CN111529718B - 一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用 - Google Patents
一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用 Download PDFInfo
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Abstract
本发明提供了一种阳离子微泡‑rAAV‑miRNA病毒复合物及其制备方法以及应用,涉及生物学技术领域。包括如下步骤:制备阳离子微泡;合成带有负电荷的rAAV‑miRNA病毒;将所述阳离子微泡与所述rAAV‑miRNA病毒按照一定体积比混合,并在室温下孵育后形成所述阳离子微泡‑rAAV‑miRNA病毒复合物。本发明的复合物具有病毒的持续表达性和一定的靶向性,是一种miRNA干预的有效试剂。另外,本发明结合超声靶向微泡破坏(UTMD)技术介导阳离子微泡‑rAAV‑miRNA病毒复合物在心脏处靶向释放rAAV‑miRNA病毒以干预miRNA。在UTMD的作用下,使阳离子微泡‑rAAV‑miRNA病毒复合物爆破靶向释放rAAV‑miRNA病毒,能够进一步提高病毒的感染效率,从而增强miRNA的干预作用使miRNA的表达水平降低或上调。
Description
技术领域
本发明涉及生物学技术领域,尤其是涉及一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用。
背景技术
MicroRNA(miRNA)是一类在转录后水平调控基因表达的小分子非编码RNA,大小约为22个核苷酸。miRNA的调控方式主要通过靶向结合mRNA,进而抑制或阻断翻译过程,抑制靶基因表达。目前很多研究已证实,miRNA在转录后水平调控参与心肌细胞各种病理生理学过程,是很多心血管疾病的干预靶点。
目前实现miRNA心肌干预的途径主要包括核酸直接注射法、病毒转染、核酸-脂质体心脏局部注射和超声靶向微泡破坏技术(UTMD)等。其中,核酸直接注射法是通过外周循环直接将miRNA的核酸静脉注射,这种方法虽然操作最简单,但是半衰期较短,需要多次重复给药,存在一定的副作用风险,且直接注射miRNA干预试剂(核酸)在循环过程中容易被血液中核酸酶降解,从而进一步降低其作用效果。病毒转染法可使质粒进入心肌细胞,实现miRNA的长效持续干预,尤其是腺相关病毒(AAV)具有较少的免疫源性和炎症反应,但是通过病毒携带miRNA进行干预依赖于病毒的感染效率,而在确保安全性的前提下,低剂量病毒的感染效率较低,导致miRNA干预作用较弱。核酸-脂质体心脏局部注射是通过脂质体包裹miRNA局部心脏注射,可实现局部靶向治疗,但该方法需要开胸有创操作,不利于向临床转化。另外,超声靶向微泡破坏技术(UTMD)是通过在特定部位给予超声辐照,击破微泡,被动靶向释放所携带的药物或基因,是一种安全、高效的靶向释放技术。例如:Kwekkeboom,R.F等人利用二硬脂酰基磷脂酰胆碱(DSPC)、1,2-硬脂酰基-3-三甲基氯化铵(DSTAP)制备了阳离子微泡,并与负电荷的miRNAantagomir(miRNA小分子核酸抑制剂)静电结合,在UTMD作用下增强了miRNAantagomir的心肌富集。但这种方法UTMD介导miRNA的直接干预小分子在体内作用时间有限,在48h以内,且在心肌缺血再灌注模型中,该UTMD介导miRNAantagomir方法无明显增强miRNA干预效果。因此,对于心肌miRNA干预目前缺乏一种高效、持续且靶向的方法。
发明内容
本发明的目的在于提供一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用。本发明提供的诸多技术方案中的优选技术方案所能产生的诸多技术效果详见下文阐述。
为实现上述目的,本发明提供了以下技术方案:
本发明提供的一种阳离子微泡-rAAV-miRNA病毒复合物的制备方法,所述制备方法包括如下步骤:
制备阳离子微泡;
合成带有负电荷的rAAV-miRNA病毒;
将所述阳离子微泡与所述rAAV-miRNA病毒按照一定体积比混合,并在室温下孵育后形成所述阳离子微泡-rAAV-miRNA病毒复合物。
根据一种优选实施方式,所述合成带有负电荷的rAAV-miRNA病毒的步骤包括:
采用分子克隆方法合成rAAV-miRNA质粒;
利用H293T细胞系对所述rAAV-miRNA质粒进行转染,以合成rAAV-miRNA病毒;
分离纯化rAAV-miRNA病毒。
根据一种优选实施方式,所述采用分子克隆方法合成rAAV-miRNA质粒的步骤包括:
基于miRBase数据库和UCSC基因数据库分别查找特定miRNA的成熟序列和前体序列;
基于碱基互补原则设计所述特定miRNA的成熟序列的功能抑制性片段,其中,功能抑制性片段前后末端用PCR方法分别连接AgeI和RsrII限制性内切酶片段。优选地,功能抑制性片段含2个重复的所述特定miRNA的完全碱基互补序列,抑制性片段中间用ATCT四个碱基分隔,上下游连接两个“茎结构”(为碱基互补序列),使功能抑制性片段转录产物具有稳定的“茎环”二级结构。或者,基于PCR方法设计所述特定miRNA的前体序列及其上下游至少100bp的功能过表达片段。优选地,功能过表达片段应含有所述特定miRNA的全长序列,以及其基因组上下游至少100bp碱基序列以达到和体内真实转录miRNA相似的二级结构。功能过表达片段前后末端用PCR方法分别连接AgeI和RsrII限制性内切酶片段;
采用限制性内切酶法,以AgeI和RsrII两个酶切位点为插入点,在所述rAAV-miRNA质粒的U6启动子下游插入所述功能抑制性片段或所述功能过表达片段;
验证所插入的目的片段是否正确。
根据一种优选实施方式,所述利用H293T细胞系对所述rAAV-miRNA质粒进行转染的步骤包括:
培养H293T细胞系;
待细胞融合率达70-90%时,用PEI聚乙烯亚胺试剂按照DNA:PEI为1:2-1:4的比例共转染Rep/Cap包壳质粒、pHGTI/deltaF6辅助质粒和所述rAAV-miRNA质粒;
待转染8-10小时后更换无血清培养基进行转染;
继续转染60-72小时后收集细胞及培养基上清;
利用细胞裂解液裂解所收集的细胞及培养基上清,然后利用核酸酶降解杂质核酸,进行离心后收集富含病毒的上清液。
根据一种优选实施方式,所述制备阳离子微泡的步骤包括:
所制备的阳离子微泡的脂质成分为二硬脂酰基磷脂酰胆碱、2,3-二油酰基丙基三甲基氯化铵和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000,且所述二硬脂酰基磷脂酰胆碱、所述2,3-二油酰基丙基三甲基氯化铵和所述二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的摩尔比为5:4:1;
将磷脂混悬液置于-50℃的低温浴中进行快速冷冻,然后置于冻干机中制成冻干粉并对冻干粉进行密封保存。
根据一种优选实施方式,所述制备阳离子微泡的步骤包括:所述制备阳离子微泡的步骤包括:
将一定摩尔比例的二硬脂酰基磷脂酰胆碱和2,3-二油酰基丙基三甲基氯化铵和一定质量的棕榈酸混合置于圆底烧瓶中,并加入氯仿进行溶解;
将上述混合物置于60℃水浴中进行旋转减压蒸干,待有机溶剂完全清除后,磷脂在所述圆底烧瓶内形成脂质膜;
向所述圆底烧瓶内加入一定摩尔比例的二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000,设置旋转蒸发仪对上述混悬液进行加热;
将上述磷脂混悬液分装于西林瓶中,并置于-50℃的低温浴中进行快速冷冻,然后置于冻干机内制成冻干粉,使用换气装置向西林瓶内充入全氟丙烷气体,将制成的冻干粉密封,即制备成所述阳离子微泡。
本发明还提供了一种阳离子微泡-rAAV-miRNA病毒复合物,所述复合物按照所述的制备方法制备而成。
根据一种优选实施方式,所述阳离子微泡-rAAV-miRNA病毒复合物能够抑制干预miRNA。
本发明还提供了阳离子微泡-rAAV-miRNA病毒复合物在制备治疗心肌血管疾病的药物中的应用。
根据一种优选实施方式,所述应用包括结合超声靶向微泡破坏技术介导阳离子微泡-rAAV-miRNA病毒复合物在心脏处靶向释放rAAV-miRNA病毒以干预miRNA。
基于上述技术方案,本发明的一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用至少具有如下技术效果:
本发明提供的阳离子微泡-rAAV-miRNA病毒复合物将制备的阳离子微泡与通过分子克隆方法制备的带有负电荷的rAAV-miRNA病毒通过静电结合形成阳离子微泡-rAAV-miRNA病毒复合物,其中,本发明所制备的阳离子微泡通过调整二硬脂酰基磷脂酰胆碱、2,3-二油酰基丙基三甲基氯化铵和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的比例,使得制备的阳离子微泡粒径更小,所携带正电荷更高,能够使其更好的与带有负电荷的rAAV-miRNA病毒结合。另外,本发明所制备的阳离子微泡采用了冻干粉,使得混合磷脂外壳的微泡稳定性更好,而且冻干粉也易于长期保存。本发明所制备的阳离子微泡-rAAV-miRNA病毒复合物既克服了单纯低剂量的病毒感染效率低、干预miRNA作用有限的缺点,同时相对于单纯使用miRNA核酸干预试剂,本发明的复合物具有病毒的持续表达性和一定的靶向性,是一种miRNA干预的有效试剂。
另一方面,本发明还提供了阳离子微泡-rAAV-miRNA病毒复合物在制备治疗心肌血管疾病的药物中的应用,并且,该应用结合超声靶向微泡破坏(UTMD)技术介导阳离子微泡-rAAV-miRNA病毒复合物在心脏处靶向释放rAAV-miRNA病毒以干预miRNA。从而在UTMD的作用下,使阳离子微泡-rAAV-miRNA病毒复合物爆破靶向释放rAAV-miRNA病毒,能够进一步提高病毒的感染效率,从而增强miRNA的干预作用,使miRNA的表达水平降低或上调。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术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的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例1中制备的阳离子微泡的zeta电位图;
图2是本发明实施例1中制备的阳离子微泡的粒径分布图;
图3是本发明实施例1中制备的阳离子微泡的DiO荧光染色图;
图4A是本发明实施例1中一种优选实施例的miRNA功能抑制性插入序列;
图4B是本发明实施例1中一种优选实施例的miRNA功能过表达插入序列;
图4C是本发明实施例中rAAV-miRNA质粒图谱;
图5是本发明的rAAV-miRNA病毒复合物在UTMD介导作用下(UTMD组)与单纯病毒注射组相比对小鼠感染效率的共聚焦显微镜图;
图6是本发明的rAAV-miRNA病毒复合物在UTMD介导作用下(UTMD组)与对照组的miR-199a荧光定量PCR结果分析图。
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,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
下面结合说明书附图对本发明的技术方案进行详细说明。
实施例1
本实施例提供了阳离子微泡-rAAV-miRNA病毒复合物的制备方法,其中制备方法包括如下步骤:
步骤一:制备阳离子微泡。
优选地,本实施例的阳离子微泡的脂质成分为二硬脂酰基磷脂酰胆碱(DSPC)、2,3-二油酰基丙基三甲基氯化铵(DOTAP)和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)。优选地,二硬脂酰基磷脂酰胆碱(DSPC)、2,3-二油酰基丙基三甲基氯化铵(DOTAP)和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)的摩尔比为5:4:1。
优选地,本实施例制备阳离子微泡的步骤包括:
称取8mgDSPC并按照上述摩尔比加入DOTAP和500mg棕榈酸,置于圆底烧瓶中,加入3ml氯仿进行溶解,然后将圆底烧瓶置于60℃水浴中进行旋转减压蒸干15min,使有机溶剂完全清除。而磷脂在烧瓶内壁上形成一层很薄的脂质膜。
待反应完成后再按照上述摩尔比加入对应质量的DSPE-PEG2000和3.6ml叔丁醇,设置旋转蒸发仪于60℃温度下对混悬液进行加热15min。将上述磷脂混悬液快速分装于10ml西林瓶中,并置于-50℃的低温浴中1min进行快速冷冻,再置于冻干机中过夜,制成冻干粉。
使用换气装置向西林瓶内充入全氟丙烷气体,将制成的冻干粉密封,即制备成阳离子微泡。使用时注入5ml生理盐水轻微摇匀,即可使用。
如图1和图2所示,图1示出了上述方法制备的阳离子微泡的zeta电位图;图2示出了上述方法制备的阳离子微泡的粒径分布图。从图1和图2可以看出,本发明制备的阳离子微泡经zeta电位仪(ZetasizerNanoZS,Malvern)检测的平均电荷为+41.6±8.25mV。经库尔特颗粒技术仪(Multisizer4E,Beckman)检测平均粒径为1.23±0.49μm,浓度为8.1×108MBs/ml。图3示出了经DiO(3,3’-Dioctadecyloxacarbocyanineperchlorate,细胞膜绿色荧光分子探针)染色后微泡荧光图。从图3可以看出,微泡浓度较为合适。
因此,实验结果表明,通过本实施例的制备方法制备的阳离子微泡其表面带41.6mV的正电荷,且微泡的粒径为微米级,浓度适中,经申请人进行多次实验,能够满足后续结合UTMD技术的要求。
本发明所制备的阳离子微泡与现有技术相比具有如下优点:1、由于采用了合适的DSPC:DOTAP:DSPE-PEG2000配方,使得所制备的阳离子微泡粒径与现有技术中Kwekkeboom,R.F等人制备的微泡粒径为1.77μm相比,本发明所制备的阳离子微泡粒径更小,正电荷更高,能够在应用时更好地与负电荷rAAV-miRNA病毒结合;2、本发明将阳离子微泡制作成冻干粉,使其混合磷脂外壳微泡稳定性更好,且冻干粉易于长期保存。
步骤二:合成带有负电荷的rAAV-miRNA病毒。
优选地,包括如下步骤:
1、采用分子克隆方法合成rAAV-miRNA质粒。
通过miRBase数据库(www.mirbase.org)和UCSC基因组数据库(genome.ucsc.edu)分别查找到特定miRNA的成熟序列和前体序列pre-miRNA。其中,所述的特定miRNA是指与心肌肥厚和心肌衰竭疾病相关的特定miRNA,例如miR-199a。
通过碱基互补原则按照图4A设计特定miRNA的成熟序列的功能抑制性片段。见图4A中的茎环结构的功能抑制性片段转录产物,以大鼠rno-miR-199a-5p为例,所设计的功能抑制性片段基因序列见表1。图4A中茎结构1和茎结构2为茎环结构中起稳定作用的互补碱基序列,miRNA结合位点即环结构为功能序列,可以与靶基因竞争性结合miRNA,有效抑制miRNA功能。功能抑制性片段前后末端用PCR方法分别连接AgeI和RsrII限制性内切酶片段。或者通过PCR方法设计特定miRNA的前体序列pre-miRNA及其上下游至少100bp的含AgeI、RsrII限制性内切酶的功能过表达片段,功能过表达片段能够使miRNA表达上升。如图4B所示,其具体基因序列见表1。其中,图4B中AgeI、RsrII为限制性内切酶位点,能够通过分子克隆方法插入rAAV9-miRNA质粒。
通过限制性内切酶法,以AgeI、RsrII两个酶切位点为插入点,在rAAV-miRNA质粒U6启动子下游插入功能抑制性片段或功能过表达片段,如图4C所示。
通过测序验证是否正确插入目的片段。
表1 rAAV-miRNA针对大鼠rno-miR-199a-5p的过表达或功能抑制性插入片段基因序列
2、利用H293T细胞系对所述rAAV-miRNA质粒进行转染,以合成rAAV-miRNA病毒。
利用DMEM高糖培养基培养H293T细胞系,待细胞融合率(cellconfluency)达到70-90%时,用PEI(Polyethylenimine,聚乙烯亚胺)试剂按照DNA:PEI为1:2-1:4的比例共转染Rep/Cap包壳质粒、pHGTI/deltaF6辅助质粒和前述步骤中合成的rAAV-miRNA质粒,转染8-10小时后换无血清培养基,转染60-72h后用细胞刮收集细胞及培养基上清。
通过细胞裂解液lysisbuffer(20mMTrisPH8.0,150mMNaCl,1mMMgCl2)裂解细胞,Benzonase核酸酶降解杂质核酸,离心后收集富含病毒的上清。
3、分离纯化rAAV-miRNA病毒。
通过分层超高速离心法分离纯化病毒:将前述步骤中的上清转移至Optiseal超高速离心管,分别加入17%、25%、40%和60%不同浓度的lodixanol(碘克沙醇,密度分层剂),并对25%和60%层使用酚红标记,在Beckman超高速离心机以48,000-50,000转速离心分层液90-120分钟,富含病毒的上清会被离心至40%层。然后用注射器抽取40%液体,在Amicon50ml过滤管中多次离心过滤,最终纯化为100-500μl体积的rAAV-miRNA病毒,于-80℃保存。
步骤三:将步骤一制备的阳离子微泡与步骤二制备的rAAV-miRNA病毒按照1:1体积比混合,并在室温下孵育后形成所述阳离子微泡-rAAV-miRNA病毒复合物。
优选地,将1-1.5×1011Vg的rAAV-miRNA-GFP病毒和阳离子微泡按照一定体积比混合,室温下孵育10-15分钟,形成病毒-微泡复合物。
实施例2
本实施例将实施例1制备的阳离子微泡-rAAV-miRNA-GFP病毒复合物应用于大鼠进行实验。具体如下:
通过大鼠尾静脉,以约3ml/h的速度微泵连续、缓慢推注阳离子微泡-rAAV-miRNA病毒复合物1ml(约20分钟),使用GEVIVID7心脏超声仪高频10S探头定位心脏位置,再使用低频M3S探头以如下爆破参数:每4个心动周期进行一次爆破,使用较低频率和大鼠心脏合适深度(约2-2.5cm),使爆破时机械指数MI不低于1.3,在心脏收缩末期爆破微泡,靶向释放rAAV-miRNA-GFP病毒。
与对照组相比,其中对照组只尾静脉注射相同剂量rAAV-miRNA-GFP病毒,不使用UTMD,即不使用超声靶向微泡破坏技术,rAAV-GFP在左心室的感染效率比对照组有所增加,说明UTMD提高了病毒的感染效率,参考图5,而由于rAAV-miRNA病毒携带干预miRNA片段,病毒感染效率的提高会增强miRNA干预作用。图5示出了采用UTMD介导rAAV-miRNA-GFP与单纯使用病毒注射组的感染情况,其中黑色线条代表WGA(麦胚凝集素)标记的细胞膜;黑色不规则形状代表病毒表达GFP荧光的细胞,用黑色箭头指出。
一月后,取材各组心脏标本,miRNAqPCR定量检测结果显示,与对照组相比,miR-199a表达水平在UTMD实验组有明显下降,结果见图6,图6中UTMD组为UTMD介导阳离子微泡-rAAV-miR199a-TuD抑制性病毒复合物组,*p<0.05。上述实验结果表明,UTMD介导阳离子微泡-rAAV-miR199a-TuD抑制性病毒复合物起到了对miR-199a的抑制作用。
本发明还提供了阳离子微泡-rAAV-miRNA病毒复合物在制备治疗心肌血管疾病的药物中的应用。优选地,所述应用包括结合UTMD技术介导阳离子微泡-rAAV-miRNA病毒复合物在心脏处靶向释放rAAV-miRNA病毒以干预miRNA。
本发明采用UTMD技术介导阳离子微泡-rAAV-miRNA病毒复合物在心脏处靶向释放rAV-miRNA病毒,干预miRNA,既具有病毒对miRNA干预作用的持续表达性,又克服了低剂量病毒感染效率低的弱点,是一种miRNA干预的高效、持续和靶向技术。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
SEQUENCE LISTING
<110> 四川大学华西医院
<120> 一种阳离子微泡-rAAV-miRNA病毒复合物及其制备方法以及应用
<130> 2023.4.17
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 427
<212> DNA
<213> 未知
<400> 1
accggttgct gcaaatgtgc cacgtcaaga ctggaaatca cagcccttta gtgtgtctca 60
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gctccgtcgc cccagtgttc agactacctg ttcaggacaa tgccgttgta cagtagtctg 240
cacattggtt agactgggca agggccagca acgccatgga cggctgggga caaaatgtgc 300
tgtttccaag gagaggacag ctgggtgctc ctttgttgag tctcatatcc agtgtttctc 360
ttcacaggtt ttgcaaacta gggaccagaa agccctcagg gactctgcca ggggagaaga 420
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aagtattctg gtcacagaat acaacgaaca ggtagtctat ctgaacactg ggcaagatga 120
tcctagcgcc gtcttttttc ggaccg 146
Claims (8)
1.一种阳离子微泡-rAAV-miRNA病毒复合物的制备方法,其特征在于,所述制备方法包括如下步骤:
制备阳离子微泡:
所述制备阳离子微泡的步骤包括:
将一定摩尔比例的二硬脂酰基磷脂酰胆碱和2,3-二油酰基丙基三甲基氯化铵和一定质量的棕榈酸混合置于圆底烧瓶中,并加入氯仿进行溶解,得到混合物;
将上述混合物置于60℃水浴中进行旋转减压蒸干,待有机溶剂完全清除后,磷脂在所述圆底烧瓶内形成脂质膜;
向所述圆底烧瓶内加入一定摩尔比例的二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000,得到磷脂混悬液,设置旋转蒸发仪对上述磷脂混悬液进行加热;
将加热后的磷脂混悬液分装于西林瓶中,并置于-50℃的低温浴中进行快速冷冻,然后置于冻干机内制成冻干粉,使用换气装置向西林瓶内充入全氟丙烷气体,将制成的冻干粉密封,即制备成所述阳离子微泡;
其中,所述二硬脂酰基磷脂酰胆碱、所述2,3-二油酰基丙基三甲基氯化铵和所述二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的摩尔比为5:4:1;
合成带有负电荷的rAAV-miRNA病毒;
将所述阳离子微泡与所述rAAV-miRNA病毒按照一定体积比混合,并在室温下孵育后形成所述阳离子微泡-rAAV-miRNA病毒复合物。
2.根据权利要求1所述的制备方法,其特征在于,所述合成带有负电荷的rAAV-miRNA病毒的步骤包括:
采用分子克隆方法合成rAAV-miRNA质粒;
利用H293T细胞系对所述rAAV-miRNA质粒进行转染,以合成rAAV-miRNA病毒;
分离纯化rAAV-miRNA病毒。
3.根据权利要求2所述的制备方法,其特征在于,所述采用分子克隆方法合成rAAV-miRNA质粒的步骤包括:
基于miRBase数据库和UCSC基因数据库分别查找所述miRNA的成熟序列和前体序列;
基于碱基互补原则设计所述miRNA的成熟序列的功能抑制性片段,其中,所述功能抑制性片段其前后末端用PCR方法分别连接AgeI和RsrII限制性内切酶片段;或者,基于PCR方法设计所述miRNA的前体序列及其上下游至少100bp的功能过表达片段,其中,所述功能过表达片段前后末端用PCR方法分别连接AgeI和RsrII限制性内切酶片段;
采用限制性内切酶法,以AgeI和RsrII两个酶切位点为插入点,在所述rAAV-miRNA质粒的U6启动子下游插入所述功能抑制性片段或所述功能过表达片段;
验证所插入的目的片段是否正确。
4.根据权利要求3所述的制备方法,其特征在于,所述利用H293T细胞系对所述rAAV-miRNA质粒进行转染的步骤包括:
培养H293T细胞系;
待细胞融合率达70-90%时,用聚乙烯亚胺PEI试剂按照DNA:PEI为1:2-1:4的比例共转染Rep/Cap包壳质粒、pHGTI/deltaF6辅助质粒和所述rAAV-miRNA质粒;
待转染8-10小时后更换无血清培养基进行转染;
继续转染60-72小时后收集细胞及培养基上清;
利用细胞裂解液裂解所收集的细胞及培养基上清,然后利用核酸酶降解杂质核酸,进行离心后收集富含病毒的上清液。
5.一种阳离子微泡-rAAV-miRNA病毒复合物,其特征在于,所述复合物按照权利要求1至4任一项所述的制备方法制备而成。
6.根据权利要求5所述的阳离子微泡-rAAV-miRNA病毒复合物,其特征在于,所述阳离子微泡-rAAV-miRNA病毒复合物能够干预miRNA。
7.根据权利要求5或6所述的阳离子微泡-rAAV-miRNA病毒复合物在制备治疗心肌血管疾病的药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述应用包括结合超声靶向微泡破坏技术介导阳离子微泡-rAAV-miRNA病毒复合物在心脏处靶向释放rAAV-miRNA病毒以干预miRNA。
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