人干扰素α-2b基因重组卡介苗的构建与鉴定

目的 构建能自动分泌表达人干扰素α-2b(IFNα-2b)的重组卡介苗(rBCG-IFNα-2b)菌株,并对其进行鉴定.方法 分别从人外周血和BCG基因组中提取DNA,聚合酶联反应(PCR)扩增人IFNα-2b基因和BCGAg85B信号肽基因,将该两片段插入质粒pMV261,构建分泌型卡介苗穿梭表达载体pMV261-Ag85B-IFNα-2b.电穿孔将该载体导入BCG中,构建rBCG-IFNα-2b.分别用PCR扩增和Western blot检测rBCG-IFNα-2b中人IFNα-2b基因和蛋白的表达情况,酶联免疫吸附(ELISA)检测rBCG-IFNα-2b培养上清中IFNα-2b蛋白的表达情况.结果 采用酶切、PCR扩增及DNA测序对pMV261-Ag85B-IFNα-2b进行鉴定,结果 显示BCG Ag85B信号肽片段和人IFNα-2b片段与文献结果 一致,连接方向正确.以rBCG-IFNα-2b DNA为模板、IFNα-2b引物进行PCR扩增后得到与理论上大小相同的扩增片段,Western blot显示rBCG-IFNα-2b的培养上清和菌体中均可检测到IFNα-2b蛋白的表达,且培养上清中蛋白的表达量明显多于菌体,ELISA法可检测到培养上清中有高表达的IFNα-2b蛋白(301.45 pg/ml).结论 成功构建了能自动分泌表达人IFNα-2b的新型重组卡介苗菌株rBCG-IFNα-2b,为进一步研究其免疫活性和抗膀胱肿瘤疗效奠定了基础.

Construction of recombinant bacillus Calmette-Guérin vaccine secreting human interferon-alpha 2b

Objective To construct a recombinant bacillus Calmette-Guérin vaccine(rBCG)secreting human interferon-alpha 2b(IFNα-2B).a recombinant bacillus Calmette Guérin vaccine(rBCG)were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction(PCR),respectively.IFNα-2b gene was cloned in E.coli-BCG shuttle-vector pMV261 to get pMV261-IFNα-2b.A new recombinant plasmid pMV261-IFNα-2b was constructed by inserting BCG Ag85B signal sequence into pMV261-Ag85B-IFNα-2b.Then,BCG was transformed with this recombinant plasmid by electroporation.and designated as rBCG-IFNα-2b.The DNA and protein expressions of IFNα-2b gene in rBCG were determined by PCR and Western blot respectively.Also the quantity of IFNα-2b protein secreted by rBCG in cuhure supernatants was determined by enzyme linked immunosorbent assay(ELISA).Results By partial nucleotide sequencing,the DNA sequences of human IFNα-2b and BCG Ag85B were consistent with that in the GeneBank,and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFNα-2b.BCG was successfully transformed with this recombinant plasmid by electroporation and the recombinant BCG(rBCG-IFNα-2b)was capable of synthesizing and secreting cytokine IFNα-2b.The concentration of IFNα-2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45 pg/mL Conclusions Recombinant BCG secreting human IFNα-2b(rBCG-IFNα-2b)was constructed successfully and the specific IFNα-2b protein can be expressed highly and steadily by rBCG vaccine.