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为制备检测新城疫病毒的实时荧光RT-PCR病毒样颗粒内标,扩增MS2噬菌体的成熟酶蛋白和衣壳蛋白序列,将其定向插入pET32a载体的相应位置,双酶切和PCR鉴定,构建pET32a-MS2重组质粒;根据新城疫病毒实时荧光RT-PCR检测的目标片段,采用化学合成和klenow酶连接技术制备内标片段,将内标片段插入pET32a-MS2重组质粒,构建pET32a-MS2-IC重组质粒,转化大肠杆菌BL21,IPTG诱导表达,经RNase和DNase消化后,采用荧光RT-PCR检测耐核糖核酸酶病毒样颗粒内标的含量。结果表明,制备了高表达量耐核糖核酸酶病毒样颗粒内标,可用于新城疫病毒实时荧光RT-PCR检测方法的建立。
To prepare real-time fluorescent RT-PCR virus-like particle internal standard for detection of Newcastle disease virus, the mature enzyme protein and the capsid protein sequence of MS2 phage were amplified and inserted into the corresponding position of pET32a vector, double-digested and PCR identified According to the target fragment detected by real-time RT-PCR of Newcastle disease virus, the internal standard fragment was prepared by chemical synthesis and klenow enzyme ligation. The internal fragment was inserted into pET32a-MS2 recombinant plasmid to construct pET32a-MS2-IC Recombinant plasmids were transformed into E.coli BL21 and induced by IPTG. After digestion with RNase and DNase, the contents of internal standard of resistant ribavirin-like particles were detected by fluorescent RT-PCR. The results showed that the internal standard of ribavirin-like particles with high expression level was prepared and could be used for the detection of Newcastle disease virus by real-time fluorescence RT-PCR.