CN112779293A - 一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法 - Google Patents
一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法,包括检测细胞的培养;将检测细胞置于底层胶为LMA,顶层胶为X‑gal、LMA混合液的培养介质中进行培养,观察培养介质中胶色变化,选择胶色不变化的检测细胞作为所述LacZ标记基于山羊痘病毒的宿主细胞;提取检测细胞RNA进行RT‑PCR,对得到的PCR扩增产物测序,并将测序DNA与LacZ基因进行同源性分析鉴定。本发明方法不光适用于乳仓鼠肾细胞、羊肾细胞、羔羊睾丸原代细胞,作为LacZ基因标记重组病毒的宿主细胞都可使用该方法进行筛选,通过简单的变色观察即可筛选出适合LacZ标记重组病毒的宿主细胞,为后续便于挑蓝色病毒蚀斑纯化,排除细胞本身产生蓝斑对LacZ基因标记重组病毒蓝斑造成干扰。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法。
背景技术
山羊痘活疫苗在国内推广应用多年,在防控山羊痘的过程中起到了关键性作用,制备山羊痘活疫苗的弱毒株(Gotapox virus,GPV)安全性和有效性得到充分证明。山羊痘病毒同时还是一个重要的活病毒载体。20世纪80年代,重组痘苗病毒表达主要免疫蛋白的成功建立,让以GPV为载体研制新型疫苗成为可能。由于重组GPV可实现了表达载体与免疫载体的有机融合,作为活载体疫苗优势十分明显:(1)作为痘病毒的一种,GPV具有良好的免疫原性,病毒在体内增殖过程中产生的外源蛋白可刺激机体产生免疫应答,不仅刺激机体产生体液免疫,还可诱导很强的细胞免疫反应,一次免疫接种就能获得长期的免疫效果。(2)GPV宿主范围明确,主要为山羊、绵羊和牛等反刍兽,对人类不具感染性和致病性,以GPV作为疫苗载体并不会引入新的病毒。(3)表达病毒保护性抗原的重组GPV不但可以有效预防病毒感染,而且不会干扰这些传染病的常规血清学监测。(4)山羊痘病毒基因组结构背景明确,具有高度的遗传稳定性,对外源基因插入耐受性强,可以允许大片段的基因丢失或删除,至少可容纳长达25kb外源片段而不丧失感染力。
迄今为止,以GPV作为载体表达外源蛋白可保持蛋白的天然构象和生物学活性,其有效性已被大量研究所证实。构建方法主要是将山羊痘疫苗弱毒株作为亲本毒,通过同源重组重组的方式,表达反刍和非反刍动物病毒的主要免疫蛋白基因编码的蛋白。由于同源重组具有保真性高、但发生概率低的特点,导致阳性病毒筛选纯化十分困难。LacZ基因是一种重要的Reporter基因,其产物是β-半乳糖苷酶,LacZ基因广泛用作分子标记研究。为了构建纯化LacZ基因标记的山羊痘病毒(Goatpox Virus,GPV),目前的策略是基于LacZ基因表达β-半乳糖苷酶,让β-半乳糖苷酶与底物5-溴-4-氯-3-吲哚-β-D-半乳糖苷(5-Bromo-4-chloro-3-indolylβ-D-galactopyranoside,X-gal)发生水解反应生成蓝色。通过挑选蓝色病毒蚀斑,作持续多轮细胞培养,最后实现对LacZ基因标记病毒的纯化。
由于β-半乳糖苷酶是细胞溶酶体中的一种水解酶,此酶广泛存在于微生物、植物及动物细胞中,许多生物自身可以合成β-半乳糖苷酶。因此,在构建LacZ基因标记重组GPV之前,分析低熔点琼脂糖胶固定化培养的宿主细胞在X-gal环境中的显色反应,选择不产生蓝色且适合培养病毒的宿主细胞系,对后续快速筛选、纯化重组病毒,加快山羊痘病毒新型疫苗的研发有重要意义。迄今为止,已有多种细胞被选择用于LacZ基因标记重组山羊痘病毒或者研发山羊痘病毒新型疫苗,但对于相关宿主细胞在底物X-gal环境中的培养显色情况鲜见报道。
发明内容
有鉴于此,本发明实际要解决的问题是提供一种筛选适合LacZ标记山羊痘病毒宿主细胞的方法来实现纯化LacZ基因标记的山羊痘病毒。
为了解决上述技术问题,本发明采用以下技术方案:
针对当前常用于培养山羊痘病毒的乳仓鼠肾细胞系(Baby Hamster SyrianKidney,BHK21)、羊肾细胞系(Sheep Kidney,SK)、羔羊睾丸原代细胞(Lamb Testis,LT),将它们置于含底物X-gal的低熔点琼脂糖胶(Low Melting Point Agarose,LMA)的DMEM培养基中固定化染色,持续观察各细胞在此环境中的显色情况。同时测定液体培养基培养细胞产生的β-半乳糖苷酶。筛选出不表达或低表达β-半乳糖苷酶、酶与底物X-gal反应不足以产生蓝色且适合GPV生长繁殖的宿主细胞,为后续便于挑蓝色病毒蚀斑纯化,排除细胞本身产生蓝斑对LacZ标记重组病毒蓝斑造成的干扰。该方法不光适用于上述乳仓鼠肾细胞、羊肾细胞、羔羊睾丸原代细胞,只要作为LacZ标记重组病毒的宿主细胞都可使用该方法进行筛选。
一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法,将检测细胞置于底层胶为LMA固定,顶层胶为X-gal、LMA混合液的培养介质中进行培养,观察所述培养介质中胶色变化,选择胶色不变化的检测细胞作为所述LacZ基因标记山羊痘病毒的宿主细胞。
进一步,所述方法包括如下步骤:
1)检测细胞的培养;
2)利用权利要求1所述方法筛选所述检测细胞;
3)提取所述检测细胞RNA进行RT-PCR扩增得到扩增DNA后测序;
4)将步骤3)所述扩增DNA测序后与LacZ基因相应段进行同源性分析。
进一步,所述LMA质量分数为1.0%;所述X-gal浓度为200μg/ml。
进一步,所述检测细胞为羊肾细胞、仓鼠肾成纤维细胞、羔羊睾丸细胞或其他可作为LacZ基因标记山羊痘病毒的细胞。
进一步,步骤1)前可进行细胞复苏,从液氮中取出所述检测细胞冻存管,快速置于37℃水中水浴,不停摇动直至管内全部融化。将融化液加入到含10%小牛血清DMEM培养液的细胞瓶中,置于37℃5%CO2培养箱中作复苏培养。待细胞长满瓶、弃细胞培养液,取1×PBS Buffer洗涤之后用胰酶消化,终止消化、轻轻吹打、洗脱贴壁细胞、低速离心收集,用10%小牛血清DMEM培养液均匀重悬细胞。
进一步,步骤1)所述检测细胞的培养条件为10%小牛血清DMEM培养液、37℃、5%CO2细胞培养箱。
进一步,步骤1)中将所述检测细胞培养至单层状态。
进一步,步骤3)所述RT-PCR扩增所使用的引物对为序列SEQ ID NO.1和SEQ IDNO.2所示。
进一步,步骤3)具体步骤为将PCR扩增得到的所述扩增DNA用1×TAE buffer、1.0%琼脂糖凝胶、120V条件下电泳,染色观察电泳胶,切胶目标条带,回收DNA连接至pMD18-T载体,转化感受态E.coli DH5a,挑单克隆菌培养检测,阳性克隆菌的质粒测序。
进一步,步骤4)所述同源性分析具体步骤为将测序后的所述扩增DNA在NCBI上作BLAST搜索,进行同源性比较。
进一步,步骤4)中所述扩增DNA与所述LacZ基因相应段相似性为100%。
本发明的有益效果在于:
1.本发明将检测细胞置于含底物X-gal的低熔点琼脂糖胶LMA的DMEM培养基中固定化染色,筛选出不表达或低表达β-半乳糖苷酶、酶与底物X-gal反应不足以产生蓝色且适合GPV生长繁殖的宿主细胞,为后续便于挑蓝色病毒蚀斑纯化,排除细胞本身产生蓝斑对LacZ标记重组病毒蓝斑造成的干扰。
2.本发明方法不光适用于上述乳仓鼠肾细胞、羊肾细胞、羔羊睾丸原代细胞,只要作为LacZ标记重组病毒的宿主细胞都可使用该方法进行筛选。
3.本发明方法通过简单的变色观察即可筛选出适合LacZ标记重组病毒的宿主细胞,避免了过多的检测步骤以及操作,为筛选宿主细胞节约了工作量。
附图说明
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
图1:不同处理BHK21细胞、SK细胞、LT细胞和空白孔在0h、24h、48h和72h的显色结果;
图2:不同处理BHK21细胞、SK细胞、LT细胞和空白孔在72h时显微镜观察的结果;
图3:单层细胞培养72h产β-半乳糖苷酶检测结果;
图4:RT-PCR产物的电泳图;M2000:DNA相对分子量标准;1:重组质粒LacZ扩增片段;2,3,4:BHK21、SK、LT细胞cDNA为模板扩增片段。
具体实施方式
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
实施例1
1.1质粒、细胞和病毒
含LacZ基因重组质粒(pMD19-LacZ)、BHK21细胞为重庆市兽用生物制品工程技术研究中心保存,LT参照《中华人民共和国兽用生物制品规程》(2005年)制作,SK细胞、山羊痘病毒疫苗弱毒株(GPV AV41株)为重庆澳龙生物制品有限公司惠赠,测定病毒TCID50为1×10-5/0.1ml。
1.2主要试剂和引物
RNA提取试剂盒、反转录试剂盒(PrimeScriptTM RT-PCR Kit)、LMA、X-gal、T4 DNA连接酶,PCR Master Mix(2×)、pMD18-T vecter购自TaKaRa公司,DMEM细胞培养液购自Gibico公司。β-半乳糖苷酶ONPG定量检测试剂盒购自北京百奥莱博科技有限公司。反转录试剂盒(PrimeScriptTM RT-PCR Kit)、质粒小提试剂盒、胶回收试剂盒购自OMEGA公司。LacZ基因检测引物(F:LZjcf:5-GTGACTACCTACGGGTAACA-3;LZjcr:5-ATTTGATCCAGCGATACAGC-3)参考LacZ基因(GenBank ID:V00296.1)设计并送上海生工公司合成。
1.3主要仪器
细胞培养箱为Thermo,倒置显微镜为Olympus,低速离心机为生工HICO21,酶标仪为SynergyMX,电泳仪为上海比朗,PCR仪为ABI Veriti TM,核酸蛋白浓度测定仪为Eppendorf BioPhotometer等。
实施例2
2.1细胞复苏、铺板与培养
从液氮中取出BHK-21、LT和SK细胞冻存管,快速置于37℃水中水浴,不停摇动直至管内全部融化。将融化液加入到含10%小牛血清DMEM培养液的细胞瓶中,置于37℃5%CO2培养箱中作复苏培养。待细胞长满瓶、弃细胞培养液,取1×PBS Buffer洗涤之后用胰酶消化数分钟,终止消化、轻轻吹打、洗脱贴壁细胞、低速离心收集,用10%小牛血清DMEM培养液均匀重悬细胞。取12孔细胞培养板,按BHK-21、SK、LT细胞和培养液(空白)4列布局,将重悬细胞加到各自的孔中。置细胞板于37℃5%CO2培养箱培养,直到细胞长至单层。
2.2固定化处理细胞及其培养
取一块长至单层状态的细胞板,倾去培养液用1×PBS洗涤3次,细胞板各行分别作不同固定化处理。第一行取0.1MOI 200μL GPV孵育2.0h后洗去残液,先加入200μL 1.0%LMA溶液作底层胶固化处理,再用1.0%LMA溶液200μL作顶层胶固定(简称:GPV-Cell+LMA);第二行取0.1MOI 200μL GPV孵育2.0h后洗去残液,先以200μL 1.0%LMA作底层胶固化处理,后用200μg/ml X-gal的1.0%LMA混合液200μL作顶层胶固定染色(简称:GPV-Cell+LMA+X-gal);第三行先以1.0%LMA底层胶固化单层细胞,后用200μg/ml X-gal的1.0%LMA混合液200μL作顶层胶固定染色(简称:Cell+LMA+X-gal)。固定化处理结束(即0h)后将细胞板于37℃5%CO2培养箱中倒置培养,持续观察胶变色及细胞生长情况。
2.3液体培养细胞及β-半乳糖苷酶检测
取另一块长至单层状态的细胞板,以2%小牛血清DMEM溶液继续培养,72h后倾去培养基,各孔中细胞用1×PBS洗涤3次;按照β-半乳糖苷酶ONPG定量检测试剂盒说明书检测BHK21、SK、LT细胞的产酶。同时,再取一块长至单层状态的细胞板,倾去培养液后各孔中加入200μg/ml X-gal的2%小牛血清DMEM液继续培养,持续观察各细胞孔培养液的颜色及其变化。
2.4RT-PCR扩增、克隆和测序
采用RNA提取试剂盒抽提细胞的RNA,用反转录试剂盒制备cDNA:第一步准备变性及退火反应液(10μl):dNTP mix(10mM)1.0μl,Random 6mers 1.0μl,RNA模板2.0μl,RNasefree dH2O 6.0μl;65℃反应5min。第二步进行反转录(20μl):变性退火反应液10.0μl,5×PrimeScript Buffer 4.0μl,RNase Inhibitor(40U/μL)0.5μl,PrimeScript RTase(for2Step)0.5μl,RNase Free dH2O 5.0μL;30℃10min,42℃30min,95℃5min。获得cDNA置于-20℃保存备用。以cDNA为模板、LacZ基因检测引物作PCR,反应体系(25μl):2×Taq PCRMaster Mixes 12.50μL,模板4.0μL,Primer-F(10μM)1.0μL,Primer-R(10μM)1.0μL,ddH2O6.50μL。反应条件:94℃预变性5min;94℃1min,55℃50sec,72℃50sec,共35个循环;72℃延伸10min。同时,将重组质粒pMD19-LacZ的PCR作阳性对照。
将PCR产物用1×TAE buffer、1.0%琼脂糖凝胶、120V条件下电泳。染色观察电泳胶,切胶目标条带,回收DNA连接至pMD18-T载体,转化感受态E.coli DH5a涂LB平板板,挑单克隆菌培养检测,对阳性克隆菌质粒测序鉴定。
2.5同源性比较
将测序DNA在NCBI上作BLAST搜索,进行DNA序列的同源性分析。
实施例3
结果分析
3.1.固定化细胞培养显色结果
对固定化处理的细胞板,在0h、24h、48h和72h等时间点摄像,获得不同细胞孔的显色情况(图1)。
1)第一行中的,随着培养时间的延长,一直表现浅黄色,未出现蓝色。说明在未有底物X-gal存在的培养环境中,无论细胞/病毒是否表达β-半乳糖苷酶,均无法完成催化反应,进而不会出现蓝色。
2)第二行中的,其中BHK21细胞、LT细胞培养孔,随着时间的延长,蓝色从无到有、并逐渐加深,且细胞显色不受病毒感染影响;表明这两种细胞均表达了β-半乳糖苷酶,酶量足够与底物X-gal发生催化反应生产蓝色。与此相反,SK细胞、病毒+X-gal-LMA孔,一直呈浅黄色,均未见蓝色。
3)第三行中的,尽管未感作GPV病毒,但BHK21细胞、LT细胞孔随时间延长,仍然出现了蓝色;SK细胞、X-gal-LMA空白孔,一直未出现蓝色。
2.2.镜检固定化细胞
经过LMA固定化处理、培养72h的各细胞孔,在显微镜下观察结果(图2)。由图可知:在无底物X-gal的环境中,BHK21细胞、SK细胞、LT细胞以及空白孔均无蓝色细胞斑点出现。但是在含X-gal的固定化培养环境中,BHK21细胞、LT细胞孔中均出现了一些明显可见的蓝色细胞斑点(如箭头所示);而SK细胞和空白无可见的蓝斑点。
2.3.细胞产β-半乳糖苷酶检测
对β-半乳糖苷酶的定量检测显示,三种细胞均能够产生β-半乳糖苷酶,但各细胞产酶能力有显著差异,表现为BHK21细胞>LT细胞>SK细胞(图3)。产酶差异顺序与固定化细胞在X-gal环境中培养显色蓝色、且蓝色由强变弱乃至不可见的现象相一致。
但是,对于已经长至单层的细胞,用含X-gal的液体培养基继续培养,随着时间延长,结果发现BHK-21、SK、LT细胞孔溶液颜色一直没有变化,且均未出现蓝色。表明细胞产生的β-半乳糖苷酶被培养基稀释后,与底物X-gal反应产生蓝色不足以被观察到。
2.4DNA序列的鉴定分析
RT-PCR产物经琼脂糖凝胶电泳检测,结果显示在DNA分子量标准大约700bp处出现预期的、单一的目标DNA条带;该DNA带与LacZ基因阳性质粒PCR产物DNA条带分子量大小相一致(图4,箭头所示)。对目标条带测序鉴定获得678bp的DNA序列;相似性分析发现3种细胞cDNA序列与LacZ基因(GenBank ID:V00296.1)相应段的核苷酸相似性值达到100%。
许多生物自身可合成β-半乳糖苷酶,观察病毒宿主细胞在底物X-gal环境中培养的显色情况,对后续研制LacZ基因标记病毒十分关键。本研究结果显示:
(1)原代LT细胞在固定化培养初期,胶孔未显示蓝色,随着时间延长,固定胶孔蓝色越来越明显。分析原因可能是大多数原代细胞被认为仅有有限代次的分裂能力,初期有正常分裂能力的细胞不表达β-半乳糖苷酶。但随着细胞逐渐老化,在不能分裂后就进入衰老状态。此时细胞仍然是存活的,但细胞的基因和蛋白的表达谱发生了很大改变;表达了高酶活性的β-半乳糖苷酶,该酶是衰老细胞的检测标志物之一。由于此酶与X-gal发生水解反应生成蓝色,进而将固定胶染成蓝色。
(2)传代BHK21细胞在固定化培养过程中,胶很快就被染成蓝色,说明该细胞能够较快地产生β-半乳糖苷酶,产酶量足够与底物X-gal发生水解反应生成蓝色并将胶染蓝。分析原因可能是受到培养条件改变或其他相关因素的影响,诱导了永生化细胞衰老,进而诱导表达衰老相关β-半乳糖苷酶的活性。只有SK细胞在含X-gal的环境里,在固定化培养过程中一直未出现蓝色,说明羊肾细胞适应了试验培养条件,表达的β-半乳糖苷酶极低,与底物X-gal发生水解反应蓝色不被观察到。
此外,RT-PCR产物鉴定结果显示三种细胞均产生了LacZ基因的cDNA片段,表明BHK21、SK和LT细胞都生成了β-半乳糖苷酶基因的转录本。定量检测试验细胞的产酶情况,进一步证实三种细胞均产生了β-半乳糖苷酶,只是产酶差异明显,尤以SK细胞产酶量最低,导致酶与X-gal反应变蓝程度不同。本结果与大多生物能自身合成β-半乳糖苷酶的文献报道相符。
分析羊肾细胞、仓鼠肾成纤维细胞、羔羊睾丸细胞在X-gal环境中显色反应,明确了羊肾细胞是一种较为适宜用于研制LacZ基因标记GPV的宿主细胞。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
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<110> 重庆市畜牧科学院
<120> 一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法
<160> 2
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<211> 20
<212> DNA
<213> Artificial sequence
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Claims (10)
1.一种筛选LacZ基因标记山羊痘病毒的宿主细胞的方法,其特征在于,将检测细胞置于底层胶为LMA,顶层胶为X-gal、LMA混合液的培养介质中进行培养,观察所述培养介质中胶色变化,选择胶色不变化的检测细胞作为所述LacZ基因标记山羊痘病毒的宿主细胞。
2.根据权利要求1所述的方法,其特征在于,所述方法包括如下步骤:
1)检测细胞的培养;
2)利用权利要求1所述方法筛选所述检测细胞;
3)提取所述检测细胞RNA进行RT-PCR扩增,得到扩增DNA后测序;
4)将步骤3)所述扩增DNA测序后与LacZ基因进行同源性分析。
3.根据权利要求1所述的方法,其特征在于,所述LMA质量分数为1.0%;所述X-gal浓度为200μg/ml。
4.根据权利要求1或2所述的方法,其特征在于,所述检测细胞为羊肾细胞、仓鼠肾成纤维细胞、羔羊睾丸细胞或其他可作为LacZ基因标记山羊痘病毒的细胞。
5.根据权利要求1或2所述的方法,其特征在于,所述检测细胞的培养条件为10%小牛血清DMEM培养液、37℃、5%CO2。
6.根据权利要求2所述的方法,其特征在于,步骤1)中将所述检测细胞培养至单层状态。
7.根据权利要求2所述的方法,其特征在于,步骤3)所述RT-PCR扩增所使用的引物对为序列SEQ ID NO.1和SEQ ID NO.2所示。
8.根据权利要求2所述的方法,其特征在于,步骤3)具体步骤为将PCR扩增得到的所述扩增DNA用1×TAE buffer、1.0%琼脂糖凝胶、120V条件下电泳,染色观察电泳胶,切胶目标条带,回收DNA连接至pMD18-T载体,转化感受态E.coli DH5a,挑单克隆菌培养检测,对阳性克隆菌的质粒测序。
9.根据权利要求2所述的方法,其特征在于,步骤4)所述同源性分析具体步骤为将测序后的所述扩增DNA在NCBI上作BLAST搜索,进行同源性比较。
10.根据权利要求2所述的方法,其特征在于,步骤4)中所述扩增DNA与所述LacZ基因相应段相似性为100%。
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