Objective To construct a recombinant bacillus Calmette-Gutrin vaccine (rBCG) secreting hu-man B7-2. Methods The DNA fragment encoding human hB7-2 was amplified from the plasmid containing hB7-2 by using polymerase chain reaction (PCR) and then inserted into the shuttle expression vector pYL-MCS. The re-combinant plamid pYL-hB7-2 was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing, pYL-hB7-2 was electroperated into BCG to get rBCG. The DNA of hB7-2 gene in rBCG were deter-mined by PCR and nucleotide sequencing. The protein expression of hB7-2 was detected by SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) respectively. Results The hB7-2 sequence derived from rBCG by PCR was the same as that reported previously. The recombinant plamid pYL-hB7-2 was constructed successfully and con-firmed by restriction endonuclease analysis, PCR detection and nueleotide sequencing analysis, pYL-hB7-2 was suc-cessfully transformed into BCG by electroporation and was capable of synthesizing and secreting hB7-2 by SDS-PAGE. The level of hB7-2 in the culture supernatant of rBCG was 3.8U/ml. Conclusions The constructed recom-binant BCG strain produces and secretes costimulator human B7-2, which will provide a laboratory foundation for po-tential clinical use for bladder cancer immunotherapy.%目的 构建分泌表达人共刺激分子B7-2(hB7-2)的重组卡介苗(rBCG).方法 采用聚合酶链反应(PCR)以含hB7-2的质粒为模板扩增出hB7-2活性序列,利用基因重组技术插入pYL-MCS质粒构建出分泌型卡介苗穿梭表达载体pYL-hB7-2.用电穿孔方法将该质粒转入卡介苗得到rBCG,利用PCR扩增和测序检测其hB7-2的DNA表达,分别SDS-PAGE和ELISA法测定rBCG上清液中和菌内hB7-2蛋白.结果 酶切鉴定、PCR扩增和DNA测序均表明rBCG含有的hB7-2 DNA与文献结果一致,SDS-PAGE检测出有相应蛋白表达,ELISA法测定rBCG上清液中分泌的B7-2蛋白为3.8 U/ml.结论 构建的重组BCG可以分泌表达人共刺激分子hB7-2,能够提供激活免疫反应的共刺激信号,将会成为膀胱肿瘤免疫治疗的一种新型方法.
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