic inhibition test.Results Sequencing,PCR and restriction enzyme analysis proved that the gene sequences encoding Ag85B signal peptide and mIFN-γmature peptide were successfully cloned and inserted into the corresponding sites ofpBCG3000.The mIFN-γcontent secreted by the recombinant BCG reached 1234.1 pg/ml,and the mIFN-γactivity against VSV in L929 cells was 64 U/ml.Conclusion The recombinant BCG secreting bioactive mIFN-γwas constructed successfully,which laid a foundation of further research on its immune effects.更多还原