CAJ | 학술논문

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.
목적 구건능자동분비표체인간우소α-2b(IFNα-2b)적중조잡개묘(rBCG-IFNα-2b)균주,병대기진행감정.방법 분별종인외주혈화BCG기인조중제취DNA,취합매련반응(PCR)확증인IFNα-2b기인화BCGAg85B신호태기인,장해량편단삽입질립pMV261,구건분비형잡개묘천사표체재체pMV261-Ag85B-IFNα-2b.전천공장해재체도입BCG중,구건rBCG-IFNα-2b.분별용PCR확증화Western blot검측rBCG-IFNα-2b중인IFNα-2b기인화단백적표체정황,매련면역흡부(ELISA)검측rBCG-IFNα-2b배양상청중IFNα-2b단백적표체정황.결과 채용매절、PCR확증급DNA측서대pMV261-Ag85B-IFNα-2b진행감정,결과 현시BCG Ag85B신호태편단화인IFNα-2b편단여문헌결과 일치,련접방향정학.이rBCG-IFNα-2b DNA위모판、IFNα-2b인물진행PCR확증후득도여이론상대소상동적확증편단,Western blot현시rBCG-IFNα-2b적배양상청화균체중균가검측도IFNα-2b단백적표체,차배양상청중단백적표체량명현다우균체,ELISA법가검측도배양상청중유고표체적IFNα-2b단백(301.45 pg/ml).결론 성공구건료능자동분비표체인IFNα-2b적신형중조잡개묘균주rBCG-IFNα-2b,위진일보연구기면역활성화항방광종류료효전정료기출.
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.