CN105821040B - 联合免疫基因抑制高危型HPV表达的sgRNA、基因敲除载体及其应用 - Google Patents
联合免疫基因抑制高危型HPV表达的sgRNA、基因敲除载体及其应用 Download PDFInfo
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- CN105821040B CN105821040B CN201610134003.3A CN201610134003A CN105821040B CN 105821040 B CN105821040 B CN 105821040B CN 201610134003 A CN201610134003 A CN 201610134003A CN 105821040 B CN105821040 B CN 105821040B
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Abstract
本发明提供联合免疫基因抑制高危型HPV表达的sgRNA、基因敲除载体及其应用:具体包括选择适合CRISPR‑Cas9靶向剪辑的人乳头瘤病毒16型基因和人PD‑1基因的sgRNA序列,将其与CRISPR‑Cas9核酸酶基因的表达载体转入HPV16+的人宫颈癌细胞、荷HPV16+移植瘤小鼠,可以明显降低HPV16的表达,并抑制肿瘤的生长。本发明制备的基因表达载体方法步骤简单、sgRNA靶向性好、CRISPR‑Cas9系统的敲除效率高,并且不仅能够精确靶向剪接高危型HPV16型和PD‑1基因,高效降低高危型HPV16型的基因表达,联合应用还能明显抑制肿瘤的生长。
Description
技术领域
本发明属于基因工程及生物医药领域,涉及CRISPR/Cas9特异性修饰高危型HPV16及PD-1多个基因靶点的设计方法,通过靶向敲除高危型HPV16的癌基因表达预防人宫颈癌,并应用联合免疫基因治疗策略获得增强靶向剪接高危型HPV16的癌基因治疗HPV16+小鼠移植瘤的良好效果。
背景技术
近年来,基因组编辑工具广泛应用于生物医学领域,而成簇的规律间隔的短回文重复序列(CRISPR)技术已成为基因组编辑的热点。CRISPR是自然存在于细菌DNA中的序列,与CRISPR相关(Cas)核酸酶结合,具有指导RNAs保护细菌基因组免受侵入性噬菌体中检测到的靶向特异性序列攻击的作用。CRISPR/Cas9技术被Nature、Science杂志分别评为2013年前10位的明星技术之一,并位居2015年科学杂志评选出来的十大发现之首。该项技术将成为功能基因组学和系统生物学领域中强有力的研究工具。
全世界每年有50多万的女性罹患宫颈癌,约26万人死于该病,成为女性患者死亡的第二位原因。宫颈癌是少数几个致病原因清楚的人类肿瘤之一,高危型人乳头瘤病毒(HPV)感染与其发生明确相关。HPV是一种极为常见的病毒,高达75%的女性在一生的某一阶段会感染HPV,大多数感染可通过自发的免疫反应迅速清除,但少部分感染会持续,并成为宫颈癌以及宫颈上皮内瘤变(CIN)的主要病因。高危型HPV包括HPV-16,HPV-18,HPV-31,HPV-33,HPV-45等类型。大量的流行病学资料显示,HPV16型为目前诱发宫颈癌的高危亚型,可在50~60%的宫颈癌组织中检测到此型HPV,其E6和E7基因为明确的致癌基因。目前,已有针对高危型HPV感染的预防性疫苗在发达国家广泛应用,具有预防高危型HPV感染的效果。但是临床上仍然使用手术和放化疗治疗宫颈癌,其对中晚期宫颈癌患者的治疗效果还很不理想,而且对解除高危型HPV感染持续存在也还缺乏有效的方法和措施。因此,对人正常宫颈上皮细胞感染高危型HPV后的病毒清除以及中晚期人宫颈癌治疗都有待于出现新的特异性药物和新的治疗方法。
研究表明,人宫颈癌患者的免疫耐受涉及先天性免疫和获得性免疫两个环节。首先,参与先天免疫反应的抗原提呈细胞(APC)、巨噬细胞和NK细胞数量下降,其次参与获得性免疫的Th1/Th2细胞平衡被打破。目前还没有发现十分明确的宫颈癌特异性免疫耐受标志物,但研究表明,宫颈癌患者外周免疫系统中PD-1显著上调。故可以从肿瘤通用的免疫调节途径如PD-1/PD-L1共抑制分子入手,将PD-1基因作为人宫颈癌治疗的干预靶点。
脂质体是具有良好的生物相容性的传递载体,其表面修饰配体是构建主动靶向脂质体的重要方式。细胞穿膜肽(TAT)能够穿过与之接触的任何细胞的细胞膜,而对细胞膜没有损伤,叶酸受体存在于多种人细胞表面,包括人宫颈上皮细胞及宫颈癌细胞,因而可以赢应用设计的靶向肿瘤细胞给药(如叶酸修饰包裹靶向HPV16基因的sgRNA质粒的脂质体)方式抑制HPV16基因的表达,预防癌症的发生。
现在人正常宫颈上皮高危型HPV感染清除和中晚期宫颈癌治疗存在的问题:(1)药物不能有效清除细胞内感染的高危型HPV;(2)其他方法特异性敲除HPV E6、E7基因的效率低,降低HPV基因表达效果较差;(3)靶向人宫颈癌的治疗药物很少,效果有限,副作用较明显;(4)利用多种治疗方法联合治疗宫颈癌的策略需要提高治疗效果。
发明内容
本发明的目的在于提供联合免疫基因抑制高危型HPV表达的sgRNA、基因敲除载体及其应用。
为达到上述目的,本发明采用了以下技术方案:
联合免疫基因抑制高危型HPV表达的sgRNA,该抑制高危型HPV表达的sgRNA包括在CRISPR-Cas9特异性修饰人PD-1基因中可特异性靶向人PD-1基因的sgRNA,该sgRNA的序列如SEQ.ID.NO.4或SEQ.ID.NO.5所示。
所述抑制高危型HPV表达的sgRNA还包括与所述可特异性靶向人PD-1基因的sgRNA联用的可特异性靶向高危型HPV16的sgRNA,该sgRNA的序列如SEQ.ID.NO.6、SEQ.ID.NO.7或SEQ.ID.NO.8。
联合免疫基因抑制高危型HPV表达的基因敲除载体,该基因敲除载体选自重组质粒pGL3-U6-PD1 sgl、pGL3-U6-PD1 sg2中的一种,pGL3-U6-PD1 sgl由序列如SEQ.ID.NO.4所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-PD1 sg2由序列如SEQ.ID.NO.5所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.4或SEQ.ID.NO.5所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内,或者,该基因敲除载体为构建于用于表达核酸酶Cas9的质粒基础上的重组质粒,所述用于表达核酸酶Cas9的质粒的多克隆位点内分别插入有如SEQ.ID.NO.4、SEQ.ID.NO.5所示序列中的一种或两种以及如SEQ.ID.NO.6、SEQ.ID.NO.7、SEQ.ID.NO.8所示序列中的一种、两种或三种。
联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,该组合物包括pGL3-U6-PD1质粒,所述pGL3-U6-PD1质粒选自重组质粒pGL3-U6-PD1 sgl、pGL3-U6-PD1 sg2中一种或两种,pGL3-U6-PD1 sgl由序列如SEQ.ID.NO.4所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-PD1 sg2由序列如SEQ.ID.NO.5所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.4或SEQ.ID.NO.5所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内。
所述pGL3-U6-PD1 sgl与pGL3-U6-PD1 sg2的质量比为(1~2):(1~2)。
所述组合物还包括pGL3-U6-HPV16 sg质粒,所述pGL3-U6-HPV16 sg质粒选自重组质粒pGL3-U6-HBV16-P sg、pGL3-U6-HPV-E6 sg、pGL3-U6-HPV16-E7 sg中的一种、两种或三种,pGL3-U6-HBV16-P sg由序列如SEQ.ID.NO.6所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-HPV-E6 sg由序列如SEQ.ID.NO.7所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-HPV-E7 sg由序列如SEQ.ID.NO.8所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.6、SEQ.ID.NO.7或SEQ.ID.NO.8所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内。
所述pGL3-U6-HPV16 sg质粒与pGL3-U6-PD1质粒的质量比为(1~3):(1~2);所述pGL3-U6-HPV16 sg质粒采用pGL3-U6-HPV16-P sg与pGL3-U6-HPV-E6 sg或pGL3-U6-HPV16-E7 sg联用,pGL3-U6-HPV16-P sg与pGL3-U6-HPV-E6 sg或pGL3-U6-HPV16-E7 sg的质量比为(1~2):(1~2);或者,所述pGL3-U6-HPV16 sg质粒采用pGL3-U6-HBV16-P sg与pGL3-U6-HPV-E6 sg以及pGL3-U6-HPV16-E7 sg联用,pGL3-U6-HPV16-P sg:pGL3-U6-HPV-E6 sg:pGL3-U6-HPV16-E7 sg的质量比为(1~2):(1~2):(1~2)。
所述组合物还包括用于表达核酸酶Cas9的质粒,所述用于表达核酸酶Cas9的质粒:pGL3-U6-HPV16 sg质粒:pGL3-U6-PD1质粒的质量比为(1~4):(1~3):(1~2),所述用于表达核酸酶Cas9的质粒:pGL3-U6-PD1质粒的质量比(1~2):(1~2)。
上述联合免疫基因抑制高危型HPV表达的sgRNA在制备用于抗HPV16+人宫颈癌高危型人乳头瘤病毒药物或者治疗人宫颈癌药物中的应用。
上述联合免疫基因抑制高危型HPV表达的基因敲除载体或载体组合物在制备用于抗HPV16+人宫颈癌高危型人乳头瘤病毒药物或者治疗人宫颈癌药物中的应用。
本发明的有益效果体现在:
本发明提出了适合CRISPR-Cas9靶向剪辑的人PD-1基因的sgRNA序列,其与适合适合CRISPR-Cas9靶向剪辑的人乳头瘤病毒(HPV)16型基因,可用于构建表达抑制高危型人乳头瘤病毒16型基因(P、E6、E7)和人PD-1基因的sgRNA质粒载体,共同转入荷HPV16+移植瘤小鼠体内可以明显降低HPV16的表达,并抑制肿瘤的生长。本发明制备的基因表达载体方法步骤简单、sgRNA靶向性好,CRISPR-Cas9系统的敲除效率高。
本发明制备的特异性靶向高危型HPV16型和PD-1基因的sgRNA载体,不仅能够精确靶向剪接高危型HPV16型和PD-1基因,高效降低高危型HPV16型的基因表达,联合应用可以明显抑制肿瘤的生长,既显示出免疫基因方法治疗恶性肿瘤的优效性,还将成为制备靶向治疗高危型人乳头瘤病毒16+宫颈癌新型药物的核心成分。
本发明可应用CRISPR/Cas9快速、简便、高效、特异性剪接高危型宫颈癌HPV16基因和人PD-1基因的方法,并为将来采用叶酸与细胞穿膜肽等共修饰包裹靶向HPV16基因的sgRNA脂质体及其他给药方式奠定了物质基础,展现出可能有效清除人正常宫颈上皮内持续存在高危型HPV16型,并能解决目前宫颈癌治疗中存在问题的明显优势。本发明具有(1)概念新,利用CRISPR/Cas9剪辑高危型HPV16抑制其病毒复制,继而预防宫颈癌的发生;(2)效率高,在体内、外实验可明显降低E6,E7的表达,可以清除高危型HPV的感染,预防肿瘤的发生,还可抑制肿瘤的生长;(3)多靶点,可以同时敲除修饰多个靶点基因的突出特点。
附图说明
图1为Cas9实现定点切割导致DNA及双链断裂过程示意图;
图2为sgRNA/Cas9介导的HPV特异性切割造成HPV16E6(a)、E7(b)表达变化情况;
图3为MTT比色法检测刺激指数的结果;
图4为观察肿瘤及体积的变化的结果;1、对照组;2、单独敲低PD1组;3、单独敲低HPV16组;4、联合敲低PD1+HPV16组;
图5为用流式细胞仪计数小鼠体内CD8+T细胞的变化情况;1、对照组;2、单独敲低PD1组;3、单独敲低HPV16组;4、联合敲低PD1+HPV16组;
图6为载体PgL3-U6-sgRNA的结构;
图7为载体pST1374-NLS-flag-cas9-ZF的结构。
具体实施方式
下面结合附图和实施例对本发明作详细说明。
参见图1,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技术,以高危型HPV16+的人宫颈癌细胞和荷HPV16+移植瘤小鼠模型为研究对象,采用靶致病基因HPV16,以及协同靶向肿瘤免疫调节分子PD-1的基因编辑策略。首先,通过体内外筛选针对HPV16基因的gRNA序列,实现HPV16基因的有效敲除;然后,通过体内外筛选针对PD-1的gRNA序列,通过共转染实现多基因协同编辑效应,进而考察联合干预不同靶点的治疗策略是否对HPV16+小鼠移植瘤治疗具有“1+1>2”的协同效应。本发明是采用cas9能够实现多重基因敲除的优势,采取特异性靶向“单一基因”或“协同作战”的方法,针对疾病的内外因涉及的主要分子靶点同时进行干预,能够联合发挥被动清除病毒致癌基因和主动免疫的治疗作用,从而为人宫颈上皮高危型HPV16基因清除和宫颈癌的有效治疗提供新的策略。
本发明在直接靶向剪接高危型HPV16基因的基础上,利用CRISPR/Cas9联合特异性敲除HPV16和人PD-1基因的方法,对小鼠HPV16+的移植瘤实施免疫基因治疗的策略。首先分别设计和合成特异性靶向HPV16基因的sgRNA1和特异性靶向PD-1基因的sgRNA2,将sgRNA1和sgRNA2与线性的pGL3-U6-sgRNA质粒连接成pGL3-U6-HPV16 sg质粒和pGL3-U6-PD1质粒。在验证将pGL3-U6-HPV16 sg质粒和pST1374-NLS-flag-Cas9-ZF质粒转入HPV16+的Siha细胞后可以明显抑制HPV16基因表达的基础上,将pGL3-U6-HPV16 sg质粒和pGL3-U6-PD1质粒联合转入荷HPV16+移植瘤的小鼠体内,获得高效敲除HPV16基因表达并明显抑制移植瘤生长的效果。
一、靶向HPV16的sgRNA1和靶向PD1的sgRNA2寡核苷酸的设计和选择,如无特殊说明,文中sgRNA1是靶向HPV16的序列;sgRNA2是靶向人PD-1的序列。
1、在HPV16基因上选择5’-GGN(19)GG的序列,如果没有5’-GGN(19)GG的序列,5’-GN(20)GG或者5’-N(21)GG也可以。PD-1基因上选择5’-GGN(19)GG的序列,如果没有5’-GGN(19)GG的序列,5’-GN(20)GG或者5’-N(21)GG也可以。
2、sgRNA1在HPV16的靶向位点分别位于HPV16的启动子,E6和E7区域。sgRNA2在人PD-1基因上的靶点位于基因的外显子,这样更容易引起片段的缺失或移框突变,从而达到基因完全失活的目的。sgRNA2在人PD-1基因上的靶位点位于不同的各种剪切形式的共有外显子上。
3、在UCSC数据库中用BLAT或NCBI数据库中用BLAST,确定sgRNA1和sgRNA2的靶序列是否唯一,减少潜在的脱靶位点。
4、如果用针对HPV16不同区域的sgRNA1来实现联合靶向HPV16基因,即可更有效的敲低HPV基因。
5、如果用靶向HPV16不同区域的三个sgRNA1联合靶向人PD-1基因的sgRNA2,能更有效的抑制小鼠体内荷HPV16+移植瘤的生长。
二、构建sgRNA的寡聚核苷酸双链
根据选择的sgRNA1s和sgRNA2s,在其5’加上CCGG得到正向寡核苷酸(Forwardoligo),如果序列本身在5’端已经有1或者2个G,那么就对应的省略1或者2个G;根据选择的sgRNA,获得其对应DNA的互补链,并且在其5’加上AAAC得到反向寡核苷酸(Reverse oligo);分别合成上述正向寡核苷酸和反向寡核苷酸,将合成的sgRNA寡聚核苷酸的forward oligo和reverse oligo成对退火。
退火反应体系如下:
在PCR仪中按照以下touch down程序运行:95℃,5min;95-85℃at-2℃/s;85-25℃at-0.1℃/s;hold at 4℃
退火之后形成可以连入U6真核表达载体的双链,序列如下:
Forward oligo:5’-CCGGNNNNNNNNNNNNNNNNNN
Reverse oligo:NNNNNNNNNNNNNNNNNNCAAA-5’。
三、sgRNA寡聚核苷酸质粒的构建
1.线性化pGL3-U6-sgRNA质粒(如图6所示,Addgene(Cambridge,MA,USA))酶切体系和条件如下:2μg pGL3-U6-sgRNA(400ng/μL);1μL CutSmart Buffer;1μL BsaI(NEB).补水至50μL,37℃孵育3-4小时;酶切完成后用AxyPrep PCR Clean up Kit(AP-PCR-250)纯化回收至20-40μL灭菌水中。
2.将退火的sgRNA1寡聚核苷酸双链和退火的sgRNA2寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒分别连接,获得pGL3-U6-HBV16 sg质粒和pGL3-U6-PD1质粒。
3.转化大肠杆菌感受态细胞,并涂Amp+平板(50μg/mL)。
4.用SEQ.ID.NO.1的通用引物U6测序的方法鉴定阳性克隆。
5.37℃摇床摇菌过夜培养阳性克隆并用AxyPrep Plasmid Miniprep Kit(AP-MN-P-250)抽提pGL3-U6-HPV16 sg质粒和pGL3-U6-PD1质粒。
四、转染人宫颈癌SiHa(HPV16+)细胞
1.按Lipofectamine 2000Transfection Reagent(Invitrogen,11668-019)的操作手册,将分别带有对应HPV16基因的sgRNA寡聚核苷酸的pGL3-U6-HPV16 sg质粒(单独靶向HPV16启动子、E6或E7)与pST1374-NLS-flag-Cas9-ZF质粒(结构如图7所示,Addgene(Cambridge,MA,USA)混匀,共转染SiHa细胞。
2.为提高基因敲除效率,在靶向HPV16基因的sgRNA寡核苷酸设计、选择和合成之后,将靶向HPV16基因的sgRNA1寡聚核苷酸(即靶向HPV启动子、E6或E7的sgRNA)分别与线性化pGL3-U6-sgRNA质粒连接获得含靶向HPV16启动子,E6和E7的sgRNA寡聚核苷酸的载体pGL3-U6-HPV16 sg,按如下操作转染SiHa细胞:按照Lipofectamine 2000TransfectionReagent(Invitrogen,11668-019)的操作手册,分别将两组(第一组:靶向HPV16启动子区的sgRNA1-P寡聚核苷酸的载体pGL3-U6-HPV16-P sg(对应的sgRNA为序列表的SEQ.ID.NO.6)和靶向HPV16 E6区的sgRNA1-E6寡聚核苷酸的载体pGL3-U6-HPV-E6 sg(对应的sgRNA参照序列表中的SEQ.ID.NO.7);第二组:靶向HPV16启动子区的sgRNA1-P寡聚核苷酸的载体pGL3-U6-HPV16-P sg和靶向HPV16 E7区的sgRNA1-E7寡聚核苷酸的载体pGL3-U6-HPV16-E7sg(对应的sgRNA参照序列表中的SEQ.ID.NO.8),每组内的两种质粒混合比例按1:1(病人类型不同,对应的靶点的量也可调整),混合质粒与pST1374-NLS-flag-Cas9-ZF质粒混匀,共转染SiHa细胞。
在转染后二天,提取细胞RNA,用RT-PCR的方法检测E6、E7基因的表达。
参见图2,对照组(gRNA empty vector)是转入了没有切割活性的sgRNA载体pGL3-U6-HPV16 sg(对应的sgRNA为SEQ.ID.NO.2),处理组是单独(图2a及图2b中E6、E7)或者联合加入(图2a及图2b中Promoter+E6、Promoter+E7)针对HPV16的sgRNA载体pGL3-U6-HPV16sg。用RT-PCR方法检测单独或者联合sgRNA1转染细胞后,E6,E7基因的表达情况。图2中的Blank是不加任何质粒的细胞,图2结果表明:相比较空白组和对照组,单独敲除可以有效降低E6或E7的表达,联合敲除相比较单独敲除有更好的效果。
五、敲除人PD-1基因,采用MTT比色法检测其刺激指数
1.DC细胞的制备和培养
(1)断颈处死小鼠,浸没在75%酒精消毒5min,无菌条件下分离取出小鼠两股骨和胫骨,剔除肌肉和筋膜,于75%酒精消毒30s,置于生理盐水中备用;
(2)用剪刀剪去胫骨和股骨的两端,暴露骨髓管,用10mL针筒吸取生理盐水,分别插入骨髓两端,冲出骨髓于50mL离心管;
(3)于1500rpm离心10min,弃上清,沉淀加入2ml红细胞裂解液,吹打均匀作用约2min,再加入约40mL生理盐水沉淀混匀终止;
(4)于1000rpm离心10min,弃上清,加入新鲜1640培养液5mL,镜下计数细胞量,以1x106mL细胞密度种入6孔培养板,每孔2mL;
(5)置37℃、5%CO2培养箱培养2h,生理盐水轻轻洗去未贴壁细胞;
(6)加入1640培养液,并加入rmGM-CSF(20ng/mL),rmIL-4(20ng/mL),非必需氨基酸(1:100),丙酮酸钠(1:100),FCS(10%),每孔约2mL,最后置于培养箱培养,隔天半量换液。然后分为对照组和转染PD-1-CRISPR/Cas9组(带有对应PD-1的sgRNA寡聚核苷酸的pGL3-U6-PD1 sg质粒与pST1374-NLS-flag-Cas9-ZF质粒混匀)。
2.T淋巴细胞分离和纯化
(1)无菌条件下分离出小鼠脾脏,用生理盐水冲洗干净,用剪刀反复剪碎,经200目细胞滤器过滤,去掉血管及一些结缔组织,收集细胞悬液至50ml离心管;
(2)于1500rpm离心15min,弃上清,沉淀加入4ml红细胞裂解液(NH4CI),吹打均匀作用约2min,再加入约40mL生理盐水混匀终止;
(3)于1000rpm离心10min,弃上清,重悬于RPMI-1640培养液(含10%FCS)备用;
(4)尼龙毛用0.2M的HCI侵泡过夜,用双蒸水洗净HCI,烘干后仔细撕开梳整,取约0.3g均匀装入5mL的一次性注射器,高压灭菌;
(5)用预温至37℃的RPMI-1640培养基润洗尼龙毛3遍,再浸润后置于37℃培养箱静止1h;
(6)放出注射器里的液体,立即加入2x107/mL细胞悬液(置于含约10%FCS的RPMI1640培养基中)约2mL,再加约0.5mL含10%FCS的RPMI-1640培养基,置于培养箱静止1h;
(7)收集滤液,速度控制在1滴/S,然后用预温至37℃、含10%FCS的RPMI1640培养基15-20mL洗柱3遍,收集洗液,同样速度控制在1滴/s;
(8)收集到的液体于1000rpm离心10min,得到的沉淀即为T淋巴细胞,将部分T细胞冻存,用于后续的实验。
3、MTT比色法检测刺激指数
(1)将上述冻存的T细胞复苏,重悬于含10%胎牛血清的RPMI-1640完全培养基;
(2)取步骤(1)培养到第9天的DC细胞,分别用完全RPMI-164培养液悬浮为1×106个/mL,加入丝裂霉素25μg/mL,37℃水浴30min去增殖后,用不完全RPM I-1640培养液洗涤3次,再用含10%胎牛血清的RPMI-1640完全培养基悬浮细胞;
(3)按1∶10、1∶20、1∶50的DC与T细胞比例加入96孔圆底培养板中,终体积为200μL,每组各设3复孔,在37℃、5%CO2培养箱培养4d;
(4)培养结束前4h,每孔加入5mg/mL的MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)15μL,继续在37℃、5%CO2培养箱培养;
(5)4h后弃去全部上清(1000r Pmin离心5min),每孔加入DMSO 100μL,充分振荡10min,测定波长490nm的吸光度(A)值,结果以3孔均值表示。
参见图3,对照组是与pST1374-NLS-flag-Cas9-ZF质粒共同转入的没有切割活性的sgRNA载体pGL3-U6-PD1 sg(对应的sgRNA为SEQ.ID.NO.3,gRNA-empty vector),处理组是联合加入针对PD1的sgRNA载体pGL3-U6-PD1 sgl和pGL3-U6-PD1 sg2(对应的sgRNA为SEQ.ID.NO.4和SEQ.ID.NO.5,gRNA-PD1-(sg1+sg2)组,1:1,病人类型不同,对应的靶点的量也可调整),以及仅加一种针对PD1的sgRNA载体pGL3-U6-PD1 sgl(gRNA-PD1-sg1组)或pGL3-U6-PD1 sg2(gRNA-PD1-sg2组)。在同一靶效比的条件下,CRISPR/Cas9-PD1组DC刺激T淋巴细胞增殖的能力明显强于对照组。
六、建立小鼠荷HPV16+移植瘤模型,观察联合靶向剪接HPV16和PD1基因抑制肿瘤的效果
1、建立小鼠荷HPV16+移植瘤模型
培养小鼠TC-1细胞,按每个注射点2×106个细胞的数量接种Balb/c小鼠背部皮下,总共20只。
2、分组体内联合免疫基因治疗实验
待移植瘤长至2mm3大小,将其分为分为4组,即对照组、敲低HPV16组、敲低PD1组以及联合敲低PD1+HPV16组。采用电穿孔注射靶向HPV16和人PD1基因质粒的方法,每组给的剂量分别为对照组:40μg pST1374-NLS-flag-Cas9-ZF+20μg空gRNA;敲低HPV16组:40μgpST1374-NLS-flag-Cas9-ZF+10μg pGL3-U6-HPV16-P sg+10μg pGL3-U6-HPV16-E6 sg+10μg pGL3-U6-HPV16-E7 sg;单独敲除人PD1组:40μg pST1374-NLS-flag-Cas9-ZF+10μgpGL3-U6-PD1 sg1+10μg pGL3-U6-PD1 sg2;联合敲低PD1+HPV16组:40μg pST1374-NLS-flag-Cas9-ZF+10μg pGL3-U6-HPV16-P sg+10μg pGL3-U6-HPV16-E6 sg+10μg pGL3-U6-HP16V-E7+10μg pGL3-U6-PD1 sg1+10μg pGL3-U6-PD1 sg2。
3、观察移植瘤生长与HPV16基因表达改变
于给药后3d/7d/14d分别用游标卡尺测量抑制瘤大小(3次),计算、观察移植瘤变化情况,在给药后于22d脱臼处死小鼠,称量移植瘤,并测定HPV16E6和E7基因的表达改变。参见图4,相比较于对照组,单独敲除PD1或HPV有很好的效果;联合敲除PD1+HPV相比较对照组和单独敲除组有更好的抑制肿瘤生长的作用。
4、测定CD8+T细胞数量变化
处死小鼠时留取其脾细胞,分离淋巴细胞;调整收集的淋巴细胞密度至3×106/mL,取1μg anti-CD8-FITC对应106细胞,冰浴、避光染色30min后,用流式细胞术测定CD8+T细胞数量变化。参见图5,相比较于对照组和单独敲除组,联合敲除PD1+HPV可以显著提高体内CD8+T细胞的数量。
Claims (10)
1.联合免疫基因抑制高危型HPV表达的sgRNA,其特征在于:该抑制高危型HPV表达的sgRNA包括在CRISPR-Cas9特异性修饰人PD-1基因中可特异性靶向人PD-1基因的sgRNA,该sgRNA的序列如SEQ.ID.NO.4或SEQ.ID.NO.5所示。
2.如权利要求1所述联合免疫基因抑制高危型HPV表达的sgRNA,其特征在于:所述抑制高危型HPV表达的sgRNA还包括与所述可特异性靶向人PD-1基因的sgRNA联用的可特异性靶向高危型HPV16的sgRNA,该sgRNA的序列如SEQ.ID.NO.6、SEQ.ID.NO.7或SEQ.ID.NO.8所示。
3.联合免疫基因抑制高危型HPV表达的基因敲除载体,其特征在于:该基因敲除载体选自重组质粒pGL3-U6-PD1 sgl、pGL3-U6-PD1 sg2中的一种,pGL3-U6-PD1 sgl由序列如SEQ.ID.NO.4所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-PD1 sg2由序列如SEQ.ID.NO.5所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.4或SEQ.ID.NO.5所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内,或者,该基因敲除载体为构建于用于表达核酸酶Cas9的质粒基础上的重组质粒,所述用于表达核酸酶Cas9的质粒的多克隆位点内分别插入有如SEQ.ID.NO.4、SEQ.ID.NO.5所示序列中的一种或两种以及如SEQ.ID.NO.6、SEQ.ID.NO.7、SEQ.ID.NO.8所示序列中的一种、两种或三种。
4.联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,其特征在于:该组合物包括pGL3-U6-PD1质粒,所述pGL3-U6-PD1质粒选自重组质粒pGL3-U6-PD1 sgl、pGL3-U6-PD1sg2中一种或两种,pGL3-U6-PD1 sgl由序列如SEQ.ID.NO.4所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-PD1 sg2由序列如SEQ.ID.NO.5所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.4或SEQ.ID.NO.5所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内。
5.如权利要求4所述联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,其特征在于:所述pGL3-U6-PD1 sgl与pGL3-U6-PD1 sg2的质量比为(1~2):(1~2)。
6.如权利要求4所述联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,其特征在于:所述组合物还包括pGL3-U6-HPV16 sg质粒,所述pGL3-U6-HPV16 sg质粒选自重组质粒pGL3-U6-HBV16-P sg、pGL3-U6-HPV-E6 sg、pGL3-U6-HPV16-E7 sg中的一种、两种或三种,pGL3-U6-HBV16-P sg由序列如SEQ.ID.NO.6所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-HPV-E6 sg由序列如SEQ.ID.NO.7所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,pGL3-U6-HPV-E7 sg由序列如SEQ.ID.NO.8所示的sgRNA的寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得,通过连接SEQ.ID.NO.6、SEQ.ID.NO.7或SEQ.ID.NO.8所示序列插入至pGL3-U6-sgRNA质粒的多克隆位点内。
7.如权利要求6所述联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,其特征在于:所述pGL3-U6-HPV16 sg质粒与pGL3-U6-PD1质粒的质量比为(1~3):(1~2);所述pGL3-U6-HPV16 sg质粒采用pGL3-U6-HPV16-P sg与pGL3-U6-HPV-E6 sg或pGL3-U6-HPV16-E7 sg联用,pGL3-U6-HPV16-P sg与pGL3-U6-HPV-E6 sg或pGL3-U6-HPV16-E7 sg的质量比为(1~2):(1~2);或者,所述pGL3-U6-HPV16 sg质粒采用pGL3-U6-HBV16-P sg与pGL3-U6-HPV-E6 sg以及pGL3-U6-HPV16-E7 sg联用,pGL3-U6-HPV16-P sg:pGL3-U6-HPV-E6 sg:pGL3-U6-HPV16-E7 sg的质量比为(1~2):(1~2):(1~2)。
8.如权利要求4、6中任一权利要求所述联合免疫基因抑制高危型HPV表达的基因敲除载体组合物,其特征在于:所述组合物还包括用于表达核酸酶Cas9的质粒,所述用于表达核酸酶Cas9的质粒:pGL3-U6-HPV16 sg质粒:pGL3-U6-PD1质粒的质量比为(1~4):(1~3):(1~2),所述用于表达核酸酶Cas9的质粒:pGL3-U6-PD1质粒的质量比(1~2):(1~2)。
9.如权利要求1、2中任一权利要求所述联合免疫基因抑制高危型HPV表达的sgRNA在制备用于抗HPV16+人宫颈癌高危型人乳头瘤病毒药物或者治疗人宫颈癌药物中的应用。
10.如权利要求4、6、8中任一权利要求所述联合免疫基因抑制高危型HPV表达的基因敲除载体组合物在制备用于抗HPV16+人宫颈癌高危型人乳头瘤病毒药物或者治疗人宫颈癌药物中的应用。
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