CN118256422A - 一种异育银鲫皮肤细胞系及其应用 - Google Patents
一种异育银鲫皮肤细胞系及其应用 Download PDFInfo
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Abstract
本发明涉及一种异育银鲫皮肤细胞系GiCS的构建方法及其应用。该细胞系于2024年1月9日保藏于中国典型培养物保藏中心(CCTCC)，保藏号为CCTCC NO:C202422。GiCS主要由上皮样形态的细胞组成，核型为3n＝156，占所观察分裂相染色体数目的54％。该细胞系传代倍增时间为3‑5天，对多种水生动物病毒具有易感性，特别是对鲫疱疹病毒表现出高度敏感性，并且可进行遗传操作。本发明是在本领域中首次得到异育银鲫皮肤细胞系，也是首次获得CaHV的敏感细胞系，填补了CaHV敏感细胞系的空白。该细胞系可用于表达外源蛋白、分离培养水生动物病毒，特别是CaHV，为感染鲫的病毒提供了重要的研究工具。同时，也可作为水体中的水生动物病毒的监测工具，以及用于检测水生动物是否感染病毒。
Description
技术领域
本发明涉及淡水鱼类细胞领域，更特别地是，涉及一种异育银鲫皮肤细胞系及其应用。
背景技术
异育银鲫(Carassius gibelio)隶属于鲤形目、鲤科、鲫属，是我国淡水养殖鱼类中的一个重要品种，具有多种繁殖模式。然而，异育银鲫产量的增加以及养殖环境的恶化，导致其养殖场中病害频繁爆发，尤其是病毒性病原引起的病害。鲫疱疹病毒(Carassiusauratus herpesvirus，CaHV)引起鲫急性鳃出血和高死亡率，严重威胁异育银鲫养殖产业。目前为止，尚没有CaHV高度敏感的细胞系，严重影响了对CaHV的深入研究和防控。
不同种类的鱼类细胞系的建立可作为分离、鉴定、和增殖鱼类病毒以及研究潜在致病机制的重要工具，也可为病毒的疫苗研究和流行病学调查及防治等方面奠定坚实基础。目前已构建的异育银鲫细胞系只有异育银鲫尾鳍细胞系和脑细胞系，严重阻碍了对异育银鲫病毒病原的研究。已测试的细胞系均对鲫疱疹病毒CaHV不敏感，极大地限制对CaHV的研究进展。
作为物理和生理屏障，皮肤组织是鱼类抵御病原微生物的第一道重要防线，但鱼类皮肤组织相关的细胞系并不多见。迄今为止，还未有来源于异育银鲫的皮肤细胞系的报道。
发明内容
发明人在工作中构建了一种异育银鲫皮肤细胞系GiCS，于2024年1月9日保藏于中国典型培养物保藏中心(CCTCC)，保藏号为：CCTCC NO:C202422。GiCS主要由上皮样形态的细胞组成，已经连续稳定传代至少60代，核型为3n＝156，占所观察分裂相染色体数目的54％，染色体数目正确。该细胞系传代倍增时间为3-5天，可进行转染。对水生动物病毒例如鲫疱疹病毒(Carassius auratus herpesvirus，CaHV)、沼泽绿牛蛙病毒(Rana gryliovirus，RGV)、大鲵蛙病毒(Andrias davidianus ranavirus，ADRV)、草鱼呼肠孤病毒(Grasscarp reovirus,GCRV)和鲤鱼春季病毒血症病毒(Spring viremia of carp virus，SVCV)等多种水生动物病毒具有高度敏感性，并且可进行遗传操作。
本发明还提供了上述异育银鲫皮肤细胞系在蛋白表达中的应用。由于GiCS细胞可进行遗传操作，并且外源基因可在GiCS细胞中进行有效表达，因此，GiCS细胞可用于进行蛋白表达、生产蛋白制品、制备疫苗等。
由于GiCS对多种水生动物病毒具有易感性，因此，本发明还提供了上述异育银鲫皮肤细胞系GiCS在分离或培养水生动物病毒，特别是鲫疱疹病毒CaHV中的应用，从带毒动物或水体中分离或者培养这些水生动物易感病毒，可提供相应的生物制品以满足研究或生产的需要。
此外，本发明还提供了上述异育银鲫皮肤细胞系GiCS在检测水体中水生动物病毒中的应用。
本发明还提供了一种检测水生动物病毒的方法，包括以下步骤：
S1：将水生动物组织匀浆后或添加待检水样过滤除菌至上述异育银鲫皮肤细胞系GiCS的培养环境中；
S2：观察S1培养的异育银鲫皮肤细胞系GiCS的特征变化。
在一个具体实施方案中，所述特征变化包括细胞形态变化和/或病毒基因表达变化。异育银鲫皮肤细胞系GiCS接种多种水生病毒，在接毒24h后，GiCS的细胞形态可发生明显的病变，因此，可将细胞形态的变化作为待检样品中是否存在水生动物病毒的评价指标之一。
细胞系的保藏
本发明所提到的异育银鲫皮肤细胞系已于2024年1月9日保藏于湖北省武汉市武昌区八一路299号武汉大学的中国典型培养物保藏中心(CCTCC)，保藏号为：CCTCC NO:C202422。
附图说明
图1为异育银鲫皮肤细胞系GiCS的显微照片,bar＝100μm。
图2为异育银鲫皮肤细胞系GiCS在不同温度条件下(20℃、25℃和30℃)的生长曲线图。
图3为异育银鲫皮肤细胞系GiCS的核型分析。其中，A为100个GiCS细胞的染色体数统计分布图；B为代表性细胞的染色体核型图，bar＝200μm。
图4为异育银鲫皮肤细胞系GiCS易受CaHV的感染情况。其中，A为异育银鲫皮肤细胞系GiCS接种CaHV后不同天数的显微照片，bar＝100μm；B为扩增CaHV MCP基因检测CaHV感染GiCS不同天数的PCR结果图；C-D为电镜观察CaHV感染的GiCS细胞，bar＝2μm和bar＝500nm。
图5为异育银鲫皮肤细胞系GiCS接种不同水生动物病毒后的显微照片,bar＝100μm。
图6为在异育银鲫皮肤细胞系GiCS中转入EGFP基因后24h和48h的荧光显微照片。EGFP为绿色荧光蛋白，bar＝200μm。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
1、异育银鲫皮肤细胞系GiCS的构建
取健康异育银鲫A+品系一尾(约300g)进行皮肤细胞的原代培养。具体步骤为：将此鱼处死后，用蒸馏水冲洗干净，洗净表面的脏污，然后置于75％酒精的烧杯中消毒。小心将背部两侧的皮肤和肌肉剥离，随即浸泡在含有10％青霉素-链霉素双抗(1000单位/ml)的L-15培养基中，浸泡三次，每次30分钟。将浸泡后的皮肤组织切成1mm3的小块，随后转移到T25细胞培养瓶中。细胞培养瓶在25℃条件下倒置4小时，然后加入5毫升含有20％胎牛血清和抗生素(100μg/ml链霉素和100U/ml青霉素)的L-15完全培养基，放置25℃培养箱中培养。每隔4-5天更换50％的生长培养基，直至细胞单层长出。
当细胞从组织块中迁移出来并形成完整的单层时，移除旧培养基，进行传代培养。使用0.25％的胰蛋白酶-EDTA溶液将单层细胞分散成单个细胞，按照1:2的比例传代，仍使用20％胎牛血清和抗生素(100μg/ml链霉素和100U/ml青霉素)的L-15培养基。待传到第十代时，将培养基中的血清含量从20％降至10％。待细胞长满后仍按此方法传代。
异育银鲫皮肤细胞系GiCS的细胞形态如图1所示，主要以上皮样细胞形态为主，经过反复传代和筛选，最终获得稳定传代的异育银鲫皮肤细胞系。
2、GiCS细胞系的保藏
将获得的GiCS细胞系于2024年1月9日保藏于湖北省武汉市武昌区八一路299号武汉大学的中国典型培养物保藏中心(CCTCC)，保藏号为：CCTCCNO:C202422。
3、GiCS的培养和生长曲线测定
将GiCS细胞消化，传代至多个12孔培养板中，起始浓度为1.5×105细胞/孔，随后将培养板分别放置于20℃、25℃和30℃的培养箱中培养。每天用胰酶消化，收集细胞，然后用血球计数板计数，测定细胞数量和密度。如图2所示，该细胞系在20℃-30℃的温度范围内均能够稳定生长，其中最适生长温度为25℃。
4、GiCS核型分析
将异育银鲫皮肤细胞GiCS传代至T25细胞培养瓶，置于25℃培养箱中。培养24小时后，加入预先配置好的秋水仙素溶液使其终浓度达到200μg/mL，继续在25℃培养5-6小时。胰酶消化，收集细胞，重悬于低渗溶液(0.075MKCl)中。水浴(37℃)45分钟，然后加入Carnoy’s固定液(甲醇：乙酸＝3:1)，室温放置20分钟。离心收集细胞，再次重悬于新鲜的Carnoy’s固定液中，室温放置20-25分钟。离心收集再次固定过的细胞，重悬于0.3毫升的新鲜固定液中。将重悬液滴到干净的预冷载玻片上，干燥后使用10％吉姆萨染液染色2小时,随后冲洗晾干。在倒置显微镜下观察至少100个分裂中期的细胞并拍照。如图3所示，异育银鲫皮肤细胞GiCS的核型为3n＝156，占所观察分裂相染色体数目的54％。
5、GiCS对鲫疱疹病毒敏感性的测定
使用鲫疱疹病毒(Carassius auratus herpesvirus，CaHV)进行病毒敏感性测定。将收集的新鲜病毒悬液(0.5mL)接种到铺满单层GiCS细胞的T25细胞培养瓶中。在25℃吸附1小时，去除未结合的病毒，然后加入5毫升含有5％胎牛血清的L-15培养基，接毒的细胞继续在25℃培养箱中培养，每天用倒置显微镜拍照观察。当GiCS细胞出现80-90％的病变效应时收获上清液，然后将上清液在4℃以5000rpm离心20分钟以去除细胞碎片，并按上述方法用于感染新鲜的GiCS细胞。此过程重复三次，仍能够观察到在第四次连续感染中的第四天开始出现典型的细胞病变情况，包括出现细胞皱缩、变圆、细胞融合和空泡化的情况，结果如图4A所示。在感染后0、1、3、5、7天(dpi)收获第四次连续感染CaHV的GiCS细胞，通过提取样品的RNA，逆转录得到其cDNA，通过扩增CaHV MCP基因以检测病毒基因表达情况。结果如图4B所示，在CaHV感染的GiCS细胞中，从感染的第1天开始就能检测到与MCP相对应的清晰条带，一直到感染的第7天，表达量均在持续增加。
为了进一步明确病毒感染，制备了CaHV感染GiCS细胞的超薄切片并用透射电镜观察。结果如图4C-D所示，CaHV感染的GiCS细胞呈现不规则性状的细胞核，在细胞核中能观察到大量的病毒核衣壳，直径为90-100nm，显示CaHV在GiCS细胞中大量增殖。以上结果表明GiCS细胞对CaHV非常敏感。
6、GiCS对其他四种水生动物病毒敏感性的测定
使用4种水生动物病毒，沼泽绿牛蛙病毒(Rana gryliovirus，RGV)、大鲵蛙病毒(Andrias davidianus ranavirus，ADRV)、草鱼呼肠孤病毒(Grass carp reovirus，GCRV)和鲤鱼春季病毒血症病毒(Spring viremia of carp virus，SVCV)，进行病毒敏感性测定。将GiCS传代至12孔培养板中，置于25℃培养箱中待其长满单层。将这4种病毒分别以0.01MOI的剂量接入GiCS细胞。接毒的细胞继续在25℃培养箱中培养，并在不同的时间点(0、12、24、36、48和72h)拍照观察。
如图5所示，4种病毒(RGV、ADRV、GCRV和SVCV)均能引起GiCS细胞的细胞病变，包括细胞皱缩、细胞裂解及脱落等，且四种病毒的致细胞病变效应在接种GCRV病毒24h、接种SVCV病毒36h以及接种RGV和ADRV病毒48h后就非常明显。同时PCR分析显示，RGVMCP、ADRVMCP和SVCV P基因相对应的条带在感染后12h均可检测到，且这些条带均逐渐增加。在感染的36h时可以观察到GCRV VP7基因所对应的条带。结果表明GiCS细胞对这4种病毒均高度敏感，并在感染后表现出形态和基因表达方面的变化。由此可见，GiCS可广泛地对多种水生动物病毒具有易感性，并产生感染的表型。
本实验表明，GiCS可用于接种并繁殖水生动物病毒，特别是鲫疱疹病毒CaHV。此外，由于GiCS对多种水生动物病毒具有易感性，因此可用于水环境监测尤其是淡水鱼类养殖水域的水环境监测，检测水体中是否存在例如异育银鲫等鱼类易感的水生动物病毒，或检测水生动物是否携带病毒。
7、外源基因在GiCS中的表达测试
将编码绿色荧光蛋白的外源基因编码质粒pEGFP-N3转染到GiCS细胞中，观察其表达情况。具体步骤为：首先将GiCS细胞传代至12孔培养板中，置于25℃培养箱中培养，待GiCS细胞密度为80-90％时进行外源基因转染。取pEGFP-N3质粒1μg与2μl转染试剂(TransIT)分别加入100μlOpti-MEM无血清培养基中，混匀并在室温下放置15-20min。然后加入12孔培养板的GiCS细胞中，培养6-8小时后，弃去含有转染试剂的旧培养基，换上新鲜的L-15培养基。转染24h和48h后于荧光显微镜下观察并拍照。
如图6所示，转染后24h，可观察到部分GiCS细胞发出绿色荧光，转染后48h绿色荧光逐渐变强，且分布在整个细胞中。这说明，我们构建的GiCS细胞系可方便地进行遗传操作，在细胞中表达外源基因。因此，如果有需要，可将目的基因表达框转入GiCS中，进行目的蛋白的表达。
以上所述仅为本发明的较佳实施例，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、改进等，均应包含在本发明的保护范围之内。
Claims (7)
1.一种异育银鲫皮肤细胞系，其特征在于，2024年1月9日保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO:C202422。
2.权利要求1所述的异育银鲫皮肤细胞系在蛋白表达中的应用。
3.权利要求1所述的异育银鲫皮肤细胞系在分离或培养水生动物病毒中的应用。
4.权利要求1所述的异育银鲫皮肤细胞系在检测水生动物病毒中的应用。
5.根据权利要求3或4所述的应用，其特征在于，所述水生动物病毒包括但不限于：鲫疱疹病毒(Carassius auratus herpesviru)、沼泽绿牛蛙病毒(Rana gryliovirus)、大鲵蛙病毒(Andrias davidianus ranavirus)、草鱼呼肠孤病毒(Grass carp reovirus)和鲤鱼春季病毒血症病毒(Spring viremia of carp virus)。
6.一种检测水生动物病毒的方法，其特征在于，包括以下步骤：
S1：将待检测的样品添加至权利要求1所述的异育银鲫皮肤细胞系的培养环境中；
S2：观察S1培养的异育银鲫皮肤细胞系的特征变化。
7.根据权利要求6所述的方法，其特征在于，所述特征变化包括细胞形态变化和/或病毒基因表达变化。
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