摘要
目的应用反义RNA沉默技术,构建针对murA基因的超敏工程菌。方法将不同murA基因片段插入带有paired termini结构的反义表达载体pHN678中,获得重组质粒,导入大肠埃希菌宿主诱导表达。通过生长表型考察不同反义片段对murA沉默效果的影响,并用靶向MurA抑制剂磷霉素验证反义工程菌E.coli DH5α/pHNM2的敏感性及靶向性。结果反义片段murA1和murA2具有较好的沉默效果,E.coli DH5α/pHNM2对MurA抑制剂磷霉素具有靶标特异性和敏感性。结论成功获得靶向murA反义工程菌E.coli DH5α/pHNM2,并有望进一步应用于MurA特异性抑制剂高通量筛选模型的构建。
Objective To construct murA-specific antisense engineering strains by using antisense RNA technology. Methods Three murA asRNA fragments were inserted into the paired-termini antisense RNA expression vector pHN678 respectively. The constructed plasmids were then transferred into E.coli DH5a and different transformants were obtained. Gene silencing efficacy was investigated by observing growth phenotype. MurA-specific inhibitory fosfomycin was used to validate the sensitivity and target specificity of antisense engineering strain E.coli DH5a/pHNM2. Results Fragments mural and murA2 showed the better silencing effect. E.coli DH5α/pHNM2 displayed prominent specificity and sensitivity to fosfomycin. Conclusion A sensitive engineering strain E.coli DH5α/pHNM2 was obtained and may be applied to MurA-specific screening model establishment.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2014年第6期413-417,共5页
Chinese Journal of Antibiotics
基金
国家重大新药创制专项(2012ZX09301002-003)
关键词
反义RNA技术
murA
靶向筛选
Antisense RNA technology
murA
Target-specific screening
