卡介苗穿梭表达质粒pMSIL-2的构建及鉴定

Construction and identification of recombinant shuttle-plasmid with secretory interleukin-2

目的 构建分泌性表达白介素 (IL) 2的重组卡介苗 (BCG)。方法 分别以BCG和IL 2cDNA为模板 ,通过PCR扩增得到约 117bp的BCG抗原 85B(BCG Ag85B)信号肽序列和 399bp的IL 2基因序列。将BCG Ag85B信号肽序列克隆至大肠杆菌 BCG穿梭质粒 pMV2 6 1,得到重组质粒 pMS。再将IL 2基因克隆至 pMS中 ,得到重组质粒pMSIL 2。 结果 质粒 pMSIL 2经双酶切和PCR扩增及测序鉴定证实 ,克隆基因BCG Ag85B信号肽和IL 2正确插入载体 pMV2 6 1。结论 重组质粒 pMSIL 2可望在BCG中分泌性表达细胞因子IL 2 ,该质粒的构建成功为改造BCG、发展新型抗膀胱肿瘤疫苗奠定了基础。

Objective To construct a recombinant secretory BCG Ag85B fused human interleukin (IL) 2. Methods BCG Ag85B signal sequence and IL 2 gene were amplified from the genome of BCG and IL 2 by PCR, respectively. BCG Ag85B signal sequence was cloned in E.coli BCG shuttle vector pMV261 to obtain pMS. Then a new recombinant plasmid pMSIL 2 was constructed by inserting the IL 2 gene into pMS. Results The cloned genes BCG Ag85B and IL 2 were correctly inserted into the vector pMV261,which had been confirmed by restriction endonuclease digestion and PCR amplification of IL 2 and gene sequencing, respectively. Conclusion pMSIL 2 is expected to possess expression of secretory IL 2 in BCG providing the basis for the development of a new anti bladder cancer vaccine.