【病毒外文文獻】2013 Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protei

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Journal of Virological Methods 187 2013 413 420 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods jou rn al h om epage Immunoreactivity characterisation of the three coronavirus OC43 nucleocapsid protein by diagnosis of coronavirus infection Fang Ying Liang a b 1 Leng Chieh Lin c d 1 Tsung Ho Ying Tswen Kei a h i a b c Department of Emergency Medicine Chang Gung Memorial Hospital Puzih City Chiayi Taiwan d Departments of Nursing and Respiratory Care Chang Gung Institute of Technology Puzih City Chiayi Taiwan e Department of Obstetrics and Gynaecology School of Medicine College of Medicine Chung Shan Medical University Taichung Taiwan f Department of Pathology Tri Service General Hospital National Defense University Taipei Taiwan g Department of Medical Laboratory Science and Biotechnology Yuanpei University Hsinchu Taiwan h College of Life Science National Chung Hsing University Taichung Taiwan i Article Received Received Accepted Available online 19 November 2012 Keywords Human OC43 Nucleocapsid Antigenicity Polyclonal Virus NP E Chung fax 0166 0934 http dx doi org 10 1016 j jviromet 2012 11 009 Institute of Genomics and Bioinformatics National Chung Hsing University Taichung Taiwan history 26 December 2011 in revised form 15 October 2012 8 November 2012 coronavirus strain protein antibody infection a b s t r a c t Previous studies have reported that a prokaryotic expressed recombinant nucleocapsid protein NP is a suitable reagent for the epidemiological screening of coronavirus infection In this study soluble recombinant human coronavirus OC43 HCoV OC43 NP was produced to examine the antigenicity of the HCoV OC43 NP of betacoronavirus Using the purified recombinant NP as an antigen a polyclonal antibody from rabbit serum with specificity for HCoV OC43 NP was generated this antibody reacts specifically with HCoV OC43 NP and does not cross react with other human CoV NPs including those of SARS CoV and HCoV 229E by Western blot Sera from 26 young adults 17 middle aged and elderly patients with respiratory infection and 15 cord blood samples were also tested Strong reactivity to the NPs of HCoV OC43 was observed in 96 82 and 93 of the serum samples from the young adults respiratory patients and cord blood samples respectively To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum the antigenicities of three protein fragments including the N terminal domain aa 1 173 the central linker region aa 174 300 and the C terminal domain aa 301 448 were evaluated by Western blot The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central linker region and the C terminal domain than to the N terminal domain Three different patterns for the immunoreactivities of the three structural regions of HCoV OC43 NP were observed in human serum suggesting variability in the immune responses that occur during HCoV OC43 infection in humans The central linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection 2012 Elsevier B V All rights reserved Abbreviations RNP ribonucleoprotein HCoV OC43 human coronavirus OC43 nucleocapsid protein IPTG H9252 d 1 thiogalactopyranoside S spike M matrix envelope SPR surface plasmon resonance Corresponding author at Institute of Genomics and Bioinformatics National Hsing University Taichung 402 Taiwan Tel 886 4 22840450 x6131 886 4 22861905 E mail address mhho nchu edu tw M H Hou 1 These authors contributed equally to this work 1 Introduction HCoV OC43 was identified in the 1960s and is responsible for the majority of common colds in humans St Jean et al 2004 Vabret et al 2003 Although HCoV OC43 infections are generally mild more severe upper and lower respiratory tract infections such as bronchiolitis and pneumonia which are particularly severe in infants elderly individuals and immunocom promised patients have been documented El Sahly et al 2000 Gagneur et al 2002 St Jean et al 2004 There have also been reports of clusters of HCoV OC43 infections that cause pneumonia in adults Vabret et al 2003 Wenzel et al 1974 In addition a see front matter 2012 Elsevier B V All rights reserved Tang g 1 Yi Wen Chen f Ming Hon Hou Biotechnology Center National Chung Hsing University Taichung Taiwan Department of Food Science Nutrition and Nutraceutical Biotechnology Shih Chien University structural regions of the human Western blot Implications for the e 1 Chen Wen Yao f 1 Taipei Taiwan 414 F Y Liang et al Journal of Virological Methods 187 2013 413 420 previous study has reported that the neurotropism and neuroin vasion of HCoV are associated with multiple sclerosis Arbour et al 2000 In recent years several emerging human coronaviruses have been discovered Skowronski et al 2005 Vabret et al 2005 2006 and between 2003 and 2004 the SARS CoV outbreak caused a worldwide epidemic that had a significant economic impact in countries where the disease outbreak occurred Skowronski et contains the NL63 bronchiolitis navirus infections ing and third in mary as tein 2003 studies in Che that mature make infection produced antibody patients ern To the and immunoreactivity nised foster 2 USA synthesised gel 2 1 by Taiwan fied pGENT Nde The BL21 RIL grown induced 1 thiogalactopyranoside the pellets pH 7 3 150 mM NaCl and 15 mM imidazole The soluble pro teins were collected from the supernatant following centrifugation 27 200 g 30 min 4 C to remove the precipitate The methods for protein purification have been described previously Chen et al 2010 Hung et al 2012 Full length and truncated HCoV OC43 NPs have a His 6 tag at the N terminus and the C terminus and the expressed proteins were purified using a Ni NTA column Novagen al 2005 Phylogenetic analyses have shown that SARS CoV sequences that are closely related to sequences found in betacoronaviruses In 2004 another alphacoronavirus HCoV which was isolated from a 7 month old child suffering from and conjunctivitis was reported in the Netherlands Vabret et al 2005 Woo et al 2005 described a novel betacoro HKU1 which was found in patients with respiratory tract Woo et al 2005 The RNA genomes of coronaviruses include the genes encod the structural proteins S spike M matrix E envelope N nucleocapsid Additionally some coronaviruses encode a glycoprotein HE hemagglutinin esterase which is present most of the betacoronaviruses Lai and Cavanagh 1997 The pri function of CoV NP is to recognise a stretch of RNA that serves a packaging signal leading to the formation of the ribonucleopro RNP complex during viral assembly Huang et al 2004 Lai Navas Martin and Weiss 2004 Nelson et al 2000 Previous have shown that the NPs are the immunodominant domain hosts infected with several coronaviruses Chan et al 2005 et al 2005 Lau et al 2004 Additionally it has been shown NPs can accumulate intracellularly before being packaged in viruses Garoff et al 1998 Together these characteristics the NP a suitable candidate for early diagnosis of coronavirus Chan et al 2005 Mourez et al 2007 In this study a purified soluble full length HCoV OC43 NP was and characterised using highly specific rabbit polyclonal Sera from young healthy adults respiratory infection and cord blood samples were also analysed using West blot assays using the purified recombinant NP as an antigen identify the immunodominant regions of the HCoV OC43 NP antigenicities of three structural regions aa 1 173 aa 174 300 aa 301 448 were analysed These results which defined the patterns of the three structural regions recog by the rabbit polyclonal antibody and human serum may the development of a diagnostic test for CoV infection Materials and methods Drugs and reagents were purchased from Sigma St Louis MO All oligoribonucleotides or oligodeoxyribonucleotides were using an automated DNA synthesiser and purified by electrophoresis Expression and purification of recombinant NPs The templates for the HCoV OC43 NP were kindly provided the Institute of Biological Chemistry Academia Sinica Taipei To generate recombinant NPs the NP gene was ampli by the polymerase chain reaction PCR from the plasmid using various primers The PCR products were digested with I and XhoI and the DNA fragments were cloned into pET28a recombinant plasmid was transformed into Escherichia coli cells using the heat shock method and the cells were at 37 C in Luria Bertani medium Protein expression was at an OD 600 of 0 6 by the addition of 1 mM isopropyl H9252 d IPTG at 10 C for 24 h After harvesting bacteria by centrifugation 3500 g 30 min 4 C the bacterial were lysed with lysis buffer 50 mM Tris buffered solution with an elution gradient ranging from 15 to 300 mM imidazole The pure fractions were collected and dialysed against a low salt buffer The protein concentrations were determined using the Bradford method with Bio Rad protein assay reagents The SARS CoV and HCoV 229E NPs were expressed in E coli and purified as described previously Tang et al 2005 2 2 Immunisation of rabbits Soluble and purified recombinant NPs prepared as described above were used as antigens for immunisation and the immunoas says All animal procedures described were approved by the Animal Care Use Committee of National Chung Hsing Univer sity Rabbits New Zealand White strain weighing approximately 3 kg were immunised by an intrasplenic injection with 400 H9262g of recombinant HCoV OC43 NP per immunisation The antigen was administered together with an equal amount of Gold TiterMax adjuvant CytRx Norcross GA USA Additional booster immuni sations were administered to obtain a high titre of the antisera and high titre polyclonal antibodies were obtained in 6 8 weeks The rabbit antisera were used without purification for the majority of the subsequent experiments The titre of rabbit sera was deter mined for the NP antigen by Western blot 2 3 Serum collections Twenty six human serum specimens denoted H1 to H26 from young healthy adults approximately 18 26 years of age were col lected at random from Shih Chien University Seventeen serum samples denoted R1 to R17 were collected from patients that were approximately 50 80 years of age who had reported to the Emer gency Department of the Medicine Chang Gung Memorial hospital with symptoms of respiratory tract infections Fifteen cord blood samples denoted C1 to C15 were collected from the Department of Obstetrics and Gynaecology Chung Shan Medical University The criteria used for the selection of human serum samples from the middle aged and elderly patients with respiratory infections were largely based on clinical symptoms No symptoms indicative of respiratory infection were noted by physicians in the young healthy adults or the newborns from when cord blood was obtained All sera were stored at 20 C All of the human serum collections were approved by Institutional Review Boards 2 4 Western blot immunoassay using the rabbit polyclonal antibody The NPs were solubilised in sample buffer containing 50 mM Tris HCl pH 7 5 0 1 CHAPS 10 glycerol 150 mM NaCl and 10 mM dithiothreitol and boiled for 5 min Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS PAGE was performed using a discontinuous buffer system and the polypep tide bands were revealed by staining the gel with Coomassie Brilliant Blue G 250 For immunoblotting polypeptides separated by SDS PAGE were electrotransferred onto a PVDF membrane with transfer buffer containing 50 mM Tris 384 mM glycine and 20 v v methanol pH 8 3 Electrotransfer was conducted at 65 V for 1 h The PVDF membrane was then incubated for 1 h in PBS buffer containing 0 05 Tween 20 PBS T After washing three times in PBS T the membrane was incubated for 2 h at room temperature F Y Liang et al Journal of Virological Methods 187 2013 413 420 415 Fig induced pH of 15 with tion a the peroxidase allowed membrane the 2 5 to 50 fold 1 A Soluble protein fraction of recombinant HCoV OC43 NP from IPTG cells prepared using different concentrations of CHAPS in 50 mM Tris HCl 7 3 and 150 mM NaCl buffer B Expression and purification of OC43 NP Extract un induced cells lane 1 extract of induced cells lane 2 protein eluted with mM imidazole lane 3 and purified NP lane 4 rabbit antisera The three washes were followed by the addi of horseradish peroxidase conjugated goat anti rabbit IgG at dilution of 1 10 000 After a 2 h incubation at room temperature membrane was washed three times and covered with the substrate 3 3 prime diaminobenzidine DAB The blot was to develop and the reaction was stopped by washing the in distilled water Quantitation of the protein bands on gel was achieved using the Uniphoto Band Tool software Western blot immunoassay using human serum An identical protocol to that described above was used test human sera by Western blotting Each well contained ng of recombinant NP and human sera were diluted 200 in blocking solution The secondary antibody horseradish Fig 2 A Titre analysis of the rabbit polyclonal antibody against HCoV OC43 NP using Western blotting B SDS PAGE analysis of the NPs from HCoV OC43 HCoV 229E and SARS CoV purified by Ni NTA chromatography C Specificity analysis of the rabbit polyclonal antibody against HCoV OC43 HCoV 229E and SARS CoV NPs by Western blot peroxidase conjugated goat anti human antibody IgG IgM was used and detected as described above 3 Results 3 1 Purification of recombinant HCoV OC43 NP To express soluble HCoV OC43 NP as a set of fusion proteins in E coli the NP gene was cloned into the pET 28a expression vector CHAPS which demonstrates low light absorbance in the ultraviolet region of the electromagnetic spectrum facilitates the character isation of protein properties when using CD and UV VIS spectra To solve the problem of the solubility of the NP buffers containing CHAPS were used to obtain NP His 6 in a soluble form for subsequent analysis Fig 1A shows that NP His 6 was soluble in Tris buffer in the presence of 0 1 0 2 and 0 4 CHAPS Large scale expression and purification of the His tag NP in the presence of 0 1 CHAPS from E coli was then performed Fig 1B shows that the His tag NP was purified from the soluble fraction using Ni NTA column chromatography resulting in a single band of the expected mass of 55 kDa The final yield of NP His 6 was approximately 10 mg l from the E coli culture The NP is responsible for recognising a stretch of RNA that serves as a viral packaging signal leading to the formation of the ribonucleoprotein RNP complex during viral assembly Lai and Cavanagh 1997 Previous surface plasmon resonance SPR data showed that purified recombinant HCoV OC43 NP can bind 416 F Y Liang et al Journal of Virological Methods 187 2013 413 420 Fig NP and single stranded is 3 2 antibodies HCoV OC43 lowing full length Western clearly specificity against Western motifs anti HCoV OC43 antibody ing Previous same 3 A Conformational analysis of HCoV OC43 NP by the VL3 BA predictor of the PONDR aa 1 173 aa 174 300 and aa 301 448 purified by Ni NTA chromatography Antigenicity aa 301 448 against the rabbit polyclonal antibody at dilutions of C 1 50 000 and D RNA suggesting that recombinant HCoV OC43 NP folded properly Huang et al 2009 Production and antigenicity analysis of rabbit polyclonal against HCoV OC43 NP Rabbits were immunised with soluble recombinant full length NP to produce a high quality polyclonal antibody Fol three booster immunisations the titre of antibody against HCoV OC43 NP in the rabbit serum was analysed using blotting Western blot data showed that 50 ng NP were detected at dilutions of at least 1 1 900 000 Fig 2A The and cross reactivity of the rabbit polyclonal antibody HCoV 229E and SARS CoV NPs were also determined by blot analysis Despite the presence of several conserved in the NPs from these coronaviruses the specificity of the NP polyclonal antibody was excellent and the did not cross react with other human CoV NPs includ SARS CoV and HCoV 229E at 1 150 000 dilutions Fig 2B studies have suggested that the NPs of CoVs share the modular organisation Fig 3A Huang et al 2009 The program B SDS PAGE analysis of three truncated fragments of HCoV OC43 analysis of three truncated fragments of HCoV OC43 NP aa 1 173 aa 174 300 1 450 000 PONDR program a predictor of naturally disordered regions predicts three structural regions in HCoV OC43 NP aa 1 173 the N terminal domain aa 174 300 the central linker region and aa 301 448 the C terminal domain Romero et al 2004 Fig 3A To analyse the antigenicity of these three structural regions in the NP fragments encoding aa 1 173 aa 174 300 and aa 301 448 which together span the entire coding region were cloned expressed and successfully purified as single bands on SDS PAGE gels Fig 3B The results demonstrated that the rabbit polyclonal antibody reacted strongly with the regions aa 174 300 and aa 301 448 whereas the aa 1 173 region showed low reactivity with the antibody at dilutions of 1 50 000 and 1 450 000 Fig 3B D In Fig 3C a minor band slightly below the major band is apparent indicating that aa 174 300 was partially degraded 3 3 Serological assay and antigenicity analysis of the NP antibody in humans The recombinant protein based Western blot assay was used to screen human serum from 26 young adults 17 middle aged and elderly patients with respiratory infection symptoms and 15 cord F Y Liang et al Journal of Virological Methods 187 2013 413 420 417 Fig 4 A Western blot analysis of the individual human sera obtained from young healthy adults and patients with respiratory infection symptoms tested against HCoV OC43 and SARS CoV NPs denoted by H3 and R2 respectively at an antibody dilution of 1 1000 B Type I C Type II and D Type III immunoreactivity patterns of three structural regions of HCoV OC43 NP aa 1 173 aa 174 300 and aa 301 448 against human antisera at a 1 1000 dilution determined by Western blotting above The bar diagram represents the relative density of each of the three truncated fragments determined by immunoreactivity analysis for human sera aa 174 300 was used as a control below Table 1 Screening of human sera for HCoV OC43 NP antibodies Group Total number of individuals screened HCoV OC43 positive HCoV 229E positive SARS CoV positive Immunoreactivity patterns for the three structural regions of HCoV OC43 NP Type I Type II Type III 18 25 years 26 24 92 3 21 80 7 0 9 8 7 50 80 years a 17 14 82 3 13 76 4 0 7 3 4 Cord blood 15 14 93 3 c 13 86 6 0 b Total 58 52 89 7 47 81 0 0 16 11 11 a These specimens were taken from patients with respiratory tract infection symptoms b Not determined due to the limited quantities of cord blood collected per sample c Sera from cord blood samples were analysed using purified NP as an antigen 418 F Y Liang et al Journal of Virological Methods 187 2013 413 420 blood samples Fig 4A The serum samples from young adults middle aged and elderly patients with respiratory infection and cord blood samples were 92 3 82 3 and 93 3 seropositive for HCoV OC43 respectively Table 1 None of the samples reacted with the SARS CoV NP Interestingly approximately 81 of the serum samples reacted to the HCoV 229E NP Thirty eight human sera that tested positive for HCoV OC43 NP in the recombinant pro tein examine of immunoreactivity were each immunoreactivity bit 174 300 activities was regions three the experiments test aged To 1 2000 1 64 000 1 8000 1 64 000 showing young toms in 2012 11 009 4 transmitted route The catarrh mately colds the et and reverse transcription et reaction opment 2005 SARS gens to aviruses Wu recombinant fied culture umn protein by showed the purified recombinant NP was folded properly The prokaryotic expression system used in this study is high yield inexpensive and highly efficient does not require viral cultures and is nontoxic Despite these advantages viral proteins expressed in prokaryotic cells lac