CN106039285A - 359aa多肽及其载体和应用 - Google Patents

359aa多肽及其载体和应用 Download PDF

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CN106039285A
CN106039285A CN201610353341.6A CN201610353341A CN106039285A CN 106039285 A CN106039285 A CN 106039285A CN 201610353341 A CN201610353341 A CN 201610353341A CN 106039285 A CN106039285 A CN 106039285A
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recombination
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沈爱国
刘晓娟
张超
吴翔
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Abstract

本发明公开了359aa多肽及其载体和应用。本发明利用生物工程技术，基因重组一段359个氨基酸多肽对应的DNA序列到pcDNA3.1真核表达载体。经酶切和序列分析证明重组成功后，将此真核表达重组多肽转染到心肌细胞中，免疫印迹证明多肽的蛋白表达，实现了多肽的重组。接着对多肽的抗心肌IR后凋亡功能进行了研究，细胞学实验表明此多肽具有抑制心肌IR后细胞凋亡的重要保护功能。该多肽类似物高效表达，纯化工艺简单，有利于进一步大规模制备。可为寻找IR后细胞损伤有效的干预靶点并开发新的治疗药物提供理论依据。对于应用于IR的临床治疗实现心脏血管再通后的远期预后具有十分重要的开发前景。

Description

359aa多肽及其载体和应用
技术领域
本发明涉及生物医药技术领域，具体是359aa多肽及其载体和应用。
背景技术
心肌缺血作为多种心脏疾病尤其是急性ST段抬高型心肌梗死(ST-segmentelevation myocardial infarction,STEMI)产生的结果，是世界范围内致死致残的主要原因之一，严重危害人类的健康，而尽早恢复血流是减轻缺血心肌损伤的基础。近年来，随着溶栓治疗术、经皮穿刺冠状动脉腔内血管成形术和冠状动脉旁路手术等治疗方法的出现，由STEMI所导致的心肌缺血性损伤得到有效控制，但同时伴随血管再通所产生的心肌缺血再灌注(ischemia/reperfusion,IR)则利弊相辅，因其本身会相反地对心肌产生进一步损伤，进而阻碍心肌从缺血中的恢复和影响血管再通疗效，因此日益被人们所重视并成为目前全球研究的热点。近年来研究表明，心肌IR进程中发生心肌细胞凋亡、坏死和坏死性凋亡，其中细胞凋亡发挥着关键的作用，而细胞凋亡过程受多种信号通路的调控。因此，加强拮抗心肌细胞凋亡的研究、探索治疗靶点具有十分重要的意义。
发明内容
发明目的：针对现有技术中存在的不足，本发明的目的是提供一种359aa多肽，具有抗心肌细胞凋亡的应用价值。本发明的另一目的是提供上述359aa多肽的应用。
技术方案：为了实现上述发明目的，本发明采用的技术方案为：
359aa多肽在制备用于抑制心肌IR后细胞凋亡的药物中的应用，所述的359aa多肽的氨基酸序列如SEQ ID NO.1所示。
编码所述的359aa多肽的基因，其DNA序列如SEQ ID NO.2所示。
含有所述的359aa多肽的编码基因的DNA序列的载体。
所述的载体，将359aa多肽的DNA序列连接进pcDNA3.1真核表达载体，构建出重组表达质粒。
有益效果：与现有技术相比，本发明利用生物工程技术，基因重组一段359个氨基酸(amino acid)多肽对应的DNA序列到pcDNA3.1真核表达载体。经酶切和序列分析证明重组成功后，将此真核表达重组多肽转染到心肌细胞中，免疫印迹证明多肽的蛋白表达，实现了多肽的重组。接着对多肽的抗心肌IR后凋亡功能进行了研究，细胞学实验表明此多肽具有抑制心肌IR后细胞凋亡的重要保护功能。该多肽类似物高效表达，纯化工艺简单，有利于进一步大规模制备。可为寻找IR后细胞损伤有效的干预靶点并开发新的治疗药物提供理论依据。对于应用于IR的临床治疗实现心脏血管再通后的远期预后具有十分重要的开发前景。
附图说明
图1是359aa多肽对应DNA序列免疫印迹蛋白表达结果结果图；
图2是LDH检测359aa多肽对心肌细胞活性的抑制结果图；
图3是TUNEL实验检测359aa多肽抗进心肌细胞凋亡结果图；
图4是免疫荧光技术检测多肽片段的核转位情况结果图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著，黄培堂等译，科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件进行。
实施例1 359aa多肽重组
提取人的mRNA，逆转录为cDNA，以cDNA为模板，应用PCR技术成功扩增出对应的mRNA序列(上游-Forward：5'-CGAATTCGGATGGTGAACTTCACAGTAGATCAGA-3'；下游-Reverse:5'-CCTCTCGAGGTCCCACAGCTTCTTCATCATG-3')，其序列如SEQ ID NO.1所示，对应表达的氨基酸序列如SEQ ID NO.2所示。扩增得到的片段通过水平电泳进行分离，使用回收试剂盒获得该片段纯化产物。在37℃下使用ECOR1\Xho1酶对片段纯化产物以及pcDNA3.1-HA载体质粒，进行酶切2小时。通过水平电泳分离，使用回收试剂盒获得暴露粘性末端的片段产物。在16℃水浴下，将片段与载体以核酸量(3～10)：1的比例进行连接12小时。将连接产物转入感受态大肠杆菌中，涂布于Amp+琼脂平板上，37℃培养12小时。在含Amp+琼脂平板上挑选克隆，以碱裂解法小提重组质粒后，以ECOR1\Xho1酶切鉴定。并同时构建eEF2△1-795和eEF2△795-1500质粒，分别标记为eEF2-1，eEF2-2。将提取的重组质粒送至上海桑尼生物技术有限公司进行测序，结果在NCBI核酸比对网站进行核对，结果显示正确。
将正确连接的359aa多肽真核表达载体使用Lipofectamine 2000TransfectionReagent转染入H9c2细胞，48小时后搜集样品，RIPA细胞裂解液裂解，加入上样缓冲液后，100℃水浴15分钟，13,000rpm离心15分钟，进行免疫印迹检测，孵育HA一抗，小鼠来源二抗，Odyssey Infrared Imaging成像系统成像，免疫印迹结果证明了此多肽的表达，如图1所示。
实施例2 重组肽抗凋亡功能的检测
1)LDH检测证明重组肽细胞水平的抗心肌缺血再灌组后细胞损伤的效应
将心肌细胞H9c2分别以20000个/孔的密度接种于96孔培养板，利用Lipofectamin 2000kit将359aa多肽真核表达载体和其它对照组转染H9c2细胞，36小时后进行缺氧/复氧刺激，取细胞培养液，4℃下13000rpm离心后，使用LDH检测试剂盒检测(南京建成)，在450nm测量吸收值。吸收值大小反映了细胞损伤情况，根据实验数据，以时间为横坐标，吸收值为纵坐标做图。LDH检测结果如图2所示，显示与空载对照相比，359aa多肽真核表达实验组明显减轻了细胞损伤。
2)TUNEL证实重组肽细胞水平的抗心肌缺血再灌注后细胞凋亡的效应
将心肌细胞H9c2分别以10000个/孔的密度接种于24孔培养板，利用Lipofectamin 2000kit将359aa多肽真核表达载体和其它对照组转染H9c2细胞，36小时后进行缺氧/复氧刺激，去除培养液，冰上用预冷PBS洗涤，使用4％多聚甲醇固定40分钟，预冷PBS洗涤，使用罗氏TUNEL检测试剂盒检测细胞凋亡情况。TUNEL检测结果如图3所示，显示与空载对照相比，359aa多肽真核表达实验组明显拮抗了细胞凋亡的发生。
3)细胞免疫荧光证实重组肽细胞水平的胞内定位未发生了改变
将心肌细胞H9c2分别以10,000个/孔的密度接种于24孔培养板，利用Lipofectamin 2000kit将359aa多肽真核表达载体和其它对照组转染H9c2细胞，36小时后进行缺氧/复氧刺激，去除培养液，冰上用预冷PBS洗涤3次，使用4％多聚甲醇固定40分钟，预冷PBS洗涤，1％Triton-100通透10分钟，PBS洗涤后5％BSA固定2小时。使用一抗HA(1:50)孵育10小时，PBS洗涤后二抗R绿、DAPI(1:1000)孵育2小时。Leica荧光显微镜下观察，ImageJ进行图像后处理。如图4所示，免疫荧光显示359aa多肽真核表达未发生入核的现象。

Claims (4)

1.359aa多肽在制备用于抑制心肌IR后细胞凋亡的药物中的应用，所述的359aa多肽的氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的359aa多肽的基因，其DNA序列如SEQ ID NO.2所示。
3.含有权利要求2所述的359aa多肽的编码基因的DNA序列的载体。
4.根据权利要求3所述的载体，其特征在于：将359aa多肽的DNA序列连接进pcDNA3.1真核表达载体，构建出重组表达质粒。
CN201610353341.6A 2016-05-25 2016-05-25 359aa多肽及其载体和应用 Pending CN106039285A (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102105164A (zh) * 2008-06-11 2011-06-22 Atyr医药公司 酪氨酰-trna合成酶多肽的血小板生成活性

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102105164A (zh) * 2008-06-11 2011-06-22 Atyr医药公司 酪氨酰-trna合成酶多肽的血小板生成活性

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AGNES S. KIM等: "A small molecule AMPK activator protects the heart against Ischemia-reperfusion injury", 《JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY》 *
GEORG RAPP等: ""Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2"，第247-250页", 《BIOL. CHEM. HOPPE-SEYLER》 *
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Application publication date: 20161026