利用反义技术抑制大肠杆菌β-内酰胺酶基因表达的实验研究

目的探讨利用反义表达质粒抑制细菌耐药基因表达的可行性。方法根据GenBank中β-内酰胺酶TEM-1基因序列设计一对寡核苷酸引物，通过PCR扩增获取TEM—1基因，将其反向克隆入质粒pBK—CMV的多克隆位点构建反义表达质粒，将此反义表达质粒导入携带TEM-1型β-内酰胺酶的大肠杆菌，Western—blotting检测TEM-1表达情况。结果成功构建了TEM-1基因的反义表达质粒，将其导入携带TEM-1型β-内酰胺酶的大肠杆菌后，Western-blotting检测发现TEM-1表达显著下调。结论利用反义表达质粒可抑制大肠杆菌β-内酰胺酶TEM-1基因表达。

Down-regulation of β-lactamase TEM-1 Gene expression by antisense RNA expressing plasmid in E.coli

Objective To determine the feasibility of introducing antisense RNA expressing plasmid to suppress the expression of the drug-resistant gene in E. coli. Methods TEM-1 gene fragments were generated by polymerase chain reaction （PCR）. A recombinant plasmid was constructed by inserted the TEM-1 gene into the multiple cloning site of pBK-CMV vector in the antisense direction. The recombinant plasmid was transformed into E.coli carrying TEM-1 gene. TEM-1 expression was determined by Western-blotting. Results A recombinant plasmid expressing antisense TEM-1 RNA was successfully constructed. TEM-1 gene expression was inhibited by the antisense RNA expressing plasmid in E.coli. Conclusions Antisense technique has potential as novel strategy for blocking the expression of drug-resistant gene in E.coli.