CN113122551B - 一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 - Google Patents
一种柯萨奇病毒a组19型vp1基因重组表达蛋白、多克隆抗体的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种柯萨奇病毒A组19型VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用,属于生物工程技术领域。本发明的技术方案要点为:一种柯萨奇病毒A组19型VP1基因,其碱基序列如序列表SEQ ID NO.1所示;一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其氨基酸序列如序列表SEQ ID NO.2所示;一种基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。本发明利用原核表达CV‑A19VP1基因重组蛋白制备免疫原,方法简便易行,具有生物安全性。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种柯萨奇病毒A组19型(coxsackievirus A19,CV-A19)VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用。
背景技术
CV-A19属于小核糖核酸病毒科(Picornaviridae)肠道病毒属(Enterovirus)肠道病毒C组(Enterovirus C,EVC)[1]。CV-A19的原型株为源于日本的NIH-8663(GenBank登录号为AF499641),可以引起吉兰-巴雷综合征(Guillain-Barrésyndrome,GBS)[1]。GBS,又称格林巴利综合征、急性炎症性脱髓鞘性多发性神经病,通常发生在免疫反应产生抗体的传染病之后,抗体与神经膜上的神经节苷脂发生交叉反应,是一种潜在的威胁生命的感染后疾病,是急性弛缓性麻痹(acute flaccid paralysis,AFP)的常见病因,其特征是迅速进展,四肢对称性无力,在4周内达到最严重程度[2]。目前已有研究报道表明,从呼吸道和胃肠道疾病[3-5]、无菌性脑膜炎[6]、AFP[7]患者的标本和废水[8]样本中可以检测到CV-A19。澳大利亚Isaacs等[9]对收集自遗传风险病毒(Viruses in the Genetically at Risk,VIGR)前瞻性队列研究参与者的临床粪便样品进行近全长EV基因组的扩增与测序,亦扩增到CV-A19。本发明于2019年秋冬季从新乡市手足口病患儿的粪便中检测到一种肠道病毒,经过序列测定与分析发现,其VP1核苷酸序列与原型株8663(GenBank登录号为AF499641)的相似性为86.4%。该毒株与GenBank中已有的CV-A19毒株存在差异,其VP1核苷酸序列与Coxsackievirus A19 strain BBD26 polyprotein gene,complete cds(GenBank登录号为KC785531)相似性最高,为93.1%,其全长核苷酸序列与Coxsackievirus A19 strainBBD26polyprotein gene,complete cds(GenBank登录号为KC785531)相似性最高,为89.5%,而目前尚未见CV-A19检测与诊断的方法报道。
参考文献:
[1]Pallansch M A,Oberste M S,Whitton J L.Enteroviruses:Polioviruses,Coxsackieviruses,Echoviruses,and Newer Enteroviruses[M]//Knipe D M,Howley PM.Fields Virology.6th ed.Philadelohia:LIPPINCOTT WILLIAMS&WILKINS,a WOLTERSKLUWER business.2013:510.
[2]van den Berg B,Walgaard C,Drenthen J,Fokke C,Jacobs B C,van DoornP A.Guillain-Barre syndrome:pathogenesis,diagnosis,treatment and prognosis[J].Nat Rev Neurol,2014,10(8):469-482.
[3]Lipson S M,Walderman R,Costello P,Szabo K.Sensitivity ofrhabdomyosarcoma and guinea pig embryo cell cultures to field isolates ofdifficult-to-cultivate group A coxsackieviruses[J].J Clin Microbiol,1988,26(7):1298-1303.
[4]Iritani N,Kaida A,Abe N,Kubo H,Sekiguchi J,Yamamoto S P,Goto K,Tanaka T,Noda M.Detection and genetic characterization of human entericviruses in oyster-associated gastroenteritis outbreaks between 2001and 2012inOsaka City,Japan[J].J Med Virol,2014,86(12):2019-2025.
[5]Melica G,Langlois A L,Le Goff J,Viglietti D,Glotz D,Molina J M,Peraldi M N.Acute enteritis associated with Coxsackievirus A19 in a kidneytransplant patient[J].Transpl Infect Dis,2014,16(2):344-346.
[6]Kapusinszky B,Szomor K N,Farkas A,Takacs M,Berencsi G.Detection ofnon-polio enteroviruses in Hungary 2000-2008and molecular epidemiology ofenterovirus 71,coxsackievirus A16,and echovirus 30[J].Virus Genes,2010,40(2):163-173.
[7]Donbraye E,Olasunkanmi O I,Opabode B A,Ishola T R,Faleye T O C,Adewumi O M,Adeniji J A.Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria isgeographically defined[J].J Med Microbiol,2018,67(6):854-865.
[8]Brinkman N E,Fout G S,Keely S P.Retrospective Surveillance ofWastewater To Examine Seasonal Dynamics of Enterovirus Infections[J].mSphere,2017,2(3):e00099-17.
[9]Isaacs S R,Kim K W,Cheng J X,Bull R A,Stelzer-Braid S,Luciani F,Rawlinson W D,Craig M E.Amplification and next generation sequencing of nearfull-length human enteroviruses for identification and characterisation fromclinical samples[J].Sci Rep,2018,8(1):11889.
发明内容
本发明解决的技术问题是提供了一种柯萨奇病毒A组19型(coxsackievirus A19,CV-A19)VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用。
本发明为解决上述技术问题采用如下技术方案:
一种柯萨奇病毒A组19型VP1基因,其特征在于:该柯萨奇病毒A组19型VP1基因的碱基序列如序列表SEQ ID NO.1所示,可以CV-A19基因组cDNA为模板PCR扩增获得或人工合成。
一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其特征在于:该柯萨奇病毒A组19型VP1基因重组表达蛋白的氨基酸序列如序列表SEQ ID NO.2所示。
一种基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体的制备方法,其特征在于具体过程为:将携带柯萨奇病毒A组19型VP1基因的重组载体表达柯萨奇病毒A组19型VP1重组蛋白,再经纯化后免疫ICR小鼠制备而得。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备柯萨奇病毒A组19型特异性检测试剂中的应用。
本发明所述的基于柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备小鼠后肢样品或骨骼肌细胞中柯萨奇病毒A组19型特异性检测试剂中的应用。
本发明具有以下优点:
1.本发明利用原核表达CV-A19 VP1基因重组蛋白制备免疫原,方法简便易行,具有生物安全性。
2.本发明制备的小鼠多克隆抗体anti-CV-A19 VP1能特异性地检测CV-A19感染乳鼠后肢样品中的CV-A19 VP1蛋白,说明其能够很好地用于感染组织中CV-A19的检测。
3.本发明利用制备的小鼠多克隆抗体anti-CV-A19 VP1建立了一种免疫过氧化物酶单层试验(immunoperoxidase monolayer assay,IPMA)检测新生小鼠骨骼肌细胞中CV-A19 VP1蛋白的方法,该方法特异性好,操作简便,普通光学倒置显微镜下即可观察,实用性强。
附图说明
图3是12%(m/V)SDS-PAGE分析CV-A19 VP1基因重组蛋白的表达。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌未诱导;2:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌0.5mM IPTG 37℃诱导6h。
图4是12%(m/V)SDS-PAGE分析CV-A19 VP1基因重组表达蛋白的纯化。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌未诱导;2:pET-28a(+)-CV-A19 VP1/BL21(DE3)菌诱导;3:菌体沉淀第一次破碎后9384×g离心30min获得的上清;4:沉淀第二次破碎后3000×g离心10min获得的沉淀;5:沉淀第五次破碎后3000×g离心10min获得的沉淀;6:切胶回收、液氮研磨获得的重组CV-A19VP1蛋白。
图5是蛋白质免疫印迹(Western blot)测定CV-A19 VP1基因重组表达蛋白免疫ICR小鼠血清效价。M:Thermo ScientificTM PageRulerTM Prestained Protein Ladder;1~6:一抗分别是免疫小鼠血清1:1000、1:2000、1:4000、1:8000、1:16000、1:32000稀释;8:一抗是未免疫小鼠血清1:1000稀释。二抗用AP标记羊抗小鼠IgG(1:8000),NBT/BCIP工作液显色。
图6是Western blot检测感染乳鼠后肢样品中的CV-A19 VP1蛋白。M:ThermoScientificTM PageRulerTM Prestained Protein Ladder;1:CV-A19感染乳鼠后肢样品;2:模拟感染乳鼠后肢样品。一抗用小鼠多克隆抗体anti-CV-A19 VP1(1:1000稀释),二抗用AP标记羊抗小鼠IgG(1:8000稀释),NBT/BCIP工作液显色。
图7是IPMA检测新生小鼠骨骼肌细胞中的CV-A19 VP1蛋白。左侧:模拟感染细胞;右侧:含CV-A19粪便上清感染细胞。一抗用小鼠多克隆抗体anti-CV-A19 VP1(1:1000稀释),二抗用HRP标记羊抗小鼠IgG(1:1000稀释),AEC工作液显色。
具体实施方式
下面通过实施例对本发明的上述内容做进一步阐述,这些实施例只用于说明本发明,并非用于限制本发明的范围。以下实施例中未注明具体条件的实验方法,通常按照常规条件进行。
需特别指出的是,尽管本发明阐述的是一种CV-A19 VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用,针对感染乳鼠后肢样品和新生小鼠骨骼肌细胞中的CV-A19分别进行Western blot和IPMA检测,但应用本发明对感染任何其他细胞的CV-A19的检测也包括在本发明所要求的权利范围之内。
实施例1
CV-A19 VP1基因扩增及克隆
用QIAGENViral RNA Kit提取含有CV-A19的手足口病患儿粪便标本中的病毒RNA。用TaKaRa RNA LA PCRTM Kit(AMV)Ver.1.1将RNA逆转录成cDNA。以该cDNA为模板,用TOYOBO高成功率PCR酶KOD FX扩增CV-A19 VP1基因。所用引物由生工生物工程(上海)股份有限公司合成(如表1所示,单下划线表示酶切位点BamHI,双下划线表示酶切位点XhoI)。PCR反应体系如表2所示(3管加cDNA为模板,1管加灭菌超纯水作为对照)。PCR反应条件为:94℃,2min,(98℃,10sec;55℃,30sec;68℃,1min)35个循环,68℃终延伸10min。PCR产物用0.8%(m/V)琼脂糖凝胶电泳检测有扩增基因目的大小片段后,剩余PCR产物用OmegaCycle Pure Kit纯化。琼脂糖凝胶电泳检测纯化的PCR产物(如图1所示),发现纯化的PCR产物中有比1000bp稍小的片段,与预期的产物大小相符。
表1扩增CV-A19 VP1基因的引物
表2扩增CV-A19 VP1基因的PCR反应体系
纯化的PCR产物用NEB限制性内切酶BamHI和XhoI双酶切、OmegaCyclePure Kit纯化后,采用NEB T4 DNA连接酶与BamHI和XhoI双酶切后、切胶回收的pET-28a(+)载体片段相连,热激法转化CaCl2法制备的大肠杆菌DH5α感受态细胞,菌落PCR筛选出阳性克隆,再经BamHI和XhoI双酶切鉴定(见图2)、生工生物工程(上海)股份有限公司测序验证准确无误后用于下一步实验。
实施例2
CV-A19 VP1基因重组蛋白表达及纯化
将上述测序验证后的重组表达质粒pET-28a(+)-CV-A19 VP1通过热激法转化至CaCl2法制备的大肠杆菌BL21(DE3)感受态细胞,筛选出表达CV-A19 VP1基因重组蛋白的阳性克隆。阳性克隆划线,挑取线上单个菌落,接种于5mL含10μg/mL硫酸卡那霉素的LB液体培养基,37℃振荡过夜培养。分别取2mL菌液接种于2瓶200mL含10μg/mL硫酸卡那霉素的LB液体培养基,待菌液生长至吸光度A600为0.6~0.8时,加入终浓度为0.5mM的IPTG诱导或不加入IPTG作为对照。37℃振荡培养6h后,测定A600,收集菌液,离心收集菌体,用20mM Tris(pH7.4)洗涤3次后,加入合适体积(15×A600/mL)20mM Tris(pH7.4)重悬沉淀。用12%(m/V)SDS-PAGE电泳检测重组蛋白表达情况(如图3所示),发现与未诱导菌相比,诱导菌在35~40kDa之间有明显的蛋白表达条带,其大小与预期CV-A19 VP1基因重组表达蛋白(37kDa)相符。
上述重悬的菌体沉淀反复冻融三次,冰上超声波破碎后,差速离心分离沉淀和上清。沉淀重复超声波破碎和差速离心5次,最终将4℃3000×g离心10min获得的沉淀重悬于5mL pH7.4的磷酸盐缓冲液(phosphate buffered saline,PBS)中。取最后的重悬液加入适量5×SDS-PAGE上样缓冲液后煮沸10min,用12%(m/V)SDS-PGAE分离蛋白,5%(m/V)氯化钾(KCl)溶液染色,切胶回收目标蛋白条带,加入适量PBS后,液氮研磨三次,获得纯化的CV-A19 VP1基因重组表达蛋白。取样对纯化蛋白进行12%(m/V)SDS-PAGE分析(如图4所示),发现:第一次超声破碎后,9384×g离心30min的上清中几乎看不到目标蛋白的存在;在后续超声波破碎后,3000×g离心10min的沉淀中有较多的目标蛋白存在;最后切胶回收、液氮研磨获得的目标蛋白条带单一。结果表明,CV-A19 VP1基因重组蛋白主要以包涵体形式表达,纯化后的蛋白纯度较高,可以用于免疫ICR小鼠制备多克隆抗体。
实施例3
小鼠anti-CV-A19 VP1多克隆抗体的制备和效价测定
实施例2纯化的CV-A19 VP1基因重组表达蛋白分别以200μL/只剂量腹腔注射免疫8周龄ICR小鼠,以后每隔三周进行第二次和第三次免疫,间隔7周后进行第四次免疫,加强免疫方式均为腹腔注射,剂量约为100μL/只。第四次免疫后第10天摘眼球取血,断颈处死小鼠,分离血清,用Western blot测定抗体效价(如图5所示)。结果发现,未免疫小鼠血清1:1000稀释与纯化的CV-A19 VP1基因重组表达蛋白杂交未出现条带,免疫小鼠血清1:1000~1:32000稀释后与纯化的CV-A19 VP1基因重组表达蛋白杂交均出现位于35~40kDa之间的条带,其大小与目标蛋白(37kDa)相符,说明免疫小鼠血清中含有anti-CV-A19 VP1多克隆抗体,其效价可以达到1:32000。
实施例4
小鼠anti-CV-A19 VP1多克隆抗体用于Western blot检测感染乳鼠后肢样品中的CV-A19
取出冻存于-80℃的CV-A19感染乳鼠后肢样品和模拟感染(注射0.9wt%NaCl溶液)乳鼠后肢样品,按2mL/0.1g重量加入含有1mM PMSF的RIPA裂解液(强),液氮研磨后,冰上裂解30min。4℃14000×g离心10min后,吸取上清液,加入适量5×SDS-PAGE上样缓冲液,煮沸10min。然后用12%(m/V)SDS-PAGE分离蛋白,湿法转膜将蛋白转移到PVDF膜上,用实施例3中获得的小鼠anti-CV-A19 VP1多克隆抗体作为一抗(1:1000稀释)进行Western blot分析(见图6)。结果发现,感染样品在大约35kDa处有条带,而模拟感染样品在相应位置无明显条带,说明小鼠anti-CV-A19 VP1多克隆抗体能够特异性地检测CV-A19感染乳鼠后肢样品中的CV-A19 VP1蛋白,可用于CV-A19感染乳鼠后肢样品中的CV-A19检测。
实施例5
小鼠anti-CV-A19 VP1多克隆抗体用于IPMA检测感染新生小鼠骨骼肌细胞中的CV-A19
参考Siwar Nsaibia等人(Virus Research,2007,123:30–39)的方法,略微修改,分离新生小鼠骨骼肌细胞。在无Ca2+、Mg2+D-Hanks溶液中分离新生(1-2天)小鼠后肢,去除皮肤后,将后肢肌肉等组织剪碎成肉糜状,然后吸取至50mL离心管中,轻轻摇晃2min,再静置5min,弃去含血污的上清液,沉淀加入含有100U/mL胶原酶Ⅳ、2.4U/mL中性蛋白酶和0.036mM CaCl2的D-Hanks溶液摇匀后,于37℃消化20~30min。取出静置2min后,上清液转移至无菌洁净50mL离心管中,加入1/10(V/V)胎牛血清。沉淀的肌肉组织再消化一次,同样收集上清,加入胎牛血清。两次消化获得的细胞悬液分别用70μm孔径的尼龙膜过滤,滤液300×g离心10min后,用含有25%(V/V)Medium 199、20%(V/V)FBS、1%(V/V)100×青链霉素和2μg/mL两性霉素B的MEM培养基重悬细胞。计数后,以1.6×105个细胞/孔接种于96孔板中,37℃、体积分数5%CO2培养箱中贴壁培养1h后,上清转移至新孔中继续培养过夜,第二天用含CV-A19的粪便上清液感染或用上述培养基模拟感染新孔中的细胞1h,弃感染液或培养基,加入上述培养基继续培养。待4天后,感染细胞与模拟感染细胞出现明显差异时,进行IPMA检测:首先弃上清液,加入200μL/孔PBS洗3次;然后加入100μL/孔冰冻的无水乙醇室温固定10min,再用200μL/孔PBS清洗1次;接下来用含有5%(m/V)脱脂奶粉的PBST 37℃封闭1h,弃封闭液,用PBST清洗1次;再加入用封闭液稀释的小鼠anti-CV-A19 VP1(1:1000)作为一抗,4℃孵育过夜,PBST清洗3次;然后加入封闭液稀释HRP标记羊抗小鼠IgG(1:1000)作为二抗,37℃孵育1h,PBST清洗5次;最后加入AEC工作液充分显色,弃显色液,超纯水洗2次后,加入适量超纯水,在倒置光学显微镜下观察,拍照分析(如图7所示)。结果发现,与模拟感染细胞相比,用含CV-A19的粪便上清液感染细胞部分出现聚集皱缩,染色后呈现红棕色。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
序列表
<110> 新乡医学院
<120> 一种柯萨奇病毒A组19型VP1基因重组表达蛋白、多克隆抗体的制备方法及其应用
<130> 2021
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 888
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
ggtattgatg acattataga taatgtagtg actaatgctc tgaaagtgtc catgccacag 60
gtccaagaca cacagtctag cggaccggtg aactcaaaag aagtgcctgc gttgaccgct 120
gttgaaactg gagcaactag ccaggttgac ccgtcagact taatagagac cagacatgta 180
ataaacaacc gtcttagatc agagtgcact attgaatctt tctttgggag atccgcttgt 240
gtggctataa ttggcctttc caatcagaaa cccaccgatg acaatgcagc taaacttttt 300
gctacatgga aaataagtta ccttgatacg taccagctga gaaggaaatt agaattcttc 360
acctactcca gatttgacct tgaattgacc tttgtaatat cagagagatt cttcacctcc 420
acttcagctg cagctagaga ttatgtgtac caaatcatgt acattcctcc aggagcccca 480
attccacagg catgggatga ttacacttgg cagtcgtcaa ccaatccatc aatattttac 540
accacaggga atgcatgtcc cagagtgtcc atcccttttg ttggtatcgg tgcagcgtac 600
tcccacttct acgatgggtt ctctctagtt cctttcaata ccattgatgc cggggcatcg 660
aacaggtatg gttacaccac cattaatgac tttggaacca tggctatcag gatagtgaat 720
gaatacgacc cagtcacaat cgatgctaaa gttagggtat acatgaaacc aaaacacatc 780
aaggtttggt gtcctagacc accacgggct gtggcataca atgggccaac tgtgaatttc 840
aacgaaaacc cacatgtgat gacagcagtt gcagatatta ggacttat 888
<210> 2
<211> 331
<212> PRT
<213> 人工序列(artificial sequence)
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Ile Val Asn Glu Tyr Asp Pro Val Thr Ile Asp Ala Lys Val Arg Val
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Ala Val Ala Tyr Asn Gly Pro Thr Val Asn Phe Asn Glu Asn Pro His
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Claims (6)
1.一种柯萨奇病毒A组19型VP1基因,其特征在于:该柯萨奇病毒A组19型VP1基因的碱基序列如SEQ ID NO.1所示。
2.一种柯萨奇病毒A组19型VP1基因重组表达蛋白,其特征在于:该柯萨奇病毒A组19型VP1基因重组表达蛋白的氨基酸序列如SEQ ID NO.2所示。
3.一种基于权利要求2所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体。
4.一种权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体的制备方法,其特征在于具体过程为:将携带权利要求1所述的柯萨奇病毒A组19型VP1基因的重组载体表达柯萨奇病毒A组19型VP1重组蛋白,再经纯化后免疫ICR小鼠制备而得。
5.权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备柯萨奇病毒A组19型特异性检测试剂中的应用。
6.权利要求3所述的柯萨奇病毒A组19型VP1基因重组表达蛋白的多克隆抗体在制备小鼠后肢样品或骨骼肌细胞中柯萨奇病毒A组19型特异性检测试剂中的应用。
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