CN1552734B - 重组腺伴随病毒载体介导的肿瘤坏死因子相关的细胞凋亡配体肽段及其用途 - Google Patents
重组腺伴随病毒载体介导的肿瘤坏死因子相关的细胞凋亡配体肽段及其用途 Download PDFInfo
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Abstract
本发明提供一种利用腺伴随病毒载体携带重组肿瘤坏死因子相关细胞凋亡配体(TRAIL)的第95至281的肽段，和其在体内表达三聚体的活性形式，以及在制备抗肝转移肿瘤和肺癌肿瘤药物中的应用。
Description
技术领域
本发明涉及一种腺伴随病毒(adeno-associated virus，简称AAV)表达载体携带的重组肿瘤坏死因子相关的细胞凋亡配体(tumor necrosis factor-related apoptosisinducing ligand，简称TRAIL)肽段及其制备方法、包装、在体内表达及其用途，特别是编码TRAIL第95至281位187个氨基酸残基与腺伴随病毒组成的重组表达载体(TRAIL95-281-AAV)及其用途。本发明的重组病毒可单独或与其它抗癌药物联用，用于肝转移癌和肺癌等肿瘤的基因治疗。
技术背景
TRAIL(tumor necrosis factor-related apoptosis inducing ligand)属于肿瘤坏死因子(TNF)家族成员，Wiley等于1995年克隆并命名。研究发现，TRAIL可迅速诱导乳腺癌、直肠癌、肺癌和前列腺癌等多种人类肿瘤细胞凋亡，而对正常细胞无毒副作用。在动物实验中已证明TRAIL可非常有效地抑制肿瘤的形成和生长，甚至可通过诱导肿瘤细胞的凋亡而导致肿瘤的缩小或消失，但对正常细胞不产生杀伤作用(Nat.Med.，1999，5(2)：157-163，这是目前临床上使用的大多数化疗药物“敌我不分”、具有强烈的毒副作用所不能比拟的。因此TRAIL具有良好的临床应用前景。
然而，重组表达的TRAIL在临床应用上存在一些限制，例如：1)动物实验表明静脉给药后，TRAIL重组蛋白的稳定时间只有5小时；2)在体内抑制肿瘤形成需要大量的TRAIL重组蛋白，有可能产生免疫原性，等等。因此，改善TRAIL在体内的利用度和给药途径是提高TRAIL疗效的重要研究方向。
自1990年首次进行人类疾病的基因治疗试验以来，基因疗法作为一种全新的医疗手段日益引起了人们的重视，特别是对于那些不能使用传统医疗手段，不能彻底治愈的疾病，如糖尿病、癌症和艾滋病等。目前使用的基因疗法载体主要包括病毒类和非病毒类两种载体。一般来说，病毒类载体在表达强度和时空性方面比非病毒类载体优越，特别是副腺病毒(adeno-associated virus，AAV)更为突出。 重组AAV载体用于基因治疗法具有许多优点，首先迄今尚没有野生型AAV能引起人类相关疾病的报道，而且它能整合到宿主的染色体，使外源基因长期稳定表达。外源基因表达后不仅没有细胞调节反应，而且具有广泛的组织亲和性。最近，人们已经克服了重组AAV的一些先天性缺陷，如产量低、辅助病毒污染及野生型AAV污染等，能产生重组AAV的细胞株也已获得成功，为临床应用打下了坚实基础。目前，重组AAV载体已广泛地应用于癌症、白血病、艾滋病的治疗研究，有的已进入了人体临床试验，取得了令人满意的结果(Nature，2001，412(6850)：914-917)，展示了美好的应用前景。
最近，Seol Dai-Wu等申请的美国专利(申请号为20020128438)，构建了包括三聚体形成结构域、分泌信号肽和TRAIL(114～281)序列的重组AAV表达载体，认为可用于疾病的基因治疗。但其技术中TRAIL(114～281)重组表达载体制备复杂，未报告能否在肝脏中特异性地表达并进入血液循环，也未报告能否用于肝转移癌和肺癌等肿瘤的基因治疗。
本发明涉及与其不同的AAV介导的TRAIL(95～281)重组表达载体的制备、包装方法及其用途。本发明中的重组AAV可单独或与其它抗癌药物联用，用于肝转移癌和肺癌等肿瘤的基因治疗。
发明内容
为了克服现有技术的不足之处，本发明的第一个目的在于提供一种氨基酸序列，其特征在于具有如序列表1中所述的序列。
本发明的第二个目的在于提供一种TRAIL基因序列，特别是人TRAIL95-281的序列，此序列与AAV构成的表达载体为pAM-CAG/EGR-1-pL-TRAIL95-281-WPRE-BGH polyA。
本发明的第三个目的在于提供一种细胞株，其特征在于是使用重组AAV表达载体感染所建立的细胞株。
本发明的第四个目的在于提供TRAIL95-281在制备抗肝转移癌和肺癌等肿瘤药物中的应用。
在本发明中还描述了所述重组AAV病毒的体内给药途径和方法。所述重组AAV病毒进入体内后，TRAIL95-281以同源三聚体的活性形式表达并进入血液循环系统。
所述的重组AAV病毒可以应用于肝转移癌和肺癌等肿瘤基因治疗。
为了完成本发明的目的，因此本发明特别提供一种氨基酸序列，其特征在于具有如序列表1中所述的序列。
发明还提供一种TRAIL基因表达载体，它包括编码TRAIL第95至281位氨基酸的基因序列。TRAIL基因表达载体包括：pAM(plasmid of anti-ampicilinconstruction)、ITR(adeno-associated virus 2 inverted terminal repeat sequence)、CAG(cytomegalovirus immediated-early enhancer/chicken β-actin hybrid)或EGR-1(promoter of a murine mitogen inducible zinc finger encoding gene)、pL(poly-linker)、TRAIL95-281(编码TRAIL第95至281位氨基酸的序列)、WPRE(Woodchuck HeptitisVirus Post-transcriptional Regulatory Element)、BGH(Bostaurus growth hormone)、polyA(多聚腺苷酸)和ITR构成的结构。所述的载体优选的是pAM/CAG/EGR-1-pL-TRAIL95-281-WPRE-BGH-polyA的AAV重组表达载体。
所述的载体为包装TRAIL95-281-AAV的三载体系统，该包装TRAIL95-281-AAV的三载体系统是：AAV重组表达载体、H22和pFD6。
本发明还提供一种使用重组TRAIL95-281-AAV表达载体感染所建立的细胞株在制备抗肝转移肿瘤和肺癌肿瘤药物中的应用。
使用重组AAV表达载体转移到体内6小时后病毒的滴度达3～4×106/mL，并能在肝转移的肿瘤细胞中表达TRAIL95-281，直接杀死肿瘤细胞。另外使用重组AAV表达载体转移到体内后，可在肿瘤细胞周围的正常肝细胞中表达TRAIL95-281，并杀死周围肿瘤细胞。使用AAV重组表达载体转移到体内后，TRAIL95-281以三聚体的活性形式表达并分泌到血液中。
在本发明中，采用基因工程等现代生物学技术和方法，提供了编码人TRAIL95-281的重组AAV表达载体的制备、包装、输入体内途径及其应用。通过肌肉、静脉、肝门静脉注射或口服途径，将所述重组病毒输入体内后，重组AAV介导的TRAIL95-281能够以同源三聚体的活性形式在肿瘤细胞及肝细胞中表达，直接杀灭肿瘤细胞，并分泌到血液循环系统，对肝转移癌和肺癌等肿瘤的生长和形成起明显抑制作用，并显著地延长了荷瘤小鼠的寿命。首次证明所述重组病毒可应用于肝转移癌和肺癌等肿瘤的基因治疗。
本发明涉及一种编码人TRAIL95-281氨基酸残基的重组AAV表达载体的制备、包装方法，它包括以下步骤：
以从中国正常人外周血淋巴细胞中克隆到的全长TRAIL的cDNA为模板，以人工合成的TRAIL95-281cDNA上、下游寡核苷酸序列为引物，进行PCR扩增，其上、下游引物分别为5’-TAGAATTCACCATGACCTCTGAGGAAACCATT-3’和5’-CCCAAGCTTTTAGCCAACTAAAAAGGCCC-3’；
合成的引物分别引入了Kozak序列并在两端加入限制性内切酶EcoRI和HindIII的酶切位点；PCR扩增的条件为94℃，45s；58℃，1m；72℃，1m；循环30次，最后在72℃延伸15m。PCR产物经酶切后，插入已预先用相同的内切酶消化过的pAM-CAG/EGR-1-pL-WPRE-BGH-polyA的AAV包装载体，构成TRAIL95-281-AAV重组载体；
采用三载体辅助病毒缺陷包装系统(AAV、H22和pFD6)进行包装，即采用磷酸钙法将重组载体TRAIL95-281-AAV和辅助质粒H22和pFD6共同转染HEK-293细胞，转染60～72小时后，收获细胞，分离出的重组病毒颗粒经HiTrap Heparin亲和层析(Amersham Biosciences，Sweden)纯化后，以real-time PCR确定其最终滴度；
另分别在转染TRAIL95-281-AAV的不同时间，裂解HEK-293细胞，进行Western印迹检测，定量测定表达的TRAIL95-281蛋白浓度。按照同样的方法制备、包装编码绿色荧光蛋白(EGFP)的重组AAV病毒载体(EGFP-AAV)作为对照。
重组TRAIL95-281-AAV病毒转导HEK-293细胞后，运用Vectastain ABC试剂盒(Vector Laboratories，Burlingame，CA)和DAB染色试剂盒(Zymed，South SanFrancisco)分别测定TRAIL95-281的表达。
体外实验表明TRAIL95-281在HEK-293细胞中高水平表达，并随着时间的延长而呈增加的趋势(图1A)。所表达的TRAIL95-281在细胞中表达，并可最终富集到细胞表面(图1B)。在转染的293细胞培养基中，也有TRAIL95-281蛋白存在，在转染24小时时，TRAIL95-281的表达量可达15.6±0.31ng/ml(图1C)，表明该重组病毒可介导TRAIL95-281在胞浆中表达，并可分泌到细胞外。该重组病毒分别感染小鼠淋巴瘤细胞EL-4和人淋巴细胞白血病T-细胞Jurkat后，可引起EL-4和Jurkat细胞发生明显凋亡(图1D)。
经C57BL/6小鼠门静脉注射该重组病毒后，RNA原位杂交的结果表明TRAIL95-281可在肝小叶等细胞中长期表达(图2A)，免疫化学分析进一步肯定了这一现象(图2B)，并在肝细胞裂解液和浓缩的血清中发现TRAIL95-281以同源三聚体的活性形式存在，大小约为60kD(图2C)，而且TRAIL95-281可在肝脏中长期 稳定地表达(图2D)。
经小鼠门静脉接种小鼠淋巴瘤细胞EL-4并形成肝转移瘤动物模型后，分别经门静脉注射不同剂量的该重组病毒，进行AAV介导的TRAIL95-281基因治疗，表明治疗后肝转移瘤显著缩小(图3A)，有50％的小鼠的肝转移瘤消失(图3B)，荷瘤小鼠的生存期明显延长(图3C)，表明该重组病毒具有明显的抗肿瘤活性、安全有效、无毒副作用，具有良好的临床应用前景。
同时AAV介导的TRAIL95-281基因治疗表明，治疗组移植瘤中发生了重组病毒给药量相关的细胞凋亡(图4A-G)，但不引起正常肝细胞凋亡。进一步分析表明，在此重组病毒介导的细胞凋亡过程中，Bcl-2表达下调，而caspase-3和caspase-8表达上调(图4H)。
附图说明
图1是显示重组AAV-TRAIL和AAV-EGFP载体的构建与鉴定。(A)：Western blot显示TRAIL95-281在HEK293细胞中的表达。(B)：免疫组化分析显示TRAIL95-281在HEK293细胞表面表达。(C)：ELISA显示TRAIL95-281可被分泌到培养液中。(D)：重组AAV-TRAIL可介导Jurkat和EL-4细胞凋亡。
图2显示经小鼠门静脉注射rAAV-TRAIL后，TRAIL95-281可在动物肝细胞内及表面和血浆中长期稳定表达。
图3是显示小鼠肝组织上形成的EL-4转移瘤及使用rAAV-TRAIL治疗前后的大小及生存时间。
图4是显示rAAV-TRAIL治疗引起移植癌细胞凋亡及细胞凋亡相关蛋白的表达变化。
具体实施方式
以下将结合附图详细描述本发明。
实验例1
以从中国正常人外周血淋巴细胞克隆到的TRAIL全长cDNA为模板，以人工合成的TRAIL95-281cDNA上、下游寡核苷酸序列为引物，建立PCR扩增反应。TRAIL95-281cDNA上、下游寡核苷酸引物的核苷酸序列分别为5’-TAGAATTCACCATGACCTCTGAGGAAACCATT-3’和5’-CCCAAGCTTTTAGC CAACTAAAAAGGCCC-3’。在引物中分别引入Kozak序列并在两端加入限制性内切酶EcoRI和HindIII的酶切位点。PCR扩增条件为94℃45sec，58℃1min，72℃1min，循环30次，最后在72℃延伸15min。PCR产物经EcoRI和HindIII酶切后，插入已预先用EcoRI和HindIII限制性内切酶消化的pAM/CAG/EGR-1-pL-WPRE-BGHpolyA的AAV表达载体。采用磷酸钙法将重组TRAIL95-281-AAV表达质粒连同辅助病毒缺陷包装系统(H22和pFD6)共转染HEK-293细胞，转染60-72小时后收获细胞，分离出的重组病毒(rAAV)颗粒经HiTrap Heparin affinity columns(Amersham Bioscineces，Sweden)纯化，以real-time
PCR确定重组病毒的最终滴度。以编码绿色荧光蛋白(EGFP)的重组病毒(EGFP-AAV)作为对照。
实验例2
参见图1A，Western Blot：肝组织或细胞经裂解液(含1％Nonidet P-40、0.35mg/ml PMSF、9.5μg/ml leupeptin和13.7μg/ml pepstatinA的1×PBS)裂解后，12000rpm离心去沉淀，上清液经BCA蛋白分析试剂盒(Pierce Chemical Company，Rockford，IL)测定蛋白浓度后进行SDS-PAGE凝胶电泳，转移至PVDF膜后4℃封闭过夜，加一抗(抗TRAIL多克隆抗体，1∶1000稀释于封闭液中)后，室温孵育2h、加二抗(HRP标记的兔抗羊抗体1∶3000稀释于封闭液中)后，室温孵育1h，彻底洗涤，然后利用ECL系统(Amersham Pharmacia Biotech Co.)进行蛋白显色。检测TRAIL95-281在小鼠血清中的表达情况，经小鼠尾静脉取血清，浓缩10～20倍后，同法进行Western Blot。
实验例3
参见图1B-D，TRAIL95-281的表达及其生物学活性测定：HEK-293细胞在Dulbecco’s modified essential medium(DMEM)完全培养基(即在DMEM培养基中添加10％的胎牛血清，100U/ml青霉素和100μg/ml链霉素)中培养6h以上，1×PBS洗涤后加入不同滴度的重组病毒，(培养基为DMEM添加2％的胎牛血清)，8小时后添加DMEM完全培养基。在不同时间点收集重组病毒转导的HEK-293细胞培养基，真空冷冻浓缩10～20倍，测定蛋白浓度，进行ELISA检测。将约100μg蛋白经0.05M Na2CO3(pH9.6)稀释后，于37℃包被96孔板。包被2h后，于37℃ 以封闭液(1×PBS添加0.05％Tween-20和5％脱脂奶粉)封闭0.5h。加入抗TRAIL多克隆抗体(1∶1000稀释于封闭液中，每孔添加100μl)，37℃温育2h。加入HRP标记的兔抗羊抗体(1∶1000稀释于封闭液中，每孔添加100μl)，37℃温育2h。酶反应底物为1，2-benzenediamine(邻苯二胺，OPD，Sigma)，2mg OPD加入5ml的显色缓冲液中(柠檬酸0.51g，Na2HPO4·12H2O1.2g，加水至100ml，pH4.5，溶解后加入7.5μl双氧水)，每孔添加100μl，室温放置约30m，加入50μl 1M的H2SO4终止反应，于MD/SPECTRAMAX340分光光度仪上在490nm处读取吸光值)。用此ELISA检测重组TRAIL95-281的灵敏度可达16ng/ml。另外采用免疫组化方法检测重组病毒转导的HEK-293细胞中TRAIL95-281蛋白的表达，使用Vectastain ABC(Vector Laboratories，Burlingame，CA)和DAB试剂盒(Zymed，South SanFrancisco)，最终的阳性信号呈棕红色。TRAIL95-281诱导肿瘤细胞凋亡的活性采用MTT(Richter A.M.et al.，1900)法检测，分别取104～106的EL-4、Jurkat和HepG2细胞(体积为100μl)接种到96孔板中，分别加入不同滴度的重组病毒(100μl，1×PBS稀释)，28小时后加入10mg/ml MTT(每孔20μl)，37℃温育4h后，每孔添加200μl DMSO终止反应，于MD/SPECTRA MAX340分光光度仪上读取570nm处光吸收值，计算细胞成活率(细胞成活率＝实验组光吸收值/对照组光吸收值×100％)。每一组设定三个样品孔，每组实验重复三次。以重组AAV-EGFP作为阴性对照。
实验例4
参见图2A、B，小鼠动物模型的建立：取7至8周龄、体重约20g的C57BL/6小鼠，门静脉接种2×105淋巴瘤细胞EL-4(1×PBS悬液200μl)，门静脉注射1×PBS缓冲液为对照组。以后每周称量小鼠体重，30～40天后尾静脉取血，苯巴比妥腹腔注射处死，组织样品经液氮冷冻后贮存在-70℃或在4％paraformaldehyde固定，制备切片，进行肝脏组织的形态及组织学分析。
实验例5
参见图2C、D，原位杂交：为鉴定TRAIL95-281蛋白在小鼠肝脏中的表达分布情况，取小鼠的肝脏，经4％的paraformaldehyde固定后进行石蜡包埋，制备8μm的组织切片。反义RNA探针选用位于TRAIL95-281基因下游约600bp的WPRE DNA 片段为模板进行标记，生物素标记试剂盒和杂交方法参见产品说明书(In vitrotranscription kit and nonisotopic labeling kit，mRNAlocator in situ hybridization kit，Ambion，UK)。PBS对照组的肝脏切片作为阴性对照。
实验例6
参见图3，荷瘤小鼠的实验治疗与观察：取7至8周龄、体重约20g的C57BL/6小鼠，经门静脉接种2×105淋巴瘤细胞EL-4(1×PBS悬液200μl)。一周后将实验动物分成三组：a)生理盐水对照组；b)EGFP-AAV对照组；c)不同剂量的TRAIL95-281-AAV重组病毒(病毒滴度1010～1012)治疗组。每组6只，通过门静脉注射。分别观察不同组动物肿瘤的生长情况和生存时间。做3批重复实验。
图3A显示小鼠肝组织上形成的EL-4肿瘤治疗前后的大小和组织切片鉴定的结果。A：从左至右分别为显示EGFP-AAV对照组、低剂量和高剂量TRAIL95-281-AAV治疗组的小鼠动物模型肝组织大小。B-D：从左至右分别为EGFP-AAV对照组、低剂量和高剂量TRAIL95-281-AAV治疗组的小鼠动物模型的肝组织切片H&E染色结果。箭头指示肿瘤细胞的异型核。CV：中央静脉；PV：门静脉。
图3B显示EL-4肝转移瘤小鼠治疗前后肝组织切片的免疫组化染色结果。
图3C显示EL-4肝转移瘤小鼠治疗前后的生存时间。分别显示EL-4肝转移瘤小鼠分别用PBS、EGFP-AAV、低剂量和高剂量TRAIL95-281-AAV治疗后的存活时间。其实验结果是：治疗组的小鼠寿命明显延长，证明该重组病毒具有明显的抗肿瘤活性、安全有效、无毒副作用，具有良好的临床应用前景。
实验例7
参见图4A-G，细胞凋亡测定：按照原位细胞死亡试剂盒(Roche MolecularBiochemicals)说明书进行TUNEL标记。肝脏切片经脱蜡、脱水、0.1％Trition-X100和0.1％柠檬酸钠处理后，在平衡缓冲液中封闭10分钟，加入含有TdT(terminaldeoxynucleotidyl transferase)和fluorophore标记的dUTP的反应缓冲液中，37℃孵育60分钟，加1×SSC(200μl/切片)，室温放置15分钟终止反应，用PBS彻底洗涤后，用Vectashield∶DAPI(3∶1)封片镜检。
参见图4H，Western blot：按实验例2的方法进行细胞凋亡相关蛋白的测定。
序列表
<110>中国医学科学院基础医学研究所
<120>重组腺伴随病毒载体介导的肿瘤坏死因子相关的细胞凋亡配体肽段及其用途
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<160>1
<170>PatentIn version3.1
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<211>187
<212>PRT
<213>人淋巴细胞
<400>1
Claims (2)
1.一种可溶性TRAIL的重组腺伴随病毒表达载体，其特征在于所述的可溶性TRAIL的重组腺伴随病毒表达载体为pAM-CAG/EGR-1-pL-TRAIL95-281-WPRE-BGH polyA。
2.如权利要求1所述的重组腺伴随病毒表达载体在制备抗肝转移癌和肺癌等肿瘤基因治疗药物中的应用。
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| CN1966082B (zh) * | 2006-11-03 | 2010-06-30 | 许瑞安 | 一种防治结直肠癌的基因药物及其制备方法和用途 |
| CN102250254A (zh) * | 2010-05-19 | 2011-11-23 | 江苏先声药物研究有限公司 | 肿瘤坏死因子相关凋亡诱导配体融合蛋白及其制备和应用 |
| CN102329387A (zh) * | 2011-09-23 | 2012-01-25 | 昆明理工大学 | 一种抗病毒蛋白trail114-281及其制备方法 |
| CN103288966B (zh) * | 2013-05-17 | 2015-01-21 | 华侨大学 | 一种融合受体及其用于治疗大肠癌的基因药物 |
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| US6284236B1 (en) * | 1995-06-29 | 2001-09-04 | Immunex Corporation | Cytokine that induces apoptosis |
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