CN117511946A - 特异性靶向scgII基因的sgRNA及其应用 - Google Patents
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Abstract
本发明属于生物技术领域,涉及特异性靶向scgII基因的sgRNA及其应用,本申请通过构建一组特异性的sgRNA,然后根据该靶位点,设计重组质粒,并将质粒整合到腺病毒载体中,通过sgRNA的靶向牵引,实现了对scgII基因的有效敲除,本申请构建的sgRNA有2条,与单条sgRNA相比,对基因的敲除效率更高;经过动物实验,构建好的腺病毒定向敲除小鼠的scgII基因后,我们发现:scgII基因敲除后不影响正常血管的生长,且能抑制病理性血管的生长,降低因病理性新生血管导致的致盲眼底疾病的发病率。对眼部疾病起到了预防和辅助治疗的作用,是一种针对眼部疾病的新型基因疗法。
Description
【技术领域】
本发明涉及生物技术领域,特别涉及特异性靶向scgII基因的sgRNA及其应用。
【背景技术】
常见的致盲眼底疾病均与异常血管生长有关,而血管的生长需要血管内皮生长因子(VEGF)表达上调。目前,研制的多种抗-VEGF药物上市,进入临床,应用于老年性黄斑变性、黄斑水肿等与VEGF升高相关性的疾病,得到了良好的治疗效果。但抗-VEGF药物无差别的抑制生理血管和病理血管的生长,抑制生理血管的生长会影响正常血管的发育。寻找新型的抑制病理性血管生长的药物是基础研究的重要方向,也是临床治疗的进一步需求。
现有技术中有学者研究发现:分泌粒蛋白(scg)蛋白家族与新生血管有一定的关联,但是,蛋白家族中有很多蛋白,具体哪种蛋白对视网膜的新生血管有影响?蛋白表达能促进血管生长还是抑制血管生长,并没有进行深入的研究,随着分子生物学技术的发展,基因治疗技术被应用于临床治疗。视网膜下腔注射或玻璃体内注射腺病毒进行基因治疗的手段已经成熟运用。
为了进一步研究分泌粒蛋白(scg)对视网膜的影响,我们可以利用腺病毒把携带有CRISPR敲除***的质粒带入小鼠视网膜,对造模小鼠的视网膜细胞基因进行靶向敲除,以获得能够对小鼠视网膜生理性血管生长有促进作用,对小鼠病理性血管生长有抑制作用的蛋白或病毒载体,为后续的基因疗法提供强有力的有利依据。
【发明内容】
鉴于研究进展,有必要进一步研究分泌粒蛋白(scg)对视网膜的影响,我们可以利用腺病毒把携带有CRISPR敲除***的质粒带入小鼠视网膜,对造模小鼠的视网膜细胞基因进行靶向敲除,以获得能够对小鼠视网膜生理性血管生长有促进作用,对小鼠病理性血管生长有抑制作用的蛋白或病毒载体,为后续的基因疗法提供强有力的有利依据。
为达到上述目的,本发明所采用的技术方案是:
特异性靶向scgII基因的sgRNA,所述的sgRNA由sgRNA1和sgRNA2组成,所述sgRNA1的核酸序列如SEQ ID NO.1所示;所述sgRNA2的核酸序列如SEQ ID NO.2所示。
本发明还包括一种重组载体,所述重组载体包含如权利要求1所述的sgRNA。
进一步的,所述重组载体由工具载体PMT649与目的基因片段连接而得;所述目的基因片段的核酸序列如SEQ ID NO.3所示。
本发明还包含所述的重组载体的腺病毒载体。
本发明还包括所述的腺病毒载体在制备促进生理性血管生长且抑制病理性血管生长药物上的应用。
进一步的,所述血管为视网膜血管。
本发明还包括所述的腺病毒载体在制备治疗、预防和辅助治疗眼底疾病药物上的应用。
本发明还包括所述重组载体的制备方法,所述方法为:将连接有2个sgRNA的目的基因片段***线性化的工具载体PMT649中,得到重组质粒PSE5862;所述目的基因片段的核酸序列如SEQ ID NO.3所示;所述目的基因片段的扩增引物对为Primer1/Primer2,其中,Primer1的核酸序列如SEQ ID NO.4所示,Primer2的核酸序列如SEQ ID NO.5所示。
本发明还包括所述腺病毒载体的制备方法,所述方法为:将连接有2个sgRNA的目的基因片段***线性化的工具载体PMT649中,得到重组质粒PSE5862;然后将重组质粒PSE5862转化到感受态细胞中,得到转化子,抽取阳性转化子质粒包装至腺病毒中得到所述腺病毒载体;所述目的基因片段的核酸序列如SEQ ID NO.3所示;所述目的基因片段的扩增引物对为Primer1/Primer2,其中,Primer1的核酸序列如SEQ ID NO.4所示,Primer2的核酸序列如SEQ ID NO.5所示。
本发明具有如下有益效果:
本申请通过构建一组特异性的sgRNA,然后根据该靶位点,设计重组质粒,并将质粒整合到腺病毒载体中,通过sgRNA的靶向牵引,实现了对scgII基因的有效敲除,本申请构建的sgRNA有2条,与单条sgRNA相比,对基因的敲除效率更高;经过动物实验,构建好的腺病毒定向敲除小鼠的scgII基因后,我们发现:scgII基因敲除后不影响正常血管的生长,且能抑制病理性血管的生长,降低因病理性新生血管导致的致盲眼底疾病的发病率。对眼部疾病起到了预防和辅助治疗的作用,是一种针对眼部疾病的新型基因疗法,为今后的眼部疾病提供了一种更简单高效的疗法,为该重组病毒开发成预防眼部疾病的药物提供了理论基础。
【附图说明】
图1:为工具载体PMT649结构示意图;
图2:为重组质粒PSE5862结构示意图;
图3为目的基因的PCR检测图;图中,M为2kb DNAladder Marker,从上至下依次为:2Kb、1.5kb,1Kb、750bp、500bp、250bp,100bp;
图4:为工具载体(PMT649)酶切后的线性载体;图中,M为DNAladder Marker,从上至下依次为:10kb、8Kb、6kb、5Kb、4kb、3.5Kb、3Kb、2.5kb、2Kb、1.5kb、1Kb、750bp、500bp、250bp。
图5:为Marker电泳图像示意图;
图6:为载体构建示意图;
图7:为阳性菌落的PCR检测图;图中,M为2kb DNAladder Marker,从上至下依次为:2Kb、1.5kb,1Kb、750bp、500bp、250bp,100bp;1-4分别为阳性转化子;
图8:为OIR小鼠的视网膜铺片染色图;
图9:为OIR小鼠视网膜血管染色图;
图10为OIR小鼠视网膜新生血管统计图。
【具体实施方式】
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施的限制。
实施例1:
重组载体的构建:
从基因库中找到scgII基因的基因序列,根据该序列分析设计出针对该基因的sgRNA序列,其中,sgRNA序列由sgRNA1和sgRNA2组成,sgRNA1的序列为5’-GGAGCTAAGGCGTACCGACT-3’(SEQ ID NO.1);sgRNA2的序列为5’-GCCTCTACCATACCACGACC-3’(SEQ IDNO.2),重组载体中必须同时包含有上述两个序列。
构建上述重组载体,其构建方法如图1和图2所示:将含有2个sgRNA的目的基因片段***线性化的工具载体PMT649中,得到重组质粒PSE5862;所述目的基因片段的核酸序列如SEQ ID NO.3所示;所述目的基因片段的扩增引物对为Primer1/Primer2,其中,Primer1的核酸序列为:5′-TGTGGAAAGGACGAAACACCGGAGCTAAGGCGTACCGACTGTTTTA GAGCTAGAAATAG-3′(SEQ ID NO.4);Primer2的核酸序列为:5′-TGCTATTTCTAGCTC TAAAACGGTCGTGGTATGGTAGAGGCGGGAAAG-3′(SEQ ID NO.5)。
上述PCR扩增的反应体系共50μL为:5×Buffer 10μl、2.5mM dNTP 4μl、10μMPrimer1、Primer2各1μl、DNATemplate 1μL、PrimeSTAR 0.5μl、H2O 32.5μl;PCR反应程序为98℃预变性3min;98℃变性10s,55℃退火15min,72℃延伸1min总共45个循环,72℃10min延长;
扩增得到的基因片段如序列表SEQ ID NO.3所示,目的基因扩增出来的电泳图如图3所示,图中,M为2kb DNAladder Marker,从上至下,条带依次为:2Kb、1.5kb,1Kb、750bp、500bp、250bp,100bp;a为扩增出的条带,从图中可见,在584bp处有扩增条带,说明目的基因已被扩增。
其中,本实施例的载体具体酶切方法为:以BsmBI-V2酶对工具载体(PMT649)进行酶切,得到9417bp线性载体;回收的载体片段如图4所示,图中,M为DNAladder Marker,从上至下依次为:10kb、8Kb、6kb、5Kb、4kb、3.5Kb、3Kb、2.5kb、2Kb、1.5kb、1Kb、750bp、500bp、250bp;在9417bp处有条带,说明该片段为载体片段,载体电泳图谱如图4所示。
其中,本实施例的载体连接具体方法为:将目的基因片段(SEQ ID NO.3)与线性载体以摩尔比2:1加到试管中进行连接反应,混匀后在42℃孵育30min然后转移到冰上。具体如表1所示:
表1不同实验组的连接比例
试剂 | 阳性对照(μl) | 自连对照(μl) | 连接组(μl) |
胶回收后的目的基因片段 | 4 | 4 | 4 |
线性化的表达载体 | 1 | 1 | 1 |
无缝克隆反应液 | 15 | 0 | 15 |
ddH2O | Upto20 | Upto20 | Upto20 |
上述重组质粒PSE5862由生工生物工程(上海)股份有限公司构建,工具载体PMT649由该公司提供。
实施例2:
本实施例为将实施例1的载体包装至腺病毒中,具体为:
(1)将实施例1构建的重组质粒PSE5862转移到感受态细胞DH5α中。
(2)取一支感受态细胞(每管100μl,-80℃保存)于冰上,待溶解后加入10μl连接液,轻轻旋转以混匀内容物,在冰中放置30分钟;将管放到预加温到42℃的恒温水浴锅中热激90秒;快速将管转移到冰浴中,使细胞冷却2-3分钟;每管加900μl LB培养液,然后将管转移到37℃摇床上,温育1小时使细菌复苏;取恰当量的转化菌液涂布于LB琼脂平板上(含表达载体相应的抗生素);倒置平皿,于恒温培养箱中37℃培养,16小时;菌落PCR检测阳性克隆,并把阳性转化子送测序,检测载体构建情况。如果菌落鉴定出978bp的片断则说明该菌落是阳性克隆,如菌落鉴定得到0bp的片断则说明菌落为阴性克隆;菌落鉴定的引物对为Primer ID(+)/Primer ID(-),其中,Primer ID(+)5’-TACGATACAAGGCTGTTAGAGAG-3’(SEQID NO.6);Primer ID(-)5’-CGGGCCATTTACCGTAAGTTATG-3’(SEQ ID NO.7);
上述PCR扩增的反应体系共20μL为:10×Buffer 2μl、2.5mM dNTP 1.6μl、10μM上、下游引物各0.8μl、DNATemplate 1μL、Taq酶0.1μl、H2O 13.7μl;PCR反应程序为94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min总共30个循环,72℃10min延长。
采用上述鉴定方法扩增得到的PCR图如图7所示,图中,M为2kb DNAladderMarker:2kb,1.5kb,1Kb,750bp,500bp,250bp,100bp;1~4为挑取PSE5862的4个转化子,图中可见,转化子在978bp处都有扩增条带,说明1-4均为阳性转化子。
(3)将步骤2鉴定为阳性的转化子测序后,测序正确,再抽提阳性转化子的质粒,将其包装到腺病毒中,得到包含重组载体质粒的腺病毒。
实施例3:
将实施例2的腺病毒进行动物验证:
1、实验分组
将新生小鼠随机分为C57BL/6J小鼠常氧组和C57BL/6J小鼠OIR组。
2、OIR小鼠模型的建立
将P7小鼠与母鼠共同置于体积分数(75±2)%高氧氧箱中饲养至P12以建立OIR模型,注意观察母鼠饮水进食及活动状态,必要时可在P10更换哺乳母鼠,以保证乳鼠正常生长。在P12将母鼠和小鼠共同返回常氧中饲养,常氧组小鼠则始终在正常空气中饲养。将体重范围在6-7.5g范围内的P17小鼠纳入使用,以保证视网膜病变程度的一致性。
3、P12玻璃体腔注射
(1)术前准备:术前将高氧氧箱中的P12 OIR小鼠与母鼠返回常氧中1h。对P12 OIR小鼠行1%戊巴比妥麻醉,用棉签蘸取70%酒精清洁眼睑及眼周皮肤,双眼点2.5%盐酸去氧肾上腺素滴眼液散瞳;
(2)玻璃体腔注射给药:在体式显微镜下左手持显微有齿镊轻轻夹住鼻侧球结膜,右手持33G汉密尔顿微量注射针于角膜缘外1mm处垂直进针穿孔,待瞳孔区见针尖后缓缓匀速推药1μl,左眼注射PBS,右眼注射病毒;
(3)注射完毕后,缓慢拔出针头,术后给予氧氟沙星眼膏点眼,防止眼部感染发生。
4、视网膜铺片染色
(1)取材:利用颈椎脱臼法处死小鼠,迅速用眼科剪剪开内外眦皮肤和周围组织,将弯镊沿颞侧眶壁垂直进入后再平行向鼻侧走形,至眼球下方夹住视神经,水平向上提起,轻柔完整地摘取眼球,将其置于4%PFA溶液中室温固定45min,然后将眼球转移至1x PBS(pH7.4)溶液中。接着在显微镜下用眼科剪和眼科镊去除眼周组织,沿角膜缘剪开眼球,去除眼前节、晶状体和玻璃体,从角巩膜缘处分离视网膜与脉络膜和巩膜,取出完整的神经视网膜组织,最后移除残留的玻璃体血管;
(2)固定:将视网膜置于4%PFA溶液中室温固定1h;
(3)通透封闭:将视网膜置于1ml用5%BSA配制的0.5%Triton X-100中,4℃通透封闭过夜;
(4)敷一抗:用封闭通透液配制一抗(CD31),配制比例为CD31:封闭通透液=1:500,配置好一抗后4℃孵育过夜。
(5)洗涤:隔天1x PBS室温摇床清洗视网膜3次,10min/次;
(6)敷二抗:用封闭通透液配制二抗(555),配制比例为555:封闭通透液=1:200,配置好二抗后37℃摇床孵育2h。
(7)洗涤:2h后1x PBS室温摇床清洗视网膜3次,10min/次;
(8)剪切:在显微镜下进一步清理残留的玻璃体血管,从4个径线剪开视网膜,剪开深度大约为视网膜周边至视神经的内2/3处;
(9)铺片及封片:将视网膜平铺在载玻片上,50%甘油封片;
(10)观察:使用荧光显微镜观察视网膜新生血管区、无血管区的面积并拍照用于定量分析。得到的结果如图8-10所示,其中,图8为OIR小鼠视网膜铺片荧光染色图,图中,OIR为OIR造模组,OIR+PBS为OIR造模鼠的玻璃体腔中注射PBS,OIR+V为OIR造模鼠的玻璃体腔中注射腺病毒AAV,从图中可见,对OIR造模小鼠进行玻璃体腔注射,左眼注射1μL的PBS,右眼注射1μL的腺相关病毒(AAV)。AAV带有外源质粒,质粒上含有敲除scgII基因的CRISPR***及标记基因GFP。视网膜铺片上检测到绿色荧光说明AAV成功侵入视网膜细胞并表达GFP基因。注射了病毒的视网膜上检测到绿色荧光,而OIR组和注射PBS组未检测到绿色荧光,符合实验预期结果。
图9为OIR小鼠视网膜血管染色图,图中圈起来的地方即为新生血管;经过对病理性新生血管的分析结果如图10所示:OIR小鼠的病理性血管面积占整张视网膜的16.81%±1.97%,注射了PBS的病理性血管占比为14.68%±1.01%,注射了AAV的病理性血管占比为6.52%±1.24%。OIR组和PBS注射组间没有显著差异,OIR组和AAV组间差距极显著(mean±SEM,n=3or 4,one-wayANOVAtest,***p<0.01,ns for not significant)。
由此说明,采用CRISPR/Cas9的方法敲除小鼠的scgII基因,能有效促进眼部血管新生,抑制病理性血管的生长,为后期将该腺病毒载体在制备促进生理性血管生长且抑制病理性血管生长药物上的应用或在制备治疗、预防和辅助治疗眼底疾病药物上的应用提供了一种新型的思路和新型的基因药物、基因疗法。
综上,本申请构建的腺病毒能敲除小鼠的scgII基因,且scgII基因敲除后不影响正常血管的生长,且抑制病理性血管的生长,降低因病理性新生血管导致的致盲眼底疾病的发病率。对眼部疾病起到了预防和辅助治疗的作用,是一种针对眼部疾病的新型基因疗法,为今后的眼部疾病提供了一种更简单高效的疗法。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明的保护范围应以所附权利要求为准。
Claims (9)
1.特异性靶向scgII基因的sgRNA,其特征在于,所述的sgRNA由sgRNA1和sgRNA2组成,所述sgRNA1的核酸序列如SEQ ID NO.1所示;所述sgRNA2的核酸序列如SEQ ID NO.2所示。
2.一种重组载体,其特征在于,所述重组载体包含如权利要求1所述的sgRNA。
3.根据权利要求2所述重组载体,其特征在于,所述重组载体由工具载体PMT649与目的基因片段连接而得;所述目的基因片段的核酸序列如SEQ ID NO.3所示。
4.包含如权利要求2或3任意一项所述的重组载体的腺病毒载体。
5.如权利要求4所述的腺病毒载体在制备促进生理性血管生长且抑制病理性血管生长药物上的应用。
6.根据权利要求5所述的应用,其特征在于,所述血管为视网膜血管。
7.如权利要求4所述的腺病毒载体在制备治疗、预防和辅助治疗眼底疾病药物上的应用。
8.如权利要求2或3任意一项所述重组载体的制备方法,其特征在于,所述方法为:将连接有2个sgRNA的目的基因片段***线性化的工具载体PMT649中,得到重组质粒PSE5862;所述目的基因片段的核酸序列如SEQ ID NO.3所示;所述目的基因片段的扩增引物对为Primer1/Primer2,其中,Primer1的核酸序列如SEQ ID NO.4所示,Primer2的核酸序列如SEQ ID NO.5所示。
9.如权利要求4所述腺病毒载体的制备方法,其特征在于,所述方法为:将连接有2个sgRNA的目的基因片段***线性化的工具载体PMT649中,得到重组质粒PSE5862;然后将重组质粒PSE5862转化到感受态细胞中,得到转化子,抽取阳性转化子质粒包装至腺病毒中得到所述腺病毒载体;所述目的基因片段的核酸序列如SEQ ID NO.3所示;所述目的基因片段的扩增引物对为Primer1/Primer2,其中,Primer1的核酸序列如SEQ ID NO.4所示,Primer2的核酸序列如SEQ ID NO.5所示。
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