CN116769928A - Lta4h基因或蛋白为靶点在筛选抑制口蹄疫病毒复制的药物中的应用 - Google Patents
Lta4h基因或蛋白为靶点在筛选抑制口蹄疫病毒复制的药物中的应用 Download PDFInfo
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- CN116769928A CN116769928A CN202310743827.0A CN202310743827A CN116769928A CN 116769928 A CN116769928 A CN 116769928A CN 202310743827 A CN202310743827 A CN 202310743827A CN 116769928 A CN116769928 A CN 116769928A
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Abstract
本发明属于基因工程技术领域,具体涉及一种LTA4H基因/蛋白为靶点在筛选抑制口蹄疫病毒复制的药物中的应用。具体为:①本发明首先发现抑制或沉默LTA4H基因/蛋白能够抑制口蹄疫病毒的复制,可作为靶点用于筛选抑制口蹄疫病毒复制的药物;②以LTA4H为靶点的涉及了靶向LTA4H的sgRNA,结合CRISPR‑Cas9技术在PK‑15细胞中实现了LTA4H的完全敲除,获得的细胞系对口蹄疫病毒具有抗性表型,对未来预防或抑制FMDV感染提供一些理论依据,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LTA4H基因或蛋白为靶点在筛选抑制口蹄疫病毒复制的药物中的应用。
背景技术
口蹄疫(Foot and mouth disease,FMD)是一种严重的、高度传染性的全球家畜病毒性疾病,主要影响家畜和野生偶蹄类动物,包括牛、猪、羊、山羊、骆驼和鹿。由一种小核糖核酸科病毒引起,即口蹄疫病毒(FMDV)。在感染口蹄疫病毒(FMDV)后,随后的临床症状包括发烧、食欲不振、跛行,并在口腔、鼻、乳头和足部出现水泡性病。FMDV属于小RNA病毒科,口蹄疫病毒属,是一种小型、非包膜病毒,包含一个约8500个碱基的单链正义RNA基因组,周围是一个二十面体的衣壳蛋白,四种结构蛋白VP1–4各有60个拷贝。该病毒抗原高度可变,由七个血清型组成,包括O型、A型、C型、亚洲1型、南非地区(SAT)1型、SAT2型和SAT3型,每个血清型中又有多个亚型。这七种血清型之间存在约60-70%的同源性,不同血清型之间和不同亚型之间几乎没有交叉保护,同一血清型内的不同亚型或分离株发生交叉免疫的程度均不相同,因此,该病被国际兽疫局视为最具传染性的猪病。
基因编辑技术是通过分子生物学方法对各类细胞中基因进行定向、精准的突变、修饰或编辑的技术。该技术已被广泛用于疾病治疗、抗病育种、遗传工程改造等研究领域。基因编辑技术不断发展成熟,从第一代的依赖锌指核酸酶ZFN的编辑技术,第二代依赖转录激活样效应因子核酸酶TALEN的编辑技术,到第三代依赖成簇的规律性间隔的短回文重复序列CR ISPR-Cas9的编辑技术,历经发展近30年,已经取得了突破性的进展。
聚集的规则间隔短回文重复序列(CRISPR)-Cas(CRISPR相关蛋白)系统最初在细菌和古菌中被发现,为细菌和古菌提供了适应性免疫反应,以抵抗噬菌体、质粒和转座子等移动遗传元件的入侵。其利用单一引导RNA(sgRNA)序列与基因组中的特定靶位点和Cas9核酸内切酶结合。利用sgRNA和靶DNA序列之间的同源性,Cas9核酸内切酶指向特定的靶位点,纠正、删除、添加或破坏基因,并永久修复致病突变。在预防或治疗多种疾病方面有着广泛的应用。这些系统已被重新用作非常强大的生物技术工具,用于细菌和真核生物系统中的基因编辑应用。该技术相比较其他技术更加便捷、高效、精准、稳定,而且相关使用成本费用更加低廉。为基因组定向改造、修饰、调控和应用等带来了突破性革命,在医学与生命科学各大领域具有广阔的应用前景。
白三烯A4水解酶(LTA4H)是一种属于肽酶M1家族的蛋白质,特别是那些作用于醚键(醚水解酶)的蛋白质,是一种双功能含锌酶,且是重要的抗炎靶点,具有环氧化物水解酶活性和功能不明的氨基肽酶活性。作为环氧化物水解酶,LTA4H催化环氧化物LTA4水解为促炎物质二醇白三烯B4(LTB4),其主要起化学引诱剂和炎症细胞激活剂的作用。作为一种氨基肽酶,LTA4H可以处理与炎症和宿主防御相关的肽。LTA4H参与花生四烯酸代谢,被贝斯塔丁抑制,并受到白三烯A4的自杀抑制,这是由于在Tyr-379处形成共价加合物。有研究表明,LTA4H在一些人类癌症中高度表达,包括食道腺癌、肺癌和甲状腺癌,并在动物模型中抑制肿瘤发生,具有深度的研究价值。
基于上述问题,本发明意外发现通过抑制或沉默宿主LTA4H基因,能够抑制FMDV的复制,可作为靶点用于设计和筛选抑制FMDV复制的药物。基于此,为了进一步研究LTA4H基因基因在细胞内调控病原微生物复制的分子机制,本发明设计了一种特异性靶向LTA4H基因基因的sgRNA,所述的sgRNA能够特异性靶向LTA4H基因,结合CRISPR/Cas9技术实现了LTA4H基因的完全敲除,获得的单克隆细胞系对FMDV具有抗性表型,能够显著抑制FMDV在细胞内的复制,为研究LTA4H基因在细胞内调控病原微生物复制的分子机制提供了研究工具和材料,也可用于抗FMDV的动物育种。
发明内容
针对上述技术问题,本发明首先发现通过抑制或沉默宿主LTA4H基因/蛋白,能够抑制FMDV的复制,可作为靶点用于制备抑制FMDV复制的药物;其次,本发明提供了一种特异性靶向LTA4H基因/蛋白的sgRNA,所述的sgRNA能够特异性靶向LTA4H基因/蛋白,结合CRISPR-Cas9技术实现了LTA4H基因/蛋白的完全敲除,获得的单克隆细胞系对FMDV具有抗性表型,能够显著抑制FMDV在细胞内的复制,为进一步研究LTA4H基因/蛋白在细胞内调控病原微生物复制的分子机制提供研究工具和材料。具体包括以下内容:
第一方面,本发明提供了一种LTA4H基因/蛋白为靶点在筛选用于预防或治疗口蹄疫病毒感染的药物中的应用;所述药物以LTA4H基因/蛋白为靶点,抑制或沉默LTA4H基因/蛋白的表达。
第二方面,本发明提供了一种LTA4H基因/蛋白表达抑制剂在制备预防或治疗口蹄疫病毒感染的药物、药物组合物或疫苗组合物中的应用。
优选地,所述LTA4H基因/蛋白表达抑制剂包括以LTA4H基因/蛋白为靶点设计的小干扰RNA;或所述LTA4H基因/蛋白表达抑制剂包括靶向敲除LTA4H基因/蛋白的sgRNA。
优选地,所述sgRNA包括LTA4H-sgRNA1和/或LTA4H-sgRNA2;
所述LTA4H-sgRNA1的靶向序列为:GCAGCGTCGACTTTACTCGC;
所述LTA4H-sgRNA2的靶向序列为:TTACTCGCCGGGTACTGACC。
优选地,所述LTA4H-sgRNA1由LTA4H-sgRNA1-F和LTA4H-sgRNA1-R退火形成的双链片段;所述LTA4H-sgRNA2由LTA4H-sgRNA2-F和LTA4H-sgRNA2-R退火形成的双链片段;
LTA4H-sgRNA1-F:5’-GCAGCGTCGACTTTACTCGC-3’:
LTA4H-sgRNA1-R:5’-GCGAGTAAAGTCGACGCTGC-3’:
LTA4H-sgRNA2-F:5’-TTACTCGCCGGGTACTGACC-3’:
LTA4H-sgRNA2-R:5’-GGTCAGTACCCGGCGAGTAA-3’。
第三方面,本发明提供了一种LTA4H基因功能缺失细胞系的构建方法,所述构建方法为:通过基因打靶技术使宿主细胞中LTA4H基因功能缺失。
优选地,所述方法包括以下步骤:
(1)制备特异性靶向LTA4H基因/蛋白的sgRNA;
(2)将步骤(1)制备的sgRNA寡聚核苷酸的双链片段插入到表达Cas9的慢病毒载体的多克隆位点,转染细胞得到同时表达Cas9蛋白基因和打靶sgRNA序列的重组慢病毒;
(3)将步骤(2)制备的重组慢病毒转导宿主细胞,挑取单个细胞,接种培养,获得LTA4H基因/蛋白功能缺失细胞系。
优选地,所述宿主细胞为PK-15细胞。
优选地,所述sgRNA包括LTA4H-sgRNA1和/或LTA4H-sgRNA2;
所述LTA4H-sgRNA1的靶向序列为:GCAGCGTCGACTTTACTCGC;
所述LTA4H-sgRNA2的靶向序列为:TTACTCGCCGGGTACTGACC。
所述sgRNA寡聚核苷酸的双链片段为:
LTA4H-sgRNA1-F:5’-GCAGCGTCGACTTTACTCGC-3’:
LTA4H-sgRNA1-R:5’-GCGAGTAAAGTCGACGCTGC-3’:
LTA4H-sgRNA2-F:5’-TTACTCGCCGGGTACTGACC-3’:
LTA4H-sgRNA2-R:5’-GGTCAGTACCCGGCGAGTAA-3’。
第四方面,本发明提供了一种上述第三方面所述方法构建获得的LTA4H基因功能缺失细胞系。
本发明的有益效果是:①本发明发现通过抑制或沉默宿主LTA4H基因/蛋白,能够抑制FMDV的复制,可作为靶点用于制备抑制FMDV复制的药物;②本发明提供了一种靶向LTA4H基因的sgRNA,所述sgRNA能够特异性靶向LTA4H基因,结合CRISPR-Cas9技术可实现宿主细胞中LTA4H基因的敲除,打靶准确、敲除效率高;③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LTA4H基因/蛋白功能丧失细胞系的方法,通过使LTA4H基因/蛋白功能丧失,获得的了具有FMDV抗性表型的细胞系,能够显著抑制FMDV的复制,为进一步研究POP基因/蛋白在细胞内调控病原微生物复制的分子机制提供研究工具和材料,也可用于抗FMDV的动物育种,对未来预防或抑制FMDV感染提供一些理论依据,具有旷阔的应用前景。
附图说明
图1靶向LTA4H基因组区域的sgRNA色谱示意图;
图2LTA4H-KO-1细胞系基因缺失突变型分析图;
图3LTA4H-KO-2细胞系基因缺失突变型分析图;
图4Western Blotting检测敲除细胞系中LTA4H基因的蛋白水平结果;
图5LTA4H基因功能缺失细胞系LTA4H-KOs细胞活力检测结果;
图6相对定量检测FMDV在LTA4H-WT和LTA4H-KOs细胞中的复制情况;
图7Western blot检测FMDV在LTA4H-WT和LTA4H-KOs细胞中的蛋白水平差异;
图8LTA4H-KO细胞中口蹄疫病毒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LTA4H基因靶向敲除,导致LTA4H基因编码蛋白的功能丧失,进而构建了LTA4H基因编码蛋白功能丧失的细胞系,并用于FMDV疫苗的生产。但是本发明并不局限于LTA4H基因敲除,也可通过其他技术手段使LTA4H基因编码蛋白的功能丧失,并用于构建LTA4H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”是指通过同源重组使特定靶基因失活。本发明通过基因敲除技术,使宿主细胞中的LTA4H基因敲除,获得的LTA4H基因编码蛋白功能丧失的单克隆细胞系能抑制FMDV感染后的病毒复制;本发明也可以通过对宿主细胞中的LTA4H基因突变,或插入基因片段,导致LTA4H基因编码蛋白发生移码突变,成功构建出LTA4H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LTA4H基因的sgRNA;本发明在直接靶向剪接LTA4H基因的基础上,利用CRISPR/Cas9联合特异性敲除LTA4H基因的方法,以PK-15细胞为例,进行了LTA4H基因敲除,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LTA4H基因进行敲除,获得了一种基因编码蛋白功能丧失的细胞系,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LTA4H基因敲除。
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LTA4H基因的gRNA序列,实现LTA4H基因的有效敲除,获得一种抑制FMDV病毒复制的LTA4H基因敲除的单克隆细胞系,从而为感染口蹄疫病毒做好预防或治疗提供新的策略。
利用CRISPR/Cas9基因编辑技术,通过靶向LTA4H基因的sgRNA引导Cas9蛋白结合到LTA4H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。
利用CRISPR/Cas9基因编辑技术,通过靶向LTA4H基因的sgRNA引导Cas9蛋白结合到LTA4H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。
FMDV(FMDV/O/BY/CHA/2010株)来源于中国农业科学院兰州兽医研究所口蹄疫与新发病流行病学团队与国家口蹄疫参考实验室。
以下实施例所述的LTA4H基因序列如SEQ ID NO.2所示,氨基酸序列如SEQ IDNO.1所示。
实施例1LTA4H基因敲除PK-15细胞系的构建
利用NCBI数据库查询LTA4H基因序列,根据CRISPR/Cas9设计原则,利用CRISPR软件在LTA4H基因的第1个外显子区域的327位置和339位置分别设计两条sgRNA序列(结果如图1所示):LTA4H-sgRNA1和LTA4H-sgRNA2:
所述LTA4H-sgRNA1的靶向序列为:GCAGCGTCGACTTTACTCGC(SEQ ID NO.3所示);所述LTA4H-sgRNA1是由LTA4H-sgRNA1-F(5’-GCAGCGTCGACTTTACTCGC-3’,SEQ ID NO.5所示)和LTA4H-sgRNA1-R(5’-GCGAGTAAAGTCGACGCTGC-3’,SEQ ID NO.6所示)退火形成的双链片段;
所述LTA4H-sgRNA2的靶向序列为:TTACTCGCCGGGTACTGACC(SEQ ID NO.4所示);所述LTA4H-sgRNA2是由LTA4H-sgRNA2-F(5’-TTACTCGCCGGGTACTGACC-3’,SEQ ID NO.7所示)和LTA4H-sgRNA2-R(5’-GGTCAGTACCCGGCGAGTAA-3’,SEQ ID NO.8所示)退火形成的双链片段;
sgRNA的退火: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,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BBSI内切酶酶切PX459载体,按照PX459载体5μL,BBSI 1μL、10×buffer 2μL、ddH2O 12μL,共20μL反应体系37℃酶切7h,胶回收酶切片段;
酶切载体与sgRNA的连接:T4连接酶0.5μL、10×T4连接酶buffer 0.5μL、PX459酶切片段1μL、退火后的sgRNA 2μL,ddH2O 1μL,共5μL体系,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,提取质粒,送南京擎科生物科技有限公司测序。使用BLAST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LTA4H基因不同外显子的sgRNA重组质粒,位置和序列信息如图1;重组质粒分别命名为:PX459-LTA4H-sgRNA1、PX459-LTA4H-sgRNA2。
细胞转染:根据Lipofectamine 2000(Invitrogen)的标准转染程序,将CRISPR重组质粒转染PK-15细胞系,转染24h后加入终浓度3μg/mL的嘌呤霉素筛选3天后,进行细胞计数,分别铺100个和300个细胞,一周后单个细胞克隆形成后用克隆环挑取单克隆(LTA4H-KO-1和LTA4H-KO-2),转移至48孔板,于37℃,5%CO2培养箱中,添加DMEM培养基中(10%胎牛血清和1%抗生素(penicilin-streptomycin))扩大培养。收集不同细胞克隆分别在DNA水平和蛋白水平进行鉴定;扩增产物进行Sanger测序,结果如图2和图3所示,LTA4H-KO-1在Cas9外显子1的334预定切割位置处(距离PAM基序(CGG)第4和5个碱基之间切割)有1个碱基的插入,LTA4H-KO-2在外显子1的355预定切割位置处(距离PAM基序(GGG)第4和5个碱基之间切割)有1个碱基的缺失。
蛋白水平鉴定时用LTA4H的抗体(Cat No.13662-1-AP),利用Western blot检测LTA4H的蛋白水平表达。结果如图4所示,LTA4H-KO-1和LTA4H-KO-2细胞系中均检测不到LTA4H的蛋白表达。上述结果表明,LTA4H基因敲除PK-15细胞系构建成功。
实施例2LTA4H基因敲除PK-15细胞系的活力检测
将WT型PK-15细胞、LTA4H-KO-1和LTA4H-KO-2消化,细胞消化后计数,调整细胞悬液浓度,每孔100μL接种至96孔板,置细胞培养箱正常培养一定时间;向板的每个孔加入10μL CCK-8溶液,注意不要向孔中引入气泡。放细胞培养箱继续培养1-4h,用酶标仪测定每孔的OD450 nm,分析数据。结果如图5所示,LTA4H基因敲除PK-15细胞系LTA 4H-KO-1和LTA4H-KO-2与正常型PK-15细胞的细胞活力相同,表明LTA4H基因的敲除并不影响宿主细胞的正常生长。
实施例3LTA4H-KO细胞系对FMDV复制的影响
为了研究敲除LTA4H之后对FMDV蛋白水平的影响,分别将LTA4H基因敲除PK-15细胞系LTA4H-KO-1和LTA4H-KO-2和正常型PK-15细胞(WT)铺于6孔板中,待细胞长满后感染FMDV,于0,6,12,24h分别收取细胞,用Trizol裂解法提取细胞总RNA,测定浓度后,进行反转录:RNA,4μL;ddH2O,12μL;5×super MIXⅡ,4μL。反应程序:50℃,15min;85℃,5s;4℃,保存。qPCR用2×ChamQ SYBR qPCR Master Mix(Vazyme),5μL;上下游引物各0.5μL;ddH2O,3μL;CDNA,1μL。GAPDH作为内参基因,对FMDV的相对表达水平进行定量。结果如图6所示,敲除LTA4H之后在不同时间点显抑制FMDV复制。
为了研究敲除LTA4H之后对FMDV蛋白水平的影响,分别将LTA4H基因敲除PK-15细胞系LTA4H-KO-1和LTA4H-KO-2和正常型PK-15细胞(WT)铺于6孔板中,待细胞长满后感染FMDV,于0,6,12,24h分别收取细胞,加1×上样缓冲液混匀,煮沸15min,室温静置冷却,利用Western blot检测,上样20μl,与实例1中过程一样。结果如图7所示,在FMDV接毒后收取的样品中,LTA4H基因敲除PK-15细胞系LTA4H-KO中口蹄疫病毒非结构蛋白3B的表达远低于野生型细胞,说明LTA4H基因功能缺失单克隆细胞系能够显著抑制FMDV的复制。
在LTA4H基因敲除PK-15细胞系LTA4H-KO和WT细胞中分别比较病毒滴度的差异,将FMDV分别感染LTA4H基因敲除PK-15细胞系LTA4H-KO-1和LTA4H-KO-2和WT细胞,于6,12,24h分别收取细胞样品,反复冻融三次,用无血清的DMEM将获得细胞样品进行10-2~10-7倍梯度稀释,将BHK细胞铺96孔板,用各梯度稀释毒液分别接种96孔细胞培养板,每个孔100μL毒液,100μL细胞。置于37℃、5% CO2恒温培养箱中培养,36h、48h、72h观察,并记录细胞病变(CPE)情况,根据Reed-Muench法计算扩增病毒的TCID50。结果如图8所示,在FMDV接毒后收取的样品中,LTA4H基因敲除PK-15细胞系LTA4H-KO-1和LTA4H-KO-2中口蹄疫病毒的病毒滴度远低于野生型细胞,说明LTA4H基因功能缺失单克隆细胞系能够显著抑制FMDV的复制。
综上结果表明,LTA4H基因敲除细胞系能够显著抑制小核糖核酸病毒科病毒FMDV的复制,抑制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。
Claims (10)
1.LTA4H基因/蛋白为靶点在筛选用于预防或治疗口蹄疫病毒感染的药物中的应用;所述药物以LTA4H基因/蛋白为靶点,抑制或沉默LTA4H基因/蛋白的表达。
2.LTA4H基因/蛋白表达抑制剂在制备预防或治疗口蹄疫病毒感染的药物、药物组合物或疫苗组合物中的应用。
3.如权利要求2所述的应用,其特征在于,所述LTA4H基因/蛋白表达抑制剂包括以LTA4H基因/蛋白为靶点设计的小干扰RNA;或所述LTA4H基因/蛋白表达抑制剂包括靶向敲除LTA4H基因/蛋白的sgRNA。
4.如权利要求3所述的应用,其特征在于,所述sgRNA包括LTA4H-sgRNA1和/或LTA 4H-sgRNA2;
所述LTA4H-sgRNA1的靶向序列为:GCAGCGTCGACTTTACTCGC;
所述LTA4H-sgRNA2的靶向序列为:TTACTCGCCGGGTACTGACC。
5.如权利要求4所述的应用,其特征在于,所述LTA4H-sgRNA1由LTA4H-sgRNA1-F和LTA4H-sgRNA1-R退火形成的双链片段;所述LTA4H-sgRNA2由LTA4H-sgRNA2-F和LTA4H-sgRNA2-R退火形成的双链片段;
LTA4H-sgRNA1-F:5’-GCAGCGTCGACTTTACTCGC-3’:
LTA4H-sgRNA1-R:5’-GCGAGTAAAGTCGACGCTGC-3’:
LTA4H-sgRNA2-F:5’-TTACTCGCCGGGTACTGACC-3’:
LTA4H-sgRNA2-R:5’-GGTCAGTACCCGGCGAGTAA-3’。
6.一种LTA4H基因功能缺失细胞系的构建方法,其特征在于,所述构建方法为:通过基因打靶技术使宿主细胞中LTA4H基因功能缺失。
7.如权利要求6所述的构建方法,其特征在于,所述方法包括以下步骤:
(1)制备特异性靶向LTA4H基因/蛋白的sgRNA;
(2)将步骤(1)制备的sgRNA寡聚核苷酸的双链片段插入到表达Cas9的慢病毒载体的多克隆位点,转染细胞得到同时表达Cas9蛋白基因和打靶sgRNA序列的重组慢病毒;
(3)将步骤(2)制备的重组慢病毒转导宿主细胞,挑取单个细胞,接种培养,获得LT A4H基因/蛋白功能缺失细胞系。
8.如权利要求7所述的构建方法,其特征在于,所述宿主细胞为PK-15细胞。
9.如权利要求8所述的构建方法,其特征在于,所述sgRNA包括LTA4H-sgRNA1和/或LTA4H-sgRNA2;
所述LTA4H-sgRNA1的靶向序列为:GCAGCGTCGACTTTACTCGC;
所述LTA4H-sgRNA2的靶向序列为:TTACTCGCCGGGTACTGACC。所述sgRNA寡聚核苷酸的双链片段为:
LTA4H-sgRNA1-F:5’-GCAGCGTCGACTTTACTCGC-3’:
LTA4H-sgRNA1-R:5’-GCGAGTAAAGTCGACGCTGC-3’:
LTA4H-sgRNA2-F:5’-TTACTCGCCGGGTACTGACC-3’:
LTA4H-sgRNA2-R:5’-GGTCAGTACCCGGCGAGTAA-3’。
10.如权利要求6-9任一所述方法构建获得的LTA4H基因功能缺失细胞系。
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