卡介苗穿梭表达质粒pMS MCP-1的构建与鉴定

Construction and Dentification of Recombinant Shuttle Plasmid with Secreting Monocyte Chemotactic Protein-1

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。

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.