通过大肠杆菌thyA染色体质-粒平衡致死系统构建无抗性基因表达载体

Expression Plasmid-Host Strain Using Chromosome-plasmid Balanced Lethal System Based on the Escherichia coli thyA

克隆了大肠杆菌和霍乱弧菌胸腺嘧啶合成酶基因thyA ,并以pcDNA3质粒为基础 ,分别用两种来源的thyA基因替代其氨苄抗性基因Amp,构建了不含抗性基因 ,且可在thyA营养缺陷型大肠杆菌中基于染色体 质粒平衡致死系统稳定传代的真核表达载体。该载体可有效表达红色荧光蛋白报告基因。

To construct a vector for DNA vaccine and protein expression by using chromosome plasmid balanced lethal system which was based on the thyA + gene/ΔthyA Escherichia coli . The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the ΔthyA derivative of E.coli K 12 strain DY330 TI, then two chromosome plasmid balanced systems for E.coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome plasmid balanced lethal system, a reporter gene—red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c myc. The two recombinant plasmids, pcDNATE DsRed2, pcDNATC DsRed2, were transfected into HEK293 solely and DsRed2 myc was detected by the fluorescence microscope assay and western blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations.The chromosomal plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application.