摘要
目的构建含有肠道病毒(EV)5'端非编码区(5'-UTR)部分RNA序列的耐核糖核酸酶(RNase)病毒样颗粒。方法利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-MS2。将肠道病毒5'-UTR基因片段连接到中间载体的下游,构建原核表达载体pET32a-MS2-EV。将其转化宿主菌,IPTG诱导表达,并进行RT-PCR检测和稳定性实验。结果表达载体pET32a-MS2-EV经PCR、双酶切鉴定和测序分析后证实构建成功。RT-PCR和稳定性实验结果表明,诱导产生的病毒样颗粒内含肠道病毒5'-UTR部分RNA序列,并且稳定性良好。结论成功构建了含肠道病毒5'-UTR部分RNA序列的病毒样颗粒,可作为肠道病毒检测时的标准品和质控品使用。
[Objective]To construct RNase-resistant virus-like particles containing partial RNA sequence of EV 5′-UTR. [Methods]The coat protein and maturase gene of E.coli bacteriophage MS2 was amplified by PCR, then the gene was cloned into pET32a to construct the intermediate vector pET32a-MS2. The gene fragments of EV 5′-UTR was cloned into the downstream of pET32a-MS2 to construct the prokaryotic expression vector pET32a-MS2EV. The recombinant plasmid pET32a-MS2-EV transformed into E.coli cells was induced to express with IPTG, then tested by RT-PCR and stable experiment. [Results]The virus-like particles were successfully constructed by PCR amplification, enzymolysis identification and sequencing analysis. Partial RNA sequence of EV 5′-UTR was contained in the virus-like particles ,and the virus-like particles had a good stability.[Conclusion] The virus-like particles containing partial RNA sequence of EV 5′-UTR can be used as standards and quality controls in EV virus detection.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2010年第10期1507-1511,共5页
China Journal of Modern Medicine
基金
国家自然科学基金(No.30901285)
