CN114410683B - 一种基于Cre-lox重组系统的RIM3-RNAi及其应用 - Google Patents
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Abstract
本发明适用于生物医学技术领域，提供了一种基于Cre‑lox重组系统的RIM3‑RNAi，包括一种Cre酶依赖的rAAV表达的RNAi表达载体，所述的表达载体包含顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，RIM3 RNAi发卡结构插入位点，WPRE，bGHpoly（A）signal以及AAV2ITR。本发明在生物信息学分析的基础上，发现小鼠痛模型中高表达的突触传递相关蛋白Rab3‑interacting molecule‑3(简称RIM3)，因此构建了shRNA干扰策略，可有效缓解痛模型动物的伤害性感受，本发明将为靶向镇痛治疗提供方向。
Description
技术领域
本发明属于生物医学技术领域，尤其涉及一种基于Cre-lox重组系统的RIM3-RNAi及其应用。
背景技术
1.RNA干扰技术：
RNA干扰（RNA interference, RNAi）是指在进化过程中高度保守的、由双链RNA（double-stranded RNA，dsRNA）诱发的、同源mRNA高效特异性降解的现象。由于使用RNAi技术可以特异性降低或关闭特定基因在细胞或有机体中的表达，所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。人们也基于这一现象研究了一种反向调控基因表达的遗传学机制。在动物中，RNAi可以通过U6启动表达shRNA来实现。目前的U6载体可以通过2种方式表达在动物细胞内表达：瞬时表达和稳定表达。
2.Cre-lox技术：
Cre-lox技术是20世纪80年代从P1噬菌体中发现的一种位点特异性重组技术。该技术可通过Cre重组酶和lox位点的相互作用，实现目的片段的删除、翻转、插入和易位等。该技术不需要借助任何辅助因子，可作用于多种结构的DNA底物，如线形、环状甚至超螺旋DNA等，操作简单、快速、高效，因此被广泛应用于真核生物和原核生物的基因敲除、插入、翻转和易位等研究中。目前，Cre-lox系统应用最热门的领域是基因打靶，已被证明是哺乳动物细胞和小鼠遗传操作最有用的工具，该系统和基因打靶的结合提供了实现条件基因敲除或激活的手段。
3.病毒依赖的基因重组
由于转基因动物依赖的基因重组存在耗时长、成本高、区域或者组织特异性不高等不足。采用比较灵活的方式使用Cre-lox系统，例如通过病毒引入Cre或lox元件，从而在小鼠中实现基因重组。借助病毒表达的cre-lox重组系统，可以特异性对某些细胞的标记和操控。当前运用神经示踪技术对大脑特定神经环路的结构和功能进行解析时，运用 Cre 重组酶系统和 AAV 血清型（比如 rAAV2/9、rAAV2/retro、rAAV2/1）结合，达到特异性对神经环路标记和功能研究的目的：病毒可以通过局部注射的方式保证区域特异性感染，再加上驱动 Cre 基因的特异性启动子，能够实现更强的区域和细胞特异性的基因重组。但是对于如何时空特异性地干扰特定的神经环路的功能，需要涉及一种更优的时空特异调控相关重要基因的表达工具的开发。
发明内容
本发明实施例的目的在于提供一种基于Cre-lox重组系统的RIM3-RNAi及其应用，旨在解决背景技术中提出的问题。
本发明实施例是这样实现的，一种基于Cre-lox重组系统的RIM3-RNAi，包括一种Cre酶依赖的rAAV表达的RNAi表达载体，所述的Cre酶依赖的rAAV表达的RNAi表达载体包括顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，RIM3RNAi发卡结构插入位点，WPRE， bGHpoly（A）signal以及AAV2ITR。
进一步的技术方案，所使用的RIM3 shRNA 的具体序列为：
5’-GCCTGTGTGTGGATCTCAT-3’。
对照组Scramble shRNA的具体序列为：
5’-GGTTTATATCGCGGTTATT -3’。
本发明实施例的另一目的在于，一种基于Cre-lox重组系统的RIM3-RNAi的应用，将所述的RIM3-RNAi应用于重组病毒pAAV2-CaMKIIα-DIO-(mCherry-bGH polyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA的制备。
进一步的技术方案，将所述的重组病毒pAAV2-CaMKIIα-DIO-(mCherry-bGHpolyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA 注射于小鼠基底外侧杏仁核，并将辅助病毒AAV2/1-hSyn-Cre-EGFP 注射到小鼠岛叶，待病毒表达四周后，观察到腓总神经结扎模型小鼠后足底机械痛阈值。
本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用，通过腺相关病毒依赖的Cre-lox重组系统，对特定脑区Rab3-interacting molecule-3 (简称RIM3)分子进行shRNA干扰，从而应用于镇痛治疗。本发明在生物信息学分析的基础上，发现小鼠痛模型中高表达的突触传递相关蛋白RIM3（是一种突触活性蛋白，位于在突触后，通过作用于电压依赖性的钙通道，参与调节突触前囊泡的胞吐作用，在神经系统的多个脑区都有表达），因此构建了shRNA干扰策略，可有效缓解痛模型动物的伤害性感受，本发明将为靶向镇痛治疗提供方向。
附图说明
图1为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的痛模型小鼠IC和BLA脑区RIM3分子表达对照图。
图2为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的RIM3荧光素酶检测图。
图3为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的病毒注射示意图。
图4为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的行为学检测结果示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，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的具体实现进行详细描述。
本发明一个实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi，包括一种Cre酶依赖的rAAV表达的RNAi表达载体，该表达载体包含顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，RIM3 RNAi发卡结构插入位点，WPRE，bGHpoly（A）signal以及AAV2ITR。
所使用的RIM3 shRNA 的具体序列为：5’-GCCTGTGTGTGGATCTCAT-3’；对照组Scramble shRNA的具体序列为：5’-GGTTTATATCGCGGTTATT -3’。
小鼠痛觉模型实验的具体步骤包括：
步骤1：建立小鼠腓总神经结扎（common peroneal nerve ligation，CPNL）模型：小鼠通过2%异氟醚麻醉后，在左腿做切口暴露腓总神经，无菌手术线结扎腓总神经后局部缝合消毒，待动物清醒后放回笼中饲养。假手术组仅暴露腓总神经，但不进行结扎。
步骤2：机械痛检测：动物手术前使用von-frey丝（克数为0.008g，0.02g，0.04g，0.16g，0.4g，0.6g，1g，1.4g，2g）检测左足机械痛阈值，记为第0天数据，手术后从第1天开始，每天固定时间检测左足机械痛阈值，并进行统计学分析。
步骤3：蛋白印迹分析：动物术后第7天，麻醉分离大脑，对右侧IC和BLA脑区进行取材提蛋白，对RIM3等蛋白表达进行半定量分析（结果如图1所示）。
步骤4：病毒构建与注射：委托武汉枢密公司构建重组腺相关病毒pAAV2-CaMKIIα-DIO-(mCherry-bGH-polyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA 和辅助病毒AAV2/1-hSyn-Cre-EGFP，在HEK293细胞对shRNA进行验证，结果如图2所示。随后将该病毒注射在小鼠右侧BLA (注射量为200nl，病毒滴度>1012)，将辅助病毒跨突触AAV2/1-hSyn-Cre-EGFP注射在小鼠右侧IC(注射量为200nl，为了确保跨突触，病毒滴度>1013)，注射方法如图3所示。
步骤5：动物行为学检测：病毒表达4周后，建立腓总神经结扎模型，检测小鼠左后足机械痛阈值，对各组动物痛阈进行统计学分析，如图4所示：注射shRNA（RIM3）的痛模型动物痛阈明显上升，而注射shRNA（Scramble）的痛模型动物痛阈相比于无病毒注射组没有明显改变，通过shRNA降低IC-BLA神经通路上的RIM3分子表达，可以有效镇痛。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.一种Cre酶依赖的rAAV表达的RNAi表达载体在制备镇痛药物中的应用，其特征在于，所述的Cre酶依赖的rAAV表达的RNAi表达载体包含顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，Rab3-interacting molecule-3 RNAi发卡结构插入位点，WPRE，bGHpoly（A）signal以及AAV2ITR。
2.根据权利要求1所述的Cre酶依赖的rAAV表达的RNAi表达载体在制备镇痛药物中的应用，其特征在于，所使用的Rab3-interacting molecule-3 shRNA 的具体序列为：
5’-GCCTGTGTGTGGATCTCAT-3’。
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