仙台病毒假病毒装甲RNA标准质控品的制备及其应用

Preparation and application of pseudovirus armor RNA standard quality control for Sendai virus

目的基于Ms2噬菌体制备内含仙台病毒(Sendai virus,SeV)RNA的假病毒装甲RNA标准质控品,以期应用于SeV感染的诊断及该病毒的分子生物学安全检测。方法将SeV的L基因片段插入质粒pET-28b-Ms2中,构建重组质粒pET-28b-Ms2-L(SeV)。将重组质粒转化感受态E.coli BL21(DE3),经IPTG诱导表达重组蛋白,即SeV假病毒装甲RNA标准质控品。将标准质控品置电镜下观察其形态,并进行10%SDS-PAGE分析、抗DNaseⅠ及抗RNsaeA试验、稳定性分析。采用重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术对SeV、小鼠肝炎病毒(mouse hepatitis virus,MHV)、小鼠肺炎病毒(pneumonia virus of mice,PVM)、汉坦病毒(Hantaan virus,HV)、呼肠弧病毒Ⅲ型(reovirus typeⅢ,ReoⅢ)、小鼠细小病毒(minute virus of mice,MVM)进行检测,验证标准质控品的效果。结果标准质控品于电镜下呈Ms2噬菌体假病毒颗粒结构,相对分子质量约为14000,可有效抵抗DNaseⅠ及RNsaeA的降解。标准质控品于-80℃保存6个月、-20℃保存6个月、28℃保存5、10、15 d后仍具有良好的稳定性。仅SeV可见RPA特异性扩增曲线,其他病毒均未出现扩增曲线。结论本研究构建的SeV假病毒装甲RNA标准质控品具有良好的稳定性及特异性,可作为SeV的各项分子生物学安全检测的阳性对照。

Objective To prepare a pseudovirus armor RNA standard control containing Sendai virus(SeV)RNA by Ms2 bacteriophage in order to use it in the diagnosis of SeV infection and the molecular biological safety detection of the virus.Methods The L gene fragment of SeV was inserted into plasmid pET-28b-Ms2 to construct recombinant plasmid pET-28b-Ms2-L(SeV),which was transformed into competent E.coli BL21(DE3)and induced by IPTG to express the recombinant protein,SeV pseudovirus armor RNA standard quality control.The morphology of the standard quality control was observed under electron microscope,and 10%SDS-PAGE analysis,anti-DNaseⅠand anti-RNsaeA test,as well as stability analysis were performed.SeV,mouse hepatitis virus(MHV),pneumonia virus of mice(PVM),Hantaan virus(HV),reovirus typeⅢ(ReoⅢ)and minute virus of mice(MVM)were detected by recombinase polymerase amplification(RPA)to verify the effect of the standard quality control.Results The standard quality control showed the structure of Ms2 bacteriophage pseudoviral particles under electron microscope,with a relative molecular mass of about 14000,which effectively resisted the degradation of DNaseⅠand RNsaeA.The standard quality control was stored at-80℃for 6 months,-20℃for 6 months and 28℃for 5,10 and 15 days,and still had good stability.RPA specific amplification curve was seen only in SeV,while no amplification curve was observed in other viruses.Conclusion The standard quality control of SeV pseudovirus armor RNA constructed in this study has good stability and specificity,and can be used as a positive control for various molecular biological safety detections of SeV.