CN105483130B - 一种逆转难治性癫痫的耐药性的药物 - Google Patents
一种逆转难治性癫痫的耐药性的药物 Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
本发明公开了SEQ ID NO.1所示的核苷酸序列,还公开了包含前述核苷酸序列的重组病毒及其应用。本发明重组慢病毒可以抑制耐药蛋白Pgp和耐药相关基因H I F‑1α的表达,还可以减少药物的泵出率,能够逆转难治性癫痫的耐药性,可以与其他治疗癫痫的药物联合使用,治疗难治性癫痫,临床应用前景良好。
Description
技术领域
本发明涉及一种逆转难治性癫痫的耐药性的药物。
背景技术
难治性癫痫又称之为顽固性癫痫,通常指无中枢神经系统进行性疾病或占位性病变,但临床迁延,经2年以上正规抗癫痫治疗,试用主要抗癫痫药单独或合用,达到患者能耐受最大剂量,血药浓度达到有效范围,仍不能控制发作,且影响日常生活,方可确定为难治性癫痫。难治性癫痫约占癫痫病人的20%~30%。
根据目前的研究发现,难治性癫痫之所以无法有效治疗跟其耐药有关,因此寻找药物逆转其耐药性的药物,以实现有效治疗非常重要。
在癫痫的耐药中,研究最多的是MDR1基因编码的P-糖蛋白(P-glycoprotein,P-gp),它利用分解ATP所释放的能量主动将药物泵出细胞外,导致细胞内药物浓度降低。研究发现,血脑屏障血管内皮细胞及星形胶质细胞,皮层和海马的神经元、星形胶质细胞、血管内皮细胞上均有P-gp的表达。研究证实,大多数抗癫痫药物如苯妥英钠、丙戊酸纳等都是P-gp的天然底物,P-糖蛋白通过耗能方式主动将抗癫痫药物泵出细胞外,导致癫痫病灶内药物浓度的降低,使抗癫痫药物不能有效发挥作用,这可能是引起难治性癫痫多药耐药的重要机制之一。
多项研究也发现,抑制P-gp的表达或活性可以有效逆转癫痫的耐药,实现难治性癫痫的有效治疗。因此,寻找抑制耐药蛋白Pgp过度表达的药物非常重要。
还有研究发现难治性癫痛患者脑内H I F-1α,其高表达也与难治性癫痫相关。抑制耐药蛋白H I F-1α表达的药物有望逆转难治性癫痫的耐药性。
发明内容
为了解决上述问题,发明人构建了一种可以抑制耐药蛋白Pgp过度表达的慢病毒。
本发明提供了SEQ ID NO.1所示的核苷酸序列。
本发明还提供了一种重组病毒,它包括SEQ ID NO.1所示的核苷酸序列。
优选地,它是重组慢病毒。
本发明还提供了前述重组病毒在制备逆转难治性癫痫耐药性的药物中的用途。
逆转难治性癫痫耐药性的药物:是指降低或者消除难治性癫痫耐药性的药物。
本发明还提供了前述重组慢病毒与癫痫治疗药物在制备治疗难治性癫痫的联合用药物中的用途。
本发明重组慢病毒可以抑制耐药蛋白Pgp和耐药相关基因H I F-1α的表达,还可以减少药物的泵出率,能够有效逆转难治性癫痫的耐药性,可以与其他治疗癫痫的药物联合使用,治疗难治性癫痫,临床应用前景良好。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
具体实施方式
实施例1重组慢病毒的构建
一、试验材料
DMEM培养基+10%FBS
D-Hank’s Solution
Trypsin-EDTA Solution(0.25%Trypsin+0.1mM EDTA,Hyclone)
10cm培养皿(Corning)
15cm培养皿(Corning)
RNAi-mate(上海吉玛)
无血清DMEM培养基
50ml离心管(Corning)
过滤器(Sartorius Stedim)
离心机(TGL)
DMEM培养基+10%FBS
D-Hank’s Solution
Trypsin-EDTA Solution(0.25%Trypsin+0.1mM EDTA,Hyclone)
96孔板(Corning)
Polybrene(5ug/ml Sigma)
慢病毒相关质粒,购自上海吉玛制药技术有限公司(Shanghai GenePharma Co.,Ltd)
名称 | 型号 | 厂家 |
PCR仪 | MG96+ | 杭州朗基科学仪器有限公司 |
冷冻离心机 | TGL-20M | 上海卢湘仪离心机仪器有限公司 |
恒温振荡培养箱 | HZQ-X400 | 太仓华美生化仪器有限公司 |
台式高速离心机 | TGL-14G | 上海医疗器械有限公司 |
电热恒温培养箱 | DPX-9082B-1 | 上海福玛实验设备有限公司 |
电泳仪电泳槽 | EPS300 | Tanon |
恒温磁力搅拌器 | 85-2 | 上海司乐仪器有限公司 |
电子天平 | TP1102 | 上海光正医疗仪器有限公司 |
微型旋涡混合仪 | XW-80A | 上海沪西分析仪器厂有限公司 |
数显恒温水浴锅 | HH-2 | 国华电器有限公司 |
仪器:
名称 | 货号 | 厂家 |
DNA内切酶(BamHI、EcoRI) | #ER0051、0271 | MBI Fermentas |
DNA连接酶 | #EL0011 | MBI Fermentas |
DNA marker | #SM0161 | MBI Fermentas |
琼脂糖 | RT101 | 天根生化科技有限公司 |
DNA凝胶回收试剂盒 | DP209-03 | 天根生化科技有限公司 |
中量抽提试剂盒 | AP-MD-P-25 | 杭州爱思进生物技术有限公司 |
酵母提取物 | LP0041 | OXOID |
胰蛋白胨 | LP0042 | OXOID |
琼脂 | 0201G16 | 捷瑞 |
Kanamycin | DH177-6.1 | Japan |
Ampicillin | DH022-1.1 | Sigma |
SDS | S0227 | BBI |
无乙水醇、异丙醇、丙三醇 | 北京国药集团化学试剂有限公司 | |
氯化钠 | 10019318 | 北京国药集团化学试剂有限公司 |
无水氯化钙 | 10005861 | 北京国药集团化学试剂有限公司 |
氢氧化钠 | 10019718 | 北京国药集团化学试剂有限公司 |
二、制备方法
(一)构建
1、Oligo Designer3.0
合成目的基因(SEQ ID NO.1):
AATTCAAAAAAGTTGCATAGTCACAAAAGTGATCTCTCTTGAAGATCACTTTTGTGACTATGCAACG
shRNA模板中的loop结构选用了TTCAAGAGA以避免形成终止信号。正义链模板的5’端添加了GATCC,与BamHI酶切后形成的粘端互补;反义链模板的5’端添加了AATTC,与EcoRI酶切后形成的粘端互补。
正义链:
5’-GATCC-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTG-3’
反义链:
3’-G(CN18)-(AAGTTCTCT)-(N18G)-AAAAAACTTAA-5’
2、shDNA模板的退火
将DNA oligo分别用TE(pH8.0)溶解,浓度为100uM。取相应的正义链和反义链oligo溶液,按照如下配比配置退火反应体系。
在PCR仪上按照如下程序进行退火处理:95℃ 5min;85℃ 5min;75℃5min;70℃5min;4℃保存。退火处理后得到浓度为10μM的shRNA模板。将所得模板溶液稀释50倍,终浓度为200nM,用于连接反应。
3、LV3载体的线性化
取10ug LV3载体,按照如下体系进行酶切处理:
37℃酶切1小时,琼脂糖电泳,使用Agarose Gel DNA Purification Kit Ver2.0回收,电泳检测估算浓度,稀释浓度至50ng/ul。
4、合成重组慢病毒LV3-shRNA
按照如下体系进行重组慢病毒的连接反应:
上表中的shDNA template为目的基因(SEQ ID NO.1):
AATTCAAAAAAGTTGCATAGTCACAAAAGTGATCTCTCTTGAAGATCACTTTTGTGACTATGCAACG。
22℃ 1hr,transform to Top10 competent cells.
5、感受态细胞的制备:(氯化钙法)
5.1从于37℃培养16小时的新鲜平板中挑取一个单菌落,转到一个含有100ml LB培养基的1L烧瓶中。于37℃剧烈振摇培养3小时(旋转摇床,300转/分)。
5.2在无菌条件下将细菌转移到一个无菌、一次性使用的、用冰预冷的50ml聚丙烯管中,在冰上放置10分钟,使培养物冷却至0℃。
5.3于4℃,以4000转/分离心10分钟,回收细胞。
5.4倒出培养液,将管倒置1分钟,使最后残留的痕量培养液流尽。
5.5以10ml用冰预冷的0.1mol/L CaCl2重悬每份沉淀,放置于冰浴上。
5.6于4℃,,以4000转/分离心10分钟,回收细胞。
5.7倒出培养液,将管倒置1分钟,使最后残留的痕量培养液流尽。
5.8每50ml初始培养物用2ml用冰预冷的0.1mol/L CaCl2(含20%甘油)重悬每份细胞沉淀。
5.9将细胞分装成小份(100μl/支),放于-70℃冻存。
(感受态细胞制备参考:分子克隆实验指南第二版55页)
6、连接产物(重组慢病毒LV3-shRNA)的转化
6.1从-70℃中取出感受态细胞,将装有感受态细胞的离心管冰上放置4分钟,待感受态细胞解冻后,加入10μl连接产物重组慢病毒LV3-shRNA,轻柔混匀内容物,在冰中放置30分钟。
6.2将离心管放到预加温到42℃的水浴锅中放好的试管架上,放置90秒,不要摇动离心管。
6.3快速将离心管转移到冰浴中,使细胞冷却3分钟。
6.4向每支离心管加入800μl LB培养基(不含抗生素),然后将离心管转 移到37℃摇床,250转/分钟,培育45分钟使细菌复苏。
6.5取200μl培育后的细胞均匀涂布于含50μg/ml Ampicillin LB平板上。
6.6等平板上液体被吸收后,将平板倒置于37℃培养箱中,培养16小时。
7、阳性克隆的鉴定与测序
7.1从每块平板上挑取5个菌落,接种到含50ug/ml Ampicillin的LB培养基中,37℃培养16小时。
7.2使用碱裂解法抽提质粒:
7.2.1取1.5ml培养物入微量离心管中,室温离心12000g×30sec,弃上清,将离心管倒置,使液体尽可能流尽。
7.2.2将细菌沉淀重悬于100μl预冷的溶液Ⅰ中,剧烈振荡,使菌体分散混匀。
7.2.3加200μl新鲜配制的溶液Ⅱ,颠倒数次混匀(不要剧烈振荡),并将离心管室温放置2-3min,使细胞裂解(溶液Ⅱ为裂解液,故离心管中菌液逐渐变粘稠变清)。
7.2.4加入150μl预冷的溶液Ⅲ,将管温和颠倒数次混匀,见白色絮状沉淀,可在冰上放置3-5min。溶液Ⅲ为中和溶液,此时质粒DNA复性,染色体和蛋白质不可逆变性,形成不可溶复合物,同时K+使SDS-蛋白复合物沉淀。
7.2.5加入450μl的苯酚/氯仿/异戊醇,振荡混匀,4℃离心12000g×5min。
7.2.6小心移出上清约400μl于一新微量离心管中,加入2倍体积预冷的无水乙醇,混匀,-20℃放置20min,4℃离心12000g×15min,弃上清。
7.2.7用1ml预冷的70%乙醇洗涤沉淀1-2次,4℃离心8000g×7min,弃上清,将沉淀在室温下晾干。
7.2.8沉淀溶于50μl TE(含RNase A 20μg/ml),室温放置10min以降解RNA,-20℃保存备用。
7.3所得质粒用EcoRI进行单酶切鉴定,酶切结果表明,被EcoRI切开的可能是阳性克隆,将克隆进行测序鉴定。
7.4测序正确的菌株采用高纯度质粒中量抽提试剂盒抽提,所得质粒可以用于常规的分子生物学实验和细胞学实验。如果在用于细胞转染时细胞毒性较大,请重新转化至大肠杆菌Top10中,然后用试剂盒或CsCl超速离心法制备更高纯度质粒。
(二)慢病毒包装
1.293T细胞在10cm培养皿中培养至80-90%融合时,接种15cm培养皿。
2.倾去培养液,用1ml D-Hank’s solution洗涤细胞两次。
3.加入1ml Trypsin-EDTA solution,混匀后,37℃放置2-3分钟。
4.小心吸去胰酶溶液,加入2ml含10%FBS的DMEM培养液,吹打使细胞形成单细胞悬液。
5.将细胞悬液接种15cm培养皿,加入18ml含10%FBS的DMEM培养液,混匀后37℃5%CO2培养过夜。
6.在一支无菌的5ml离心管中加入1.5ml无血清DMEM,按比例加入含目的基因的重组质粒和包装质粒(pGag/Pol、pRev、pVSV-G),混匀,取另一支无菌的5ml离心管,加入1.5ml无血清DMEM,再加入300μl RNAi-mate,混匀,室温放置5分钟后将两管混合,室温放置20~25分钟。
7.除去15cm培养皿中的培养液,加入8ml无血清的DMEM培养液。
8.将转染混合物逐滴加入15cm培养皿中,轻轻地前后摇晃培养皿以混匀复合物,在37℃5%CO2培养箱中温育4-6小时。
9.吸弃转染液,加入18ml含10%FBS的DMEM培养液。37℃5%CO2继续培养72小时。
(三)慢病毒收集
1.将培养皿中细胞上清液吸到50ml离心管中,4℃,4000rpm,4min。
2.低速离心后,将离心管上清液倒入50ml注射器内,用0.45um过滤器过滤。
3.滤液在离心机中进行超速离心,4℃,20000rpm,2h。
4.将浓缩液收集分装至出货管中。
5.分装的病毒液(即本发明重组慢病毒LV3)贴上标签,-80℃冰箱保存。
三、慢病毒滴度检测
1.293T细胞在10cm dish中培养至80-90%融合时,倾去培养液,用3ml D-Hank’ssolution洗涤细胞两次。
2.加1ml Trypsin-EDTA solution,混匀后,小心吸去胰酶溶液,37℃放置3-5分钟。
3.在加入2ml含10%FBS的DMEM培养液,吹打使细胞形成单细胞悬液。
4.按3×104细胞/孔的浓度接种96孔板,混匀后于37℃5%CO2培养24h。
5.将慢病毒原液(10-20ul),用10%FBS的DMEM培液十倍稀释3-5个梯度(根据细胞状态,如有必要可加入终浓度为5ug/ml的Polybrene)。
6.吸去96孔板中的培养液,每孔加入100μl稀释的病毒液,同时设立空白对照组,于37℃5%CO2培养24h。
7.吸弃96孔板中的稀释病毒液,每孔加入100μl 10%FBS的DMEM培液,(根据细胞状态,如有必要可分出1/3-1/5)于37℃5%CO2继续培养72h。
8.通过荧光显微镜或FACS计数荧光细胞,结合稀释倍数计算病毒滴度。
四、检测结果
检测结果显示,本发明制备得到的重组慢病毒的滴度为1x109TU/ml(注:此病毒侵染时间均为72h。)
试验结果说明,本发明方法制备得到的包含SEQ ID NO.1所示目的基因的重组慢病毒,且其滴度高。
以下用试验例的方式来说明本发明的有益效果:
试验例1本发明重组慢病毒逆转难治性癫痫耐药性的效果实验
马桑内酯点燃SD大鼠脑血管内皮细胞、星形胶质细胞及马桑内酯致痫耐药基因整体动物模型事是公认癫痫细胞模型和动物模型,脑组织中均检测到Pgp和HIF-1a过度表达,并证实其是癫痫耐药的重要机制。
本实验以马桑内酯点燃SD大鼠脑血管内皮细胞、星型胶质细胞(两种细胞按照文献Lei Chen,Xinwang Cheng,Linyu Tian,Tianhua Yang,Stefan Hermann,DongZhou.Inhibition of P-glycoprotein Over-expression by shRNA-mdr1bin RatAstrocytes.Neurochem Res(2009)34:411–417的方法构建)及马桑内酯致痫耐药基因整体动物模型为研究对象,从细胞水平到机体水平探讨其对耐药相关基因、蛋白HIF-1a和P-gp的表达抑制效率及癫痫耐药的逆转:
1、细胞模型
通过罗丹明蓄积及外排实验模拟抗癫痫药物来检测慢病毒感染前后细胞Pgp功能的变化,从而了解是否癫痫耐药能够得到逆转。
取实施例1构建的本发明包含SEQ ID NO.1所示目的基因的重组慢病毒、空白慢病毒,分别感染星型胶质细胞和微血管内皮细胞72h后,用胰酶消化收集细胞,细胞加入红色罗丹明染料500ug/mL,在5%二氧化碳37℃恒温孵箱培养45min,收集清洗后300g离心10min,用500uLPBS重悬细胞立即行流式细胞检测,以检测罗丹明在细胞内的蓄积。用Elite型(Coulter公司)流式细胞仪在λex=488nm,λem=525nm处检测罗丹明荧光强度。
对比重组慢病毒感染组和空白慢病毒感染组细胞对罗丹明的泵出率,发现前者的泵出率降低约70%,说明本发明重组慢病毒可以减少药物的排出,能够逆转癫痫耐药。
2、动物模型(马桑内酯致痫耐药基因整体动物模型)
(1)试验方法
模拟人类难治性癫痫的化学点燃耐药癫痫模型—马桑内酯点燃的颞叶癫痫模型,检测其中的耐药基因的表达水平:
模型构建:大鼠肌肉注射不同剂量的马桑内酯,确定阈下刺激剂量。大鼠每72h肌注阈下剂量的马桑内酯一次,固定给药时间,对照组用等量生理盐水肌注。发作参照Racine的5级分级标准判定。点燃标准:连续5次给药后3h内5级发作伴EEG显示海马及皮层持续性高波幅癫痫样波,给药间期1-3级发作伴EEG阵发局部棘波、尖波,棘-尖、棘-慢复合波。达到点燃标准后以每6天给药一次维持发作。一.5~3个月模型构建成功。模型构建中应用成熟的大鼠模型脑电监测方法,见下:1%戊巴比妥(30mg/kg,腹腔注射)麻醉后,固定在定位仪上,剪开头顶皮肤,暴露颅骨顶部,按照大鼠脑图谱,选定双侧海马坐标(前囟后3.6mm,中线旁开4.9mm,颅骨表面下0.5mm),在颅骨相应位置上钻一直径为0.8mm的小孔,分别埋入单芯电极,以牙托粉固定,作为记录皮层脑电图的引导电极。
检测方法:
选取SD大鼠耐药癫痫模型分为两组,分别给与本发明实施例1构建的包含SEQ IDNO.1所示目的基因的重组慢病毒和空白慢病毒侧脑室注射,在12天取脑组织,分别通过免疫组织化学方法进行颞叶HIF-1α,MDR1表达蛋白Pgp检测,荧光显微镜下观察各自的表达,最后用RT-PCR和Western-Blot技术验证上述两种耐药相关基因在大鼠模型颞叶的表达量和表达蛋白的表达量,说明本发明重组慢病毒有逆转耐药的作用。具体检测技术见下:
免疫组织化学(IHC)检测:
取石蜡包埋脑组织冠状面连续切片(Leica,RM2135),厚度为4μm,相邻切面分别行HE染色及免疫组织化学检测。图片获取使用Olympus图像采集系统。
IHC采用Envision法步骤:
1)切片脱蜡;
2)梯度酒精水化组织切片;
3)蒸馏水漂洗3min*2次,PBS洗5min*2次;
4)3%H2O2阻断内源性过氧化物酶,室温10min;
5)蒸馏水漂洗3min*2次,PBS洗5min*2次;
6)滴加一抗(HIF-1α小鼠单克隆抗体,MDR1/Pgp兔多克隆抗体,PBS代替一抗作为阴性对照)37℃孵育1小时;
7)PBS漂洗5min*3次;
8)滴加二抗(通用型多聚物HRP酶标二抗),孵育30min;
9)PBS漂洗5min*3次;
10)DAB试剂盒显色5-10分钟(镜下掌握染色程度);
11)蒸馏水漂洗3min*2次;
12)苏木素复染1~2min;
13)蒸馏水漂洗3min*2次;
14)树脂封片。
RQ-PCR检测:
组织提取采用Trizol法。两个目标基因分别以β-actin为内参标准化。
1)组织50mg用Trizol(Invitrogen公司)裂解;
2)异丙醇沉淀、乙醇洗涤提取总RNA;
3)1%琼脂糖凝胶电泳检测RNA的完整性;
4)逆转录合成cDNA第一链,反应条件:30℃10min,42℃30min,99℃5min,5℃5min;
5)RQ-PCR:反应体系包括:Taq DNA聚合酶(5u/μl)*0.3μl、PCR反应缓冲液(10×)3μl、MgCl2溶液(25mM)*3μl、dNTP(25mM)*0.36μl,cDNA 2μl,上、下游引物各*1μl、TaqMan探针*1μl(引物合成及探针修饰:上海生工生物工程有限公司)以双蒸水(ddH2O)配至30μl。探针及引物序列详见表2。PCR扩增条件:94℃2分钟,1个循环;94℃20秒,50℃(HIF-1α)/54℃(MDR1、β-actin)20秒,60℃30秒,循环45次(FTC2000PCR系统)。
6)随机选取15个PCR扩增产物通过2.0%琼脂糖凝胶电泳定性检测。
根据Livak[33]推导得出公式:相对表达率(R)=2-ΔΔCt,ΔCT=CT(目标基因)–CT(β-actin);whereΔΔCT=ΔCT(实验组)–ΔCT(对照组)。其中CT代表热循环仪检测到反应体系中荧光信号时的循环数;R表示实验组目的基因表达相对于对照组基因表达倍数。
表2.HIF-1α、MDR1及β-actin探针及引物序列
Western-Blot检测:
两个目标蛋白HIF-1α、Pgp均以β-actin为内参标准化,步骤如下:
1)组织100mg于裂解液(含PBS、1%SDS)中超声裂解匀浆(冰上操作);
2)4℃离心(12000g),15分钟,取上清液;
3)用BCA蛋白定量检测试剂盒(Pierce公司)测蛋白浓度,调整浓度至2μg/μl;
4)蛋白样品煮沸,10min,按需取用,余低温储存备用;
5)各标本取15μL蛋白样品经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)浓缩分离蛋白(浓缩胶4%、PH6.8,分离胶10%、PH8.8)(Amersham:80-6418-77,160V恒压);
6)分离完成后湿转蛋白至PVDF膜(电压100V,恒流300mA,1小时,Amersham TE22);
7)封闭液室温封闭1.5小时,加稀释度1:1000一抗,4℃过夜,洗涤;
8)加HRP标记山羊抗小鼠(HIF-1α)、抗兔(Pgp)二抗(1:10000,Pierce)室温孵育1小时,洗涤;
9)以化学发光显影底物(Millipore)室温孵育2min;
10)转移至暗箱,胶片曝光定影;
灰度值的测定采用Image-J分析软件(NIH)。不同膜上的样本相对表达分值以对照组中的同一个样本标准化。
(2)试验结果
大鼠模型颞叶检测表达量如下表所示:
△CT值越大,代表mRNA表达水平越低。
检测结果发现,本发明重组慢病毒感染大鼠耐药癫痫模型后,模型的海 马、颞叶组织中与耐药相关基因HIF-1α,Pgp及其蛋白表达量显著下降(有统计学差异,P<0.05)。
综上,本发明成功构建得到了包含SEQ ID NO.1所示目的基因的重组慢病毒,且证明了其可以有效降低耐药癫痫模型中耐药相关蛋白Pgp和HIF-1α的表达,还可以减少药物的泵出率,能够有效逆转难治性癫痫的耐药性,可以与其他治疗癫痫的药物联合使用,治疗难治性癫痫。
Claims (4)
1.一种核酸,其特征在于:它的核苷酸序列如SEQ ID NO .1所示。
2.一种重组病毒,其特征在于:它包括SEQ ID NO .1所示的核苷酸序列。
3.根据权利要求2所述的重组病毒,其特征在于:它是重组慢病毒。
4.权利要求2或3所述的重组病毒在制备逆转难治性癫痫的耐药性的药物中的用途。
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