CN113913463A - 抑制sost基因表达的重组质粒及其骨靶向重组腺相关病毒与应用 - Google Patents
抑制sost基因表达的重组质粒及其骨靶向重组腺相关病毒与应用 Download PDFInfo
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- CN113913463A CN113913463A CN202111127433.XA CN202111127433A CN113913463A CN 113913463 A CN113913463 A CN 113913463A CN 202111127433 A CN202111127433 A CN 202111127433A CN 113913463 A CN113913463 A CN 113913463A
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Abstract
本发明属于生物医药技术领域,具体公开了一种抑制SOST基因表达的重组质粒及其骨靶向重组腺相关病毒与应用。通过构建一种可以靶向到骨细胞,降低骨细胞Sclerostin(Sost)的表达和分泌的,具有抗骨质疏松作用,给药途径包括肌肉注射、静脉输注,方便应用,为制备治疗或预防骨质疏松症上的应用提供了有力的证据。
Description
技术领域
本发明属于生物医药领域,具体涉及抑制SOST基因表达的重组质粒及其骨靶向重组腺相关病毒与应用。
背景技术
骨质疏松是一种会导致骨量减少、骨密度降低,骨微结构发生改变的全身性骨代谢疾病,骨质疏松根据发病原因可以分为原发性和继发性。继发性一般是由其他疾病或药物引发的骨骼疾病,而原发性是随着年龄的增长必然发生的一种生理衰退型病变,包括绝经性、老年性和特发性三种类型的骨质疏松。绝经后骨质疏松多发生于55岁-65岁中老年女性,而老年性骨质疏松多发生于70岁以上老年人群,特发性骨质疏松多见于青少年和成年,多伴有家族遗传史。骨质疏松的发病率逐年增加,之前的文献报道,全球大约有2亿人患骨质疏松,在中国,绝经女性占总人口的比例约占17%,这意味着患骨质疏松的绝经女性数量将大幅度增加。骨质疏松的主要特点为骨量大量丢失,包括骨矿物质与其基质比例减少;骨微结构由于骨组织形成和吸收失衡导致的退变,表现为骨小梁数量减少,骨小梁变细或发生断裂;骨的脆性增加、骨的力学强度降低,容易发生骨折等。这严重影响正常老年人的生活质量,也给社会的经济带来严重负担。
经典Wnt/β-catenin信号传导通路主要通过激活细胞内的β-catenin活性,使其转移至细胞核内,从而发挥生物学功能。成骨细胞的β-catenin的激活对骨形成至关重要,在小鼠的成骨细胞核骨细胞敲除β-catenin可显著降低松质骨和皮质骨的骨量。但奇怪的是,成骨细胞的细胞数并没有明显上升,而破骨细胞的细胞数却明显增加。破骨细胞的增加的原因认为是β-catenin调控了OPG基因,造成了骨保护素的降低。经典Wnt信号通路主要在细胞膜上通过与低密度脂蛋白供受体家族成员-细胞膜上供受体(LRP5/6)以及F-跨膜卷曲蛋白受体结合,促进糖原合成激酶3β磷酸化,抑制β-catenin退化,进而激活Wnt/β-catenin通路,使β-catenin蛋白因子大量聚集并移向核内,与核内转录因子(TCF/TEF)结合,激活成骨细胞核内下游靶基因cyclin-D1与c-myc从而使成骨细胞增殖加速。可见β-catenin蛋白在骨形成过程中具有重要作用。
Sclerostin是由sost基因编码表达翻译的糖蛋白,在成年以前,发现骨细胞、肝脏细胞、心脏、血管内皮细胞都可以分泌,在成年以后,sost基因主要由骨细胞、软骨细胞表达,分泌于血液中调控外周骨量。之前的研究发现,sclerostin是Wnt信号的特异性抑制因子,可以与细胞表面LRP5/6结合,激活了下游的GSK-3β活性,从而增加β-catenin的磷酸化水平,促进其蛋白降解,从而起到抑制Wnt信号作用。然而,虽然sclerostin目前已被FDA批准上市,但其具有相当程度的心血管风险,所以并未被广泛应用。
RNA干扰是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA搞笑特异性降解的现象。简单的说是指一种分子生物学上由双链RNA诱发的基因沉默现象。当细胞中导入与内源性mRNA编码区同源的双链RNA时,该mRNA发生降解而导致基因表达沉默,是一种特异性的转录后基因沉默,因此其可以特异的将特定的基因沉默,从而获得基因功能丧失或基因表达降低的作用,因此可以作为功能基因组学的一种强有力的研究工具。其中shRNA包括两个短反向重复序列,克隆到shRNA表达载体中的shRNA包括两个短反向重复序列,中间由一个颈环(Loop)序列分隔的,组成发夹结构,由polIII启动子控制,随后在连上5-6个T作为RNA聚合酶III的转录终止子。当送入动物体内时,该发夹序列被表达出来,形成一个“双链RNA”,并被RNAi通道处理。然而由于其打入体内具有一定的肝毒性,所以并未被广泛应用。因此增加组织靶向性将成为未来RNAi体内治疗的优势。
shRNA作为一种基因编辑技术,可以将靶细胞或靶组织中特异的基因沉默,从而获得基因功能丧失或基因表达降低的功能,因此可以作为功能基因组学的一种强有力的研究工具。Sclerostin是由sost基因编码表达翻译的糖蛋白,可以与细胞膜表面Lrp5/6受体结合,抑制细胞内Wnt/β-catenin通路,具有抑制骨形成的作用,因此Sost基因的shRNA可以作为一种基因层面治疗骨质疏松的潜在作用方式,然而由于Sost大多由骨细胞分泌,而骨细胞的shRNA治疗更为复杂,因此在动物层面无有效干扰骨细胞中Sost技术。同时由于在小鼠中发现shRNA技术具有一定的肝脏毒性作用,因此目前尚未用于临床验证。
发明内容
针对现有技术的不足,本发明提供了一种抑制SOST基因表达的重组质粒及其骨靶向重组腺相关病毒与应用,本发明通过构建一种可以靶向到骨细胞,降低骨细胞Sclerostin(Sost)分泌的,具有抗骨质疏松作用,给药途径包括肌肉注射、静脉输注,方便应用,为制备治疗或预防骨质疏松症上的应用提供了有力的证据。
为解决现有技术问题,本发明采取如下技术方案:
一种抑制SOST基因表达的重组质粒,所述重组质粒通过shRNA技术将编码SOST基因的核苷酸序列转入至pLKD-CMV-EGFP-2A-Puro-U6-shRNA载体上,使得124-142位点沉默,所述SOST基因的氨基酸序列如SEQ NO.1所示,所述124-142位点的氨基酸为SEQ NO.3所示:
SEQ NO.1如下所示:
1 mqpslapcli cllvhaafca vegqgwqafr ndatevipgl geypepppen nqtmnraeng
61 grpphhpyda kgvseyscre lhytrfltdg pcrsakpvte lvcsgqcgpa rllpnaigrv
121 kwwrpngpdf rcipdryraq rvqllcpgga aprsrkvrlv asckckrltr fhnqselkdf
181 gpetarpqkg rkprpgarga kanqaelena y
SEQ NO.3如下所示:
GCCTTCAGGAATGATGCCA
作为改进的是,所述shRNA的DNA序列如SEQ NO.2如下所示:GCCTTCAGGAATGATGCCATTCAAGAGATGGCATCATTCCTGAAGGC。
上述抑制SOST基因表达的重组质粒在抑制小鼠骨细胞中SOST基因表达中的应用。
一种靶向抑制SOST基因的重组腺相关病毒,表达权利要求1所述的抑制SOST基因表达的重组质粒的腺相关病毒,所述腺相关病毒经过骨靶向改造处理。
上述靶向抑制SOST基因的重组腺相关病毒的构建方法,包括以下步骤:
步骤1,将编码骨靶向肽基础序列DSS(AspSerSer)6插入于腺相关病毒中进行改造处理;
步骤2,构建权利要求1所述的抑制SOST基因表达的重组质粒;
步骤3,骨靶向包裹Sost-shRNA病毒的制备。
上述重组腺相关病毒在制备治疗或预防骨质疏松症相关产品上的应用。
一种药物组合物,其活性成分为上述重组腺相关病毒以及药学上可接受的载体或稀释剂。
作为改进的是,所述药物组合物的给药途径为肌肉注射或静脉输注。
有益效果:
与现有技术相比,本发明一种骨靶向病毒包裹干扰sost基因片段及其在制备治疗或预防骨质疏松症上的应用,构建一种可以靶向到骨细胞,降低骨细胞Sclerostin(Sost)分泌的,并借助药理实验以证明骨靶向病毒包裹sost干扰基因在抗骨质疏松中的用途。经实验验证,本发明表现出较好的骨靶向特性,同时促进骨形成增加骨量。本发明主要基于骨靶向病毒包裹sost干扰质粒所制备出来的新靶向技术,实验证明具有显著抑制骨细胞中sost表达,降低血清中sclerostin浓度,从而增加骨形成和缓解骨质疏松的作用,可用于进一步制备治疗骨质疏松的药物或保健品。
附图说明
图1为rAAV9-DSS-Nter骨靶向病毒设计原理;
图2为小鼠下肢骨活体荧光检测骨靶向组荧光显著增加;
图3为小鼠股骨组织切片Sclerostin免疫荧光检测骨靶向组骨细胞中荧光数量显著增加;
图4为不同组织蛋白中GFP蛋白检测骨靶向组骨组织中EGFP蛋白增加显著。
图5为Sost-shRNA2(Y10681)转染进入293T细胞中,可以显著降低细胞内SostmRNA表达水平,*:p<0.05,**:p<0.01,***:p<0.001,****:p<0.0001
图6为rAAV9-DSS-Nter骨靶向病毒包裹sost-shRNA设计原理;
图7为骨靶向治疗组血清中sclerostin蛋白浓度显著降低;
图8为骨靶向治疗组骨组织切片sclerostin IHC阳性点数量显著降低;
图9为骨靶向治疗组股骨组织骨小梁数量显著增加;
图10为骨靶向治疗组股骨组织切片中成骨细胞数量/骨表面积显著增加;
图11为骨靶向治疗组股骨组织切片中成骨细胞表面积/骨表面积显著增加;
图12为骨靶向治疗组中股骨骨质疏松显著缓解;
图13为骨靶向治疗组中股骨骨密度显著增加;
图14为骨靶向治疗组中BV/TV的变化情况;
图15为骨靶向治疗组中Tra.Num变化情况;
图16为选用的可以插入sost-shRNA干扰序列的载体及
pLKD-CMV-EGFP-2A-Puro-U6-shRNA载体图谱。
具体实施方式
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
另外,以下实施例中所用的质粒,载体都为常规市售产品,无需额外交代。
实施例1
1.1骨靶向病毒的制备
采用腺相关病毒2/9型(rAAV2/9)进行改造得骨靶向病毒,简单描述,构建编码骨靶向肽基础序列DSS(AspSerSer)6的DNA序列进行密码子优化,将表达AAV2 Rep基因和AAV9Cap基因的质粒(pAAV2/9)插入到AAV9 Cap基因的密码子中,获得Q588衣壳(DSS-588)。
之后构建DSS-Nter衣壳,首先将pAAV2/9中VP2的起始密码子突变(ACG→ACC),仅表达VP1和VP3(pAAV2/9.novp2),在另一个质粒中,将DSS序列融合到AAV9-VP2 ORF的N端,将Kozak序列和ATG起始密码子直接放置在DSS序列的上游,允许CMV启动子驱动表达(pcDNA.DSS-VP2(AAV9)),所构建的pAAV2/9.novp2和pcDNA.DSS-VP2(AAV9)则用于rAAV的生产。具体改造工作由上海合元生物技术公司完成。
1.2骨靶向病毒包裹EGFP的制备
EGFP靶基因序列(如SEQ NO.4示):
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT
TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC
CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC
GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC
GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT
CCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG
AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACG
GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACAT
GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
将EGFP靶基因按照常规步骤转载加入pcDNA3.1质粒中合成EGFP过表达质粒,再将EGFP过表达质粒与骨靶向病毒进行包装,构成骨靶向包裹EGFP病毒体系。具体包装过程如下:
第一步,把EGFP基因克隆进合适的载体,本实施例选用pcDNA3.1载体(上海合元生物);
第二步,重组表达质粒同pHelper(携带腺相关病毒来源的基因)和pAAV-RC(携带AAV复制和衣壳基因,此处包括未改造的rAAV2/9和改造后的rAAV2/9-DSS-Nter)共转染进AAV-293细胞,转染2-3天后重组AAV在包装细胞中组装完成;
第三步,从被感染AAV-293细胞中收集AAV病毒颗粒,一般AAV颗粒会富集在包装细胞中,所以收集细胞而后裂解释放AAV颗粒到上清中可以回收大部分的AAV颗粒;这一步得到的病毒上清液随后用于感染各种哺乳动物类细胞系的感染实验,同时上清中的病毒也可以浓缩保留;
第四步,浓缩并纯化第三步的病毒上清液,原上清液里面包含了许多细胞蛋白分子和碎片,通过2次CsCl密度梯度离心和1次超滤可以去除绝大部分的细胞蛋白和残留的CsCl离子。动物实验都需要纯化后的病毒才能够进行,否则会达不到所需剂量并引起副作用。感染宿主细胞后,基因表达前,单链病毒必须变成双链病毒;这个转变是重组基因表达的限制步骤,可以通过腺相关病毒双重感染或依托泊苷(喜树碱或丁酸钠)来加速。然而,加速基因表达的试剂对目标细胞是有毒的,如果残留到细胞上会杀死目标细胞。因此依托泊苷只能短期使用或为了提高病毒滴度时使用;
第五步,用定量PCR法测定所得到病毒的滴度。
1.3 Sost-shRNA的制备
根据shRNA文库,筛选出Sost-shRNA靶点有三个:
根据shRNA构建模式(上海合元生物)进行sost-shRNA靶基因插入载体中,简单如下:
所选用的干扰载体pLKD-CMV-EGFP-2A-Puro-U6-shRNA载体图谱见图13。用Age I和EcoR I酶切切去U6启动子下游的ccdB毒性基因,插入待构建的shRNA序列。根据MouseSost基因的转录本设计3个siRNA靶点,安排引物合成。将单链的引物退火成双链oligo序列,连接入双酶切线性化的RNA干扰载体,替换掉原来的ccdB毒性基因。菌落PCR筛选转化子,筛选的阳性克隆进行测序验证。测序验证正确的克隆,进行高纯度质粒抽提。实验共分以下8个主要步骤:
1.干扰靶点设计和引物合成:
根据shRNA设计的一般原则,设计3个siRNA靶点,安排引物合成(序列见1.3 sost-shRNA靶点表TargetSeq序列)。
2.引物退火形成带粘性末端的双链片段:
将合成好的oligo用oligo annealing buffer溶解成20μM,互补单链各取30μl混合,再将oligo混合物在水浴锅中95℃加热5min,然后水浴锅开盖置室温中自然冷却至室温,形成双链oligo片段;取1μl用于后续的连接反应,其余-20℃保存。
3.线性化表达载体的制备:
用限制性内切酶对表达载体进行酶切,酶切反应体系为:质粒2μg,10x反应Buffer5μl,限制性内切酶各1μl,去离子水补足50μl,于37℃水浴锅中孵育2h以上,酶切产物进行琼脂糖凝胶电泳检测酶切效果,并把目的载体条带从琼脂糖凝胶电泳后的胶中割下来,用TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0做胶回收,具体步骤参考试剂盒说明书。
4.干扰片段连接入表达载体:
连接反应体系,并于16℃连接过夜。
说明:阳性对照所加的退火的双链oligo是以前退火好的验证好用的片段,和连接组所加的退火的双链oligo长度一样,但序列无关。
5.感受态细胞的转化:
DH5α感受态细胞的转化,详见《精编分子生物学实验指南》。
6.菌落PCR鉴定阳性转化子:
挑取平板上长出的转化子重悬于10μl LB培养液中,取1μl做模板进行菌落PCR鉴定。反应体系和PCR循环条件如下:
①.按照下列组分配置PCR反应液:
②.PCR反应条件设置如下:
7.阳性克隆送测序:
菌落鉴定得到的阳性克隆,送测序公司进行测序验证,用Vector NTI软件比对测序结果,对测序结果进行分析。
8.质粒小提:
经测序验证正确的阳性克隆,安排质粒小提。具体步骤见试剂盒说明书。
1.4骨靶向包裹Sost-shRNA病毒的制备
将sost-shRNA质粒与1.1所提腺相关病毒进行包装,构成骨靶向包裹EGFP病毒体系。包装过程简单如下:
第一步:是把外源基因克隆进合适的载体。本项目装载pLKD-CMV-EGFP-2A-Puro-U6-shRNA(上海合元生物)。
第二步:重组表达质粒同pHelper(携带腺相关病毒来源的基因)和pAAV-RC(携带AAV复制和衣壳基因,此处为改造后的rAAV2/9-DSS-Nter)共转染进AAV-293细胞。转染2到3天后重组AAV在包装细胞中组装完成。
第三步:从被感染AAV-293细胞中收集AAV病毒颗粒,一般AAV颗粒会富集在包装细胞中,所以收集细胞而后裂解释放AAV颗粒到上清中可以回收大部分的AAV颗粒。这一步得到的病毒上清液随后用于感染各种哺乳动物类细胞系的感染实验。同时上清中的病毒也可以浓缩保留。
第四步:浓缩并纯化第三步的病毒上清液,原上清液里面包含了许多细胞蛋白分子和碎片,通过2次CsCl密度梯度离心和1次超滤可以去除绝大部分的细胞蛋白和残留的CsCl离子。动物实验都需要纯化后的病毒才能够进行,否则会达不到所需剂量并引起副作用。感染宿主细胞后,基因表达前,单链病毒必须变成双链病毒。这个转变是重组基因表达的限制步骤,可以通过腺相关病毒双重感染或依托泊苷(喜树碱或丁酸钠)来加速。然而,加速基因表达的试剂对目标细胞是有毒的,如果残留到细胞上会杀死目标细胞。因此依托泊苷只能短期使用或为了提高病毒滴度时使用。
第五步:用定量PCR法测定所得到病毒的滴度。
实施例2
为了验证骨靶向病毒在感染骨细胞过程中的有效性,将构建骨靶向包裹EGFP病毒(AAV9-DSS-Nter-EGFP)(图1),和对照病毒,及rAAV2/9-EGFP病毒。
通过尾静脉打入10周小鼠体内,浓度为4*1011。2月后对小鼠进行活体荧光检测、组织学GFP免疫荧光染色,不同组织GFP蛋白定量检测,明确骨靶向病毒是否可以到达骨细胞以及其作用效率。
进行活体荧光检测发现,骨组织中荧光强度显著高于普通型(图2),骨组织切片免疫荧光检测发现,在打入骨靶向病毒后,骨细胞表达EGFP的水平显著高于对照组(图3),提取各个组织蛋白进行Western Blot检测发现,骨组织中EGFP蛋白含量在骨靶向组中显著高于对照组(图4)。这表明我们构建的骨靶向病毒可以显著作用于骨细胞中,在功能上具有靶向骨细胞的功能。
实施例3
为了验证筛选可用的sost-shRNA质粒,将shSost转入293T细胞中,24小时后检测细胞内Sost mRNA表达水平。筛选出有效片段进行骨靶向病毒包装。
我们通过Lipo2000转染系统,将构建到的三种Sost-shRNA(见实施例1中1.3,及Y10680:GCCTCATCTGCCTACTTGT;Y10681:GCCTTCAGGAATGATGCCA;Y10682:CCATCCCTATGACGCCAAA)干扰质粒转染进入293T细胞中,24小时后提取细胞mRNA进行检测发现,Sost-shRNA2干扰序列(maker:Y20681)具有显著抑制293T细胞内Sost表达功能(图5)。因此选择其作为病毒包裹质粒进行后续研究。
实施例4
为了明确骨靶向包裹Sost-shRNA是否具有抗骨质疏松作用,选择15月龄小鼠进行尾静脉注射,浓度为4*1011。2个月后检测小鼠血清中sclerostin蛋白浓度,以及骨组织切片sclerostin免疫组化染色、骨组织切片马松三色、骨组织Micro-CT组织学评价。明确骨靶向包裹Sost-shRNA是否具有在衰老过程中治疗骨质疏松的作用。
构建好的骨靶向包裹的Sost-shRNA通过尾静脉注射入15月龄小鼠体内(如图6),2月后检测血清中Sclerostin蛋白浓度发现其显著下降(图7),骨细胞表达sclerostin水平显著降低(图8)。对骨量进行检测发现,股骨松质骨量增加明显(图9),成骨细胞数量显著增加(图10),成骨细胞表面积增加明显(图11)。MicroCT分析发现(图12),rAAV9-DSS-NtershSost组显著增加衰老小鼠骨密度(图13),同时对骨骼微结构具有显著的缓解作用(图14和图15)。因此,本发明可以具有靶向降低骨细胞Sost基因表达抑制sclerostin分泌,从而促进骨形成的作用。
综上所述,本发明通过改造rAAV9具有靶向骨细胞作用,在其包裹Sost-shRNA干扰质粒后,具有显著降低骨细胞Sost基因表达抑制sclerostin分泌促进骨形成的作用,从而具有抗骨质疏松的作用。
以上实验例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 郭保生
石天舒
蒋青
<120> 抑制SOST基因表达的重组质粒及其骨靶向重组腺相关病毒与应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 211
<212> PRT
<213> 氨基酸序列(Amino Acid Sequence)
<400> 1
Met Gln Pro Ser Leu Ala Pro Cys Leu Ile Cys Leu Leu Val His Ala
1 5 10 15
Ala Phe Cys Ala Val Glu Gly Gln Gly Trp Gln Ala Phe Arg Asn Asp
20 25 30
Ala Thr Glu Val Ile Pro Gly Leu Gly Glu Tyr Pro Glu Pro Pro Pro
35 40 45
Glu Asn Asn Gln Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro
50 55 60
His His Pro Tyr Asp Ala Lys Gly Val Ser Glu Tyr Ser Cys Arg Glu
65 70 75 80
Leu His Tyr Thr Arg Phe Leu Thr Asp Gly Pro Cys Arg Ser Ala Lys
85 90 95
Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu
100 105 110
Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly Pro
115 120 125
Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu
130 135 140
Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val
145 150 155 160
Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu
165 170 175
Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gln Lys Gly Arg Lys
180 185 190
Pro Arg Pro Gly Ala Arg Gly Ala Lys Ala Asn Gln Ala Glu Leu Glu
195 200 205
Asn Ala Tyr
210
<210> 2
<211> 19
<212> DNA
<213> 核苷酸序列(Nucleotide sequence)
<400> 2
gccttcagga atgatgcca 19
<210> 3
<211> 47
<212> DNA
<213> 核苷酸序列(Nucleotide sequence)
<400> 3
gccttcagga atgatgccat tcaagagatg gcatcattcc tgaaggc 47
<210> 4
<211> 720
<212> DNA
<213> 核苷酸序列(Nucleotide sequence)
<400> 4
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gcctcatctg cctacttgt 19
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gccttcagga atgatgcca 19
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccatccctat gacgccaaa 19
Claims (8)
1.抑制SOST基因表达的重组质粒,其特征在于,所述重组质粒通过shRNA技术将编码SOST基因的核苷酸序列转入至pLKD-CMV-EGFP-2A-Puro-U6-shRNA载体上,使得124-142位点沉默,所述SOST基因的氨基酸序列如SEQ NO.1所示,所述124-142位点的氨基酸为SEQNO.3所示。
2.根据权利要求1所述的抑制SOST基因表达的重组质粒,其特征在于,所述shRNA的DNA序列如SEQ NO.2如下所示
GCCTTCAGGAATGATGCCATTCAAGAGATGGCATCATTCCTGAAGGC。
3.权利要求1或2所述的抑制SOST基因表达的重组质粒在抑制小鼠骨细胞中SOST基因表达中的应用。
4.一种靶向抑制SOST基因的重组腺相关病毒,其特征在于,表达权利要求1所述的抑制SOST基因表达的重组质粒的腺相关病毒,所述腺相关病毒经过骨靶向改造处理。
5.基于权利要求4所述的一种靶向抑制SOST基因的重组腺相关病毒的构建方法,其特征在于,包括以下步骤:
步骤1,将编码骨靶向肽基础序列DSS(AspSerSer)6插入于腺相关病毒中进行改造处理;
步骤2,构建权利要求1所述的抑制SOST基因表达的重组质粒;
步骤3,骨靶向包裹Sost-shRNA病毒的制备。
6.基于权利要求4或5所述的重组腺相关病毒在制备治疗或预防骨质疏松症相关产品上的应用。
7.一种药物组合物,其特征在于,其活性成分为权利要求5或权利要求6所述的重组腺相关病毒以及药学上可接受的载体或稀释剂。
8.根据权利要求7所述的药物组合物,其特征在于,所述药物组合物的给药途径为肌肉注射或静脉输注。
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