CN112725340A - 小分子干扰核糖核酸及其重组腺病毒与应用 - Google Patents
小分子干扰核糖核酸及其重组腺病毒与应用 Download PDFInfo
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Abstract
本发明公开了小分子干扰核糖核酸及其重组腺病毒与应用。所述小分子干扰核糖核酸,由正义链、反义链和环状序列共同组成的shRNA,所述正义链和反义链选自以下任一组:1)序列如SEQ ID№.1所示的正义链和序列如SEQ ID№.2所示的反义链;2)序列如SEQ ID№.3所示的正义链和序列如SEQ ID№.4所示的反义链;3)序列如SEQ ID№.5所示的正义链和序列如SEQ ID№.6所示的反义链。环状序列可采用TTCAAGAGA或其他常规间隔序列代替。该小分子干扰核糖核酸可用于制备重组腺病毒,用于抑制猪Rab32基因的表达。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及抑制Rab32基因表达的小分子干扰核糖核酸,含有该小分子干扰核糖核酸的重组腺病毒,以及应用。
背景技术
Rab蛋白是小分子GTP结合蛋白家族(small GTP-binding proteins)中最大的亚家族,目前发现的Rab家族成员已达60多种,其中一些Rab蛋白(如Rab5,Rab7,Rab10和Rab18等)参与自噬的调控,并影响脂质代谢,关于Rab32确切的功能仍在研究当中。前期研究显示Rab32在胞内定位于线粒体。
RNA干扰(RNA interference,RNAi)是将与靶基因编码区同源的双链RNA(double-stranded RNA,dsRNA)导入细胞内,使靶基因的表达降低或沉默,从而对基因功能进行研究。细胞内转录的shRNA与多种酶和蛋白组成RNA诱导的沉默复合物(RNA-inducedsilencing complex,RISC),识别靶基因降解靶RNA。腺病毒载体目前在基因功能研究中广泛应用,其转染效率高,对分裂期细胞和非分裂期细胞均具有感染能力强,安全性,且其带有抗生素标记的载体可以在细胞中持续抑制靶基因的表达,持续数星期甚至更久。
发明内容
本发明目的在于提供小分子干扰核糖核酸及其重组腺病毒与应用,以解决现有技术中所存在的一个或多个技术问题,至少提供一种有益的选择或创造条件。
本发明一方面提供的小分子干扰核糖核酸,由正义链、反义链和环状序列共同组成的shRNA,所述正义链和反义链选自以下任一组:
1)序列如SEQ ID №.1所示的正义链和序列如SEQ ID №.2所示的反义链;
2)序列如SEQ ID №.3所示的正义链和序列如SEQ ID №.4所示的反义链;
3)序列如SEQ ID №.5所示的正义链和序列如SEQ ID №.6所示的反义链。
shRNA中的环状序列可采用TTCAAGAGA或其他常规间隔序列代替。
本发明第二方面提供了含有上述小分子干扰核糖核酸的原核、真核穿梭载体、腺病毒载体。
本发明第三方面提供了含有上述小分子干扰核糖核酸的重组腺病毒。该重组腺病毒可用于抑制猪Rab32基因表达。
本发明第三方面提供了上述重组腺病毒的制备方法,包括步骤:
A.合成小分子干扰核糖核酸;
B.所述小分子干扰核糖核酸与穿梭载体进行连接,获得抑制Rab32基因表达的重组穿梭质粒;
C.将步骤B所得重组穿梭质粒线性化,转化入制备好的含有腺病毒骨架质粒pAdEasy-1的感受态细胞,通过细胞同源重组获得抑制Rab32基因表达的重组腺病毒载体;
D.将步骤C所得重组腺病毒载体线性化后用脂质体转染慢病毒感染细胞,获得抑制Rab32基因表达的重组腺病毒。
在本发明的一些实施方式中,所述慢病毒感染细胞为293T。
附图说明
图1为感染细胞72小时是细胞状态及GFP表达情况图;
图2为抑制Rab32基因表达后成脂分化细胞的油红染色结果图;
图3为抑制Rab32基因表达后的各例表达量对比图。
具体实施方式
下面将结合具体实施例,对本发明实施例中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前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1.shRNA的设计和质粒的构建:
(1)根据猪的转录本设计Rab32敲低的靶序列,经过分析和设计获得3组能够实现目的的siRNA序列,具体如下所示:
siRNA1序列:CAGCCCTATTCCTGCTGTTCTCTTA(SEQ ID №.1);
siRNA2序列:CCTATTCCTGCTGTTCTCTTAGCTA(SEQ ID №.3);
siRNA3序列:GCTGTTCTCTTAGCTAACAAGTGTG(SEQ ID №.5)。
将以上三个靶点的序列设计入病毒载体构建框架,具体序列如下:
shRNA1序列:
Top strand:
tcgagGCAGCCCTATTCCTGCTGTTCTCTTATTCAAGAGATAAGAGAACAGCAGGAATAGGGCTGTTTTTTA(SEQ ID №.7);
Bottom strand:
agcttAAAAAACAGCCCTATTCCTGCTGTTCTCTTATCTCTTGAATAAGAGAACAGCAGGAATAGGGCTGCC(SEQ ID №.8)。
shRNA2序列:
Top strand:
tcgagGCCTATTCCTGCTGTTCTCTTAGCTATTCAAGAGATAGCTAAGAGAACAGCAGGAATAGGTTTTTTA(SEQ ID №.9);
Bottom strand:
agcttAAAAAACCTATTCCTGCTGTTCTCTTAGCTATCTCTTGAATAGCTAAGAGAACAGCAGGAATAGGCC(SEQ ID №.10)。
shRNA3序列:
Top strand:
tcgagGCTGTTCTCTTAGCTAACAAGTGTGTTCAAGAGACACACTTGTTAGCTAAGAGAACAGCTTTTTTA(SEQ ID №.11);
Bottom strand:
agcttAAAAAAGCTGTTCTCTTAGCTAACAAGTGTGTCTCTTGAACACACTTGTTAGCTAAGAGAACAGCC(SEQ ID №.12)。
安排生物公司合成初始序列。
(2)引物退火形成带粘性末端的双链片段:
目的在于扩增实施例1所得的初始序列。扩增引物是由生物公司合成PAGE纯化的oligo序列,分别稀释至100uM。
其体系如下(20uL):
退火程序:
(3)载体酶切:
根据如下表中顺序依次加入每种试剂,轻轻吸打混匀,置于37℃水浴锅中反应1-2h;酶切结束之后进行琼脂糖凝胶电泳,回收目的片段;
载体酶切体系如下:
(4)干扰片段与载体连接:
连接反应体系(20uL)如下:
以上连接液在22℃连接1-2h,或者16℃连接过夜。
(5)转化
1)DH5α感受态细胞从-80℃冰箱拿出来之后,要立刻放到冰上融化,感受态分装过程操作轻柔,减轻对其机械破坏;
2)待感受态融化后,以每管50μL的体积分装(对于质粒转化,20μL就足够了),分装之后以不超过感受态体积1/10的量加入连接产物(目前加5μL连接产物),冰上放置20-30min;
3)42℃热激90s(这个时间要非常严格),热激完之后立刻插入冰上冰育2-3min;
在超净台中,加入500μL LB培养基(注意一定是无抗的LB培养基),轻柔的上下颠倒3-5次;
5)37℃、230rpm震荡培养45-60min;
6)将菌液涂到相应抗性的固体平板上,涂布均匀,然后将板子倒放37℃恒温箱培养12-16h;
(6)菌液PCR鉴定
1)菌液PCR鉴定体系:
2)菌液PCR鉴定程序
扩增产物经测序,结果与目的序列一致,目的质粒构建成功。测序成功之后进行菌液扩增,然后抽提质粒纯化。抽提的质粒经验证合格之后用于转染细胞。
实施例2.重组腺病毒载体
1、穿梭质粒构建成功之后,选择合适的酶切位点进行线性化,并进行琼脂糖胶回收;
2、使用制备好的含有腺病毒骨架质粒pAdEasy-1的E.coli BJ5183感受态细胞,将回收好的线性化穿梭质粒进行转化,进行细胞内重组;
3、转化之后,涂板,37℃,培养12-16h;
4、选取单克隆进行培养,抽提质粒进行PacI酶切验证;
5、验证正确的重组质粒转化E.coli stbl3感受态,得到高纯度的重组质粒;
6、重组质粒PacI线性化;
7、包装慢病毒:293T;
8、慢病毒感染细胞。
得到的病毒滴度为1.99×1010PFU/mL。
实施例3.验证重组腺病毒载体
取实施例2制得的重组腺病毒载体,稀释105感染猪SVF(血管基质组分)细胞。如图1所示,72h后观察发现全部均可表达GFP(含有shRNA1、shRNA2、shRNA3重组腺病毒载体的样本分别对应图中shRab32-1-72h、shRab32-2-72h、shRab32-3-72h),且不影响细胞的生长。
低表达Rab32的SVF细胞成脂分化:待感染后的细胞长满后,再接抑制生长两天,换成诱导分化成白脂的诱导培养基培养4天,第5天时吸走一半,补了相应体积的维持培养基,第6天时全部换成维持培养基,第8天时收细胞进行定量检测,Rab32的mRNA表达量降低,如图3以及下表所示。
样本名称 | 平均值 | 标准差 |
shc | 1.000469 | 0.068577 |
shRab32-1 | 0.136698 | 0.012687 |
shRab32-2 | 0.067617 | 0.004371 |
shRab32-3 | 0.121867 | 0.016762 |
对细胞进行油红染色,转染了含有shRNA1的重组腺病毒载体的SVF细胞标记为sub96-shR1,同理,其余分别为sub 96-shR2和sub96-shR3,染色结果见图2。证明所设计的shRNA序列均能降低细胞内Rab32的表达。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
SEQUENCE LISTING
<110> 佛山科学技术学院
<120> 小分子干扰核糖核酸及其重组腺病毒与应用
<130> 2021
<160> 12
<170> PatentIn version 3.5
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Claims (6)
1.小分子干扰核糖核酸,其特征在于,由正义链、反义链和环状序列共同组成的shRNA,所述正义链和反义链选自以下任一组:
1)序列如SEQ ID№.1所示的正义链和序列如SEQ ID№.2所示的反义链;
2)序列如SEQ ID№.3所示的正义链和序列如SEQ ID№.4所示的反义链;
3)序列如SEQ ID№.5所示的正义链和序列如SEQ ID№.6所示的反义链。
2.含有如权利要求1所述的小分子干扰核糖核酸的原核、真核穿梭载体、腺病毒载体。
3.含有权利要求1所述的小分子干扰核糖核酸的重组腺病毒。
4.如权利要求3所述的重组腺病毒的应用,其特征在于:所述重组腺病毒在抑制Rab32基因表达中的应用。
5.如权利要求3所述的重组腺病毒的制备方法,其特征在于包括步骤:
A.合成权利要求1所述小分子干扰核糖核酸;
B.所述小分子干扰核糖核酸与穿梭载体进行连接,获得抑制Rab32基因表达的重组穿梭质粒;
C.将步骤B所得重组穿梭质粒线性化,转化入制备好的含有腺病毒骨架质粒pAdEasy-1的感受态细胞,通过细胞内同源重组获得抑制Rab32基因表达的重组腺病毒载体;
D.将步骤C所得重组腺病毒载体线性化后用脂质体转染慢病毒感染细胞,获得抑制Rab32基因表达的重组腺病毒。
6.根据权利要求5所述的制备方法,其特征在于,所述慢病毒感染细胞为293T。
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