基于Qβ噬菌体的甲肝病毒装甲RNA标准参考样品的研制
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摘要
【背景】目前食品中甲肝病毒分子的检测缺乏安全、稳定的RNA标准参考样品,影响了检测结果的科学性与准确性。【目的】基于Qβ噬菌体装甲RNA技术构建内含甲肝病毒检测靶标的装甲RNA (Hepatitis A virus armored RNA,AR-HAV),并开展初步定值、均匀性、稳定性研究,为HAV分子检测提供标准参考样品。【方法】人工合成包含Qβ噬菌体成熟酶编码基因、衣壳蛋白编码基因、包装位点、HAV检测靶标cDNA序列的核酸片段QGBHAV,并亚克隆到pET-28a(+)中构建重组质粒pET-QGBHAV,转入大肠杆菌BL21(DE3)感受态细胞进行原核表达,利用超速离心、丙烯葡聚糖凝胶层析柱纯化AR-HAV后电镜观察。通过实时荧光RT-PCR对AR-HAV进行初步定值及均匀性和稳定性研究。【结果】SDS-PAGE结果表明重组质粒在大肠杆菌中有约14.1 kD的目的蛋白表达;纯化后的AR-HAV无杂蛋白和残留质粒;电镜下可见结构完整、大小约为25nm的病毒样颗粒;定值结果显示,AR-HAV中检测靶标RNA的含量为(2.57±0.12)×107 copies/μL;均匀性分析结果为F=1.23 [Background] The stable RNA reference material without biohazard to improve the accuracy and reliability of the detection result, is urgently needed for hepatitis A virus(HAV) detection in food. [Objective] To construct armored RNA reference material containing target RNA of HAV(HAV armored RNA, AR-HAV) based on Qβ bacteriophage, and to test its homogeneity, valuation and stability. [Methods] DNA fragment named QGBHAV containing matures coding gene, capsid protein coding gene, packing site of Qβ bacteriophage, and detection target sequence of HAV in GB/T 22287-2008 was synthesized, and subcloned into pET-28 a(+) expression vector to construct the recombinant plasmid pET-QGBHAV, and then transformed into Escherichia coli BL21(DE3) competent cells and expressed with isopropyl-β-thiogalactopyranoside(IPTG) induction. The expression product, virus like particles of Qβ bacteriophage containing RNA of HAV, named AR-HAV, was analyzed by SDS-PAGE. AR-HAV was centrifuged and purified by CsCl density gradient ultracentrifugation and Sephacry molecular sieve chromatography. The morphology of AR-HAV was observed by transmission electron microscopy. The valuation, homogeneity and stability of AR-HAV were tested according to the GB/T 15000.3-2008. [Results] SDS-PAGE analysis showed that the molecular mass of the expressed protein was about 14.1 kD. The virus like particles of AR-HAV, 25 nm in diameter, with typical morphology could be observed under electron microscope. AR-HAV samples prepared in this study had no other proteins nor recombinant plasmid DNA residual contamination, were valued as(2.57±0.12)× 107 copies/μ L and behaved well in the homogeneity test, F=1.23
引文
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[23]Saldanha J,Heath A,Lelie N,et al.A World Health Organization International Standard for hepatitis a virus RNAnucleic acid amplification technology assays[J].Vox Sanguinis,2005,89(1):52-58
[24]Janner A.Form,symmetry and packing of biomacromolecules.IV.Filled capsids of cowpea,tobacco,MS2 and pariacoto RNAviruses[J].Acta Crystallographica Section A:Foundations and Advances,2011,67(Pt6):517-520
[25]Pan Y,Zhang Y,Jia TT,et al.Development of a microRNAdelivery system based on bacteriophage MS2 virus-like particles[J].FEBS Journal,2012,279(7):1198-1208
[26]ElSawy KM,Caves LSD,Twarock R.The impact of viral RNAon the association rates of capsid protein assembly:bacteriophage MS2 as a case study[J].Journal of Molecular Biology,2010,400(4):935-947
[27]Zhang D,Sun Y,Jia TT,et al.External quality assessment for the detection of measles virus by reverse transcription-PCRusing armored RNA[J].PLoS One,2015,10(8):e0134681
[28]Karatayl?E,Altuno?lu Y?,Karatayl?SC,et al.A one step real time PCR method for the quantification of hepatitis delta virus RNAusing an external armored RNA standard and intrinsic internal control[J].Journal of Clinical Virology,2014,60(1):11-15
[29]Lin GG,Zhang K,Zhang D,et al.Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus[J].Clinica Chimica Acta,2017,466:138-144
[2]Lanini S,Minosse C,Vairo F,et al.A large ongoing outbreak of hepatitis a predominantly affecting young males in Lazio,Italy;August 2016-March 2017[J].PLoS One,2017,12(11):e0185428
[3]Provost K,Dancho BA,Ozbay G,et al.Hemocytes are sites of enteric virus persistence within oysters[J].Applied and Environmental Microbiology,2011,77(23):8360-8369
[4]Wu QP,Kou XX,Zhang JM.Foodborne viruses and its detection methods[J].Microbiology China,2004,31(3):101-105(in Chinese)吴清平,寇晓霞,张菊梅.食源性病毒及其检测方法[J].微生物学通报,2004,31(3):101-105
[5]Lopalco PL,Malfait P,Menniti-Ippolito F,et al.Determinants of acquiring hepatitis a virus disease in a large Italian region in endemic and epidemic periods[J].Journal of Viral Hepatitis,2005,12(3):315-321
[6]Pontrelli G,Boccia D,Di Renzi M,et al.Epidemiological and virological characterization of a large community-wide outbreak of hepatitis a in southern Italy[J].Epidemiology&Infection,2008,136(8):1027-1034
[7]Yoon EL,Sinn DH,Lee HW,et al.Current status and strategies for the control of viral hepatitis a in Korea[J].Clinical and Molecular Hepatology,2017,23(3):196-204
[8]PintóRM,Costafreda MI,Bosch A.Risk assessment in shellfish-borne outbreaks of hepatitis A[J].Applied and Environmental Microbiology,2009,75(23):7350-7355
[9]Polo D,Varela MF,Romalde JL.Detection and quantification of hepatitis a virus and norovirus in Spanish authorized shellfish harvesting areas[J].International Journal of Food Microbiology,2015,193:43-50
[10]Li JM.The role of nucleic acid reference materials in detection of HBV DNA and HCV RNA[J].Chinese Journal of Laboratory Medicine,2007,30(8):850-852(in Chinese)李金明.标准物质在乙型和丙型肝炎病毒核酸检测标准化中的重要性[J].中华检验医学杂志,2007,30(8):850-852
[11]Li JM.Research progress on control products and standards for RNA virus amplification[J].Chinese Journal of Laboratory Medicine,2004,27(12):873-874(in Chinese)李金明.RNA病毒扩增检测的质控品和标准品研究进展[J].中华检验医学杂志,2004,27(12):873-874
[12]Zhang K,Wei YX,Li JM.RNA phage virus-like particle packaging mechanism and its application research progress[J].Journal of Microbes and Infection,2008,3(2):111-114,123(in Chinese)张括,魏玉香,李金明.RNA噬菌体病毒样颗粒包装机制及其应用研究进展[J].微生物与感染,2008,3(2):111-114,123
[13]Cielens I,Ose V,Petrovskis I,et al.Mutilation of RNA phage Qβvirus-like particles:from icosahedrons to rods[J].FEBSLetters,2000,482(3):261-264
[14]Zhang Q,Yao L,Jiang YH,et al.Development of armored RNAreference material of norovirus based on Qbeta bacteriophage[J].China Biotechnology,2018,38(1):42-50(in Chinese)张奇,姚琳,江艳华,等.基于Qbeta噬菌体装甲RNA技术的诺如病毒RNA标准参考样品的研制[J].中国生物工程杂志,2018,38(1):42-50
[15]General Administration of Quality Supervision,Inspection and Quarantine of the People’s Republic of China.GB/T22287-2008 Detection of hepatitis a virus in shellfish by contentional RT-PCR and real-time fluorescence RT-PCR[S].Beijing:China Standard Press,2008(in Chinese)中华人民共和国国家质量监督检验检疫总局.GB/T22287-2008贝类中甲型肝炎病毒检测方法普通RT-PCR方法和实时荧光RT-PCR方法[S].北京:中国标准出版社,2008
[16]The State Bureau of Quality and Technical Supervision.JJF1059-1999 Evaluation and expression of uncertaintv in measurement[S].Beijing:China Measurement Press,2004(in Chinese)国家质量技术监督局.JJF 1059-1999测量不确定度平定与表示[S].北京:中国计量出版社,2004
[17]Standardization Administration of China.GB/T 15000.3-2008Directives for the work of reference materials-Reference materials-General and statistical principles for certification[S].Beijing:China Standard Press,2008(in Chinese)国家标准化管理委员会.GB/T 15000.3-2008标准样品工作导则(3)标准样品定值的一般原则和统计方法[S].北京:中国标准出版社,2008
[18]WHO.WHO position paper on hepatitis a vaccines[J].Weekly Epidemiological Record,2012,28/29(87):261
[19]Lemon SM,Ott JJ,van Damme P,et al.Type a viral hepatitis:a summary and update on the molecular virology,epidemiology,pathogenesis and prevention[J].Journal of Hepatology,2018,68(1):167-184
[20]Mo XM,Gao DW.SYBR GreenⅠreal-time polymerase chain reaction for the detection of HAV in strawberry[J].Food Science,2010,31(14):153-157(in Chinese)莫雪梅,高东微.运用SYBR GreenⅠ荧光实时RT-PCR法检测草莓中甲肝病毒[J].食品科学,2010,31(14):153-157
[21]Hu Y,Arsov I.A rapid single-tube protocol for HAV detection by nested real-time PCR[J].Food and Environmental Virology,2014,6(3):189-195
[22]Wang DY,Fang ZD,Xie CX,et al.Standard construction for detection of rotavirus using the real-time fluorescence quantitative PCR[J].Journal of Logistical Engineering University,2013,29(4):66-73(in Chinese)王大勇,方振东,谢朝新,等.轮状病毒荧光定量PCR标准品的构建[J].后勤工程学院学报,2013,29(4):66-73
[23]Saldanha J,Heath A,Lelie N,et al.A World Health Organization International Standard for hepatitis a virus RNAnucleic acid amplification technology assays[J].Vox Sanguinis,2005,89(1):52-58
[24]Janner A.Form,symmetry and packing of biomacromolecules.IV.Filled capsids of cowpea,tobacco,MS2 and pariacoto RNAviruses[J].Acta Crystallographica Section A:Foundations and Advances,2011,67(Pt6):517-520
[25]Pan Y,Zhang Y,Jia TT,et al.Development of a microRNAdelivery system based on bacteriophage MS2 virus-like particles[J].FEBS Journal,2012,279(7):1198-1208
[26]ElSawy KM,Caves LSD,Twarock R.The impact of viral RNAon the association rates of capsid protein assembly:bacteriophage MS2 as a case study[J].Journal of Molecular Biology,2010,400(4):935-947
[27]Zhang D,Sun Y,Jia TT,et al.External quality assessment for the detection of measles virus by reverse transcription-PCRusing armored RNA[J].PLoS One,2015,10(8):e0134681
[28]Karatayl?E,Altuno?lu Y?,Karatayl?SC,et al.A one step real time PCR method for the quantification of hepatitis delta virus RNAusing an external armored RNA standard and intrinsic internal control[J].Journal of Clinical Virology,2014,60(1):11-15
[29]Lin GG,Zhang K,Zhang D,et al.Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus[J].Clinica Chimica Acta,2017,466:138-144