科研之友
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摘要
Our objective was to construct a recombinant bacillus Calmette-Gu,rin vaccine (rBCG) that secretes human interferon-alpha 2b (IFN alpha-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637. The signal sequence BCG Ag85B and the gene IFN alpha-2b were amplified from the genome of BCG and human peripheral blood, respectively, by polymerase chain reaction (PCR). The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFN alpha-2b. BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFN alpha-2b. Mononuclear cells were isolated from human peripheral blood (PBMCs) and stimulated with rBCG-IFN alpha-2b or wild type BCG for 3 d, and then cultured with human bladder cancer cell lines T24 and T5637. Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFN alpha-2b by electroporation and the recombinant BCG (rBCG-IFN alpha-2b) was capable of synthesizing and secreting cytokine IFN alpha-2b. PBMC proliferation was enhanced significantly by rBCG-IFN alpha-2b, and the cytotoxicity of PBMCs stimulated by rBCG-IFN alpha-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG. A recombinant BCG, secreting human IFN alpha-2b (rBCG-IFN alpha-2b), was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637. This suggests that rBCG-IFN alpha-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.
