CN106148289A - 一种纯化戊型肝炎病毒的方法 - Google Patents
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Abstract
本发明公开了一种戊型肝炎病毒在体外纯化的方法;该方法通过添加含有甲基纤维素的血清维持细胞生长,利用流行病学调查分离到的HEV毒株接种HepG2细胞,噬斑法挑取病变的细胞,最后用RT‑PCR检测HEV;该发明在HEV体外纯化方面有了大的突破,为进一步研究HEV的生物学及免疫学特性,抗HEV药物筛选与疫苗的研发奠定了良好的基础。
Description
技术领域
本发明属于生物技术领域,涉及一种戊型肝炎病毒毒株在体外成功纯化并提高病毒纯度的新方法。
背景技术
戊型肝炎病毒(Hepatitis E Virus,HEV)是一种经肠道传播的病毒性肝炎病原体,可以跨种间感染人和多种动物。HEV为无囊膜单股正链RNA病毒,球型病毒颗粒大小为27~34nm,表面有突起和缺刻,内部密度不均匀,基因组全长约为7.3 kb,由3个开放阅读框组成。全球HEV主要有4种基因型,在中国主要以IV型HEV毒株常见。HEV传播途径主要经粪-口途径,也可通过其他途径传播,包括血液传播,接触传播,垂直传播,以及器官移植进行传播。
HEV是导致许多发展中国家戊型肝炎暴发性流行的病原体,HEV感染后,一般患者在4-6周内自愈,免疫缺陷病人及老年人易发展成为慢性肝炎。据世界卫生组织估计,全球每年大约有2010万人感染HEV,造成70万人死亡和3000胎儿宫内死亡,感染HEV的非孕妇和孕妇患者死亡率分别为1.9%和19.8%孕妇感染HEV,发病率高,病情严重,尤其以妊娠晚期最为严重,病死率可高达25%,常发生胎儿早产,流产,死胎及孕妇产后急性肝坏死等症状。戊型肝炎的防控形势已经迫在眉睫,发展有效的戊型肝炎疫苗已成为关系公众健康的急为紧迫的问题,而在这一过程中病毒培养至关重要。
病毒纯化是指利用物理、化学的方法,以不使病毒受损伤和失活为前提,去除宿主细胞组分等非病毒杂质,提取出高纯度浓缩的病毒样品。病毒提纯是病毒学研究的重要前提,且病毒微细形态结构的研究、病毒抗原蛋白的分离提纯、病毒化学成分及其遗传物质的研究都需要高纯度的病毒样品。
发明内容
本发明的目的在于提供一种戊型肝炎病毒(HEV)在体外纯化的方法,该方法通过添加含有甲基纤维素的胎牛血清对较高滴度的HEV毒株维持在体外的细胞层面上,能够使细胞发生病变,从而挑选出单个的戊肝病毒颗粒,为后续研发戊肝疫苗,深入研究HEV生物学、免疫学特性及致病性提供必不可少的前提。
本发明戊型肝炎病毒体外纯化方法,采用如下步骤进行:
(1)将HEV病毒溶液经0.22μm滤膜过滤除菌后,经Real-time qPCR测定其病毒拷贝数为2×105~2×106拷贝数/mL,加入400U/mL青霉素和1000U/mL链霉素4℃处理1h,-80℃保存备用;
(2)将HepG2细胞按1×104/孔接种至96孔板中,用含质量百分比10%胎牛血清的DMEM培养基在37℃、5% CO2培养箱中静止培养细胞,直至细胞长成单层;
(3)取100μL步骤(1)的HEV病毒悬液利用DMEM依次稀释至10-11,按每孔100μL接种至单层细胞中,置37℃孵育2小时,每15min 轻微摇匀一次,使病毒充分吸附到细胞上;弃上清,1×PBS洗涤细胞3次,加入含有质量百分比1%甲基纤维素、质量百分比2%胎牛血清的DMEM培养基中,置于37℃、5% CO2培养箱中继续培养;
(4)待HepG2细胞出现病变时,利用噬斑法挑取病变的细胞,溶于150μL DMEM,取100μL病毒液进行RT-PCR检测HEV,剩余50μL于-80℃保存待用;
HEV检测引物:
外套上游引物(P1): 5′-AATTATGCYCAGTAYCGRGTTG-3′ ;外套下游引物 (P2): 5′-CCCTTRTCYTGCTGMGCATTCTC-3′;
内套上游引物(P3): 5′-GTWATGCTYTGCATWCATGGCT-3′;内套下游引物(P4): 5′-AGCCGACGAAATCAATTCTGTC-3′;
逆转录程序:42℃,60min;72℃,10min;PCR程序:94℃,3 min;94℃,30s;50℃,30s;72℃,30s;35个循环。总延伸72℃,2 min;
(5)检测为阳性病毒克隆剩余的50μL接种至铺有单层HepG2细胞层的96孔板内;
(6)待细胞发生病变后,按步骤(4)挑取病变细胞;
(7)重复步骤(4)、(5)两次;
(8)蚀斑纯化三轮后的HEV病毒液检测其中是否存在肠道病毒(A群、B群和C群)的污染。
目前全世界仅有少数几株HEV毒株可以进行体外培养,因此尚无戊肝病毒纯化的报道。本发明采用戊型肝炎病毒在HepG2细胞中培养,添加甲基纤维素起到半固定作用,防止病毒液向四周扩散,使发生病变的细胞形成清晰的病毒蚀斑,便于挑取,纯化。
附图说明
图1是本发明中使用的HepG2细胞感染HEV的示意图;其中A图为正常HepG2细胞,B图为HEV(稀释倍数为10-1)感染2天后的HepG2细胞,C图为HepG2细胞感染HEV的电镜照片;
图2是本发明中利用RT-nPCR法检测第一轮挑取蚀斑HEV RNA的部分样品检测结果电泳示意图;
图3为部分HEV阳性蚀斑是否存在肠道病毒A、B、C群污染的检测结果电泳示意图;
图4为本发明中利用RT-nPCR法检测接种人HEV挑取的蚀斑中HEV RNA检测的部分PCR产物电泳示意图;
图5为部分人HEV阳性蚀斑是否存在肠道病毒A、B、C群污染的检测结果电泳示意图。
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;实施例中主要采用常规的细胞生物学方法,这些方法是本领域普通技术人员所熟知的。按照以下实施例,不难根据具体情况略作修改和变换而成功实施本发明,这些修改和变换均落在本申请权利要求的范围内,实施例中如无特殊说明的百分比均为质量百分比。
实施例1:
1、HepG2细胞培养
自液氮中取出HepG2细胞(购买于美国ATCC,编号CCL–185)迅速放入37℃的温水中,使细胞悬液快速融化;然后加入含有10%胎牛血清DMEM(购买于GIBCO InvitrogenCorporation公司)培养液,调pH至7.2于37℃培养4h左右,弃去全部培养液,加入新鲜的培养液继续培养,待长成致密单层后,用PBS(购买于GIBCO Invitrogen Corporation公司)洗细胞,胰酶–EDTA(购买于GIBCO Invitrogen Corporation公司)消化,加入上述培养液,置于37℃、5% CO2培养箱中继续培养,如图1A正常HepG2细胞所示。
2、HEV接种HepG2细胞并维持培养
(1)将分离的HEV阳性粪便制备重量体积比10% 的PBS(pH7.4)粪便悬液,剧烈震荡,使粪便乳化,经4℃,12000g离心10min,收集上清,0.22μm滤膜过滤除菌,加入病毒溶液体积2%的双抗溶液(400U/mL青霉素和1000U/mL链霉素),4℃处理1h,-80℃保存备用,经Real-timeqPCR测定其病毒拷贝数为2×106拷贝数/mL;
分离自中国云南昆明市HEV RNA阳性猪粪便样品,基因型为4型,GenBank数据库编号为No.JF747598;
(2)将步骤1中细胞(HepG2细胞)按1×104/孔接种至96孔板中,用质量百分比10%胎牛血清的DMEM培养基在37℃、5% CO2培养箱中静止培养细胞,当细胞长至单层的70~80%,弃上清培养基,如图1A所示,每孔加入100μL HEV病毒稀释液(10-1-10-11),置37℃孵育2小时,每隔15min 轻微摇晃一次,使病毒充分吸附到细胞上,同时添加终浓度为0.01mM MgCl2增加病毒的吸附能力;弃上清,1×PBS洗涤细胞3次,加入含有质量百分比1%甲基纤维素、质量百分比2%胎牛血清的DMEM培养基,置于37℃、5% CO2培养箱中继续培养;HEV感染2天后稀释倍数为10-1孔板内的细胞发生病变(如图1B所示),利用噬斑法挑取病毒颗粒,溶于150 μLDMEM溶液中,收获病毒。
3、对应用上述培养方法获得的HEV病毒进行以下系统鉴定:①运用RT-nPCR检测挑取蚀斑中是否存在阳性HEV颗粒(参见图2),并且筛选出未被肠道病毒污染的病毒颗粒(参见图3);
取100μL上述挑取的蚀斑病毒液,利用Trizol法提取总RNA,经M-MLV逆转录酶逆转录成cDNA,逆转录程序为:42℃,60min;72℃,10min。取5μl cDNA作为PCR扩增模板,各加入1μlHEV检测引物,按照下述PCR程序检测HEV RNA,剩余50μL于-80℃保存待用;
HEV检测引物:
外套上游引物(P1): 5′-AATTATGCYCAGTAYCGRGTTG-3′ ;外套下游引物 (P2): 5′-CCCTTRTCYTGCTGMGCATTCTC-3′;
内套上游引物(P3): 5′-GTWATGCTYTGCATWCATGGCT-3′;内套下游引物(P4): 5′-AGCCGACGAAATCAATTCTGTC-3′;nPCR程序为:94℃,3 min;94℃,30s;50℃,30s;72℃,30s;35个循环,总延伸72℃,2 min。PCR产物经琼脂糖凝胶电泳检测,DNA marker作为参考,出现348 bp目的条带的蚀斑判定为阳性,同时设置阴性和阳性对照。
将检测为HEV RNA阳性的病毒液对应的50μL接种至长成单层HepG2细胞层的96孔板内,待细胞发生病变后,挑取病变细胞并检测;重复上述步骤蚀斑纯化病毒两次;蚀斑纯化三轮后仍为HEV阳性的病毒液检测是否存在肠道病毒的污染;
表1. 肠道病毒检测引物
②利用电镜观察,确认在HepG2细胞形成的蚀斑中存在HEV子代病毒(参见图1C HEV电镜照片,箭头所示);总之,从感染HEV的HepG2细胞发生明显细胞病变、挑取蚀斑中HEV RNA阳性、电镜观察到蚀斑挑取的病毒颗粒中存在HEV子代病毒等方面对蚀斑纯化的HEV进行了不同层次的鉴定;结果证实,噬斑法可应用于HEV的纯化。
采用本实施例方法对分离自中国云南昆明市HEV RNA阳性猪粪便样品的一株HEV病毒,基因型为4型,GenBank No.JF747598的纯化;该毒株能在体外培养,培养上清中未经浓缩的病毒滴度达2×106基因拷贝数/mL,病毒在-80℃冰箱保存6年生物活性不变,仍能感染猪和恒河猴。
实施例2:
1、HepG2细胞培养
自液氮中取出HepG2细胞(购买于美国ATCC,编号CCL–185)迅速放入37℃的温水中,使细胞悬液快速融化;然后加入含有10%胎牛血清DMEM(购买于GIBCO InvitrogenCorporation公司)培养液,调pH至7.2于37℃培养4h左右,弃去全部培养液,加入新鲜的培养液继续培养,待长成致密单层后,用PBS(购买于GIBCO Invitrogen Corporation公司)洗细胞,胰酶–EDTA(购买于GIBCO Invitrogen Corporation公司)消化,加入上述培养液,置于37℃、5% CO2培养箱中继续培养。
2、HEV接种HepG2细胞并维持培养
(1)经ELISA检测IgM阳性,且RT-PCR检测HEV RNA阳性的人血清,经0.22μm滤膜过滤除菌后-80℃保存备用;
毒株为分离自中国云南昆明市HEV RNA阳性血清样品,基因型为4型,GenBank数据库编号为KR872417。
(2)将步骤(1)中细胞(HepG2细胞)按1×104/孔接种至96孔板中,用质量百分比10%胎牛血清的DMEM培养基在37℃、5% CO2培养箱中静止培养细胞,当细胞长至单层的70~80%,弃上清培养基,每孔加入100μL HEV病毒稀释液(10-1),置37℃孵育2小时,每隔15min轻微摇晃一次,使病毒充分吸附到细胞上,同时添加终浓度为0.01mM MgCl2增加病毒的吸附能力;弃上清,1×PBS洗涤细胞3次,加入含有质量百分比1.5%甲基纤维素、质量百分比2%胎牛血清的DMEM培养基,置于37℃、5% CO2培养箱中继续培养;维持5d后细胞发生病变后收获病毒。
(3)将RT-PCR检测为阳性的毒株扩大培养。
3、对应用上述培养方法获得的HEV病毒进行以下系统鉴定:
运用RT-nPCR检测挑取蚀斑中是否存在阳性病毒(参见图4),并且筛选出未被肠道病毒污染的病毒颗粒(参见图 5);
取100μL上述挑取的蚀斑病毒液,利用Trizol法提取总RNA,经M-MLV逆转录酶逆转录成cDNA,逆转录程序为:42℃,60min;72℃,10min。取5μL cDNA作为PCR扩增模板,各加入1μLHEV检测引物,按照下述PCR程序检测HEV RNA,剩余50μL于-80℃保存待用;
HEV检测引物、nPCR程序同实施例1;
将检测为HEV RNA阳性的病毒液对应的50μL接种至长成单层HepG2细胞层的96孔板内,待细胞发生病变后,挑取病变细胞并检测;重复上述步骤蚀斑纯化病毒两次;蚀斑纯化三轮后仍为HEV阳性的病毒液检测是否存在肠道病毒的污染;肠道病毒检测引物如表1,PCR程序同实施例1。
采用本实施例方法对分离自中国云南昆明市HEV RNA阳性人血清样品的一株HEV病毒,基因型为4型,GenBank No. KR872417的纯化;该毒株能在体外培养,培养上清中未经浓缩的病毒滴度达2×106基因拷贝数/mL,病毒在-80℃冰箱保存2年生物活性不变,仍能感染猪和恒河猴。
Claims (1)
1.一种纯化戊型肝炎病毒的方法,其特征在于按如下步骤进行:
(1)将HEV病毒溶液经0.22μm滤膜过滤除菌后,经Real-time qPCR测定其病毒拷贝数为2×105~2×106拷贝数/mL,加入终浓度为400U/mL青霉素和1000U/mL链霉素,4℃处理1h,-80℃保存备用;
(2)将HepG2细胞按1×104/孔接种至96孔板中,用含质量百分比10%胎牛血清的DMEM培养基在37℃、5% CO2培养箱中静止培养细胞,直至细胞长成单层;
(3)取100μL步骤(1)的HEV病毒悬液利用DMEM培养基依次稀释至10-11,按每孔100μL接种至单层细胞中,置37℃孵育2小时,每15min 轻微摇匀一次,使病毒充分吸附到细胞上;弃上清,1×PBS洗涤细胞3次,加入含有质量百分比1-1.5%甲基纤维素、质量百分比2%胎牛血清的DMEM培养基,置于37℃、5% CO2培养箱中继续培养;
(4)待HepG2细胞出现病变时,利用噬斑法挑取发生病变的细胞,溶于150μL DMEM培养基中,取100μL病毒液进行RT-PCR检测HEV,剩余50μL于-80℃保存待用;
HEV检测引物:
外套上游引物(P1): 5′-AATTATGCYCAGTAYCGRGTTG-3′ ;外套下游引物 (P2): 5′-CCCTTRTCYTGCTGMGCATTCTC-3′;
内套上游引物(P3): 5′-GTWATGCTYTGCATWCATGGCT-3′;内套下游引物(P4): 5′-AGCCGACGAAATCAATTCTGTC-3′;
逆转录程序:42℃,60min;72℃,10min;PCR程序:94℃,3 min;94℃,30s;50℃,30s;72℃,30s;35个循环,总延伸72℃,2 min;
(5)将检测为HEV阳性的病毒液对应的50μL接种至长成单层HepG2细胞层的96孔板内;
(6)待细胞发生病变后,按步骤(4)挑取病变细胞并检测;
(7)按照步骤(4)、步骤(5)重复蚀斑纯化病毒两次;
(8)蚀斑纯化三轮后仍为HEV阳性的病毒液检测是否存在肠道病毒的污染。
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