分泌人白介素-2的重组卡介苗构建、表达与鉴定

Construction,Expression and Identification of Recombinant BCG secreting Human interleukin-2

目的构建分泌人白介素-2(IL-2)的重组卡介苗(BCG)。方法采用PCR技术从BCG基因组中扩增出Ag85B的信号肽基因序列,从pBIL-2质粒中扩增出IL-2cDNA全长序列。利用DNA重组技术将以上两个片段插入质粒pMV261结核杆菌HSP60启动子下游,构建一个分泌型的卡介苗穿梭表达质粒pMSIL-2。利用电穿孔技术将质粒pMSIL-2转入BCG得到重组BCG,分别用PCR扩增和Western blotting检测重组BCG菌体和培养上清中IL-2的DNA和蛋白表达,酶联免疫吸附法(ELISA)检测重组BCG培养上清中IL-2的含量。结果酶切鉴定、PCR扩增和DNA测序分析结果均表明,所克隆的信号肽DNA片段和IL-2片段与文献结果完全一致,pMSIL-2的连接方向正确,阅读框与预期完全一致。以重组BCG质粒DNA为模板,通过PCR扩增得到IL-2的基因片段,Western blot证实重组BCG分泌的蛋白能与人IL-2的抗体特异性结合,ELISA法检测到重组BCG培养上清中高含量的IL-2。结论构建的重组BCG能够分泌具有功能活性的细胞因子IL-2,有望成为膀胱肿瘤免疫治疗的一种有效方法。

Objective To construct a recombinant BCG vaccine secreting interleukin-2 (IL-2). Methods BCG Ag85B signal sequence was amplified from the genome of BCG by using polymerase chain reaction (PCR) and cloned in E.coli-BCG shuttle-vector pMV261 to get pMS. The cDNA fragment encoding human IL-2 was amplified from the plasmid pBIL-2 by using PCR and inserted into the shuttle expression vector pMV261. The recombinant plamid pMSIL-2 was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing. pMSIL-2 was electroporated into BCG to get rBCG. The DNA and protein expressions of IL-2 gene in rBCG were determined by PCR and Western blotting respectively. IL-2 in the culture supernatant of rBCG was detected by an enzyme-linked immunosorbent assay (ELISA). Results The recombinant plamid pMSIL-2 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and nucleotide sequencing analysis. pMSIL-2 was successfully transformed into BCG by eletroporation and were capable of synthesizing and secreting cytokine IL-2. Western blotting revealed that the secretive proteins could specially combine with antibody against human IL-2. the level of IL-2 in the culture supernatant of rBCG was higher than control group by ELISA assay. Conclusions The constructed recombinant BCG strain produce and secrete human IL-2 and it will be used for the treatment of superficial bladder cancer.