卡介苗穿梭表达质粒pMS MCP-1的构建与鉴定 |
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引用本文: | 李杜渐,徐耀庭,韦芳,李惠民,黄汝强,许晓文,顾炜,顾伟平.卡介苗穿梭表达质粒pMS MCP-1的构建与鉴定[J].医学理论与实践,2012,25(20):2478-2479,2482. |
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作者姓名: | 李杜渐 徐耀庭 韦芳 李惠民 黄汝强 许晓文 顾炜 顾伟平 |
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作者单位: | 李杜渐 (上海市第一人民医院分院泌尿外科,200081) ; 徐耀庭 (上海市第一人民医院分院泌尿外科,200081) ; 韦芳 (上海交通大学附属上海市第一人民医院实验中心) ; 李惠民 (上海交通大学附属上海市第一人民医院实验中心) ; 黄汝强 (上海市第一人民医院分院泌尿外科,200081) ; 许晓文 (上海市第一人民医院分院泌尿外科,200081) ; 顾炜 (上海市第一人民医院分院泌尿外科,200081) ; 顾伟平 (上海市第一人民医院分院泌尿外科,200081) ; |
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基金项目: | 上海市卫生局课题经费资助项目,上海市虹口区卫生局课题经费资助项目 |
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摘 要: | 目的:构建分泌性表达单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)的重组卡介苗。方法:分别以卡介苗(BCG)和MCP-1cDNA为模板,通过PCR扩增得到120bp的BCG Ag85B信号肽序列和300bp的MCP-1基因序列。将BCG Ag85B信号肽序列插入大肠杆菌-卡介苗穿梭质粒pMV261,得到重组质粒pMS。再将MCP-1基因序列克隆至pMS中,得到重组质粒pMS MCP-1。结果:质粒pMS MCP-1用双酶切和PCR扩增及测序鉴定证实,克隆基因BCG Ag85B和MCP-1正确插入载体pMV261。结论:重组质粒pMS MCP-1可望在BCG中分泌性表达细胞因子MCP-1,从而协同加强对肿瘤的杀伤作用,该质粒的成功构建为改造卡介苗、发展新型抗膀胱肿瘤疫苗奠定了基础。
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关 键 词: | 卡介苗 单核细胞趋化因子-1 多聚酶链反应 |
Construction and Dentification of Recombinant Shuttle Plasmid with Secreting Monocyte Chemotactic Protein-1 |
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Institution: | LI Dujian,XU Yaoting,WEI Fang,et al.Department of Urology,Branch of Shanghai First People’s Hospital Affiliated To Shanghai Jiaotong University,ShangHai 200081 |
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Abstract: | Objective:To construct a recombinant Bacillus Calmette Guérin(BCG) secreting BCG Ag85B fused human monocyte chemotactic protein-1(MCP-1).Methods:BCG Ag85B signal sequence and MCP-1gene were amplified from the genome of BCG and MCP-1 by PCR respectively.BCG Ag85B signal sequence was cloned in E.coli-BCG shuttle vector pMV261 to get pMS.Then a new re-combinant plasmid pMS MCP-1 was constructed by inserting MCP-1 gene into pMS.Results:The cloned genes BCG Ag85B and MCP-1 were correctly inserted into the vector pMV261,which was confirmed by restriction endonuclease digestion and PCR amplification of MCP-1 and gene sequencing,respectively.Conclusion:pMS MCP-1was expected to secretively express MCP-1 of cytokine in BCG.This study provides the possibility of further researches on the development of new anti bladder cancer vaccine. |
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Keywords: | BCG MCP-1 PCR |
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