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目的构建可分泌表达人共刺激分子B7-2的重组BCG,并观察其对膀胱癌细胞的作用。方法利用电穿孔技术将pYL-hB7-2穿梭质粒转导入野生型BCG中;卡那霉素抗性、PCR及测序筛选重组BCG菌株(rBCG-hB7-2);SDS-PAGE和ELISA检测重组BCG的表达产物B7-2分子,并测定rBCG-hB7-2对淋巴细胞的增殖作用及对膀胱癌细胞的杀伤作用。结果获得的阳性菌株rB-CG-hB7-2具有卡那霉素抗性,其PCR产物测序后与已发表的人B7-2基因cDNA序列一致。通过SDS-PAGE可见29200的目的蛋白条带含量增加,ELISA法检测出培养上清液及菌体内均有B7-2分子表达,上清液中含量为3.8U/ml,菌体内为10.5U/ml。体外实验中,重组BCG较野生型BCG刺激淋巴细胞增殖数量增加34.8%,重组BCG所激活淋巴细胞对膀胱癌细胞的杀伤能力是野生型的2.14倍。结论成功构建了可分泌具有共刺激活性B7-2分子的基因重组BCG菌株,为该菌株进一步临床应用提供了实验依据。
Objective To construct recombinant BCG secreting human costimulatory molecule B7-2 and observe its effect on bladder cancer cells. Methods The shuttle plasmid pYL-hB7-2 was transfected into wild-type BCG by electroporation. The recombinant BCG strain (rBCG-hB7-2) was screened by PCR and sequencing with kanamycin resistance. The recombinant BCG The expression of B7-2 molecules, and determination of rBCG-hB7-2 on lymphocyte proliferation and cytotoxicity of bladder cancer cells. Results The positive strain rB-CG-hB7-2 was resistant to kanamycin. The PCR product was sequenced and was consistent with the published cDNA sequence of human B7-2 gene. The SDS-PAGE showed that the content of the target protein of 29200 was increased, and the expression of B7-2 was detected by ELISA in the culture supernatant and in the cell body. The content of the supernatant in the supernatant was 3.8 U / ml and that in the cell was 10.5 U / ml. In vitro, recombinant BCG increased lymphocyte proliferation by 34.8% compared with wild-type BCG, and cytotoxicity of recombinant BCG-activated lymphocytes to bladder cancer cells was 2.14 times that of wild-type. Conclusion The recombinant BCG strain secreting the costimulatory B7-2 gene was successfully constructed and provided an experimental basis for further clinical application of this strain.