CN118127055A - KLK5 RNAi慢病毒载体的构建方法及其在膀胱癌基因治疗中的应用 - Google Patents
KLK5 RNAi慢病毒载体的构建方法及其在膀胱癌基因治疗中的应用 Download PDFInfo
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- CN118127055A CN118127055A CN202410318602.5A CN202410318602A CN118127055A CN 118127055 A CN118127055 A CN 118127055A CN 202410318602 A CN202410318602 A CN 202410318602A CN 118127055 A CN118127055 A CN 118127055A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种KLK5RNAi慢病毒载体的构建方法及其在膀胱癌基因治疗中的应用,构建方法包括以下步骤:S1.根据人KLK5基因的mRNA序列,设计得到3个RNAi靶点序列,3个RNAi靶点序列如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示,针对各RNAi靶点序列设计合成相应的shRNA寡核苷酸单链;S2.将S1中合成的3对所述shRNA寡核苷酸单链退火后形成3种含有RNAi靶点序列的双链shRNA,分别将3种所述双链shRNA插入到表达载体pcDNATM6.2‑GW/EmGFPmiR中,转化感受态大肠杆菌DH5α,培养后挑取单克隆菌落PCR鉴定,测序验证,得到含有KLK5的shRNA的慢病毒载体。本发明通过搭载特定RNAi靶点序列,有效抑制膀胱癌细胞的发生发展,为膀胱癌症患者症状缓解及后续治疗提供了新的指引。
Description
技术领域
本发明属于基因工程领域,涉及一种KLK5 RNAi慢病毒载体的构建方法及其在膀胱癌基因治疗中的应用。
背景技术
膀胱癌是指发生在膀胱黏膜上的恶性肿瘤。是泌尿系统最常见的恶性肿瘤,也是全身十大常见肿瘤之一。占我国泌尿生殖系肿瘤发病率的第一位,而在西方其发病率仅次于膀胱癌,居第2位。2012年全国肿瘤登记地区膀胱癌的发病率为6.61/10万,列恶性肿瘤发病率的第9位。膀胱癌可发生于任何年龄,甚至于儿童。其发病率随年龄增长而增加,高发年龄50~70岁。男性膀胱癌发病率为女性的3~4倍。膀胱肿瘤具有生物侵袭性和特异性放化疗耐药性,导致患者的高复发率和进展率。大多数膀胱癌可经尿道膀胱肿瘤电切术(TUR)治愈,但术后3~5年内复发率高达60%~90%。
癌症的生长和转移是依托癌细胞的增殖和迁移实现的,而癌细胞的增殖和迁移往往伴随着一些基因的过度表达。而对这些过度表达的基因进行敲除和沉默,则可以抑制肿瘤的生长和转移,进一步抑制癌症的发展。因此,对基因敲除和沉默的慢病毒载体构建技术显得尤为重要。
慢病毒载体(Lentivirus)是一类改造自人免疫缺陷病毒(HIV)的病毒载体,是逆转录病毒的一种,基因组是RNA,其毒性基因已经被剔除并被外源性目的基因所取代,属于假型病毒,可将外源基因整合到基因组中实现稳定表达,具有感染分裂期与非分裂期细胞的特性。慢病毒基因组进入细胞后,在细胞浆中反转录为DNA,形成DNA整合前复合体,进入细胞核后,DNA整合到细胞基因组中。整合后的DNA转录成mRNA,回到细胞浆中,表达目的蛋白或产生小RNA。慢病毒介导的基因表达或小RNA干扰作用持续且稳定,并随细胞基因组的分裂而分裂。同时,慢病毒的感染具有整合特性,能够将外源基因有效地整合到宿主染色体上,达到持久性表达,因而可构建稳定细胞株,用于基因的细胞功能研究。
发明内容
本发明的目的在于提供一种KLK5 RNAi慢病毒载体的构建方法及其在膀胱癌基因治疗中的应用,旨在解决上述的问题。
本发明提供了一种KLK5 RNAi慢病毒载体的构建方法,包括以下步骤:
S1.根据人KLK5基因的mRNA序列,经BLAST同源性比对证实特异性后应用RNAstructure 4.4软件对靶mRNA序列的二级结构进行评估,设计得到3个RNAi靶点序列,3个RNAi靶点序列如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示,针对各RNAi靶点序列设计合成相应的shRNA寡核苷酸单链;
S2.将S1中合成的3对所述shRNA寡核苷酸单链退火后形成3种含有RNAi靶点序列的双链shRNA,分别将3种所述双链shRNA插入到表达载体pLV-U6-shRNA-CMV-EGFP(2A)Puro中,转化感受态大肠杆菌DH5α,培养后挑取单克隆菌落PCR鉴定,测序验证,得到含有KLK5的shRNA的慢病毒载体。
进一步的,在S2后还包括:S3.将S2构建得到的慢病毒载体、三质粒的转染体系中的三种质粒以及转染试剂共转染至HEK293T细胞,换液,收集病毒液,即得重组慢病毒载体。
进一步的,在S3后还包括:使用注射器和滤器对收集的病毒液进行过滤。
进一步的,所述S2中插入到表达载体中的具体操作为:使用2个酶切位点将表达载体切开,回收载体骨架部分;使用连接酶连接所述双链shRNA和回收的载体骨架部分。
进一步的,所述三种质粒为VSVG、PMD2.G和RRE,其比例为4:3:1,所述干扰载体与所述三种质粒的配比为1:1,所述转染试剂为PEI。
本发明还提供一种KLK5 RNAi慢病毒载体,采用上述的构建方法构建而得。
本发明还提供一种KLK5 RNAi慢病毒载体在敲低KLK5表达中的应用,使用上述的慢病毒载体感染细胞。
进一步的,所述细胞为膀胱癌细胞。
进一步的,所述细胞为T24细胞和/或5637细胞。
本发明还提供一种KLK5 RNAi慢病毒载体在膀胱癌基因治疗中的应用,使用上述的慢病毒载体感染膀胱癌细胞以敲低KLK5表达而发挥治疗作用。
与现有技术相比,本发明的有益效果为:
本发明基于生信数据库进行分析和筛选,筛选出具有治疗作用的目的基因KLK5,作为作用靶点,设计出针对KLK5进行RNA干扰的重组慢病毒载体,细胞感染效率高达80%以上,且细胞状态正常,具有较高的安全性和稳定性,能有效敲减目的基因KLK5表达,通过不同的实验验证,包括细胞增殖、转移等确定可以影响膀胱癌的发生发展,从而实现对膀胱癌的治疗作用。本发明通过搭载特定RNAi靶点序列,有效抑制膀胱癌细胞的发生发展,为膀胱癌症患者症状缓解及后续治疗提供了新的指引。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例的描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为生信分析KLK5表达;
图2为生信分析KLK5表达;
图3为使用qPCR检测KLK5在不同膀胱癌细胞系中的表达结果;
图4为T24细胞感染慢病毒的结果;
图5为5637细胞感染慢病毒的结果;
图6为T24细胞感染慢病毒后qPCR靶点筛选结果;
图7为5637细胞感染慢病毒后qPCR靶点筛选结果;
图8为CCK8实验检测T24细胞感染慢病毒后细胞增殖的结果;
图9为CCK8实验检测5637细胞感染慢病毒后细胞增殖的结果;
图10为流式细胞术检测细胞凋亡结果(T24);
图11为流式细胞术检测细胞凋亡结果(5637);
图12为细胞划痕结果(T24);
图13为细胞划痕结果(5637);
图14为Transwell迁移结果(T24);
图15为Transwell迁移结果(5637);
图16为pMD2.G质粒图谱;
图17为VSV-G包膜表达质粒图谱。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例及附图,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。以下结合具体实施例对本发明作具体的介绍。
实施例1基因生信数据库筛选出KLK5在膀胱癌中的表达情况
通过生信数据库筛选出KLK5在膀胱癌中的表达情况,结果如图1和2所示,结果表明,KLK5在膀胱癌中的表达高于癌旁,KLK5基因的表达与膀胱癌的总生存期(Overallsurvival)显著相关,即随着患者KLK5基因的表达升高,生存期减短。
实施例2KLK5 RNAi慢病毒载体的构建与鉴定
根据KLK5 mRNA全长序列(使用的人KLK5基因mRNA序列Genebank登录号为NM_001077491.2),分别设计RNAi靶点序列(网址:https://www.sigmaaldrich.cn/CN/zh/semi-configurators/shrna?activeLink=prod uctSearch),RNAi靶点序列为RNAi-13267、RNAi-13268、RNAi-13269,其片段编码序列分别如SEQ ID NO:1(ATCAAACTGAACAGAAGAATT)、SEQ ID NO:2(CCAGGAAACCATCCAGGCCAA)和SEQ ID NO:3(ATCAGCGTGCTAAGTCAGAAA)。
根据每条RNAi靶点序列,设计合成其相应的1对shRNA寡核苷酸单链,如表1,将3对shRNA寡核苷酸单链退火后形成双链shRNA,分别将3种双链shRNA插入到表达载体pLV-U6-shRNA-CMV-EGFP(2A)Puro中,构建含有KLK5的shRNA的慢病毒载体,并转化感受态细胞DH5α。
表1KLK5基因的3对shRNA寡核苷酸单链
注:划线部分为碱基互补区域(从5’→3’),粗体部分为RNAi靶点序列,序列的5’端前若干个碱基为限制性内切酶位点。
退火和连接的具体方法如下:
1、寡核苷酸退火:
先将3对合成的寡核苷酸用双蒸水(ddH2O)分别溶解成100μM,互补单链各取5μL后两两混合,加入退火缓冲液(oligo annealingbuffer)2μL,以ddH2O补足,配为总反应体积20μL。将3对shRNA寡核苷酸的反应混合物95℃加热5min,然后放置室温自然冷却。
2、载体连接
使用限制性内切酶将表达载体上的2个酶切位点(AgeI和EcoRI)切开,回收载体骨架部分;将胶回收的shRNA稀释成10nM浓度,按下表2配制反应体系,室温下连接反应30min。
表2连接pcDNATM6.2-GW/EmGFP载体体系
连接体系 | 用量(μL) |
连接缓冲液 | 2 |
pLV-U6-shRNA-CMV-EGFP(2A)Puro载体骨架 | X(≥50ng) |
双链的shRNA | 4 |
T4连接酶 | 1 |
ddH2O | 13-X |
注:其中X在载体骨架回收浓度为50ng/uL时,为2。
每个转化平板挑取转化后3个单克隆,去摇菌进行菌落PCR,来验证是否有阳性克隆,菌检得到的阳性克隆进行测序,验证重组克隆中插入片段序列是否与设计的寡核苷酸序列一致,得到含有KLK5的shRNA的慢病毒载体。
实施例3KLK5的shRNA的慢病毒载体的包装
处于对数生长期的HEK293T细胞,细胞消化计数后,每个10cm的细胞培养皿接种3×106个细胞,37℃,5%的CO2培养箱中培养过夜;
将实施例1得到的目的质粒与骨架质粒桉比例1:1进行配制得到转染质粒,其中骨架质粒为三质粒的转染体系的三种质粒(VSVG(图谱见图17)、PMD2.G(图谱见图16)和RRE,按4:3:1的比例进行配制),转染质粒的总质量按20μg/孔计算,加入PEI,PEI的量为转染质粒的2.5-3倍,之后加入500μL的DMEM无血清无双抗培养基/20μg转染质粒,记为A液;
取PEI(一般配置成1μg/mL)50-60μg(也就是50-60μL)添加到总体积500μL的DMEM无血清无双抗培养基中,记为B液,移液枪混匀20次;
将B液逐滴滴加到A液中,移液枪轻柔混匀66次,室温静置15-20min;将静置好的转染试剂(A+B)缓缓滴加到恢复状态20h的中;
换液:转染后的第18h,将含有转染试剂的HEK293T上清更换为10mL DMEM完全培养基;
收集病毒液:在转染后的48-72h,为病毒生产的高峰期,收集转染后48h(换液后30h,产毒30h)的病毒液用于实验;收集病毒于15mL离心管中,500g,5mL离心去细胞碎片和杂质,随后使用注射器和滤器进行过滤。收集后的病毒上清可以用来进行感染和储存。
实施例4感染细胞的选择
使用qPCR检测KLK5在膀胱癌细胞系中的表达。具体方法如下:
使用Trizol法提取RNA,将RNA逆转录获得cDNA,之后进行qPCR检测,其中,上游引物序列为:CAAAGTGCTTGGTGTCTGGC(SEQ ID NO:10),下游引物序列为GTCTCGGGTAAGCATCCTCG(SEQ ID NO:11),按下表3配制反应体系,两步法进行Real-TimePCR,并制作熔解曲线,程序如下表4:
表3qPCR反应体系
试剂 | 每管加入量(μL) |
SYBRGreenmastermix | 10 |
上游引物(10μM) | 0.4 |
下游引物(10μM) | 0.4 |
cDNA | 2.0 |
RNase-FreeH2O | 7.2 |
Total | 20 |
表4Real-Time PCR程序
结果见图3所示,可以看出,相对于膀胱上皮细胞,基因KLK5在细胞5637、T24、TCCSUP、RT-112中的表达相对较高,RT-112的表达量与在膀胱上皮细胞的表达量没有明显差异。因此优先选择膀胱癌细胞T24、5637进行后续研究。
实施例5慢病毒感染膀胱癌细胞
为了检测目的基因KLK5的感染的情况,在膀胱癌细胞(T24、5637)中感染实施例3得到的病毒液以敲减KLK5的表达,具体的方法如下:
a)处于对数生长期的T24、5637细胞分别胰酶消化,制备成细胞悬液;
b)将细胞悬液进行铺板6孔板,分别加入T24、5637细胞悬液,每孔细胞量1×105左右
c)24小时后,细胞状态良好。弃培养基,加入感染液,同时加入实施例3得到的病毒液进行感染;
d)感染18-20小时后,更换新制备的培养基;
e)72h后进行在倒置荧光显微镜下进行拍照。
实验结果显示(如图4和图5,图4和图5中的shNC为阴性组,阴性组为空载慢病毒(即没有加入目的基因的序列的慢病毒)进行感染的分组,图4中shKLK5为慢病毒感染T24的结果,图5中shKLK5为慢病毒感染5637的结果),在T24和5637细胞中,慢病毒感染细胞后,各组细胞感染效率达到80%以上,感染效果良好,可进行后续相关实验。
实施例6筛选KLK5在膀胱癌细胞系有效靶点
在T24细胞、5637细胞中感染慢病毒(3个靶点),48h后使用qPCR检测KLK5的表达(具体操作参照实施例4中的qPCR检测),结果见图6和图7,图6为慢病毒感染T24细胞的结果,图7为慢病毒感染5637细胞的结果,其中shNC为阴性组,阴性组为空载慢病毒(即没有加入目的基因的序列的慢病毒)进行感染的分组,病毒载体为pLV-U6--nonsensesequenceCMV-EGFP(2A)Puro,下述shNC与此相同,shKLK5-1组感染搭载RNAi靶点序列RNAi-13267的慢病毒,shKLK5-2组感染搭载RNAi靶点序列RNAi-13268的慢病毒,shKLK5-3组感染搭载RNAi靶点序列RNAi-13269的慢病毒。
从图6结果可以看出,在T24细胞中,慢病毒感染细胞后,相对于shNC组,shKLK5-1组KLK5基因敲减效率为83.3%(P<0.01),shKLK5-2组KLK5基因敲减效率为74.0%(P<0.01),shKLK5-3组KLK5基因敲减效率为45.0%(P<0.05)
从图7结果可以看出,在5637细胞中,慢病毒感染细胞后,相对于shNC组,shKLK5-1组KLK5基因敲减效率为87.5%(P<0.01),shKLK5-2组KLK5基因敲减效率为79.9%(P<0.01),shKLK5-3组KLK5基因敲减效率为43.3%(P<0.05)(图5)。
根据qPCR结果筛选shKLK5-1(以下为shKLK5)作为后续研究对象。
实施例7KLK5对膀胱癌细胞活性的影响评价
CCK8实验:
为了探究KLK5对膀胱癌细胞增殖的影响,发明人在膀胱癌细胞系(T24、5637)中感染实施例3得到的病毒液以敲减KLK5的表达,CCK-8实验检测KLK5对膀胱癌细胞活性的影响,具体操作如下:
(1)将处于对数生长期的各实验组细胞胰酶消化后,完全培养基重悬成细胞悬液,并利用细胞计数仪进行计数;
(2)根据细胞生长快慢决定铺板细胞密度(2000cell/well),每孔100μl,每组3-5孔重复,铺5块96孔板;
(3)统一铺好后,待细胞完全沉淀下来后,在显微镜下观察各实验组的细胞密度,并放入细胞培养箱中培养;
(4)从铺板后第二天开始,培养终止前加入10μL CCK-8试剂于孔中,无需换液;
(5)1-3h后96孔板置于振荡器上振荡2-5min,酶标仪450nm检测OD值。
结果显示(如图8和9所示),在T24细胞中,相对于shNC组,shKLK5敲减组下调(P<0.01,fold change=1.26);在5637细胞中,相对于shNC组,shKLK5敲减组下调(P<0.01,fold change=1.31)。
流式细胞术:
前面已经探究对细胞增殖的影响,发明人进一步通过流式细胞术来验证KLK5是否影响膀胱癌的生长情况,在膀胱癌细胞(T24、5637)中感染实施例3得到的病毒液以敲减KLK5的表达,流式细胞术检测KLK5对膀胱癌细胞凋亡的影响,具体操作如下:
(1)将处于对数生长期的各实验组细胞胰酶消化后,完全培养基重悬成细胞悬液,并利用细胞计数仪进行计数;
(2)待细胞生长至覆盖率为70%时,且在显微镜下观察细胞有死亡现象;
(3)收集培养上清于5ml离心管中,洗涤细胞一次,并收集于同一5ml离心管中,胰酶消化细胞,培养上清终止消化,收集细胞于同一5ml离心管中;
(4)1500rmp 5min离心,弃去上清;
(5)PBS洗涤细胞沉淀一次,1500rmp 3min离心,收集细胞;
(6)1×binding buffer洗涤细胞沉淀一次,1500rmp 3min离心,收集细胞;
(7)1ml(使细胞悬液最终密度为1×106to 1×107cell/ml)1×细胞染色缓冲液重悬细胞沉淀;
(8)取细胞悬液200μL(1×105to 1×106细胞),加入5μL annexin V-APC和5μLPI染色(40×PI染液稀释10倍使用)进行细胞染色;
(9)避光保存样品,在15min内进行流式上机。
结果显示,从细胞凋亡结果来看,在T24细胞中(图10),相对于shNC组,shKLK5敲减组促进细胞凋亡(P<0.001,fold change=2.95),在5637细胞中(图11),相对于shNC组,shKLK5敲减组促进细胞凋亡(P<0.05,fold change=1.30)。
细胞划痕实验:
为了探究癌症发生发展的机制,发明人进行转移实验来探究KLK5对细胞转移的影响。在膀胱癌细胞(T24、5637)中感染实施例3得到的病毒液以敲减KLK5的表达,划痕实验检测KLK5对膀胱癌细胞增殖能力的影响,具体操作如下:
(1)首先使用marker笔在6孔板背后,用直尺比着,均匀的划横线,大约每隔0.5~1cm一道,横穿过孔。每孔至少穿过5条线。划线时注意线不要太粗;
(2)将处于对数生长期的各实验组细胞胰酶消化后,完全培养基重悬成细胞悬液,并利用细胞计数仪进行计数;
(3)根据细胞大小决定铺板细胞密度(大多数细胞铺板数设定为500000cell/well),以次日细胞达到90%以上汇合度为准;
(4)第二天用枪头,垂直与细胞平面,沿着投一天划在平板背面的线在细胞层上进行划痕(不同孔之间最好使用同一只枪头或牙签);
(5)划痕完成后,使用无菌PBS洗细胞3次,洗去不贴壁的细胞,即划线时划线的细胞,是划线后留下的间隙清晰可见,然后更换新鲜无血清培养基;
(6)将细胞放入37℃5%CO2培养箱,培养。然后在24h取出细胞,显微镜线观察并测量划痕的宽度,并拍照;
(7)使用Image J软件打开图片后,随机划取6至8条水平线,计算细胞间面积的均值,并进行数据分析。
划痕实验结果表明,在T24细胞中(图12),相比shNC组,shKLK5组24h细胞迁移率显著降低29%(P<0.05);在5637细胞中(图13),相比shNC组,shKLK5组24h细胞迁移率显著降低49%(P<0.05)。
Transwell实验:
发明人在膀胱癌细胞(T24、5637)中感染实施例3得到的病毒液以敲减KLK5的表达,Transwell实验检测KLK5对膀胱癌细胞迁移能力的影响,具体操作如下:
(1)取所需数量小室于一空24孔板中,加100μL无血清培养基到小室内,培养箱放置1~2h进行水化;
(2)将处于对数生长期的各实验组细胞胰酶消化后,低血清培养基重悬成细胞;
(3)在步骤(1)完成后,从小室内小心移去培养基;
(4)加600μL含30%FBS培养基到下室中;
(5)用无血清培养基按一定比例稀释细胞,加该细胞悬液(含100000cell)100uL到每个小室中;
(6)用镊子将小室转移入含30%FBS培养基的下室中;
(7)在组织培养箱中培养4-24h;
(8)将上室中培养基去除,4%FPA固定液进行细胞固定,室温10-30min;
(9)去除4%FPA固定液,1×PBS清洗上室细胞1-2遍;
(10)将小室浸泡在染色液中5-10min,在膜的下表面染色转移细胞。
(11)倒扣小室于吸水纸上以去除培养基,用棉拭子轻轻移去非转移细胞;
(12)用ddHO2对上室进行清洗数次;
(13)空气中晾干;
(14)显微镜拍照膜。
Transwell实验结果表明,在T24细胞中(图14),相比shNC组,shKLK5组Transwell转移率显著降低36%(P<0.001);在5637细胞(图15),相比shNC组,shKLK5组Transwell转移率显著降低81%(P<0.001)。
上述结果证明KLK5不仅影响膀胱癌细胞增殖也可以影响细胞转移,通过感染实施例3得到的病毒液能够降低KLK5的表达,从而影响膀胱癌细胞增殖也可以影响细胞转移。
上述描述仅是对本发明较佳实施例的描述,并非对本发明范围的任何限定,本发明领域的普通技术人员根据上述揭示内容做的任何变更、修饰,均属于权利要求书的保护范围。
Claims (9)
1.一种KLK5 RNAi慢病毒载体的构建方法,其特征在于,包括以下步骤:
S1.根据人KLK5基因的mRNA序列,分别设计RNAi靶点序列,得到3个RNAi靶点序列,3个RNAi靶点序列如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示,针对各RNAi靶点序列设计合成相应的shRNA寡核苷酸单链;
S2.将S1中合成的3对所述shRNA寡核苷酸单链退火后形成3种含有RNAi靶点序列的双链shRNA,分别将3种所述双链shRNA插入到表达载体pLV-U6-shRNA-CMV-EGFP(2A)Puro中,转化感受态大肠杆菌DH5α,培养后挑取单克隆菌落PCR鉴定,测序验证,得到含有KLK5的shRNA的慢病毒载体。
2.根据权利要求1所述的构建方法,其特征在于,在S2后还包括:S3.将S2构建得到的慢病毒载体、三质粒的转染体系中的三种质粒以及转染试剂共转染至HEK293T细胞,换液,收集病毒液,即得重组慢病毒载体。
3.根据权利要求2所述的构建方法,其特征在于,在S3后还包括:使用注射器和滤器对收集的病毒液进行过滤。
4.根据权利要求1所述的构建方法,其特征在于,所述S2中插入到表达载体中的具体操作为:使用2个酶切位点将表达载体切开,回收载体骨架部分;使用连接酶连接所述shRNA和回收的载体骨架部分。
5.根据权利要求2所述的构建方法,其特征在于,所述三种质粒为VSVG、PMD2.G和RRE,其比例为4:3:1,所述慢病毒载体与所述三种质粒的配比为1:1,所述转染试剂为PEI。
6.一种KLK5 RNAi慢病毒载体,其特征在于,采用权利要求1-5任一项所述的构建方法构建而得。
7.一种KLK5 RNAi慢病毒载体在敲低KLK5表达中的应用,其特征在于,使用权利要求6所述的慢病毒载体感染细胞。
8.根据权利要求7所述的应用,其特征在于,所述细胞为膀胱癌细胞。
9.根据权利要求8所述的应用,其特征在于,所述细胞为T24细胞和/或5637细胞。
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