CN117587126A - Psme3及其表达产物的应用 - Google Patents
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Abstract
本发明涉及生物医药领域,提供一种PSME3及其表达产物的应用;提供检测PSME3及其表达产物的试剂在制备诊断软骨肉瘤的试剂盒中的应用;本发明提供的PSME3基因在软骨肉瘤组织与正常组织表达上存在显著差异,显著影响患者的预后和生存期,表明PSME3基因为制备诊断软骨肉瘤的试剂盒提供了靶点,并且为制备治疗软骨肉瘤的药物提供了新的靶点,也为软骨肉瘤的基因治疗提供依据。
Description
技术领域
本发明涉及生物医药领域,尤其涉及PSME3及其表达产物的应用。
背景技术
软骨肉瘤是一种异质性软骨形成的恶性肿瘤,现已是发病率第二的原发恶性骨肿瘤。软骨肉瘤起源于骨髓髓腔内,可发生在骨盆,股骨,肱骨和髂骨等部位,晚期有肺转移倾向。软骨肉瘤被认为化疗和放疗不敏感,手术切除是主要的治疗手段,很少有病例报道其对常规化疗药物有反应。当发生远处转移或术后复发难以切除时,往往只能选择姑息治疗。软骨肉瘤术后的复发以及对化疗药物的耐药是影响患者生存率的重要原因,探索软骨肉瘤发生发展的机制,开发新的靶向药物是目前亟需解决的难点。基因治疗作为新世纪以来重要的研究手段,探寻新的基因治疗靶点有望突破临床上治疗软骨肉瘤的难题。
发明内容
本发明的目的是针对现有技术中的不足,提供一种PSME3及其表达产物的应用。
为实现上述目的,本发明采取的技术方案是:
本发明的第一方面是提供检测PSME3及其表达产物的试剂在制备诊断软骨肉瘤的试剂盒中的应用。
优选地,所述PSME3为核苷酸序列如SEQ ID NO:1(GCAGTTTCCGGCGTGAGCGGCGAAAGCCGGGAGGGCGAGCGAGAGAGC AAGCAGGCAGCAGGCTGCCGGCGGGCGGGCGGACGGCACAGAGGGAGGGAGCGAGCGAGCAGTGAGTAAGCCAGCAAGGGCGGTCGGGTCCCGAGGTCAGCCGAGATTTCTCAGGTCCCTCCGGCCCCCTCCCTGGAGTCCACAGCGCCTCCGGTGTCCAGAGGATCGGACACGGCCCGGCCCGGCCATGGCCTCGTTGCTGAAGGTGGATCAGGAAGTGAAGCTCAAGCTCTGAGCTTCCGCTCGGCT
CAGCCCAGCCCCCAACTCCCGGGGTCGGCTTCGCGGGAGAGGAAAATATA
GGATGGCTTTCTGTACCGCGTCTGATGATGGAGAGTCCCGGGGACACCTC
CGCGGACACGTGTTGGACTCAGGGCTTTAGGCCCGTGACAGCTGACAGCT
TCAGACACAGGAGGAAGGGAGGGAAGGGGGAGGACAGAGGAGACAGGC
AGGTGCTGTCCTCAAAGCCCTGCGTTCTTCTGAGATGGAAAAATGGATCCT
CAAAAAAATAAAGTATTTGCAGTCTGGGGGCCTCTCAGCTTCTTATTACAG
TTACAAGGTTGATTCTTTCAGGGAGCGGATCACAAGTGAGGCAGAAGACT
TGGTGGCAAATTTTTTCCCAAAGAAGTTATTAGAACTTGATAGTTTTCTGA
AGGAACCAATCTTAAACATCCATGACCTAACTCAGATCCACTCTGACATG
AATCTCCCAGTCCCTGACCCCATTCTTCTCACCAATAGCCATGATGGACTG
GATGGTCCCACTTATAAGAAGCGAAGGTTGGATGAGTGTGAAGAAGCCTT
CCAAGGAACCAAGGTGTTTGTGATGCCCAATGGGATGCTGAAAAGCAACC
AGCAGCTGGTGGACATTATTGAGAAAGTGAAACCTGAGATCCGGCTGTTG
ATTGAGAAATGTAACACGGTCAAAATGTGGGTACAGCTCCTGATTCCCAG
GATAGAAGATGGAAACAACTTTGGGGTGTCCATTCAGGAGGAAACAGTTG
CAGAGCTAAGAACTGTTGAGAGTGAAGCTGCATCTTATCTGGACCAGATT
TCTAGATATTATATTACAAGAGCCAAATTGGTTTCTAAAATAGCTAAATAT
CCCCATGTGGAGGACTATCGCCGCACCGTGACAGAGATTGATGAGAAAGA
ATATATCAGCCTTCGGCTCATCATATCAGAGCTGAGGAATCAATATGTCAC
TCTACATGACATGATCCTGAAAAATATCGAGAAGATCAAACGGCCCCGGA
GCAGCAATGCAGAGACTCTGTACTGAGGCCAGGGCCAGGGCCAGGGGAC
TCTGTGAGTCTGGCTCAAGACCGACATTGCCTTGGTTTGTTACATGACTAT
CGTGATGGGGAAACTGGCTGGAAATAGTAATCACACCTCTCTGTTTTTAGT
TAGAGTCTAATGAAACTCTCATCTAGTTCTGTGATGTGTTTACCTCTTTTTT
CAGGCCTCAGGAACTCTTCTATTTCCTTCCCTAATACCCCACACCCAACCT
GTCGTAATTTCTGGAGAACTCCAGGTTTGTGTGTGCAGGATGTTGGCACAA
AAATACCTGTGTTTTCATTCTCCCCCTCTCTCCCTCCTGTGTCTTGCGCTTT
ATGTTTTCTTCCGTTTGATAATTAGTTGGTTAAAAGCTGAGGGAACCGGAA
GGAAAGTGCTAGGTGTTTTTTAGGAACTAGGGTGGCGGGGGGACGAACTT
CTCTTCCTCACATGAGGTTACTGTTTCTTTCCTCTGTGGGGCATTGGATCCT
CCCACAGTTGCCCTGGTGATGACTTAGGGCTTCCCATCTGTGTACATCCCA
CTTTGAATCTTGATCGTGACAAGAAATACCTTAGGCCTTCAGTCAATTCCG
AAGCTCCTTCAGTTGTTTTTATAATGGGCGTTTTCACATGCACATATGTGT
ATGCATGTATACGCCCATACAGACATGCACACACAGACTCCTACTCCATT
AGCTAACATACCCTCCCTCTCCACAACCCCTGTCACATACCTTTCAGGAGG
TGACAGTTGTCTTAGTTGTCATCTACCCAGACAAACGTCCTGGGCCCGTCC
TCCCTCCTGATACTGTAGCCTCTTGGTACCCAGGGTGAGTTGGTGGAGAAC
AGAGAGATGAGAAGCAGAGGGCTTGGGGAAAGCCTGTTCCTCTCTGACTC
AGCCCTTTTTGGCATTATTGCAAGAGCTTGACTCCTGGTTGCCTTTTCCCA
GCCAGTTTTCAGTTGGGGTGAAGGTTTCTGCAAGTGTGAGGTCCAGATGCT
GCTGCTCATGTTGGGCTTTCCTTTTGGGAACTATTTCTCTTTATTTATAGTG
TCGGGCTTCCGGGGAAAGCAATCATTGGTGTGTATGTGTATGTGCATGCA
CACACGTGCATATACACATTTGTGTATGTGGAAATGTGCTGGGCAAGTCA
AAACTATAGAAGAGTTGCCTCCTGTCTCTCGAATCTTCCAGAGATATCACT
TAATTGTTAACAGCTTTTGTGTTAATCCCCTTCAGCCCCTAGCTCTTTTATT
CTACCACGGCTGGAGAGTTGATACCTGCAGTCAGCCTGCCAGTGACTCTT
AGTGTCTGTTTCTGACTTATTTTTCCTGTCTCTGTCTTCCAACCCCCAATAA
TATTTCCACCGGGGATGCATCATTTTTACTCCCAATATTCTGTAGAGAGGG
AGTCAGGATGCTGTCTTCCCACGAATAGTACTCAGTAACAAACCAATTGC
ATTTTAGTTGGGCAGTGCTCCCACCCACCCTCCAGATCCCTTCCAGCTAAA
ACCCTTCCCCCTTCCCTCCATGTGTTTCTCAGTTTCCCGTTTCGTTTGTTGG
ACTGTTCCACTGCCCCTCCTCCTCACCCTATCACCCATGGATCGTAATGTA
AAATTCTTTTACCATGTCAAGAAATTATTAAAAATACAGGTACTTTGACCT
CTTTCTAAAGCCGCAGACCCTGGTGCAATGCTCTGGTGGCTAGGGATGTA
CTCATGCTCATATGTGTGCACGCTTGGACACCCACCTCCATGGACACCTAG
CCACCCTGTTGTGTGTCCTTATGCCAGTTGAGCTGAATCTTTTCCCCAGTAT
AGTGGAAAGACTGAGGCTTCTGCCTACTGAGCAAGGTTGGGTGCTTCATT
TGTGTTCAGTCTGAATTATGGGAAAGTTAGCTCTTCCCAGACCTAAGCTGC
CTTCTCTCCCTACTTTCAGAAGATCCTAGTTCCTTCCTTCCCGAGTGATACC
CATGAACTGCCAGTAGAGGCTGCTATCGTTCCATGTGTAAGGAATGAACT
GGTTCAAGGCGCGTCCTACCCAGTCATTTTCTTTACCTTATACTAATTCTTC
CTGAATAATGTCTTCAGTTTCTTGAGGAGACTCCTAGTTTTGGTTTTCAAA
TTACTTGGAGGGCTGCCTAGGAATCTATCTCCCTCTGAAATAAAGTTTCCTCATCTTCCACCTTGCAA)所示的DNA分子;或,所述PSME3为编码氨基酸序列如SEQ ID NO:2(MASLLKVDQEVKLKVDSFRERITSEAEDLVANFFPKKLLELDSFLKEPILNI HDLTQIHSDMNLPVPDPILLTNSHDGLDGPTYKKRRLDECEEAFQGTKVFVMPNGMLKSNQQLVDIIEKVKPEIRLLIEKCNTVKMWVQLLIPRIEDGNNFGVSIQEETVAELRTVESEAASYLDQISRYYITRAKLVSKIAKYPHVEDYRRTVTEIDEKEYISLRLIISELRNQYVTLHDMILKNIEKIKRPRSSNAETLY)所示的DNA分子;或,所述PSME3为在严格条件下与如SEQ ID NO:1所示的核苷酸序列杂交且编码具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子;或,所述PSME3为编码在如SEQ ID NO:2所示的氨基酸序列中经过取代或/和缺失或/和添加一个或多个氨基酸但具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子。
优选地,PSME3的表达产物为由所述PSME3编码的蛋白质。
优选地,所述试剂包括:PSME3的引物。
更优选地,所述PSME3的引物的核苷酸序列如SEQ ID NO:3(CACCGCTGATCCACCTTCAGCAACG)-SEQ ID NO:4(AAACCGTTGCTGAAGGTGGATCAGC)所示。
本发明的第二方面是提供抑制PSME3表达的试剂在制备治疗软骨肉瘤的药物中的应用。
优选地,所述PSME3为核苷酸序列如SEQ ID NO:1所示的DNA分子;或,所述PSME3为编码氨基酸序列如SEQ ID NO:2所示的DNA分子;或,所述PSME3为在严格条件下与如SEQ IDNO:1所示的核苷酸序列杂交且编码具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子;或,所述PSME3为编码在如SEQ ID NO:2所示的氨基酸序列中经过取代或/和缺失或/和添加一个或多个氨基酸但具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
本发明提供的PSME3基因在软骨肉瘤组织与正常组织表达上存在显著差异,显著影响患者的预后和生存期,表明PSME3基因为制备诊断软骨肉瘤的试剂盒提供了靶点,并且为制备治疗软骨肉瘤的药物提供了新的靶点,也为软骨肉瘤的基因治疗提供依据。
附图说明
图1为本发明一实施例中PSME3在软骨肉瘤及癌旁免疫组化典型图像;
图2为本发明一实施例中PSME3敲除成功Western Blot示例图;
图3为本发明一实施例中在HCS2/8细胞敲除PSME3基因,四天后,KO组与对照组(WT)的增殖变化图;
图4为本发明一实施例中在HCS2/8细胞KO组与WT组克隆形成差异图;图4A为克隆形成差异对比照片;图4B为差异柱状图;
图5为发明一实施例中采用Western Blot检测铁死亡通路关键蛋白变化的结果图;
图6为本发明一实施例中HCS2/8敲除PSME3基因后GSH水平变化图。
具体实施方式
下面将结合本发明实施例中的附图,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作出创造性劳动的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。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
下面结合附图和具体实施例对本发明作进一步说明,但不作为本发明的限定。
实施例
1、免疫组化实验检测PSME3基因表达情况
(1)切片:将软骨肉瘤组织及癌旁标本进行石蜡包埋切片,将切片置于65℃烘片机上烘烤60min。
(2)脱蜡:将烘烤好的玻片依次以二甲苯Ⅱ、二甲苯Ⅰ、无水乙醇与二甲苯1:1溶液分别浸泡7min脱蜡。
(3)复水:脱蜡结束后再依次以100%乙醇、95%乙醇、85%乙醇、75%乙醇、50%乙醇、双蒸水的顺序浸泡3min复水。
(4)抗原修复:提前准备好一250mL烧杯,倒入200mL EDTA溶液并放入水浴锅预热,待水浴锅加热至100℃,将复水好的切片放入烧杯中,修复抗原,20min后,将烧杯拿出,自然冷却至室温。
(5)淬灭:抗原修复后,将切片放入PBS清洗三次,每次3min;将切片置于3%过氧化氢溶液(甲醇稀释)10min,再次用PBS清洗三次,每次3min。
(6)封闭:将玻片表面上的水分擦拭干净,使用移液枪将免疫组化试剂盒中的封闭液覆盖至组织表面,封闭10min。
(7)抗体抗原反应:封闭完成后,弃去表面封闭液,按照一抗说明书的比例使用PBS稀释一抗,将一抗稀释液均匀地覆盖在组织表面,并且使用合适大小的封口膜吸附在载玻片上,防止一抗蒸干,在湿盒内4℃冰箱放置过夜。
(8)二抗与一抗结合:将切片从湿盒中拿出,放入PBS中洗去封口膜,并洗涤三次,每次3min;拭去表面水分,在组织上滴加Biotin标记的二抗,室温下结合20min。
(9)辣根过氧化物酶(HRP)标记:孵育结束后,使用PBS洗涤三次,每次3min;拭去表面水分,在组织上覆盖组化试剂盒内的HRP,室温孵育10min。
(10)DAB显色:HRP标记结束后,使用PBS洗涤切片,每次3min;使用30%H2O2溶液激活DAB,比例1:1000;在组织上滴加DAB覆盖,在光学显微镜上观察显色反应,待染色完成后,切片放入PBS终止反应。
(11)苏木精染核:DAB染色结束后,将切片置于苏木精染液中,放置5min,再在清水中漂洗切片终止染色;在1% HCl溶液中1s-2s分色,分色结束后在清水中放置30min蓝化。
(12)脱水复蜡:将蓝化后的切片依次经过50%乙醇、75%乙醇、85%乙醇、95%乙醇、100%乙醇中放置3min脱水,以无水乙醇和二甲苯1:1溶液、二甲苯Ⅰ、二甲苯Ⅱ的顺序复蜡。
(13)封片:在复蜡完成后的切片边缘滴加中性树脂,盖玻片沿边缘覆盖载玻片,尽量完全排出气泡,在通风橱下自然晾干。
(14)拍照:使用正置显微镜拍摄成像。
结果如图1所示,PSME3基因在肿瘤组织中显著高表达。
2、确定PSME3敲除成功实验
(1)确定PSME3引物,正义链的序列如SEQ ID NO:3(CACCGCTGATCCACCTTCAGCAACG)所示,反义链如SEQ ID NO:4(AAACCGTTGCTGAAGGTGGATCAGC)所示;
(2)将PSME3引物溶解成100μM浓度,按照以下体系混合:
在37℃30min,95℃5min,随后梯度降温,即从95℃,每5min降低5℃直到25℃;
(3)使用BsmbI酶切2μg的LentiCRISPRv2载体,37℃过夜,并回收载体片段;
(4)将退火引物稀释到0.1μM浓度,按以下体系连接:
(5)将连接产物转化到Stbl3感受态细胞中,涂板,挑单克隆菌落,送测确定引物连接进入载体。
(6)将构建好的质粒按照载体质粒:psPAX2:pMD2G=4:2:1的比例混入无血清DMEM中,加入4倍PEI转染试剂混匀后,室温孵育15min;
(7)将混合的质粒混合物加入70%密度293T细胞中,转染8h后换液,培养48h,收集病毒上清并使用0.45μM滤头过滤;
(8)将收集的病毒液感染HCS2/8细胞,感染24h后换液,48h后使用puro筛选细胞;
(9)筛选细胞完成后,将感染细胞稀释至100μL 1-2个细胞密度,铺在96板中,待单个细胞长成细胞团时,使用Western Blot挑选完全敲除的单株HCS2/8细胞。
结果如图2所示,结果表明HCS2/8PSME3敲除细胞构建成功。
3、PSME3基因对软骨肉瘤发生发展的影响
3.1、MTT实验
(1)将处于对数生长期的各组HCS2/8细胞用胰酶消化重悬离心,并使用血球计数法进行细胞计数;
(2)以每孔1500个细胞的密度将细胞均匀地铺至96孔板中,在37℃5%CO2的条件下培养;
(3)待细胞完全贴壁后,开始使用MTT试剂检测细胞的增殖能力,在490nm波长处检测细胞的吸光度,每24h检测一次;
(4)连续检测5天,绘制出细胞增殖曲线。
结果如图3所示,PSME3 KO细胞组增殖能力显著抑制。
3.2、克隆形成实验
(1)细胞铺板:将细胞消化重悬,使用血球计数法计数,将细胞均匀地铺在12孔板中,1500个细胞/孔;
(2)加药:次日细胞贴壁,根据实验预先设定的药物浓度处理细胞,每孔1mL培养基,每三天换一次含有药物的培养基;
(3)细胞培养:持续培养细胞7天以上,每日观察细胞克隆团数量和大小,培养至对照组克隆团之间快要互相接触;
(4)固定:将克隆形成板从培养箱中拿出,弃去上清,使用PBS清洗三次,每次3min,再加入4%多聚甲醛固定15min;
(5)染色:固定完成后,PBS清洗三次,每次3min,每孔加入300μL 0.2%结晶紫染液,染色20min;
(6)洗脱:染色结束后,回收结晶紫,使用PBS洗去多余的染液,清洗三次,每次3min,洗干后洗干水分,室温下晾干;
(7)拍照:晾干后拍照记录实验数据,分析实验结果。
结果如图4所示,相比对照组(PSME3 WT),PSME3 KO细胞组克隆数显著减少(P<0.05)。
4、Western Blot实验检测铁死亡通路关键蛋白的变化
(1)制备样品:取人软骨肉瘤细胞HCS2/8,分为两组,一组为HCS2/8PSME3 WT,另一组为敲除了PSME3基因的HCS2/8PSME3 KO;分别使用1×SDS loading裂解两组细胞,在100℃金属浴上煮沸15min使蛋白变性;
(2)配制蛋白质凝胶:清洗蛋白胶板,使用ddH2O冲洗一次;清洗干净后,放入烘箱烘干,将胶板对齐,平行夹在胶板架上,根据蛋白胶配方配制下层分离胶,使用无水乙醇压平,待下层胶凝固后,弃去无水乙醇,根据配方配制上层浓缩胶,并插入梳子,待上层胶凝固后,放入4℃冰箱保存;
(3)电泳:将制备好的蛋白样按实验设计的顺序依次从左到右上样,左右两边留好孔道上蛋白marker,使用1×loading buffer补齐;上样结束后,在跑胶槽中加入1×Running buffer,起始以80V恒压电泳,待下层胶marker分离后,以120V恒压电泳至底部;
(4)转膜:提前配好1×Transfer buffer(700mL ddH2O,200mL甲醇,100mL10×Transfer buffer),准备好转膜夹、NC膜以及滤纸,浸泡于Transfer buffer中;蛋白胶电泳结束后,使用刮板轻轻翘起薄板,切割上层胶,保留下层胶,将下层胶转移至转膜液内,按照一层海绵,三层滤纸,下层胶,NC膜,三层滤纸,一层海绵的顺序夹好转膜夹,过程中应尽量避免下层胶和NC膜之间有气泡;将转膜夹放入转膜槽中,导入转膜液,放入冰盒,以200mA恒流,冰浴的条件下转膜;
(5)封闭:转膜结束,蛋白胶上的蛋白成功转移到NC膜上,使用PBS缓冲液洗涤一次NC膜,使用7%的脱脂奶粉(PBS配制)封闭NC膜60min;
(6)一抗孵育:封闭结束后弃掉脱脂奶粉,使用PBS清洗5min,根据蛋白marker指示,裁取PSME3、SLC7A11、GPX4、ACTIN蛋白所在的条带,放入暗盒中,倒入相应的一抗,将暗盒放在4℃恒温摇床中孵育过夜;
(7)荧光二抗结合:回收一抗,使用PBST(PBS缓冲液+0.05% Tween-20)清洗条带三次,每次5min,再倒入相应的荧光二抗,在4℃恒温摇床中孵育1h;
(8)扫膜:二抗孵育结束后,使用PBST洗涤条带三次,每次5min,使用Oddesey扫膜仪扫描相应的条带,分析结果。
结果如图5所示,随着PSME3基因的敲除,铁死亡通路关键调控蛋白SLC7A11和GPX4相应下降,证明PSME3可以调控铁死亡。
5、细胞内谷胱甘肽(GSH)水平检测
(1)将HCS2/8PSME3 WT和HCS2/8PSME3 KO细胞分别铺至6孔板,长至密度为80%-90%;
(2)配制谷胱甘肽工作液:按以下配方配制工作液:6.6μL 5倍稀释谷胱甘肽还原酶,6.6μL DTNB储备液,150μL总谷胱甘肽检测缓冲液;
(3)将细胞消化离心至1.5mL EP管中,使用PBS清洗一次,弃去上清;根据细胞沉淀的体积加入3倍量的蛋白质去除试剂M溶液,吹打混匀,利用37℃水浴锅和液氮快速冻融细胞两次,以达到裂解细胞的目的,在4℃1000g条件下离心10min,收集上清至新的EP管中;
(4)每个检测孔中一次加入10μL样品,总谷胱甘肽工作液150μL,室温孵育5min,然后每孔加入50μL 0.5mg/mL NADPH,避光反应25min;
(5)待反应结束之后,使用酶标仪在412nm光谱下检测吸光度,收集并分析数据。
结果如图6所示,PSME3敲除后,HCS2/8细胞内GSH显著下降,说明细胞内GSH合成减少,细胞发生铁死亡。
综上所述,本发明提供的PSME3基因在软骨肉瘤组织与正常组织表达上存在显著差异,显著影响患者的预后和生存期,表明PSME3基因为制备诊断软骨肉瘤的试剂盒提供了靶点,并且为制备治疗软骨肉瘤的药物提供了新的靶点,也为软骨肉瘤的基因治疗提供依据。
以上所述仅为本发明较佳的实施例,并非因此限制本发明的实施方式及保护范围,对于本领域技术人员而言,应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案,均应当包含在本发明的保护范围内。
Claims (7)
1.检测PSME3及其表达产物的试剂在制备诊断软骨肉瘤的试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,所述PSME3为核苷酸序列如SEQ ID NO:1所示的DNA分子;或,所述PSME3为编码氨基酸序列如SEQ ID NO:2所示的DNA分子;或,所述PSME3为在严格条件下与如SEQ ID NO:1所示的核苷酸序列杂交且编码具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子;或,所述PSME3为编码在如SEQ ID NO:2所示的氨基酸序列中经过取代或/和缺失或/和添加一个或多个氨基酸但具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子。
3.根据权利要求2所述的应用,其特征在于,PSME3的表达产物为由所述PSME3编码的蛋白质。
4.根据权利要求1所述的应用,其特征在于,所述试剂包括:PSME3的引物。
5.根据权利要求4所述的应用,其特征在于,所述PSME3的引物的核苷酸序列如SEQ IDNO:3-4所示。
6.抑制PSME3表达的试剂在制备治疗软骨肉瘤的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述PSME3为核苷酸序列如SEQ ID NO:1所示的DNA分子;或,所述PSME3为编码氨基酸序列如SEQ ID NO:2所示的DNA分子;或,所述PSME3为在严格条件下与如SEQ ID NO:1所示的核苷酸序列杂交且编码具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子;或,所述PSME3为编码在如SEQ ID NO:2所示的氨基酸序列中经过取代或/和缺失或/和添加一个或多个氨基酸但具有如SEQ ID NO:2所示的氨基酸序列活性的DNA分子。
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