CN110927374A - 检测戊型肝炎病毒IgG抗体的胶体金试纸条及其制备方法 - Google Patents
检测戊型肝炎病毒IgG抗体的胶体金试纸条及其制备方法 Download PDFInfo
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- CN110927374A CN110927374A CN201911211859.6A CN201911211859A CN110927374A CN 110927374 A CN110927374 A CN 110927374A CN 201911211859 A CN201911211859 A CN 201911211859A CN 110927374 A CN110927374 A CN 110927374A
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- colloidal gold
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- igg antibody
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Abstract
本发明公开了一种检测戊型肝炎病毒IgG抗体的胶体金试纸条,包括反应支持物、吸水纸垫、硝酸纤维素膜、胶体金垫、金标抗原保护膜,其中胶体金垫为含有胶体金标记的戊型肝炎病毒ORF2蛋白的玻璃纤维膜,硝酸纤维素膜上带有包被山羊抗人IgG抗体、兔抗HEV多克隆抗体的两个条带;本发明能检测戊型肝炎病毒IgG抗体,该方法简便、快捷、准确,不需要特殊仪器设备,不需专业培训,结果清晰易辨,操作简单,易于推广,适合大批量检测,适用于基层筛选和流行病学调查,对戊型肝炎病毒感染的早期及中期都能起到辅助诊断的作用,对判断人体近期是否感染戊型肝炎病毒抗原具有参考意义。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种检测戊型肝炎病毒IgG抗体的胶体金试纸条及制备方法。
背景技术
人类戊型肝炎病毒(Hepatitis E Virus, HEV)是全世界范围内青壮年易感染的病原,免疫缺陷病人及老年人,孕妇也是HEV的易感人群。戊型肝炎病毒在任何国家的孕妇中都有很高的感染率和发病率。
人体感染HEV后,产生特异性IgG抗体,因此,可以通过试纸条法快速检测人体血液中戊型肝炎病毒IgG抗体作为HEV筛查感染临床诊断和HEV感染预后的初步依据。
然而,目前HEV临床诊断方法主要是通过病毒分离培养、间接免疫荧光法、RT-PCR和ELISA法检测抗原,或者利用ELISA法检测HEV特异性抗体。但是上述检测方法均需要特殊的仪器设备和专业培训人员,并且在实验室才能进行检测,费用高昂、操作复杂,耗时长,不适于临床筛查,更不适宜于现场即时检测。因此,急需建立一种快速、简便、高效,并能广泛推广的戊型肝炎病毒检测方法。
发明内容
针对现有技术的不足,本发明提供了一种简便、快捷的检测戊型肝炎病毒IgG抗体的胶体金试纸条,其包括反应支持物、吸水纸垫、硝酸纤维素膜、胶体金垫、金标抗原保护膜,胶体金垫为含有胶体金标记的戊型肝炎病毒ORF2蛋白的玻璃纤维膜,硝酸纤维素膜上带有包被山羊抗人IgG抗体、兔抗HEV多克隆抗体的两个条带。
所述金标抗原保护膜为玻璃纤维膜或滤纸纤维膜。
所述吸水纸垫为滤纸。
所述反应支持物为PVC胶板。
本发明另一目的提供上述检测戊型肝炎病毒IgG抗体的胶体金试纸条的制备方法,方法如下:
除非另有说明,本发明中所采用的百分数均为质量百分数。
(1)将山羊抗人IgG抗体、兔抗HEV多克隆抗体分别固相于硝酸纤维素膜上,形成检测条带和质控条带;将含有胶体金标记的戊型肝炎病毒ORF2蛋白包被在玻璃纤维膜上制得胶体金垫;其中固相方法及包被方法均为常规方法;
(2)依次将硝酸纤维素膜、胶体金垫、吸水纸垫、金标抗原保护膜粘贴在PVC胶板上,且相互之间有0.2mm的重叠,压紧避免留有气泡影响实验结果,使用切割机切割制得宽度在0.4~0.6cm的胶体金试纸条;置于相对湿度40~80%,温度18~25℃的含有干燥剂的铝箔袋中短期(1周)保存备用;大批量制备应该置于4℃长期(1年)保存。
使用时从冰箱取出的试纸条恢复至室温后,再开启密封袋使用。
本发明检测戊型肝炎病毒IgG抗体的胶体金试纸条的检测样本为血液或血清。
本发明的工作原理及有益效果:
1、通过在硝酸纤维素膜(NC膜)上包被含有山羊抗人IgG抗体、兔抗HEV多克隆抗体的两个条带,并结合胶体金标记的的戊型肝炎病毒ORF2蛋白,实现了用试纸条检测人戊型肝炎病毒IgG抗体的目的;克服了现有技术检测成本高、操作复杂、繁琐、耗时长、需要特殊仪器、且必需专业人员才能操作的不足。
2、本发明试纸条能检测人戊型肝炎病毒IgG抗体,该方法简便、快捷、准确,不需要特殊仪器设备,不需专业培训,结果清晰易辨,操作简单,易于推广,适合大批量检测,适用于基层筛选和流行病学调查,对戊型肝炎病毒感染的早期及中期都能起到辅助诊断的作用,对判断人体近期是否感染戊型肝炎病毒抗原具有参考意义。
附图说明
图1为本发明试纸条的俯视结构示意图;
图2为本发明试纸条的侧视结构示意图;
图3为本发明试纸条的检测结果(阳性)示意图,T线和C线显色;
图4为本发明试纸条的检测结果(阴性)示意图,C线显色;
图5为本发明试纸条的检测结果(无效)示意图,T线和C线不显色;
图6为本发明试纸条的检测结果(无效)示意图,T线显色、C线不显色;
图中:1-吸水纸垫;2-硝酸纤维素膜;3-胶体金垫;4-金标抗原保护膜;5- PVC胶板;T为检测条带,包被山羊抗人血清IgG抗体;C为质控条带,包被兔抗HEV多克隆抗体。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法;
实施例1:制备检测戊型肝炎病毒IgG抗体的胶体金试纸条
本检测戊型肝炎病毒IgG抗体的胶体金试纸条,包括反应支持物5、吸水纸垫1、硝酸纤维素膜2、胶体金垫3、金标抗原保护膜4,胶体金垫为含有胶体金标记的戊型肝炎病毒ORF2蛋白的玻璃纤维膜,硝酸纤维素膜上带有包被山羊抗人IgG抗体、兔抗HEV多克隆抗体的两个条带;其中反应支持物为6cm×0.4cm PVC胶板(购自上海金标生物技术有限公司,SM31-40);吸水纸垫为2.5×0.4cm的滤纸(购自上海金标生物技术有限公司,CH37K);2cm×0.4cm的硝酸纤维素膜(购自上海捷宁生物科技有限公司),山羊抗人IgG抗体(购自上海碧云天生物技术有限公司);胶体金垫0.8cm×0.4cm;金标抗原保护膜为1cm×0.4cm的聚酯纤维膜(购自上海金标生物技术有限公司);
1、戊型肝炎病毒ORF2蛋白的制备
1.1实验材料
在大肠杆菌中能表达戊型肝炎病毒ORF2蛋白的质粒(戊型肝炎病毒ORF2蛋白序列是提取基因4型戊型肝炎病毒RNA,以RNA经过逆转录后获得cDNA为模板,通过常规PCR扩增获得,扩增中使用的上游引物序列:CGCCATATGGCGATAGCGCTAACTCTGTTTAAT,下游引物序列为:CGCGGATCCGCGCGCAGAATGAGGTGCAAGGACACCGA,PCR产物利用限制性内切酶NdeI和BamH I酶切后***至载体pFN18the中,经测序鉴定正确后,命名为pFN18the-ORF2,戊型肝炎病毒ORF2蛋白的氨基酸序列如序列表SEQ ID NO:1所示)、卡那霉素、Rosetta大肠杆菌感受态、LB液体培养基、LB固体培养基、鼠李糖、质粒小量提取试剂盒、透析袋、HisTrapTMFF1mL预装柱等。
1.2实验步骤
(1)转化:取pFN18the-ORF2质粒0.5μL加入100μL Rosetta大肠杆菌感受态中,冰上放置20分钟,42℃热激1.5min后迅速放置冰上2min,加入1mL LB液体培养基置恒温摇床培养45min(37℃,170rpm),12000rpm离心1min,留取沉淀及上清共100μL,吹打均匀后涂布至LB固体培养基(含终浓度为0.1%的卡那霉素),37℃培养12-14h;
(2)增菌:挑取(1)中培养长出的单菌落,接种于5mL LB液体培养基(含终浓度为0.1%的卡那霉素)中,恒温摇床培养12-14h(37℃,170rpm),取500μL菌液加入500μL浓度为50%的无菌甘油-20℃保存;
(3)提取质粒酶切鉴定:使用购自北京庄盟国际生物基因科技有限公司的质粒小量提取试剂盒提取(2)中菌液的质粒,配制如下体系对所提质粒进行酶切鉴定:质粒5μL、NdeI0.5μL、BamHI0.5μL、无菌水3μL、10×cut buffer 1μL。
(4)小量增菌:将步骤(3)鉴定正确质粒的菌液保存液12000rpm离心1min,取菌体沉淀接种入5mL LB液体培养基(含终浓度为0.1%的卡那霉素)中,恒温摇床培养12-14h(37℃,170rpm);
(5)转接:取步骤(4)5mL所得菌液接种于100mL LB液体培养基(含终浓度为0.1%的卡那霉素)中,恒温摇床培养(37℃,170rpm),每30min取200μL菌液检测O.D.600值,直至达到0.4左右;
(6)诱导ORF2蛋白表达:按照5μL/mL的量在步骤(5)菌液中加入终浓度为20%鼠李糖,放置于恒温摇床培养12-14h(28℃,170rpm)至O.D.600值达到1左右;
(7)ORF2蛋白提取:取步骤(6)所得菌液12000 rpm离心10min,取菌体沉淀,用菌液原体积1/10的裂解液(300mMTris-HCl,100mMNaCl,pH=9.0)重悬,超声破菌(功率300W,工作5s,间隔10s,反复35次),2000rpm离心10min弃沉淀,上清12000rpm离心10min,留取沉淀用洗涤液(50mMTris-HCl,2mM尿素,PH=7.0)洗涤2次,12000 rpm离心10min留取沉淀,即为ORF2蛋白包涵体;
(8)ORF2蛋白纯化:使用包涵体变性液(300mMTris-HCl,100mMNaCl, 8mM尿素,pH=9.0)溶解包涵体,4℃静置1h,12000 rpm离心5min,取上清进行纯化。使用HisTrapFF1mL预装柱,用10倍体积无菌水洗涤柱子,再用5倍体积包涵体变性液(300mMTris-HCl,100mMNaCl,8mM尿素,pH=9.0)平衡柱子;将上清缓慢注入柱子,4℃静置30min;用5倍体积的包涵体变性液(300mMTris-HCl,100mMNaCl,8mM尿素,pH=9.0)洗涤柱子,再用5倍体积洗脱液(300mMTris-HCl,100mMNaCl,50mM咪唑,pH=9.0)洗脱蛋白,收集流穿液和洗脱液;SDS-PAGE检测蛋白纯度;
(9)纯化产物Western-blot分析:取纯化后的蛋白进行SDS-PAGE电泳;电泳后用PVDF膜(100%甲醇湿润)湿转法进行转膜(冰水浴,100V,350mA,90min);取PVDF膜用质量浓度10%脱脂奶溶液室温封闭2h;HEVIgG抗体阳性血清用质量浓度2%脱脂奶溶液进行10倍稀释后与PVDF膜4℃孵育12-14h;PBS(含终浓度为0.1%的Tween-20)清洗PVDF膜3次,每次5min;用质量浓度2%脱脂奶溶液将山羊抗人IgG-HRP稀释1000倍,与PVDF膜室温孵育2h;PBS(含终浓度为0.1%的Tween-20)清洗PVDF膜3次,每次5min;显影观察目的条带;
(10)透析ORF2蛋白:取步骤(9)中鉴定正确的蛋白溶液使用购自北京索莱宝科技有限公司的普通干型透析袋进行透析;透析袋使用前用无菌水煮沸10min,冷却至常温;将需透析的蛋白溶液装入透析袋,用透析夹夹紧透析袋;将透析袋完全浸泡在100倍体积的透析液(20mM,Tris-HCl,0.5mMNaCl,pH=7.0)中4℃透析24h,每3h更换一次透析液;透析所得蛋白溶液即为可用于胶体金标记的戊型肝炎病毒ORF2蛋白,-40℃保存;
2、胶体金标记戊型肝炎病毒ORF2蛋白制备方法
2.1胶体金溶液的制备
将500μL避光保存的质量浓度20%的氯金酸溶液添加在110mL的ddH2O中,避光加热至沸腾持续15min后,立即加入850μL的质量浓度1%的柠檬酸三钠溶液,持续避光加热6min浓缩至100mL,冷却至室温,4℃保存;
2.2胶体金标记戊型肝炎病毒ORF2蛋白标记物的制备
在步骤2.1中所得的胶体金溶液加入质量浓度1%的K2CO3溶液调pH至7.6;取戊型肝炎病毒ORF2蛋白加入胶体金溶液中(使ORF2蛋白终浓度为50ng/μL-100 ng/μL),10℃搅拌1h;然后加入聚乙二醇至聚乙二醇终浓度为1%,搅拌1h;4℃静置2h后2500rpm离心5min弃沉淀,9000rpm离心60min,取沉淀加入质量浓度2%的BSA至BSA终浓度为1%,沉淀即为胶体金标记的戊型肝炎病毒ORF2蛋白,4℃保存;
2.3胶体金标记的戊型肝炎病毒ORF2蛋白标记物包被玻璃纤维膜的方法
用移液枪取胶体金标记的戊型肝炎病毒ORF2蛋白滴加在玻璃纤维膜上,每条试纸使用1μL胶体金标记的戊型肝炎病毒ORF2蛋白,4℃于锡箔袋中保存。
3、兔抗HEV多克隆抗体制备
取戊型肝炎病毒ORF2蛋白5mL,生理盐水稀释3倍,分3次连续3周皮下多点注射SPF级别新西兰大白兔,每次注射1周后采血用ELISA法检测戊型肝炎病毒IgG;检测到阳性结果后加强免疫1次,2周后处死兔子,采集血液,4℃静置1-2h,4℃1000rpm离心30min,留取上层血清,1.5mL EP管分装后冻存于-40℃保存,所得血清即为兔抗HEV多克隆抗体。
4、T线(检测条带)、C线(质控条带)生物制剂固相于硝酸纤维素膜的方法
使用PBS以体积比1:200的比例稀释山羊抗人IgG抗体,-40℃备用,使用PBS以体积比1:50的比例稀释兔抗HEV多克隆抗体,-40℃备用;使用时冰上融化,在室温、湿度于40%-80%条件下用移液枪取山羊抗人IgG抗体溶液滴加于硝酸纤维膜上T线的位置,取兔抗HEV多克隆抗体溶液滴加于硝酸纤维膜上C线的位置,每条试纸T、C线生物制剂标记量为1-2μL;将标记过T、C线的硝酸纤维膜放于37℃烘箱干燥2-3h;4℃于锡箔袋中保存。
5、试纸条的制备方法
将吸水纸垫1、预先包被山羊抗人IgG抗体、兔抗HEV多克隆抗体两个条带的硝酸纤维素膜2、胶体金标记的戊型肝炎病毒ORF2蛋白的玻璃纤维3、金标抗原保护膜4依次粘贴于PVC胶板上,相互间留有约0.2mm的重叠,用力压紧,避免留有气泡;用切割机将组装好的硝酸纤维素膜切成每条宽约0.4~0.6cm的条带,置于相对湿度40~80%,温度18~25℃的含有干燥剂的铝箔袋中短期(1周)保存备用;大批量制备应该置于4℃长期(1年)保存。
实施例2:检测方法
按临床常规方法收集全血并分离血清,如样品不能于三日内检测,应存放于-20℃;取不少于150-200μl血清于小瓶中,将上述实施例制得的试纸条带有金标抗原保护膜4的一端***瓶内血清中,样品中的液体上行,10-15min后判读结果;
如样品中含有戊型肝炎病毒IgG抗体,则其经过胶体金垫时,会与金标HEVORF2蛋白结合,进而被T处的山羊抗人IgG抗体捕获,聚集而形成红色的检测线,T线和C线显色为阳性(图3),只有C线显色则为阴性(图4);无论样本中是否含有待检测的抗原,金标HEVORF2蛋白都会与C线处包被的兔抗HEV多克隆抗体结合形成红色沉淀线,即质控条带;如质控条带不出现,说明试纸条失效(图5、6)。
需要指出的是,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 昆明理工大学
<120> 检测戊型肝炎病毒IgG抗体的胶体金试纸条及其制备方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 239
<212> PRT
<213> 4型戊型肝炎病毒(Hepatitis E virus type 4)
<400> 1
Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu
1 5 10 15
Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg
20 25 30
Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser
35 40 45
Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile
50 55 60
Asp Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His
65 70 75 80
Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser
85 90 95
Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu
100 105 110
Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser
115 120 125
Asp Thr Val Thr Phe Val Asn Val Ala Thr Gly Ala Gln Gly Val Ser
130 135 140
Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr
145 150 155 160
Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly
165 170 175
Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr
180 185 190
Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala
195 200 205
Gly His Arg Val Cys Ile Ser Thr Tyr Thr Thr Asn Leu Gly Ser Gly
210 215 220
Pro Val Ser Ile Ser Ser Val Gly Val Leu Ala Pro His Ser Ala
225 230 235
<210> 2
<211> 33
<212> DNA
<213> 人工序列(Artificial)
<400> 2
cgccatatgg cgatagcgct aactctgttt aat 33
<210> 3
<211> 38
<212> DNA
<213> 人工序列(Artificial)
<400> 3
cgcggatccg cgcgcagaat gaggtgcaag gacaccga 38
Claims (4)
1.一种检测戊型肝炎病毒IgG抗体的胶体金试纸条,包括反应支持物、吸水纸垫、硝酸纤维素膜、胶体金垫、金标抗原保护膜,其特征在于:胶体金垫为含有胶体金标记的戊型肝炎病毒ORF2蛋白的玻璃纤维膜,硝酸纤维素膜上带有包被山羊抗人IgG抗体、兔抗HEV多克隆抗体的两个条带。
2.权利要求1所述的检测戊型肝炎病毒IgG抗体的胶体金试纸条,其特征在于:金标抗原保护膜为玻璃纤维膜或滤纸纤维膜。
3.权利要求1所述的检测戊型肝炎病毒IgG抗体的胶体金试纸条,其特征在于:反应支持物为PVC胶板。
4.权利要求1-3中任一项所述的检测戊型肝炎病毒IgG抗体的胶体金试纸条的制备方法,其特征在于,步骤如下:
(1)将山羊抗人IgG抗体、兔抗HEV多克隆抗体分别固相于硝酸纤维素膜上,形成检测条带和质控条带;将含有胶体金标记的戊型肝炎病毒ORF2蛋白包被在玻璃纤维膜上制得胶体金垫;
(2)依次将吸水纸垫、硝酸纤维素膜、胶体金垫、金标抗原保护膜粘贴在反应支持物上,且相互之间有0.2mm的重叠,压紧即制得宽度在0.4~0.6cm的胶体金试纸条。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109652384A (zh) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | 一种体外培养戊型肝炎病毒的方法 |
CN109943536A (zh) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | 一种戊型肝炎病毒、培养方法及其灭活疫苗的制备方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5667966A (en) * | 1993-12-22 | 1997-09-16 | Abbott Laboratories | Mouse monoclonal antibodies to Hepatitis E virus and methods for using same |
CN102253205A (zh) * | 2011-06-16 | 2011-11-23 | 昆明倍尔遵生科技有限公司 | 一种检测呼吸道合胞病毒抗体胶体金的试纸条及其制备方法 |
CN102854316A (zh) * | 2012-07-31 | 2013-01-02 | 上海博沃生物科技有限公司 | 一种甲型肝炎病毒IgM和IgG抗体胶体金法检测试剂盒 |
CN103823057A (zh) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | 一种猪hev总抗体胶体金快速诊断试纸条及其制备方法 |
CN104031144A (zh) * | 2013-03-05 | 2014-09-10 | 厦门大学 | 特异结合戊型肝炎病毒3、4型的抗体及其用途 |
CN106632618A (zh) * | 2016-11-18 | 2017-05-10 | 华南农业大学 | 一种猪戊型肝炎病毒orf2重组蛋白的制备方法及其orf2蛋白和检测试剂盒 |
CN108473540A (zh) * | 2015-11-30 | 2018-08-31 | 生物梅里埃公司 | 突变的hev多肽及其用于测定抗hev抗体的用途 |
-
2019
- 2019-12-02 CN CN201911211859.6A patent/CN110927374A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5667966A (en) * | 1993-12-22 | 1997-09-16 | Abbott Laboratories | Mouse monoclonal antibodies to Hepatitis E virus and methods for using same |
CN102253205A (zh) * | 2011-06-16 | 2011-11-23 | 昆明倍尔遵生科技有限公司 | 一种检测呼吸道合胞病毒抗体胶体金的试纸条及其制备方法 |
CN102854316A (zh) * | 2012-07-31 | 2013-01-02 | 上海博沃生物科技有限公司 | 一种甲型肝炎病毒IgM和IgG抗体胶体金法检测试剂盒 |
CN104031144A (zh) * | 2013-03-05 | 2014-09-10 | 厦门大学 | 特异结合戊型肝炎病毒3、4型的抗体及其用途 |
CN103823057A (zh) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | 一种猪hev总抗体胶体金快速诊断试纸条及其制备方法 |
CN108473540A (zh) * | 2015-11-30 | 2018-08-31 | 生物梅里埃公司 | 突变的hev多肽及其用于测定抗hev抗体的用途 |
CN106632618A (zh) * | 2016-11-18 | 2017-05-10 | 华南农业大学 | 一种猪戊型肝炎病毒orf2重组蛋白的制备方法及其orf2蛋白和检测试剂盒 |
Non-Patent Citations (1)
Title |
---|
NCBI: "BAC65254.1", 《GENBANK》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109652384A (zh) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | 一种体外培养戊型肝炎病毒的方法 |
CN109943536A (zh) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | 一种戊型肝炎病毒、培养方法及其灭活疫苗的制备方法 |
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