CN114848848B - 一种逆转非小细胞肺癌耐药性的基因药物 - Google Patents
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Abstract
本发明公开了一种用于逆转非小细胞肺癌对厄洛替尼耐药的基因药物,本发明将人LMNA基因全长编码区cDNA插入到病毒表达载体,再利用病毒感染非小细胞肺癌厄洛替尼耐药细胞株的方法过表达lamin A/C蛋白,增加lamin A/C蛋白在非小细胞肺癌中的表达,所得到的病毒载体得以高效、持续和稳定的表达lamin A/C蛋白。本发明所构建的LMNA病毒载体可以逆转非小细胞肺癌细胞对厄洛替尼的耐药性,因此具有广阔的应用前景。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种逆转非小细胞肺癌对厄洛替尼获得性耐药的基因药物。
背景技术
非小细胞肺癌(NSCLC)是临床最常见的恶性肿瘤之一,占肺癌总数的80%以上。经手术、放疗和化疗等综合治疗后,其5年总生存率仅12%~15%,严重影响人类的生存质量和期望寿命。由于治疗的持续性和高精确性,肺癌不仅给个人造成巨大经济负担,同时给社会生产力带来严重损失,影响国家经济发展。因此,深入研究NSCLC发病及耐药机制并发展新的治疗策略对人类健康和国家经济发展都具有极其重要的意义。
厄洛替尼是一种靶向表皮生长因子受体酪氨酸激酶抑制剂,与传统治疗药物(如细胞毒类药物)相比具有更强的针对性,可以增强对肿瘤的杀伤作用,并减少对正常细胞的毒副作用,对EGFR突变的晚期NSCLC患者非常有效,但是多数患者在使用厄洛替尼一段时间后(大约1年左右)会出现疾病的再次进展,根本原因是由于肺癌细胞对厄洛替尼产生了耐药性。目前已发现的耐药机制主要包括:(1)EGFR基因的二次突变(T790M突变);(2)c-MET原癌基因扩增;(3)K-Ras基因突变;(4)PTEN功能丧失;(5)组织学转化。非小细胞肺癌EGFR-TKI获得性耐药机制涉及多基因、多途径的复杂调控过程,部分耐药机制尚不明确。
核纤层蛋白A/C(Lamin A/C)由人类基因LMNA基因编码的蛋白质,可以直接与核染色质作用,或调节转录因子活性调控基因表达,在细胞信号转导、周期调控、氧化应激及DNA损伤反应等生理过程中发挥重要作用。核纤层蛋白在癌组织中的异常定位或错误表达可能成为癌症的生物靶标之一。但目前LMNA基因与肿瘤细胞的耐药性研究还不得而知,同时开发其他逆转耐药性药物克服临床治疗非小细胞肺癌厄洛替尼耐药性迫在眉睫。
发明内容
本发明的目的之一在于提供一种逆转非小细胞肺癌对厄洛替尼获得性耐药的基因药物。
本发明另一目的在于提供上述基因药物的制备方法。
本发明第三目的在于提供人LMNA基因的新应用。
本发明目的按如下技术方案实现的:
一种逆转非小细胞肺癌对厄洛替尼获得性耐药的基因药物,其特征在于,所述基因药物为携带LMNA基因的病毒载体,所述LMNA基因序列为SEQ ID NO.1 所示,所述LMNA基因受CMV启动子的调控。本发明基因药物可大量、高效地表达LaminA/C蛋白。
为了进一步实现更稳定地表达,上述病毒载体优选采用pLVX-Puro载体。
LMNA基因在制备用于逆转非小细胞肺癌对厄洛替尼耐药性的药物中的应用。
具体地,上述应用中,上述LMNA基因序列为SEQ ID NO.1 所示。
进一步,上述应用中,为了更高效地表达,上述LMNA基因受CMV启动子的调控。
更进一步,为了更高效稳定、靶向地表达,上述应用中,上述LMNA基因是与病毒载体相连,所述病毒载体优选采用pLVX-Puro载体。
上述基因药物的构建方法,其特征在于,按如下步骤进行:
1、病毒载体LMNA的构建
根据LMNA基因,核苷酸序列如SEQ ID NO.1序列设计引物,上游引物LMNA-F 核苷酸序列如SEQ ID NO.2,下游引物LMNA-R,核苷酸序列如SEQ ID NO.3,通过PCR扩增方式将酶切位点引入LMNA基因两端,PCR条件为:95℃ 预变性5min;95℃变性30s、58℃退火30s、72℃延伸2min,共35个循环; 72℃ 终延伸5min。
LMNA基因受CMV启动子的调控,同时对LMNA基因片段和pLVX-Puro载体进行
XhoI和
BamHI双酶切,回收 LMNA基因片段和pLVX-Puro载体片段,两片段连接后转化
E.coli DH5α感受态细胞,筛选、测序鉴定获得pLVX-Puro-LMNA载体;
应用宝生物公司三质粒包装系统(pMD.2G、psPAX2、pLVX-Puro-LMNA)制备慢病毒,将HEK293T细胞培养在100mm皿中,待细胞长到60-70%时可进行转染;转染体系为:取1mlDMEM无血清培养基加入2.5μg pMD.2G、7.5μg psPAX2、10μg pLVX-Puro-LMNA,混匀后加入20μl PEI (2mg/ml)溶液,与质粒载体混匀,室温静置5min,缓慢滴加到培养皿中,十字交叉混匀,培养箱中培养48h (无需更换培养基),吸取上清,4000rpm离心5min,取上清,制得病毒载体LMNA;
2、细胞株中核纤层蛋白Lamin A/C表达检测
通过浓度梯度法建立HCC827.ER耐药细胞株,利用western blot法检测亲本HCC8287和耐药细胞HCC827.ER细胞裂解物中的lamin A/C蛋白表达情况,用载体LMNA病毒对HCC827.ER细胞进行感染,感染48h后采用免疫荧光技术检测细胞中lamin A/C蛋白的表达;
3、MTT法检测各细胞对厄洛替尼的敏感性;
4、细胞骨架染色后光学显微镜观察形态与细胞骨架;
5、细胞计数仪扫描LMNA基因可以抑制肿瘤细胞克隆的形成;
6、荧光显微镜观察LMNA基因抑制细胞增殖、迁移和侵袭;
7、Western blot检测LMNA基因对细胞周期蛋白的调控。
本发明获得的有益效果:
本发明提供了一种逆转非小细胞肺癌厄洛替尼获得性耐药的基因药物。首先,本发明首次发现对厄洛替尼耐药的非小细胞肺癌细胞株中LMNA基因的甲基化明显,从而降低lamin A/C蛋白表达。并基于此科学现象本发明采用病毒载体携带人LMNA基因增加laminA/C蛋白在非小细胞肺癌中的表达,所得到的病毒载体得以高效、持续和稳定的表达laminA/C蛋白,同时本发明基因药物可更好地进入肿瘤细胞内部实现靶向表达。本发明所构建的LMNA病毒载体可以逆转非小细胞肺癌细胞对厄洛替尼的耐药性,因此具有广阔的应用前景。
附图说明
图1:载体构建电泳图,M:Tran8K DNA marker;Lane1:LMNA基因PCR片段其大小1995bp;Line2:pLVX-Puro-LMNA载体BamHI与XhoI双酶切,载体片段大小约8000bp,酶切LMNA大小1995bp与PCR片段一致。
图2:亲本株HCC827与耐药株HCC827.ER核纤层蛋白lamin A/C表达差异,A.western blot检测HCC827与HCC827.ER细胞中laminA/C表达情况,灰度值分析显示,HCC827细胞中laminA和laminC蛋白表达显著高于HCC827.ER细胞(**P<0.01);B. 免疫荧光法检测感染携带LMNA基因病毒的HCC827.ER细胞中laminA/C表达情况,结果显示感染病毒的细胞中laminA/C蛋白表达显著高于未感染细胞。
图3:细胞株DNA的硫化修饰及甲基化特异性PCR,“+”表示阳性对照,“-”阴性对照;M表示甲基化;U表示非甲基化。
图4:人LMNA基因对厄洛替尼耐药变化曲线,MTT法测定结果显示,HCC827对厄洛替尼敏感性显著高于耐药细胞HCC827.ER(##P<0.01),过表达LMNA基因能提高HCC827.ER对厄洛替尼的敏感性(*P<0.05,**P<0.01)。
图5:人LMNA基因逆转细胞形态改变,A.光学显微镜下观察HCC827、HCC827.ER、HCC827.ER-LMNA细胞形态;B. 荧光显微镜下观察细胞骨架变化,红色为细胞骨架蛋白F-actin,蓝色为细胞核。
图6:人LMNA基因抑制HCC827.ER细胞克隆形成,A.HCC827.ER与HCC827.ER-LMNA细胞在2.5μM厄洛替尼处理后细胞克隆形成图;B. 不同细胞克隆形成数量柱状图(**P<0.01)。
图7:人LMNA基因抑制细胞迁移和侵袭,A. 不同时间HCC827、HCC827.ER与HCC827.ER-LMNA细胞迁移和侵袭情况;B. 细胞划痕伤口愈合分析柱状图和细胞侵袭数量图(**P<0.01表示HCC827与HCC287.ER之间具有显著性差异;##P<0.01表示HCC827.ER与HCC827.ER-LMNA之间具有显著性差异)。
图8:人LMNA基因抑制细胞增殖,A.免疫荧光染色观察细胞增殖情况,BrdU红色代表增殖细胞,DAPI蓝色代表所有细胞。B.在不同浓度厄洛替尼下细胞增殖数量柱状分析图(*P<0.05,**P<0.01)。
图9:人LMNA基因调控细胞周期蛋白逆转厄洛替尼耐药性,NC:HCC827.ER细胞,Vector:感染了空载体的HCC827.ER细胞,LMNA:感染了pLVX-Puro-LMNA载体的HCC827.ER细胞。
具体实施方式
实施例1 病毒载体LMNA的构建及制备
根据LMNA基因,核苷酸序列如SEQ ID NO.1序列设计引物,上游引物LMNA-F 核苷酸序列如SEQ ID NO.2,下游引物LMNA-R,核苷酸序列如SEQ ID NO.3。LMNA的cDNA购于北京义翘神州生物技术有限(货号:HG12058-ANG)作为模板,PCR条件为:95℃ 预变性5min;95℃变性30s、58℃退火30s、72℃延伸2min,共35个循环; 72℃ 终延伸5min。通过PCR扩增方式将酶切位点引入LMNA基因两端,LMNA基因受CMV启动子的调控,同时对LMNA基因片段和pLVX-Puro载体(成都传世科为生物技术有限公司)进行
XhoI和
BamHI双酶切,回收LMNA基因片段和pLVX-Puro载体片段,两片段连接后转化E.coli DH5α感受态细胞,筛选、测序鉴定获得pLVX-Puro-LMNA载体,如图1所示。应用宝生物工程有限公司三质粒包装系统(pMD.2G、psPAX2、pLVX-Puro-LMNA)制备慢病毒。简要地,将HEK293T(购买于南京科佰生物科技有限公司)细胞培养在100mm皿中,待细胞长到60-70%时可进行转染。转染体系为:取1ml DMEM无血清培养基加入2.5μg pMD.2G、7.5μg psPAX2、10μg pLVX-Puro-LMNA,混匀后加入20μlPEI (2mg/ml)溶液,与质粒载体混匀,室温静置5min。缓慢滴加到培养皿中,十字交叉混匀。培养箱中培养48h (无需更换培养基),吸取上清,4000rpm离心5min,取上清,分装(1mL/EP管),-80℃保存备用。
实施例2细胞株中核纤层蛋白Lamin A/C表达检测
通过浓度梯度法建立HCC827.ER耐药细胞株,利用western blot法检测亲本HCC827和耐药细胞HCC827.ER细胞裂解物中的lamin A/C蛋白表达情况。简要步骤如下:收集细胞,加入RIPA裂解液(含蛋白酶抑制剂)破碎细胞,15000rpm离心30min,取上清;蛋白上清采用BCA法定量,上样30μg总蛋白进行SDS-PAGE电泳;采用湿转方法将PAGE胶上的蛋白转印至PVDF膜上,然后5%脱脂牛奶室温封闭2h,TBST洗涤5次,每次2min;lamin A/C抗体(1:1000,ab8984)4℃孵育过夜,TBST洗涤5次,每次2min;二抗稀释比例为1:5000,室温孵育2h,TBST洗涤5次,每次2min;ECL显影,置于化学发光成像仪中曝光,保存图片。实验结果如图2A所示,耐药细胞HCC827.ER中laminA/C蛋白表达显著低于亲本HCC827细胞,差异具有统计学意义(**P<0.01)。
收集HCC827和HCC827.ER细胞,利用基因组DNA提取试剂盒提取DNA并定量。取1μg基因组DNA,加入去离子水补足20μL,采用甲基化DNA检测试剂盒对DNA进行处理,简要步骤如下,向上述DNA样品中加入2.2μL的M-Dilution buffer,42℃水浴30min,继续加入220μLCT conversion reagent混匀,80℃避光孵育60min,再加入480μL M-buffer PA,然后通过DNA吸附柱纯化修饰后的DNA,最后溶于20μL去离子水中。以修饰后的DNA为模板,LMNA甲基化引物(M):上游引物核苷酸序列如SEQ ID NO.4,下游引物核苷酸序列如SEQ ID NO.5。LMNA非甲基化引物(U):上游引物核苷酸序列如SEQ ID NO.6,下游引物核苷酸序列如SEQID NO.7,进行甲基化特异性PCR。 PCR条件为:95℃ 预变性5min;95℃变性30s、58℃退火30s、72℃延伸30s,共35个循环;1%琼脂糖凝胶电泳,80V,30min。凝胶置于凝胶成像系统中拍照。
甲基化特异性PCR检测了laminA/C基因甲基化情况,结果如图3所示,在HCC827.ER细胞中laminA/C基因启动子区甲基化水平明显高于HCC827亲本细胞。Western blot结果显示在蛋白水平上,HCC827.ER细胞中laminA/C蛋白表达低于HCC827细胞,这表明启动子区高甲基化抑制了laminA/C的表达。
实施例1中收获的病毒对HCC827.ER细胞进行感染,感染48h后采用免疫荧光技术检测细胞中lamin A/C蛋白的表达。首先,实验设置HCC827,HCC827.ER,HCC827.ER-LMNA分组,病毒感染48h后弃掉培养上清,加入预冷的4%多聚甲醛室温固定20min,PBS缓冲液洗涤3次,每次1min;用0.1%Triton X100穿孔30min,PBS洗涤5次;加入0.5%BSA封闭液室温封闭30min,阻断抗体蛋白的非特异性结合,降低背景;吸弃封闭液,PBS洗涤5次,滴加一抗200μl(抗体稀释液稀释抗体lamin A/C 1:250),置于湿盒中4℃,过夜;PBS洗涤5次,滴加二抗(1:500),室温孵育1h;PBS洗涤5次,加入DAPI(1:1000),室温孵育5min,标记细胞核;PBS洗涤5次,荧光显微镜下观察,拍照并分析(图2B),实验结果表明,HCC827.ER细胞中laminA/C蛋白表达低于HCC827细胞,与western blot结果一致;感染LMNA过表达病毒的HCC827.ER-LMNA细胞中lamin A/C蛋白表达明显升高,说明LMNA过表达病毒构建成功。
实施例3 MTT法检测各细胞对厄洛替尼的敏感性
将HCC827、HCC827.ER、HCC827.ER-LMNA细胞计数后,调整细胞浓度为6×104个/ml,向96孔板中加入100μl细胞悬液,CO2培养箱中孵育12h使细胞贴壁;向每孔培养基中加入终浓度为0、0.0005、0.001、0.005、0.01、0.05、0.1、0.5、1、5μM的厄洛替尼处理48h;每孔中加入10μl MTT试剂后继续培养4h;弃掉上清,加入200μl DMSO,摇床80rpm振摇30min;通过酶标仪测定450nm吸光度,保存数据并分析(图4)。HCC827.ER细胞对厄洛替尼的耐受性显著高于HCC827细胞(##P<0.01),HCC827.ER细胞对厄洛替尼的耐受性比HCC827.ER细胞在厄洛替尼浓度高于0.05μM时显著降低(*P<0.05,**P<0.01)。实验结果表明,HCC827.ER细胞过表达LMNA基因对厄洛替尼的敏感性增加,提示LMNA基因能逆转HCC827.ER厄洛替尼耐药性。
实施例4 细胞形态与细胞骨架观察
将HCC827、HCC827.ER、HCC827.ER-LMNA培养于12孔板中,当融合度为70%左右时进行光学显微镜观察及细胞骨架染色。结果表明,HCC827.ER细胞变得细长,多触须,细胞分散生长;而HCC827细胞聚团生长,形态饱满,触须少(图5A);HCC827.ER-LMNA细胞形态变得与HCC827相似,触须变短减少。从细胞骨架来看,HCC827.ER细胞骨架松散,而HCC827与HCC827.ER-LMNA细胞骨架紧实(图5B)。
实施例5 LMNA基因抑制肿瘤细胞克隆的形成
将HCC827.ER与HCC827.ER-LMNA细胞计数,按3000个细胞每孔接入6孔板中,接种24h后分别加入0、2.5μM 厄洛替尼,置于37℃、5%CO2培养箱中培养约14d;弃掉上清,细胞用4%多聚甲醛固定,结晶紫染色染色30min,PBS洗涤3次,每次2min。扫描仪扫描实验结果,并进行统计分析(图6)。结果显示,过表达LMNA基因的HCC827.ER细胞在2.5μM厄洛替尼处理下细胞克隆形成数量明显低于HCC827.ER细胞(**P<0.01)。
实施例6 LMNA基因抑制细胞增殖、迁移和侵袭
将HCC827.ER与HCC827.ER-LMNA接种于12孔板的细胞爬片上,置于37℃、5%CO2培养箱中培养24h后,加入0、2.5μM 厄洛替尼处理48h,更换终浓度为10μg/ml的BrdU完全培养基继续培养1h。爬片用4%多聚甲醛固定30min,PBS洗涤3次,每次2min;加入2N的盐酸溶液200μl,37℃处理15min,PBS洗涤3次,每次2min;加入200μl 0.1%Triton X-100溶液,室温处理10min,PBS洗涤3次,每次2min;加入300μl山羊血清,37℃孵育30min,弃掉血清后,加入1:200的BrdU抗体,4℃孵育过夜,PBS洗涤3次,每次2min;将相应荧光二抗1:500稀释后,每孔加入200μl,室温孵育2h,PBS洗涤3次,每次2min;向孔里加入1:1000的DAPI溶液,室温避光孵育20min,PBS洗涤3次,每次2min;荧光显微镜下观察,拍照并分析(图7)。结果显示,HCC827.ER-LMNA细胞增殖显著低于HCC827.ER细胞,并且能增强2.5μM厄洛替尼对细胞增殖的作用(*P<0.05,**P<0.01)。
用含10% FBS的1640培养基培养HCC827、HCC827.ER与HCC827.ER-LMNA细胞。取对数生长期细胞,消化细胞,调整细胞密度至2×105个/mL,备用。将上述细胞悬液加入12孔板中,每孔体积1 mL,放入37℃,5% CO2培养箱继续培养,观察细胞待其呈融合状态,则可进行划痕,使用一个 200μL无菌枪头进行划痕,枪头摆放要与12孔板垂直,保证划痕的宽度一致。划痕完毕后,用枪缓慢吸出培养液,加入适量的 PBS 将细胞清洗 3 遍,除去细胞碎片。弃PBS,在划痕的不同时间点( 0、12、24 h) ,将12孔板置于倒置显微镜下拍照,观察划痕愈合的程度并分析(图8A)。结果显示,HCC827.ER细胞在12h和24h划痕愈合程度明显快于HCC827和HCC827.ER-LMNA细胞(**P<0.01,##P<0.01),过表达LMNA基因能抑制HCC827.ER细胞的迁移。
将Matrigel胶用预冷的1640培养基稀释10倍后包被Transwell小室底部膜的上室面,4℃操作,37℃培养箱孵育过夜,使胶原凝固。取对数生长期HCC827、HCC827.ER与HCC827.ER-LMNA细胞,无血清培养液调整细胞浓度至1×105个/mL,取上述细胞悬液100μL加入 Transwell 小室上室中,下室24孔板中每孔加入800μL 含10% FBS的1640培养基。将小室放入 24 孔板中,37℃,5%CO2培养箱常规培养24 h,取出小室,用镊子轻轻取下滤膜,4%多聚甲醛固定30 min,取出用0. 1%结晶紫染液于水平摇床上染色 30 min,PBS漂洗3次,用棉签擦掉膜上层未穿过的细胞,置于载玻片上,100倍镜下观察细胞迁移情况并拍照,200倍镜下随机选取5个视野,观察侵袭细胞数目(图8B)。结果显示,HCC827.ER细胞侵袭的数量显著高于HCC827和HCC827.ER-LMNA细胞(**P<0.01,##P<0.01),过表达LMNA基因能抑制HCC827.ER细胞的侵袭。
以上试验结果表明,LMNA基因能抑制HCC827.ER细胞的增殖、迁移和侵袭。
实施例7 LMNA基因对细胞周期蛋白的调控
将HCC827.ER与HCC827.ER-LMNA细胞接种于10cm培养皿中,待细胞生长到融合度为70%时,加入2.5μM厄洛替尼处理48h,收集细胞。Western blot过程如实施例1,其中一抗为:lamin A/C、CyclinB1、CDC2、P53、P21(图9)。结果表明,LMNA基因能通过抑制周期蛋白cyclinB1、CDC2,增加P53、P21蛋白使HCC827.ER耐药细胞周期阻滞,抑制细胞增殖,逆转HCC827.ER细胞对厄洛替尼的耐药性,起到抗肿瘤作用。
序列表
<110> 重庆文理学院
<120> 一种逆转非小细胞肺癌耐药性的基因药物
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1995
<212> DNA
<213> 人(human species)
<400> 1
atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60
ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat 120
cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180
cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240
tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc 300
cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360
accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg 420
ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480
gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540
aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg 600
aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660
cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720
ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780
aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840
aacagcaacc tggtgggggc cgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900
agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960
cgagacctgg aggactcact ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020
aaggagcggg agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag 1080
gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg caagctcttg 1140
gagggcgagg aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc 1200
cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa aaagcgcaaa 1260
ctggagtcca ctgagagccg cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320
gccgtggagg aggtggacga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag 1380
gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440
taccggttcc caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca 1500
ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca gaacacctgg 1560
ggctgcggga acagcctgcg tacggctctc atcaactcca ctggggaaga agtggccatg 1620
cgcaagctgg tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac 1680
ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740
ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc 1800
agcggctcag gagcccaggt gggcggaccc atctcctctg gctcttctgc ctccagtgtc 1860
acggtcactc gcagctaccg cagtgtgggg ggcagtgggg gtggcagctt cggggacaat 1920
ctggtcaccc gctcctacct cctgggcaac tccagccccc gaacccagag cccccagaac 1980
tgcagcatca tgtaa 1995
<210> 2
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
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ccgctcgaga tggagacccc gtcccagc 28
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cgggatcctt acatgatgct gcagttctgg ggg 33
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tttacgtttg ttaggagtaa gtcga 25
<210> 5
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<400> 5
ataaccgaca aattaacaac gct 23
<210> 6
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tttatgtttg ttaggagtaa gttga 25
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccataaccaa caaattaaca acact 25
Claims (5)
1.LMNA基因在制备用于逆转非小细胞肺癌对厄洛替尼耐药性的药物中的应用。
2.如权利要求1所述的应用,其特征在于:所述LMNA基因序列为SEQ ID NO.1 所示。
3.如权利要求1或2所述的应用,其特征在于:所述LMNA基因受CMV启动子的调控。
4.如权利要求2所述的应用,其特征在于:所述LMNA基因与病毒载体相连,所述病毒载体为pLVX-Puro载体。
5.如权利要求3所述的应用,其特征在于:所述LMNA基因与病毒载体相连,所述病毒载体为pLVX-Puro载体。
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