IL-35重组卡介苗的构建及鉴定  

Construction and identification of the IL-35-BCG recombinant vaccine

作　　者：吕平 彭万胜[1,2] 董淮富 LV Ping;PENG Wan-sheng;DONG Huai-fu(Department of Pediatrics,The First Affiliated Hospital of Bengbu Medical College,Bengbu Anhui 233004,China)

机构地区：[1]蚌埠医学院第一附属医院儿科,安徽蚌埠233004 [2]安徽省蚌埠市第一人民医院儿科

出　　处：《蚌埠医学院学报》2020年第7期849-853,858,共6页Journal of Bengbu Medical College

基　　金：安徽省自然科学青年基金项目(1608085QH188)。

摘　　要：目的:构建分泌性表达白细胞介素(IL)-35真核载体pZM03(pBudCE4.1/GLS/IL35/OriM)-重组卡介苗(BCG)。方法:在化学合成的IL-35基因片段中插入pBudCE4.1/GLS/OriM,将合成后序列插入到IL-12真核共表达载体pBudCE4.1/GLS/IL12/OriM的质粒酶切回收产物pBudCE4.1/GLS/OriM中,构建表达IL-35的穿梭/共表达质粒重组质粒pZM03(pBudCE4.1/GLS/IL35/OriM),再以重组质粒电转化到BCG,构建表达IL-35和BCG的重组pBudCE4.1/GLS/IL35/OriM-BCG。结果:重组质粒pZM03(pBudCE4.1/GLS/IL35/OriM)经PCR扩增、测序鉴定,以及在CHO 293T细胞中表达产物的Western blotting检测,从转录和翻译水平对目标基因产物进行检测验证,pBudCE4.1/GLS/IL35/OriM-BCG在细胞水平经抗酸染色验证,证实基因IL-35正确插入载体pBudCE4.1/GLS/OriM中,且pBudCE4.1/GLS/IL35/OriM-BCG构建成功。结论:重组质粒pBudCE4.1/GLS/IL35/OriM-BCG可正确表达IL-35,为进一步研究IL-35功能及其应用价值奠定了良好的基础。Objective:To construct the recombinant eukaryotic expression vector pZM03(pBudCE4.1/GLS/IL35/OriM)containing secretory protein of interleukin 35(IL-35)-bacillus calmette guerin(BCG)recombinant vaccine.Methods:The chemically synthesized IL-35 gene fragment was cloned into the pBudCE4.1/GLS/OriM,the pBudCE4.1/GLS/IL35/OriM was obtained from the pBudCE4.1/GLS/IL12/OriM,and both of above was used to construct the co-expression vector pZM03(pBudCE4.1/GLS/IL35/OriM).The recombinant plasmid pZM03 was electrotransformated into the BCG to construct the recombinant co-expression of IL-35 and BCG plasmid pBudCE4.1/GLS/IL35/OriM-BCG.Results:The results of the detection of gene,transcription,translation and cellular levels,which included the PCR,sequencing,Western blotting and acid-fast staining methods,showed that the pBudCE4.1/GLS/IL35/OriM-BCG was successfully constructed.Conclusions:The recombinant plasmid pBudCE4.1/GLS/IL35/OriM-BCG can correctly express IL-35,which lays a good foundation for further research on the function and application value of IL-35.

关 键 词：卡介苗 白细胞介素-35 重组质粒 

分 类 号：R594.3[医药卫生—内科学]