CN116497063A - 用于构建肥厚型心肌病动物模型的打靶载体及其应用 - Google Patents
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Abstract
本发明公开一种用于构建肥厚型心肌病动物模型的打靶载体及其应用，属于肥厚型心肌病动物模型构建技术领域；本发明提供了一种打靶载体，是在基础载体的基础上，通过基因编辑处理使所述基础载体上的碱基发生突变后获得；将该打靶载体和Cas9表达质粒在体外转录获得mRNA，将mRNA移植到动物受精卵中，继续培育成功获得肥厚型心肌病动物模型。
Description
技术领域
本发明涉及肥厚型心肌病动物模型构建技术领域，特别涉及用于构建肥厚型心肌病动物模型的打靶载体及其应用。
背景技术
肥厚型心肌病(hypertrophic cardiomyopathy，HCM)是遗传性心脏病的最常见形式，为常染色体显性遗传病，在人群中的发病率大概在1:500到1:200之间。HCM的特征是各种形态的左心室肥厚，伴有一系列临床表现和血流动力学异常。HCM相关症状可以分为心力衰竭、胸痛和心律失常，而HCM导致的恶性心律失常是青少年和运动性人群猝死的最主要原因。目前的医疗手段分为药物治疗和非药物治疗。药物治疗包括β受体阻滞剂、非二氢吡啶类钙通道阻滞剂、抗凝药以及其他药，比如华法林、螺内酯、地高辛等。非药物治疗包括外科室间隔切除术、心脏移植等。这些治疗手段的目的是改善症状、减少合并症和预防猝死，可临床效果并不理想，都不能完全根治肥厚性心肌病。
到目前为止，已知的HCM相关基因不下11种，相关突变更是超过1500个。其中，编码肌球蛋白结合蛋白C的MYBPC3基因是最常见的HCM相关基因，而且90％以上的MYBPC3基因突变为无义突变(non-sensemutation)，导致其出现提前终止(premature terminationcodon，PTC)。然而，MYBPC3相关HCM的确切机制仍有待解决。
2021年12月30日，浙江大学转化医学研究院/浙江大学医学院附属第一医院的梁平团队与浙江大学医学院附属邵逸夫医院的蒋晨阳团队合作在Clinical andTranslational Medicine杂志在线发表Inhibition of HSC70 alleviates hypertrophiccardiomyopathy pathologyin human induced pluripotent stem cellderivedcardiomyocytes with a MYBPC3mutation的研究论文，发现了MYBPC3相关肥厚性心肌病(MYBPC3-related HCM)的新机制以及潜在治疗靶点；并证实了临床筛查中发现的MYBPC3基因突变(c.1377del；p.Leu460fs)可导致HCM的发生，为该新发突变的致病性提供了扎实的功能学依据。
发明内容
针对以上现有技术的不足，本发明提供了腺相关病毒载体及其在治疗肥厚型心肌病的应用，具体通过以下技术实现。
用于构建肥厚型心肌病动物模型的打靶载体，所述打靶载体是在所述基础载体的基础上，通过基因编辑处理使所述基础载体上的碱基发生突变后获得；所述靶点核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
优选地，所述基础载体为pRCH载体。
本发明还提供了上述打靶载体的制备方法，是采用CRISPR/CAS9技术构建打靶载体序列，所用的引物对的正向引物和反向引物的核苷酸序列如SEQ IDNO.5和SEQ ID NO.6。
优选地，上述制备方法包括以下步骤：
S1、将所述基础载体用Not I和EcoR I双酶切，酶切完成后，凝胶回收纯化；
S2、在S1的回收产物中加入所述引物对，凝胶回收纯化；
S3、将步骤S2所得回收产物用感受态细胞进行转化和培养、提取，即可得到所述打靶载体。
本发明还提供了上述打靶载体在构建动物模型中的应用。
优选地，应用方法具体为：将打靶载体和Cas9表达质粒在体外转录获得mRNA，将mRNA移植到动物受精卵中，继续培育获得动物模型。
与现有技术相比，本发明的有益之处在于：本发明提供了一种用于构建肥厚型心肌病动物模型的打靶载体，采用这种打靶载体能够快速高效地构建肥厚型心肌病动物模型。
具体实施方式
下面将对本发明的技术方案进行清楚、完整地描述，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条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1：重组打靶载体的制备
MYBPC3基因的T2535G位点，设计确认Cas9的切割靶位点。最终打靶载体所含的核苷酸序列如SEQ ID NO.1-4所示。
然后合成含此2种sgRNA的双链寡核苷酸链，连入经酶切回收的sgRNA表达载体，构建sgRNA表达载体(即重组打靶载体)。经测序验证后选择正确的克隆，培养后提取质粒，作为体外转录的模板。
实施例2：肥厚型心肌病动物模型的构建和验证
将Cas9表达质粒和实施例1制备的重组打靶载体经酶切线性化处理后纯化，溶于无核酶水中作为模板，用于体外转录。Cas9 mRNA和sgRNA在体外利用T7 RNA聚合酶完成。把转录好的Cas9 mRNA和sgRNA混合并调整浓度至25ng/μL和12.5ng/μL/每种sgRNA，用显微注射法将RNA混合物注入通过受精卵原核显微注射法，共注射受精卵200枚，移植卵180枚，有3只假孕母鼠怀孕，获得子代小鼠15只，其中阳性鼠6只。
1、PCR鉴定小鼠的基因型
取2-4mm鼠尾以碱变性方法提取鼠尾DNA，作为PCR模板；PCR反应体系为25μL，反应条件为95℃2min；95℃30s；57℃30s；72℃30s；72℃5min；35个循环。然后用PCR纯化试剂盒纯化PCR产物，吸取5μL点样，用1.5％的琼脂糖凝胶电泳分离。另外吸取10μL，37℃条件下酶切60min，点样，挑选突变。为进一步验证突变的情况，通过TA克隆、测序进一步检测突变。
正向引物prkdc-1F-1：5′-ccaatctgtgatatctccgcatgg-3′；如SEQ ID NO.5所示；
反向引物prkdc-1R-1：5′-cctcagatactaggaagagagcag-3′；如SEQ ID NO.6所示；
用于鉴定野生型和转基因阳性小鼠。
3、转基因小鼠心脏组织内miR-27b表达水平的检测
颈椎脱臼法处死F2代转基因小鼠和野生型小鼠，后分别取出的脾脏，40μm滤网中充分研磨脾脏，收集脾脏细胞悬液，离心后，裂解红细胞。后收集脾脏细胞，提取总RNA和总蛋白，并逆转录获得cDNA，作为模板；利用引物扩增目的基因。比较转基因小鼠与野生型小鼠中mRNA的表达情况。
4、转基因小鼠表型分析
(1)心重、体重和胫骨长度的测定
小鼠称重后断颈处死，取出心脏先剪去周围大血管，用生理盐水清洗血液，滤纸吸干残余液体，用电子天平称取心脏重量，游标卡尺测量小鼠胫骨长度，计算心脏重量与体重或胫骨长度的比值，用这两个指标表示心肌肥厚的程度。经计算可以发现，阳性转基因小鼠的心脏较对照小鼠明显增大，转基因小鼠的心脏重量与体重或胫骨长度的比值较对照小鼠明显增加。
(2)小鼠心脏组织的透射电子显微镜观察
采用授权专利CN102199625B说明书第209-230段公开的方法，显示与对照小鼠相比，经过5个月饲养的阳性转基因小鼠的心脏超微结构发生明显改变，主要表现在心肌线粒体出现空泡样变化。
以上具体实施方式详细描述了本发明的实施，但是，本发明并不限于上述实施方式中的具体细节。在本发明的权利要求书和技术构思范围内，可以对本发明的技术方案进行多种简单改型和改变，这些简单变型均属于本发明的保护范围。
Claims (6)
1.用于构建肥厚型心肌病动物模型的打靶载体，其特征在于，所述打靶载体是在基础载体的基础上，通过基因编辑处理使所述基础载体上的碱基发生突变后获得；所述打靶载体所含的靶点核苷酸序列如SEQ ID NO.1-4所示。
2.根据权利要求1所述的用于构建肥厚型心肌病动物模型的打靶载体，其特征在于，所述基础载体为pRCH载体。
3.权利要求1所述的打靶载体的制备方法，其特征在于，是采用CRISPR/CAS9技术在所述基础载体进行基因编辑，使所述打靶载体上的MYBPC3基因T2535G位点由碱基T突变为碱基G，得到肥厚型心肌病小鼠模型进而使所述小鼠两条同源染色体中的一条同源染色体上的MYBPC3基因T2535G位点由碱基T突变为碱基G，得到肥厚型心肌病小鼠模型所用的引物对的正向引物和反向引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6。
4.根据权利要求3所述的打靶载体的制备方法，其特征在于，包括以下步骤：
S1、将所述基础载体用Not I和EcoR I双酶切，酶切完成后，凝胶回收纯化；
S2、在S1的回收产物中加入所述引物对，凝胶回收纯化；
S3、将步骤S2所得回收产物用感受态细胞进行转化和培养、提取，即可得到所述打靶载体。
5.权利要求1或2所述的打靶载体在构建肥厚型心肌病动物模型中的应用。
6.根据权利要求5所述的打靶载体在构建肥厚型心肌病动物模型中的应用，其特征在于，将打靶载体和Cas9表达质粒在体外转录获得mRNA，将mRNA移植到动物受精卵中，继续培育获得动物模型。
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