CN116445486B - Klhl34敲除细胞系的构建及作为小rna病毒科病毒生产细胞系的应用 - Google Patents
Klhl34敲除细胞系的构建及作为小rna病毒科病毒生产细胞系的应用 Download PDFInfo
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- CN116445486B CN116445486B CN202310322418.3A CN202310322418A CN116445486B CN 116445486 B CN116445486 B CN 116445486B CN 202310322418 A CN202310322418 A CN 202310322418A CN 116445486 B CN116445486 B CN 116445486B
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Abstract
本发明属于基因工程领域，具体涉及KLHL34敲除细胞系的构建及作为小RNA病毒科病毒疫苗生产细胞系的应用。本发明首先发现，抑制宿主细胞中KLHL34基因表达能促进小RNA病毒科病毒FMDV的复制；其次，本发明提供了一种特异性靶向KLHL34的sgRNA，所述sgRNA能够特异性靶向KLHL34基因，结合CRISPR‑Cas9技术实现了KLHL34基因的敲除，获得的单克隆细胞系能够显著促进小RNA病毒科病毒FMDV的复制，提高FMDV疫苗的生产量和抗原产量，可作为小核糖核酸病毒科病毒疫苗尤其是FMDV疫苗的生产细胞系，具有广阔的应用前景。
Description
技术领域
本发明属于基因工程领域，具体涉及一种KLHL34敲除细胞系的构建及作为小RNA病毒科病毒生产细胞系的应用。
背景技术
小核糖核酸(RNA)病毒科(Picomaviridae)是由RNA病毒中最小的类群组成的一科，主要包括肠道病毒属、鼻病毒属、心病毒属以及口疮病毒属等。口蹄疫(foot-and-mouthdis ease，FMD)是由口蹄疫病毒(foot-and-mouth disease virus，FMDV)引起的急性、热性、高度接触性传染的动物疫病，严重危害猪、牛、羊等偶蹄类动物。FMDV是单股正链RNA病毒，属于小RNA病毒科口蹄疫病毒属，有O、A、C、SAT1、SAT2、SAT3和Asia1 7个血清型，且各型之间没有交叉免疫保护，即使同一血清型不同毒株抗原之间交叉免疫保护也存在差异。因其基因组为单股正链RNA，具有很高的变异性，易产生新毒株，导致疫苗不能完全覆盖田间流行毒株，这就给口蹄疫的免疫防控带来巨大挑战。开发高效疫苗是当前防控该病最有效的举措，而选取病毒高效繁殖的细胞系是制备高效疫苗的前提，急需开展相关工作。
CRISPR-Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御系统，可用来对抗入侵的病毒及外源DNA。基于原核生物的适应性免疫防御系统研发出了CRISPR-Cas9基因编辑技术，该技术就是通过人工设计的sgRNA来识别目的基因组序列，并引导Cas9蛋白酶进行有效切割DNA双链，形成双链断裂。通常情况下，细胞会采用非同源末端连接(Non-homologous end joining,NHEJ)或同源重组修复(Homology-directedrepair,HDR)途径来对造成的断裂进行修复，实现基因的敲除与修饰。前者在修复过程中通常会发生碱基插入或缺失的错配现象，造成移码突变，使靶标基因失去功能，从而实现特定基因的敲除。
KLHL34是Kelch样(KLHL)基因家族的一员，它编码一组具有BTB/POZ结构域、BA CK结构域和5-6个Kelch基序的蛋白质。Perez-Torrado等人论述了BTB/POZ结构域促进与其他蛋白质的结合，其功能涉及多种细胞内分子机制，包括细胞骨架组织的控制、离子通道的选择等，某些KLHL基因的突变会导致孟德尔疾病或人类癌症。
本发明意外发现，抑制宿主细胞中KLHL34基因的表达能促进小RNA病毒科病毒的复制，尤其是FMDV的复制；其次，本发明提供了一种特异性靶向KLHL34基因的sgRNA，所述的sgRNA能够特异性靶向KLHL34基因，结合CRISPR-Cas9技术实现了KLHL34基因的敲除，获得的单克隆细胞系能够显著促进小RNA病毒的复制，尤其是FMDV的复制，提高小RNA病毒疫苗的生产量和抗原产量，可作为小RNA病毒疫苗的生产细胞系，具有广阔的应用前景；而且所述的KLHL34蛋白能够显著抑制口蹄疫病毒的复制，可用于预防或治疗口蹄疫病毒感染。
发明内容
针对上述问题，本发明首先发现，抑制宿主细胞中KLHL34基因的表达能促FMDV的复制；其次，本发明提供了一种特异性靶向KLHL34基因的sgRNA，所述的sgRNA能够特异性靶向KLHL34基因，结合CRISPR-Cas9技术实现了KLHL34基因的敲除，获得的单克隆细胞系能够显著促进FMDV的复制，提高FMDV疫苗的生产量和抗原产量，可作为FMDV疫苗的生产细胞系，具有广阔的应用前景。具体包括以下内容：
第一方面，本发明提供了一种KLHL34蛋白或表达KLHL34蛋白的重组载体/质粒在制备预防或治疗小RNA病毒科病毒感染药物中的应用。
优选地，所述小RNA病毒科病毒为口蹄疫病毒。
第二方面，本发明提供了一种KLHL34基因编码蛋白功能丧失细胞系作为小RNA病毒科病毒或病毒疫苗生产细胞系的应用。
优选地，所述小RNA病毒科病毒为口蹄疫病毒。
第三方面，本发明提供了一种抑制或者沉默KLHL34基因表达的试剂在制备小RNA病毒科病毒或病毒疫苗生产细胞系中的应用。
优选地，所述小RNA病毒科病毒为口蹄疫病毒。
优选地，所述试剂包括靶向敲除KLHL34基因的sgRNA，或靶向敲除KLHL34基因的sgRNA与Cas9蛋白的mRNA序列。
优选地，所述sgRNA包括KLHL34-sgRNA-1和/或KLHL34-sgRNA-2；
所述KLHL34-sgRNA-1的靶向序列为：TTGACGTGTACAACCACCGC；
所述KLHL34-sgRNA-2的靶向序列为：GGCGTCGTGTACATCTCCGG。
优选地，所述KLHL34-sgRNA-1由KLHL34-sgRNA-1-F和KLHL34-sgRNA-1-R退火形成的双链；所述KLHL34-sgRNA-2由KLHL34-sgRNA-2-F和KLHL34-sgRNA-2-R退火形成的双链；
KLHL34-sgRNA-1-F：5’-CACCGTTGACGTGTACAACCACCGC-3’；
KLHL34-sgRNA-1-R：5’-AAACGCGGTGGTTGTACACGTCAAC-3’；
KLHL34-sgRNA-2-F：5’-CACCGGGCGTCGTGTACATCTCCGG-3’；
KLHL34-sgRNA-2-R：5’-AAACCCGGAGATGTACACGACGCCC-3’。
第四方面，本发明提供了一种特异性靶向KLHL34基因的sgRNA，所述sgRNA包括KLHL34-sgRNA-1和/或KLHL34-sgRNA-2；
所述KLHL34-sgRNA-1的靶向序列为：TTGACGTGTACAACCACCGC；
所述KLHL34-sgRNA-2的靶向序列为：GGCGTCGTGTACATCTCCGG。
优选地，所述KLHL34-sgRNA-1由KLHL34-sgRNA-1-F和KLHL34-sgRNA-1-R退火形成的双链；所述KLHL34-sgRNA-2由KLHL34-sgRNA-2-F和KLHL34-sgRNA-2-R退火形成的双链；
KLHL34-sgRNA-1-F：5’-CACCGTTGACGTGTACAACCACCGC-3’；
KLHL34-sgRNA-1-R：5’-AAACGCGGTGGTTGTACACGTCAAC-3’；
KLHL34-sgRNA-2-F：5’-CACCGGGCGTCGTGTACATCTCCGG-3’；
KLHL34-sgRNA-2-R：5’-AAACCCGGAGATGTACACGACGCCC-3’。
第五方面，本发明提供了上述第四方面所述的sgRNA在敲除KLHL34基因或在制备KL HL34基因敲除细胞系中的应用。
第六方面，本发明提供了一种用于敲除KLHL34基因的试剂或试剂盒，所述试剂或试剂盒包括上述第四方面所述的sgRNA，或靶向敲除KLHL34基因的打靶载体；所述靶向敲除KLHL34基因的打靶载体包括上述第四方面所述的sgRNA和Cas9蛋白基因的编码序列。
第七方面，本发明提供了一种KLHL34基因编码蛋白功能丧失细胞系的构建方法，所述方法为：通过基因打靶技术使宿主细胞中KLHL34基因编码蛋白功能丧失。
优选地，所述方法为CRISPR-Cas9技术。
优选地，所述CRISPR/Cas9系统使用上述第四方面所述的sgRNA。
第八方面，本发明提供了一种KLHL34基因编码蛋白功能丧失PK-15细胞系，所述PK-15细胞系的构建方法包括以下步骤：
(1)制备上述第四方面所述的特异性靶向KLHL34基因的sgRNA；
(2)将步骤(1)制备的sgRNA退火并连接至PX459质粒，得到同时表达Cas9蛋白基因和打靶sgRNA序列的重组质粒；
(3)将步骤(2)制备的重组质粒转染PK-15细胞，用嘌呤霉素(puromycin)抗生素筛选以及细胞有限稀释法获得KLHL34基因功能缺失细胞系。
本发明的有益效果是：①本发明首先发现，抑制宿主细胞中KLHL34基因的表达能促进小RNA病毒科病毒FMDV的复制；②所述的KLHL34蛋白能够抑制小RNA病毒科病毒FM DV的复制，可用于预防或治疗小RNA病毒科病毒FMDV感染；③本发明提供了一种靶向KLHL34基因的sgRNA，所述sgRNA能够特异性靶向KLHL34基因，结合CRISPR-Cas9技术可实现宿主细胞中KLHL34基因的敲除，打靶准确、敲除效率高；④本发明提供了一种通过CRISPR-Cas9技术将所述sgRNA转染于宿主细胞，构建KLHL34基因编码蛋白功能丧失细胞系的方法；⑤依据本发明所述方法获得的单克隆细胞系能够显著促进小RNA病毒科病毒FM DV的复制，提高小RNA病毒科病毒FMDV疫苗的生产量和抗原产量，可作为小核糖核酸病毒科病毒或病毒疫苗的生产细胞系，具有广阔的应用前景。
附图说明
图1靶向KLHL34基因组区域的sgRNA示意图；
图2PK-15-KLHL34-KO-1细胞测序结果峰图；
图3PK-15-KLHL34-KO-2细胞测序结果峰图；
图4Western blot检测敲除细胞系中KLHL34的表达；
图5KLHL34基因功能缺失细胞系KLHL34-KOs细胞活力检测结果；
图6间接免疫荧光实验检测KLHL34-KOs细胞对FMDV复制的影响。
图7Western blot检测FMDV感染KLHL34-WT和KLHL34-KOs细胞不同时间的病毒蛋白水平差异；
图8相对定量检测FMDV感染KLHL34-WT和KLHL34-Kos细胞不同时间的mRNA水平差异；
图9FMDV在KLHL34-KOs细胞中增殖后的病毒滴度检测结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明的各实施例进行详细的阐述。然而，本领域的普通技术人员可以理解，在本发明各实施例中，为了使读者更好地理解本申请而提出了许多技术细节。但是，即使没有这些技术细节和基于以下各实施例的种种变化和修改，也可以实现本申请所要求保护的技术方案。
定义
术语“蛋白功能丧失”是指通过对编码蛋白的基因进行敲除、突变或在编码蛋白的基因片段中插入部分基因，使得基因编码蛋白发生移码突变，导致基因编码蛋白的功能丧失。本发明通过对宿主细胞中的KLHL34基因靶向敲除，导致KLHL34基因编码蛋白的功能丧失，进而构建了KLHL34基因编码蛋白功能丧失的细胞系，并用于FMDV的增殖。但是本发明并不局限于KLHL34基因敲除，也可通过其他技术手段使KLHL34基因编码蛋白的功能丧失，并用于构建KLHL34基因编码蛋白功能丧失的细胞系。
术语“基因打靶”是指利用DNA定点同源重组进行细胞或生物个体遗传信息定向改变的定向转基因技术，主要包括基因敲除、基因灭活、基因敲入、点突变、缺失突变以及染色体组大片段删除等。其中“基因敲除”是指通过一定的途径使特定靶基因失活或缺失。本发明通过基因敲除技术，使宿主细胞中的KLHL34因敲除，获得的KLHL34基因编码蛋白功能丧失的单克隆细胞系能促进FMDV的增殖；本发明也可以通过对宿主细胞中的KLHL34基因突变，或插入基因片段，导致KLHL34基因编码蛋白发生移码突变，成功构建出KLHL34基因编码蛋白功能丧失的单克隆细胞系。
术语“sgRNA”为向导RNA，是指在RNA编辑的过程中引导尿苷残基插入或缺失到动质体(kinetoplastid)中，属于一种小型非编码RNA。
本发明通过人工合成了靶向KLHL34基因的sgRNA；本发明在直接靶向剪接KLHL34基因的基础上，利用CRISPR/Cas9联合特异性敲除KLHL34基因的方法，以PK-15细胞为例，进行了KLHL34基因(KLHL34氨基酸序列如SEQ ID NO.1所示；CDS序列如SEQ ID NO.2所示)敲除，为FMDV疫苗的生产提效提供了一种策略。本发明虽然仅对PK-15细胞中KLHL34基因进行敲除，获得了一种基因敲除宿主细胞，但是本发明所述的方法可以推论并扩展到对其他动物细胞中KLHL34基因敲除，构建具有提升FMDV抗原产量额的基因敲除细胞系。
CRISPR/Cas9系统对基因的定向识别和剪切是由sgRNA和Cas9实现的，sgRNA决定了Cas9的靶向性，也决定了Cas9的切割活性。本发明旨在应用CRISPR/Cas9基因编辑技术，通过体内外筛选针对KLHL34基因的sgRNA序列，实现KLHL34基因的准确、高效地敲除，获得一种能够促进FMDV抗原产量的KLHL34基因敲除单克隆细胞系，从而为FMDV疫苗的生产提供新的策略。
利用CRISPR/Cas9基因编辑技术，通过靶向KLHL34基因的sgRNA引导Cas9蛋白结合到KLHL34基因特定序列位置对DNA双链进行切割，造成基因双链断裂，在细胞自身修复机制作用下，产生随机突变，核苷酸的缺失或插入等突变会造成基因的读码框发生改变，最终达到基因编码蛋白功能丧失的目的，获得基因编码蛋白功能丧失细胞系。
下述实施例中的实验方法，如无特殊说明，均为常规方法；下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂公司购买获得。
以下实施例中所涉及的质粒来源：购买于淼灵质粒平台。
O/BY/CHA/2010来源于中国农业科学院兰州兽医研究所国家口蹄疫参考实验室；PK-15细胞由本实验室提供，细胞培养使用含有10％胎牛血清和1％双抗的DMEM培养基进行培养；PX459载体由中国农业大学吴森教授惠赠。
大肠杆菌Trans5α感受态、T4 DNA连接酶、TB GreenⅡ购自宝生物工程(大连)有限公司；限制性核酸内切酶BbsI购自NEB公司；质粒提取试剂盒、Trizol试剂和蛋白预染Marker购自Invitrogen公司；胶回收试剂盒、微量DNA提取试剂盒购自OMEGA公司；ME M细胞培养基及0.25％ EDTA胰酶购自Gibco公司；胎牛血清(FBS)购自Biological Ind ustries(BI)公司；Polyplus jetPRIME转染试剂购自Polyplus Transfection公司；Western-抗体稀释液购自碧云天生物技术有限公司；10×PCR Buffer购自康为世纪生物科技有限公司；兔抗KLHL34多克隆抗体、鼠抗β-Actin多克隆抗体购自proteintech公司；辣根过氧化物酶(HRP)标记的山羊抗兔IgG抗体、HRP标记的山羊抗鼠IgG抗体、HRP标记的山羊抗豚鼠IgG抗体、FITC标记的山羊抗豚鼠IgG抗体购自Sigma-Aldrich(USA)公司；豚鼠抗O型FMDV多克隆抗体由本实验室制备保存。
以下实施例所述的KLHL34基因CDS序列如SEQ ID NO.2所示，氨基酸序列如SEQ IDNO.1所示。
实施例1KLHL34基因敲除PK-15细胞系的构建
1.KLHL34-sgRNA重组质粒构建
sgRNA靶点的设计：在NCBI中查询猪源KLHL34基因序列，基于张峰实验室网站http://crispr.mit.edu/设计sgRAN，在KLHL34的第1个外显子区域的1627位置和第2个外显子区域的2031位置分别设计两条sgRNA，KLHL34-sgRNA-1和KLHL34-sgRNA-2：
所述KLHL34-sgRNA-1的靶向序列为：TTGACGTGTACAACCACCGC(SEQ ID NO.3所示)；所述KLHL34-sgRNA-1由KLHL34-sgRNA-1-F：5’-CACCGTTGACGTGTACAACCA CCGC-3’(SEQ IDNO.5所示)和KLHL34-sgRNA-1-R：5’-AAACGCGGTGGTTGTACACGT CAAC-3’(SEQ ID NO.6所示)退火形成的双链；
所述KLHL34-sgRNA-2的靶向序列为：GGCGTCGTGTACATCTCCGG(SEQ ID NO.4所示)；所述KLHL34-sgRNA-2由KLHL34-sgRNA-2-F：5’-CACCGGGCGTCGTGTACATCTC CGG-3’(SEQ IDNO.7所示)和KLHL34-sgRNA-2-R：5’-AAACCCGGAGATGTACACGACG CCC-3’(SEQ ID NO.8所示)退火形成的双链。
sgRNA的退火杂交：将sgRNA上、下游序列合成产物稀释至100μmol/L后，取上、下游引物各22.5μL，并加入10×PCR buffer 5μL，共50μL体系按照99℃退火处理5min，使上下游引物形成双链；
PX459载体的酶切：利用限制性内切酶BbsI酶切PX459载体，按照10×buffer 2μL、PX459载体5μL，BbsI 1μL、ddH2O 12μL，共20μL反应体系，37℃酶切4h，胶回收酶切产物；
酶切载体与sgRNA的连接：配制10μL连接反应体系，其中，10×T4连接酶buffer 1μL、PX459酶切片段2μL、退火后的sgRNA 6μL，T4连接酶1μL，置16℃连接2h；
PX459-sgRNA连接产物转化：将连接产物转化Trans5α感受态细胞，涂布于含Amp的LB固体培养皿上，37℃过夜培养，挑取菌落振荡培养后，提取质粒，测序。使用MegAlign软件对测序结果与目的基因序列进行对比，成功构建具有靶向KLHL34基因不同外显子的sg RNA重组质粒，位置和序列信息如图1；重组质粒分别命名为：PX459-KLHL34-sgRNA-1、PX459-KLHL34-sgRNA-2。
2.细胞的转染
复苏PK-15细胞传代1-2次，当细胞性状稳定且状态良好时，用0.25％ EDTA胰酶消化细胞，按2×105个细胞/孔的密度接种至6孔板中，置含5％ CO2的37℃细胞培养箱中培养，待细胞密度生长至60％-70％时进行转染。将2μg PX459-KLHL34-sgRNA重组质粒加入100μLbuffer中轻柔混匀，再加入4μL jetPRIME转染试剂，混匀后室温静置10min，均匀滴加入培养的6孔板细胞中，将细胞培养板置于37℃、5％ CO2培养箱中培养24h。
3.嘌呤霉素加压筛选阳性细胞
为了剔除大量sgRNA阴性细胞，用0.25％ EDTA胰酶消化转染PX459-KLHL34-sgRNA重组质粒24h后的PK-15细胞，把细胞按照1：3传代比例接种到新的细胞6孔板中，同时每孔细胞中加入3μg/mL的嘌呤霉素，将细胞重新置于37℃、5％ CO2培养箱中培养加压筛选3天，更换新鲜培养基使阳性细胞正常生长。
4.亚克隆筛选阳性单克隆细胞株
挑选已通过嘌呤霉素加压筛选且状态良好的细胞孔，用0.25％ EDTA胰酶消化后，用1mL MEM培养基吹打均匀进行细胞计数，然后用MEM完全培养基将细胞稀释至10个细胞/mL，将稀释后的细胞悬液加入96孔板中，0.1mL/孔，即每孔中有1个细胞。将细胞培养板置含5％ CO2的37℃培养箱中培养5-7d后观察，剔除双克隆、多克隆细胞孔，标记单克隆且细胞状态良好的细胞孔，用0.25％ EDTA胰酶消化后，加入400μL MEM完全培养基培养基吹打混匀，传代至48孔板中继续培养，待细胞长满后依次传代至12孔板、6孔板中进行扩大培养。
5.单克隆细胞株的测序鉴定
取野生型细胞与各个待鉴定单克隆细胞株，按照细胞微量DNA提取试剂盒说明书分别提取DNA，用KLHL34鉴定引物(KLHL34-F：5’-TCAAACCCGCATCGTGTTGGTC-3’(SEQ IDNO.9所示)，KLHL34-R：5’-ATAGCAGAAGCGGTCATAGGGTA-3’(SEQ ID NO.10所示))扩增含有sgRNA靶向位点的片段，经1％琼脂糖凝胶电泳后，胶回收扩增产物，测序。将待鉴定单克隆细胞株与野生型细胞株扩增产物的测序结果进行序列分析，将显示因碱基插入或碱基缺失而导致基因序列产生移码突变的对应细胞株以及序列比对没有发生碱基改变的细胞株，分别标记为PK-15-KLHL34-KO和PK-15-KLHL34-WT。
对测序结果与目的基因序列进行比对分析，结果如图2和3所示，序列比对结果显示KL HL34敲除的PK-15细胞在sgRNA1和sgRNA2靶向目的基因的外显子处分别发生了1个碱基缺失和38个碱基缺失，并且导致了目的基因序列产生移码突变。
6.Western blot分析
分别收集经测序鉴定的细胞样品，加入适量1×SDS loading buffer充分搅拌裂解后，吸取至相应标记好的1.5mL离心管中，放入金属浴100℃变性12min，4℃，12000rpm离心2min，取上清10μL上样进行SDS-PAGE。蛋白样品经SDS-PAGE电泳后通过湿转法，在冰浴中100V恒压转膜1.5h，印记到NC膜上。5％脱脂奶粉封闭1h后，分别加入经抗体稀释液1：1000倍稀释的兔抗KLHL34抗体和鼠抗β-Actin抗体作为一抗，4℃孵育过夜；用TBST溶液重复清洗NC膜3次，置于快速摇床震荡，每次5min；再分别加入经TBST溶液1:1000倍稀释的HRP标记的山羊抗兔IgG和山羊抗鼠IgG抗体作为二抗，室温孵育2h；再次采用同样的方法清洗NC膜3次，最后用ECL化学发光显色液检测，图片经Image Lab 4.1(BI O-RAD)扫描成像。
结果如图4所示，与PK-15-KLHL34-WT相比，PK-15-KLHL34-KO1和PK-15-KLHL34-KO2细胞系中均检测不到KLHL34的蛋白表达。上述结果表明，KLHL34基因敲除PK-15细胞系构建成功，分别命名为PK-15-KLHL34-KO1、PK-15-KLHL34-KO2。
实施例2KLHL34基因敲除PK-15细胞系的活力检测
分别将野生型和KLHL34基因敲除细胞消化，调整细胞悬液密度为1-2×105个细胞/mL，接种于96孔细胞培养板中，100μL/孔，置37℃、5％ CO2细胞培养箱中培养5h；向每孔加入10μL CCK-8溶液，继续将培养板放入培养箱中孵育3h，用酶标仪测定其在450nm的吸光度，分析数据。
测定结果如图5所示，PK-15-KLHL34-KO与PK-15-KLHL34-WT的细胞活性无统计差异，表明在PK-15细胞中敲除KLHL34后没有显著影响细胞的正常生长特性。因此，构建的该敲除细胞系为猪源细胞KLHL34基因缺失的研究提供了工具。
实施例3FMDV在KLHL34-KO细胞系中的复制
1.细胞接毒感染
取成功构建的KLHL34敲除的PK-15细胞系(PK-15-KLHL34-KO1、PK-15-KLHL34-KO2)及对照细胞系(PK-15-KLHL34-WT)在35mm的细胞培养皿中培养到大约80％的汇合度时，弃掉培养基，加入1mL PBS清洗细胞2次后，按MOI＝1接种口蹄疫病毒O/BY/CHA/2010，在培养箱中孵育1h；吸弃病毒液，PBS清洗细胞2次后，加入2mL MEM培养基。置细胞培养箱中培养，然后分别在感染后不同时间收集样品，用间接免疫荧光实验、Westernblot、RT-qPCR、TCID50不同方法综合评价口蹄疫病毒的复制情况。
2.间接免疫荧光实验
将PK-15-KLHL34-KO和PK-15-KLHL34-WT细胞接种至共聚焦培养皿中，待细胞融合度约为70％时，按照上述方法进行病毒感染，37℃、5％ CO2细胞培养箱中培养9h，弃去上清，用PBS清洗细胞3次；加入4％多聚甲醛，4℃固定过夜；用PBS清洗细胞3次，5min/次；加入0.3％的Triton X-100室温通透30min；用PBS清洗细胞3次，加入5％BSA 4℃封闭4h；加入用PBS溶液1:500倍稀释的FMDV豚鼠抗体作为一抗，4℃孵育过夜；PBST清洗细胞3次，10min/次；加入用PBS溶液1:500倍稀释的FITC标记的山羊抗豚鼠IgG作为二抗，4℃避光孵育过夜；PBST清洗细胞3次，10min/次；再加入0.5mL DAPI染核15min后，用激光共聚焦显微镜观察荧光并分析。
结果如图6所示，PK-15-KLHL34-KO细胞中出现大量绿色荧光，且明显多于PK-15-KL HL34-WT细胞，说明在PK-15-KLHL34-KO细胞中FMDV复制量较野生型细胞高。
3.Western blot分析病毒蛋白的表达
培养在35mm细胞培养皿中的PK-15-KLHL34-KO和PK-15-KLHL34-WT细胞，按照上述方法进行病毒感染，于感染后7h、12h收集细胞样品，经样品处后进行SDS-PAGE，以兔抗KLHL34抗体、FMDV豚鼠抗体和鼠抗β-Actin抗体作为一抗，HRP标记的山羊抗兔IgG、HRP标记的山羊抗豚鼠IgG、HRP标记的山羊抗鼠IgG抗体作为二抗，进行Western blot分析。
结果如图7显示，感染FMDV后，不同时间点上，PK-15-KLHL34-KO细胞中FMDV的病毒蛋白丰度均明显高于对照PK-15-KLHL34-WT细胞中的蛋白丰度。
4.实时荧光定量检测病毒复制水平
取FMDV接毒后不同时间点收取的细胞，按照Trizol裂解法说明书提取总RNA，测定浓度后，利用反转录试剂盒进行反转录获得cDNA。采用Mx3005P-QPCR系统(Agilent Technologies,USA)和TB GreenⅡPremix Ex Taq 10μL，上、下游引物(FMDV-3D-F：CACTGGTGACAGGCTAAGG，SEQ ID NO.11所示；FMDV-3D-R：CCCTTCTCAGATTCCG AGT，SEQ IDNO.12所示；)各0.5μL，cDNA 1μL，ddH2O 8μL，共20μL的体系配置预混反应，并按照95℃2min；95℃10s，60℃30s(收集荧光)，共40个循环进行荧光定量PCR反应，利用GAPDH作为内参基因，对FMDV的mRNA水平进行定量，所有试验设置三个重复，用2-△△CT方法分析数据。
结果如图8所示，感染FMDV后的不同时间点，PK-15-KLHL34-KO细胞中FMDV的mRNA水平均显著高于对照细胞PK-15-KLHL34-WT。
5.病毒滴度TCID50测定
将FMDV分别感染PK-15-KLHL34-KO和PK-15-KLHL34-WT细胞，不同时间点收取病毒液，反复冻融三次后，离心取上清，用无血清的MEM培养基将待测样品进行10-2～10-8倍梯度稀释，用各稀释度毒液分别接种长满单层PK-15细胞的96孔培养板中，每个稀释度接种8个孔，100μL/孔。置于37℃、5％CO2细胞培养箱中培养3天，期间观察并记录细胞病变(CPE)情况，根据Reed-Muench法计算TCID50并分析。
结果如图9所示，PK-15-KLHL34-KO细胞不同时间点的感染样品，病毒滴度均显著高于对照细胞PK-15-KLHL34-WT，说明敲除KLHL34之后能够显著促进FMDV复制。
综上结果表明，KLHL34基因敲除细胞系能够显著促进小核糖核酸病毒科病毒FMDV的复制，促进小核糖核酸病毒科病毒的复制，可用于小核糖核酸病毒科病毒疫苗的生产；而且KLHL34能够抑制FMDV复制，可用于制备预防或治疗FMDV感染的药物。
Claims (1)
1.KLHL34基因敲除细胞系作为口蹄疫病毒疫苗生产细胞系的应用。
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