CN113425855A - 一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用 - Google Patents
一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用 Download PDFInfo
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- CN113425855A CN113425855A CN202110718784.1A CN202110718784A CN113425855A CN 113425855 A CN113425855 A CN 113425855A CN 202110718784 A CN202110718784 A CN 202110718784A CN 113425855 A CN113425855 A CN 113425855A
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Abstract
本发明提供了一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用，属于骨关节炎治疗技术领域，所述骨关节炎药物制剂，包括活性成分mRNA，所述mRNA包括编码如SEQIDNo.1～SEQIDNo.4所示重组蛋白序列中的一种或几种。本发明提供的骨关节炎药物制剂制备方法简单、快速、活性成分表达量高、免疫原性低，能够快速高效促进骨关节软骨生长，从而达到治疗骨关节炎的目的。
Description
技术领域
本发明属于骨关节炎治疗技术领域，尤其涉及一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用。
背景技术
骨关节炎(OA)是一种退行性关节疾病，系由于增龄、肥胖、劳损、创伤、关节先天性异常、关节畸形等诸多因素引起的关节软骨退化损伤、关节边缘和软骨下骨反应性增生。该病多见于中老年人群，好发于负重关节及活动量较多的关节(如颈椎、腰椎、膝关节、髋关节等)。过度负重或使用这些关节，均可促进退行性变化的发生。临床表现为缓慢发展的关节疼痛、压痛、僵硬、关节肿胀、活动受限和关节畸形等。
目前，该病缺少有效的治疗手段。当前主要采用的治疗方法是减少关节的负重和过度的大幅度活动，以延缓病变的进程。肥胖患者应减轻体重，减少关节的负荷。下肢关节有病变时可用拐杖或手杖，以求减轻关节的负担。理疗及适当的锻炼可保持关节的活动范围，必要时可使用夹板支具及手杖等，对控制急性期症状有所帮助。消炎镇痛药物可减轻或控制症状，但应在评估患者风险因素后慎重使用且不宜长期服用。软骨保护剂如硫酸氨基葡萄糖具有缓解症状和改善功能的作用，同时长期服用可以延迟疾病的结构性进展。对晚期病例，在全身情况能耐受手术的条件下，行人工关节置换术，目前是为数不多的公认消除疼痛、矫正畸形、改善功能的有效方法。然而在不进行手术的情况下，尚无有效的治疗药物。
发明内容
有鉴于此，本发明的目的在于提供一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用；本发明提供的骨关节炎药物制剂包括具有刺激软骨再生的蛋白生长因子mRNA以及编码重组蛋白的信号肽的mRNA，将所述骨关节炎药物制剂导入靶细胞后能够促进软骨细胞的生长发育，弥补了药物有效治疗骨关节炎方法的空白；所述骨关节炎药物制剂的制备方法简单、快速、活性成分表达量高、免疫原性低，能够快速高效促进骨关节软骨生长，从而达到治疗骨关节炎的目的。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种mRNA剂型的骨关节炎药物制剂，包括活性成分mRNA，所述mRNA包括编码如SEQ ID No.1～SEQ ID No.4所示的重组蛋白的mRNA序列中的一种或几种。
优选的，所述mRNA的5’端连接帽子结构和5’UTR；所述mRNA的3’端连接3’UTR和多聚A尾。
优选的，所述mRNA的序列如SEQ ID No.5～SEQ ID No.12所示；其中SEQ ID No.5或SEQ ID No.6所示的mRNA序列编码SEQ ID No.1所示的重组蛋白；SEQ ID No.7或SEQ IDNo.8所示的mRNA序列编码SEQ ID No.2所示的重组蛋白；SEQ ID No.9或SEQ ID No.10所示的mRNA序列编码SEQ ID No.3所示的重组蛋白；SEQ ID No.11或SEQ ID No.12所示的mRNA序列编码SEQ ID No.4所示的重组蛋白。
优选的，所述骨关节炎药物制剂为液体制剂。
优选的，所述骨关节炎药物制剂为注射制剂。
优选的，所述活性成分mRNA在骨关节炎药物制剂中的浓度为200～2000μg/ml。
优选的，所述活性成分mRNA的溶剂为生理盐水。
本发明提供了所述的骨关节炎药物制剂的制备方法，包括以下步骤：
1)合成转录所述的骨关节炎药物制剂中的活性成分mRNA的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒；
2)将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；
3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。
优选的，所述RNA体外合成体系以1600μl计，包括以下组分：
优选的，所述RNA体外合成的条件为36～38℃，8～12h。
本发明的有益效果：本发明提供的mRNA剂型的骨关节炎药物制剂，包括具有刺激软骨再生的蛋白生长因子mRNA，将所述骨关节炎药物制剂导入靶细胞后能够促进软骨细胞的生长发育，弥补了药物有效治疗骨关节炎方法的空白；所述骨关节炎药物制剂的制备简单、快速、表达量高、免疫原性低，能够快速高效促进骨关节软骨生长，从而达到治疗骨关节炎的目的。
根据实施例的记载，本发明提供的具有刺激软骨再生的蛋白生长因子mRNA能够在细胞内特异性的高水平表达；本发明提供的mRNA进行肌肉注射后，小鼠血清中TNFα水平显著低于TGFβ3和FGF18蛋白制剂，说明本发明提供的mRNA剂型的骨关节炎药物制剂免疫原性更低，安全性更好。
附图说明
图1为编码刺激软骨再生的蛋白生长因子mRNA的结构示意图；
图2为pRhe质粒的质粒图谱；
图3为WesternBlot检测软骨生长因子mRNA在细胞内的表达情况；
图4为小鼠注射本发明提供的mRNA剂型的骨关节炎药物制剂的TNFα水平；
图5为各实验组大鼠关节切片和番红固绿染色结果，观察软骨修复情况；
图6为各实验组大鼠关节切片OARSI评分，显示mRNA药物制剂对骨关节炎的修复作用。
具体实施方式
本发明提供了一种mRNA剂型的骨关节炎药物制剂，包括活性成分mRNA，所述mRNA包括编码如SEQ ID No.1～SEQ ID No.4所示的重组蛋白的mRNA序列中的一种或几种。
具体如下：
SEQ ID No.1(以下简称Seq 1)：
METDTLLLWVLLLWVPGSTGDGSALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS*
SEQ ID No.2(以下简称Seq 2):
MDWTWILFLVAAATRVHSDGSALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS*
SEQ ID No.3(以下简称Seq 3):
METDTLLLWVLLLWVPGSTGDEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK
SEQ ID No.4(以下简称Seq 4):
MDWTWILFLVAAATRVHSDEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK
在本发明中，所述mRNA的序列如SEQ ID No.5～SEQ ID No.12所示；具体如下：
SEQ ID No.5(以下简称Seq1-1)：
ATGGAAACCGATACATTACTTCTCTGGGTATTGCTGCTCTGGGTCCCCGGGAGTACGGGTGATGGGAGCGCATTAGATACCAACTATTGCTTTAGAAACCTCGAAGAAAATTGCTGTGTGCGCCCGCTTTATATTGACTTTCGTCAAGATCTTGGATGGAAATGGGTCCACGAGCCTAAGGGCTATTATGCAAATTTCTGTTCTGGTCCTTGCCCTTATTTAAGGTCGGCTGACACGACACACTCGACCGTATTGGGGCTTTATAACACATTGAACCCTGAAGCCTCTGCAAGTCCCTGCTGCGTCCCTCAAGATTTAGAACCCCTTACGATCTTGTATTATGTTGGTCGAACGCCGAAAGTTGAACAGTTATCCAACATGGTCGTGAAGTCGTGCAAATGTAGTTGA
SEQ ID No.6(以下简称Seq1-2)
ATAGAGACCGATACGTTGCTTTTATGGGTTCTTCTGCTCTGAGTTCCTGGTAGTACAGGGGACGGGAGTGCCTTAGATACGAACTACTGCTTCCGCAACCTGGAGGAAAACTGCTGCGTGCGGCCACTGTACATCGACTTCCGCCAGGATCTTGGATGAAAGTGGGTACACGAACCCAAAGGTTATTATGCTAACTTTTGCTCTGGGCCATGTCCATACTTGCGGTCTGCCGATACTACTCACTCTACAGTGTTAGGACTTTATAACACGTTGAACCCAGAGGCTTCTGCTAGCCCCTGCTGCGTTCCTCAGGATCTTGAGCCTCTCACCATTTTATATTATGTAGGACGCACTCCTAAAGTTGAACAATTATCAAACATGGTCGTGAAATCGTGTAAGTGTTCCTGA
SEQ ID No.7(以下简称Seq2-1)
ATGGACTGGACCTGGATATTATTTCTGGTAGCAGCTGCGACACGAGTCCATTCTGATGGCTCCGCTCTAGATACCAATTACTGCTTTCGGAACTTAGAGGAGAATTGTTGCGTGCGACCCTTATATATTGACTTTCGGCAGGACTTAGGTTGGAAGTGGGTCCACGAGCCCAAGGGATATTACGCGAACTTCTGTAGTGGTCCCTGCCCATACCTGAGGTCCGCTGATACAACTCATAGTACCGTTCTTGGGCTATATAATACTCTAAACCCAGAGGCTTCAGCTTCGCCTTGTTGTGTTCCGCAGGATCTAGAACCATTAACAATACTATATTATGTAGGGCGAACCCCTAAAGTCGAGCAACTTAGTAACATGGTGGTGAAGTCGTGCAAGTGCAGTTGA
SEQ ID No.8(以下简称Seq2-2)
ATGGACTGAACATGAATCCTTTTCTTAGTAGCAGCCGCTACGCGAGTCCACTCGGATGGCAGTGCGCTCGATACTAACTATTGTTTTCGCAATTTGGAAGAAAACTGCTGTGTACGTCCTCTATACATTGACTTTCGCCAGGATTTGGGTTGAAAATGAGTGCATGAGCCTAAAGGCTACTATGCCAATTTCTGTTCCGGCCCTTGTCCCTATCTTCGCTCTGCAGATACAACTCACTCTACGGTACTAGGGCTCTATAACACTCTTAACCCCGAAGCGTCAGCCTCGCCATGTTGTGTGCCCCAAGATCTCGAGCCCCTCACTATTCTCTACTATGTTGGTCGCACTCCTAAAGTTGAGCAACTATCAAATATGGTGGTCAAATCCTGCAAGTGTTCGTGA
SEQ ID No.9(以下简称Seq3-1)
ATGGAGACAGACACTCTCCTGCTTTGGGTTTTGCTTTTATGGGTACCAGGGTCGACCGGAGATGAAGAAAACGTCGACTTCCGTATCCACGTTGAAAATCAGACTAGAGCTAGAGACGACGTGAGTAGGAAGCAGCTTAGACTCTACCAGCTTTACTCGAGGACTAGCGGAAAGCATATACAGGTTCTTGGCCGACGTATCTCGGCCCGGGGGGAAGATGGAGACAAGTACGCGCAACTATTAGTGGAGACGGACACATTTGGTTCGCAAGTTCGAATCAAGGGGAAGGAGACTGAATTCTATTTGTGCATGAACCGCAAGGGAAAGTTAGTGGGTAAGCCGGACGGAACAAGTAAAGAATGCGTGTTTATCGAGAAGGTCCTAGAGAATAACTACACAGCCCTTATGAGCGCAAAATACTCAGGCTGGTATGTCGGTTTCACAAAAAAGGGTCGACCCCGGAAGGGCCCGAAGACTCGCGAGAATCAACAAGATGTGCACTTCATGAAACGGTACCCCAAAGGACAGCCCGAGCTACAGAAGCCTTTTAAATATACAACCGTTACGAAATGA
SEQ ID No.10(以下简称Seq3-2)：
ATGGAGACAGACACTCTGCTATTATGAGTGCTACTGTTATGAGTACCTGGATCTACTGGGGACGAGGAGAACGTCGATTTTCGCATCCACGTTGAAAATCAGACACGAGCACGCGACGATGTATCACGAAAACAGCTACGGCTCTACCAACTGTACTCGCGCACTAGCGGAAAACATATCCAGGTCCTTGGCCGGCGGATCTCAGCCCGAGGTGAAGATGGGGACAAATACGCCCAGCTACTGGTCGAAACCGACACGTTCGGCTCGCAGGTACGAATCAAGGGCAAAGAAACAGAGTTCTACCTTTGCATGAATCGGAAAGGCAAACTGGTCGGGAAGCCAGATGGTACGTCTAAGGAGTGCGTGTTCATTGAGAAAGTTCTAGAAAATAATTACACCGCCCTGATATCCGCTAAATATAGTGGGTGGTATGTTGGCTTCACAAAAAAGGGTCGTCCGCGGAAGGGGCCAAAAACACGAGAGAATCAACAGGATGTCCATTTTATAAAGCGATATCCAAAGGGGCAACCCGAGCTTCAGAAACCCTTTAAGTACACAACCGTAACTAAATGA
SEQ ID No.11(以下简称Seq4-1)
ATGGACTGGACTTGGATACTATTCTTAGTGGCTGCCGCGACTAGGGTGCATAGTGACGAGGAAAATGTAGACTTTCGTATCCACGTGGAAAACCAGACCCGGGCGCGAGATGACGTTTCACGGAAACAGCTCCGGTTATACCAGTTGTATTCACGCACAAGCGGAAAACACATTCAGGTATTAGGGAGACGGATTTCCGCTCGAGGCGAAGACGGAGACAAATATGCCCAACTACTCGTTGAGACCGACACCTTTGGTAGCCAAGTTCGTATAAAAGGGAAAGAGACGGAGTTCTATTTATGCATGAATAGGAAGGGTAAGCTTGTTGGAAAACCTGATGGGACCTCGAAGGAGTGCGTATTTATAGAGAAAGTCCTGGAAAACAACTATACGGCTCTCATGAGCGCCAAATATTCCGGTTGGTACGTTGGCTTCACCAAAAAAGGGAGACCCCGAAAAGGACCTAAGACGCGTGAGAATCAACAAGATGTGCACTTTATGAAGCGTTATCCAAAGGGCCAACCGGAGCTGCAGAAACCATTCAAATACACGACTGTTACTAAGTGA
SEQ ID No.12(以下简称Seq4-2)
ATGGATTGAACTTGGATCTTATTTCTAGTAGCAGCCGCTACTCGCGTTCACTCCGATGAGGAAAATGTTGATTTTCGTATTCATGTTGAGAACCAGACTCGCGCCCGAGACGATGTAAGCCGCAAACAACTGCGGCTCTATCAATTGTACTCACGCACTTCAGGCAAGCATATCCAAGTCTTGGGTCGGCGTATCAGTGCACGGGGCGAAGACGGTGACAAGTATGCCCAACTCTTAGTTGAAACGGATACATTTGGATCGCAGGTTCGAATCAAGGGCAAAGAAACAGAATTTTACCTATGCATAAACCGGAAAGGAAAATTGGTGGGCAAGCCAGACGGTACGTCAAAAGAATGCGTTTTTATCGAGAAAGTGCTTGAAAACAACTATACGGCACTGATATCAGCCAAGTATTCGGGTTGATATGTGGGTTTTACAAAAAAAGGGCGTCCTCGAAAGGGTCCCAAGACTCGCGAGAATCAGCAGGATGTGCATTTCATAAAACGTTACCCTAAAGGCCAACCAGAGCTCCAAAAGCCCTTCAAATACACGACAGTGACTAAATGA
在本发明中，所述mRNA的5’端优选的连接帽子结构和5’UTR；所述mRNA的3’端优选的连接3’UTR和多聚A尾。在本发明中，所述mRNA的结构示意图如图1所示。
在本发明中，所述骨关节炎药物制剂优选为液体制剂，更优选为注射制剂。在本发明中，所述活性成分mRNA在骨关节炎药物制剂中的浓度优选为200～2000μg/ml，更优选为500～1500μg/ml。所述活性成分mRNA的溶剂优选为生理盐水。本发明对所述生理盐水的制备方法没有特殊限定，采用本领域常规的生理盐水即可。本发明对所述注射制剂的制备方法没有特殊限定，满足本领域常规注射制剂的要求即可。
本发明提供了所述的骨关节炎药物制剂的制备方法，包括以下步骤：1)合成所述的骨关节炎药物制剂中的活性成分mRNA对应的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒；2)将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。
在本发明中，合成转录所述的mRNA的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒。本发明对合成所述的mRNA对应的DNA片段的方法没有特殊限定，采用本领域常规的DNA合成方法即可，在本发明具体实施过程中，优选的委托生物技术公司合成。在本发明中，所述DNA片段的具体序列根据碱基互补配对原则确定。在本发明中，所述表达质粒优选为pRhe质粒，在本发明中，所述pRhe质粒的质粒图谱如图2所示。在本发明中，优选的通过酶切连接的方法将所述DNA片段克隆至表达质粒中；在本发明中，所述DNA片段优选的通过BamHI和NheI酶进行双酶切获得酶切NDA片段；所述表达质粒优选的通过BamHI和NheI酶进行双酶切后获得酶切质粒；然后将所述酶切DNA片段和酶切质粒连接获得重组质粒。本发明对所述双酶切和连接的具体操作没有特殊限定，采用本领域常规的双酶切和连接的操作即可。
本发明在获得所述重组质粒后，将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。在本发明中，所述宿主细胞优选为大肠杆菌感受态细胞；本发明对所述转入的方法没有特殊限定，采用本领域常规的转入方法即可。本发明在获得重组细胞后，优选的进行阳性重组细胞的筛选和菌落测序。在本发明中，所述阳性重组细胞的筛选优选的在amp抗性的固体培养基上进行。在本发明中，挑选所述amp抗性的固体培养基上的单菌落进行菌落PCR，选取菌落PCR结果含目的条带的菌落进行测序。在本发明中，所述菌落PCR的引物包括引物F和引物R；所述引物F的序列如下：CTCTAGAGGATCGAACCCTT(SEQ ID No.5)；所述引物R的序列如下：AAACCCGCTGATCAGCCTCG(SEQ ID No.6)；本发明对所述菌落PCR的具体步骤没有特殊限定，采用本领域常规的菌落PCR步骤即可。
在本发明中，提取测序正确的重组细胞的质粒；本发明对所述质粒的提取方法没有特殊限定，优选的采用质粒提取试剂盒进行。在本发明中，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。在本发明中，所述PCR扩增的体系以50μl计，优选的如下：
在本发明中，所述引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μl。在本发明中，所述引物F的序列如下：CTCTAGAGGATCGAACCCTT(SEQ IDNo.5)；所述引物R的序列如下：AAACCCGCTGATCAGCCTCG(SEQ ID No.6)。在本发明中，所述PCR的扩增程序优选的如下：预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后延伸72℃，10min。
在本发明中，所述PCR扩增反应结束后，优选的对扩增产物进行琼脂糖凝胶电泳检测以确定反应是否成功；所述琼脂糖凝胶电泳检测的参数优选的如下：1.5％琼脂糖，5V/min，40min。在本发明中，琼脂糖凝胶电泳出现目的大小的条带认为是反应成功。
本发明在所述PCR扩增反应结束后，优选的将所述扩增产物进行浓缩和纯化。在本发明中，所述浓缩优选的采用Millipore 30Kd超滤管进行；所述纯化优选的采用FPLC进行；本发明在所述纯化后优选的采用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。优选260/280范围为1.8-2.1，260/230范围为大于2.0。
本发明在获得所述DNA模板后，构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。在本发明中，所述RNA体外合成体系以1600μl计，包括以下组分：
在本发明中，所述RNA体外合成的条件优选的为36～38℃，8～12h，更优选为37℃，10h。在本发明中，所述RNA体外合成优选的在恒温反应器中进行；所述RNA体外合成体系优选的置于2ml RNase-free Tube管中，一次同时反应多管；所述RNA体外合成体系中的反应试剂按照上述顺序添加。
本发明在所述RNA体外合成结束后，优选的还包括去除DNA模板、回收mRNA和纯化mRNA的步骤。在本发明中，所述去除DNA模板优选的通过DNase I消化实现；所述消化优选的包括将DNase I与RNA体外合成反应后的溶液混合后进行；所述DNase I与RNA体外合成反应后的溶液的体积比优选为3:40；所述混合优选的通过上下颠倒所述RNase-free Tube管实现，所述上下颠倒的次数优选为8～12次，更优选为10次；本发明在所述混合后，优选的进行离心将溶液收集至RNase-free Tube管底部。在本发明中，所述离心的转速优选为800～1200rpm，更优选为1000rpm；所述离心的时间优选为8～12s，更优选为10s。所述消化的温度优选为37℃；所述消化的时间优选为1h。本发明在所述消化结束后，优选的进行DNA片段残留检测。在本发明中，所述回收mRNA优选的通过将所述醋酸铵溶液沉淀实现；具体实施方法参见实施例记载；本发明在回收mRNA后，进行mRNA的质量检测；所述质量检测包括mRNA的浓度、mRNA的260/280、260/230的比值，纯mRNA的A260/A280的值为2.0～2.1，A260/A230范围为1.8～2.2。在本发明中，所述纯化mRNA通过FPLC纯化实现。本发明所述纯化mRNA后，优选的对纯化后的mRNA进行分装。
本发明提供了所述的骨关节炎药物制剂在制备治疗关节软骨损伤的药物中的应用。在本发明中，所述药物制剂的使用剂量，以每一关节计，优选为10～20μg，更优选为14～16μg，最优选为15μg。在本发明中，所述药物的使用方法为关节腔注射，本发明对所述关节腔注射的具体方法没有特殊限定，采用本领域常规的关节腔注射操作即可。本发明中，所述治疗关节软骨损伤的药物以所述骨关节炎药物制剂为活性成分或，还可以包括其他活性成分；所述药物的剂型优选为注射制剂。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
重组质粒制备：
1)合成编码seq1、seq2、seq3、seq4的目的DNA片段；
2)将目的片段和pRhe质粒分别进行BamHI和NheI双酶切，
3)使用T4连接酶进行体外连接；
4)转化大肠杆菌感受态，在amp固体培养基上培养12h；
5)菌落PCR，选取含目的条带的菌落进行测序。
6)测序正确的菌落扩大培养，质粒提取。
步骤5)中菌落PCR步骤如下：
1、单菌落挑取
把LB培养基倒入排枪槽中，然后用排枪加400μl LB培养基到48孔深孔板，用镊子拿已灭菌的小白枪头挑取平板上的单菌落并放到48孔深孔板中，同时在48孔深孔板和相应的表单上做好记录。挑好的48孔深孔板用封口膜盖好并做好相应标记(日期、板号等)，用针头在封口膜打孔，把它放在37℃摇床上摇1h。
2、菌落PCR反应
配制好如下PCR反应体系，把配好的反应液加到96孔板中，用排枪加2μl菌液到其中，按照PCR程序进行扩增：
在本发明中，所述引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μl。在本发明中，所述引物F的序列如下：CTCTAGAGGATCGAACCCTT(SEQ IDNo.5)；所述引物R的序列如下：AAACCCGCTGATCAGCCTCG(SEQ ID No.6)。在本发明中，所述PCR的扩增程序优选的如下：预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后延伸72℃，10min。
3、琼脂糖凝胶电泳
先配制1％琼脂糖凝胶(称取1g琼脂糖加入100ml的TAE溶液中)，在完成PCR反应的96孔板中加入0.5μl溴酚蓝，震荡混匀，然后点样，电泳好的琼脂糖凝胶拍照、保存。
4、判断阳性克隆并测序
根据琼脂糖凝胶电泳条带图来判断阳性克隆，把阳性克隆的菌液进行扩大培养，进行测序验证，选取具备完全正确序列的克隆进行下一步操作。
步骤6)中所述的质粒提取的步骤参考omega D6915 Endo-free PlasmidMidi Kit说明书。
得到的质粒按如下反应体系进行DNA模板的扩增：
反应体积，50μl(为单个管的反应体积，一次同时反应多管)
所述PCR扩增的体系以50μl计，优选的如下：
引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μl。引物F的序列如下：CTCTAGAGGATCGAACCCTT(SEQ ID No.5)；所述引物R的序列如下：AAACCCGCTGATCAGCCTCG(SEQ ID No.6)。所述PCR的扩增程序如下：预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后延伸72℃，10min。
反应结束后，将反应液合并于1.5ml Tube管中。取10μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖，5V/min，40min)。根据电泳目的条带的大小对反应成功与否进行确认。合格标准：电泳检测出现单一的条带，且大小正确。
测定结果：条带大小单一，大小符合要求。
DNA模板超滤
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。
DNA模板FPLC纯化
将上述超滤得到的DNA，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇＝25/24/1)，充分震荡后，12000g离心15min。
去掉沉淀，转移上清至新的离心管中，加入上清体积的1/103M NaAc(pH 5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静至30min。
4℃，12000g离心10min，弃上清。
用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的DNA模板。
用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：浓度为500ng/μl，260/280＝1.93，260/230＝1.75。
FPLC纯化后模板超滤
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。
测定结果：浓度为150ng/μl，260/280＝1.96，260/230＝1.87。
mRNA的体外合成
在恒温反应器中，进行mRNA的体外合成。
按照如下合成体系进行(反应试剂按照从上至下添加)：
反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)。
所述RNA体外合成体系以1600μl计，包括以下组分：
所述RNA体外合成的条件为37℃，10h。
DNase I消化去除DNA模板
向mRNA体外合成后的每个Tube管中各加入120μl DNase I。
上下颠倒10次混匀，1000rpm离心10s。
重新置于恒温反应器中，37℃，1h。
反应结束后，将反应液合并到RNase-free 50ml Tube管中，检测DNA片段的残留。
DNA残留检测的方法：
采用定量实时PCR检测方法，具体操作步骤如下：
(1)供试品溶液配制：取样品适量，用无酶水稀释10倍，混匀，混得。
(2)标准品溶液配制：用无酶水将标定过的质粒标准品进行稀释至1E+08copies/μl，然后再进行梯度稀释。具体操作如下：
ERC的制备：取20μl供试品溶液，加入20μl ST4，混匀，即得。
MIX反应液配制：以SuperFast Probe Mixtuure：W2306F：W2521R：W2430P：H2O＝10:0.6:0.6:0.4:7.4的比例进行配制，混匀，即得。
PCR管加样：每孔加入19μl qPCR MIX、再依次加入1μl标准曲线(ST1/ST2/ST3/ST4/ST5/ST6)、ERC、NTC、供试品溶液，混匀。每个样各做三个平行。
PCR程序设定如下：
计算公式：以标准品的Ct值对其浓度的对数值做图，线性回归分析，将样品Ct值代入方程，计算“稀释10倍后的浓度对数的检测值”，计算“DNA残留。
DNA残留浓度(copies/μl)＝检测值×稀释倍数
结果判定：三个平行孔间的Ct差值应小于1.0；Ct值大于35的样本除外。
线性相关系数R2>0.99。
无模板对照NTC应不得检出或大于标准曲线最低浓度2个Ct值。
标准规定：应不高于10ng/剂量。
mRNA沉淀回收
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。
上下颠倒10次混匀。
置于-20℃2h，沉淀。
17000g，4℃离心，30min。
去掉上清，用70％乙醇洗涤沉淀。
17000g，4℃离心，10min。
去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。
静置10min后，用枪头轻吹混匀。
用NanoDrop检测回收后的mRNA浓度为5.06μg/μl，A260/A280为1.81、A260/A230为1.95。
取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。
检测结果为，条带符合大小，片段完整。
LiCl沉淀纯化mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。
加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。
然后于-20℃静至2h。
16000g离心20min。
弃上清，用70％乙醇洗涤沉淀，16000g离心15min。
取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的mRNA。
纯化后的mRNA浓度为2μg/μl，A260/A280为1.95、A260/A230为1.84。
mRNA分装
将上一步骤中纯化后的mRNA分装至西林瓶。
实施例2
编码软骨生长因子的mRNA表达水平检测
实验方法：体外效力-Western Blot检测
293T细胞的接种
1)细胞准备：提前1-3天准备检测用细胞。取购自中科院细胞库的293T细胞，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。
2)细胞消化计数：取生长状态良好的293T细胞，去掉培养基，以10ml PBS清洗细胞后，加入体积百分含量0.25％胰酶(T75瓶加1ml 0.25％胰酶，T175瓶加3ml 0.25％胰酶)消化5min，然后加入含10％FBS的DMEM培养基(T75瓶用9ml培养基，T175瓶用17ml培养基)中和胰酶，吹打细胞并转至50ml离心管，反复吹打混匀，然后取0.3～0.5ml的细胞悬液，计数。
3)细胞稀释：取1ml细胞悬液，用含10％FBS的DMEM培养基稀释到5×105个/ml，吹打混匀。
4)细胞接种：取2ml细胞悬液加到6孔板内。每个mRNA样品需要准备2孔平行细胞，对照组样品(GFP-mRNA)需要准备1孔细胞，空白对照1孔。将6孔板放入(37±1)℃、(5±0.5)％CO2培养箱培养过夜。
细胞转染
接种完细胞后约24h，观察6孔板内的细胞状态，汇合度在90％左右。在生物安全柜内，配制所需体积的90％DMEM+10％FBS培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基(90％DMEM+10％FBS)。
a)配制转染体系：取200μl opti-MEM，加入10μg mRNA样品(SEQ ID No.1～SEQ IDNo.4)或阴性对照GFP-mRNA，用枪头轻轻吹打混匀，再加入60μl PEI(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置10min。
b)将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。
c)换液
转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。
d)收获
转染后30h收获。吸掉旧的培养基，用1ml PBS清洗一遍。
吸掉PBS，继续用1ml PBS将细胞吹打下来，收集于1.5ml离心管中，300g离心5min。将离心后的上清尽量吸去干净，沉淀的细胞用于Western blot检测。
蛋白提取
1.配制细胞裂解液：0.1％Triton X-100(sigma，P/N T9284)，150mM NaCl(sigma，P/N S5886)，50mM HEPES pH8(sigma，P/N V900477)，EDTA-free protease inhibitorcocktail(sigma，P/N 11873580001)；
2.在之前收集的细胞沉淀中加入100μL4℃预冷的细胞裂解液，重悬细胞沉淀，4℃冰浴30min；
3.将细胞样品置于冰盒上进行超声破碎(小美超声仪器(昆山)有限公司，XM-20MINI)1min：功率10W，循环模式：1s on/1s off；
4.超声后置于离心机中，4℃，20000rcf，离心10min。收集上清液。
5.BCA法测上清液蛋白浓度，BCA试剂盒(Thermo Scientific,P/N 23225)，操作步骤参见试剂盒说明书，具体如下：
A.用超纯水稀释BSA标准品，配制2000、1000、500、250、125、62.5、31.25μg/ml的标准品系列，另外加一个超纯水作为空白样品，各准备100μL。
B.取10μl上清液，加入90μl超纯水混匀。
C.配制1X工作试剂：取50份体积试剂A，加入1份体积试剂B，混匀。
D.将样品加到96孔板，每孔25μL，做3个平行。
E.每孔加入200μL工作试剂，震荡30S混匀。
F.将96孔板放置37℃孵育30min
G.孵育结束后，等待2min冷却到室温。用酶标仪对562nm读板。
H.吸光值平均扣除空白对照。标准品扣除空白吸光值对浓度作图，代入样品吸光值计算。
SDS-PAGE
1.待测蛋白样品制备：蛋白定量后，取50μg蛋白，用裂解液定容至20μL，加入5μL5×Loadingbuffer(购自生工生物工程(上海)股份有限公司，P/N C508320)，混匀后共25μL，使用95℃孵育5min，结束后室温放置；
按照表1的配方配制6％SDS-PAGE分离胶和12％SDS-PAGE分离胶(1mm厚度)
表1 6％SDS-PAGE分离胶和12％SDS-PAGE分离胶配方
6％分离胶对应4％浓缩胶，12％分离胶对应5％浓缩胶，按照表2的配方配制浓缩胶。
表2 4％SDS-PAGE浓缩胶和5％SDS-PAGE浓缩胶配方
2.配制电泳缓冲液：取100ml 10×蛋白电泳缓冲液(生工，P/N C520001)，用超纯水稀释至1000ml；
3.将凝胶夹紧到电泳槽中(Bio-rad，P/N 1658004)，灌入电泳缓冲液；
4.将25μL待测蛋白样品和5μL蛋白分子量Marker(thermo，P/N 26619)分别加到样品孔中；
5.使用80V电泳30min；
6.然后用140V电泳，对于6％分离胶，电泳至70kDa分子量Marker跑到底部但未跑出去为止；对于12％分离胶，电泳至25kDa分子量Marker跑到底部但未跑出去为止；
Western blot
7.使用半干转膜仪(Pyxis，型号SPJ-1000A)进行转膜，转膜前准备配套耗材(Pyxis，P/N SPJ-T20S)，内含Top buffer，Down Buffer，Balance buffer和滤纸，另需自行裁剪与滤纸大小相同的PVDF膜(millipore，P/N IPVH00010)；
8.将滤纸分别放入两个方形培养皿，标记好Top和Down；
9.将20ml Top、Downbuffer倒入对应的方形培养皿中，浸泡滤纸5min至完全浸润；
10.将10ml Balance倒入方形培养皿，放入PVDF膜，孵育5min；
11.用撬板把凝胶玻璃板撬开，切去浓缩胶，小心取出凝胶，防止撕裂；
12.凝胶泡入超纯水的方形培养皿；
13.取出转膜仪的转印槽，去除盖子，将Down滤纸摆好在最底层；
14.将PVDF膜小心地覆盖Down滤纸，确认无气泡；
15.将凝胶覆盖在PVDF膜上，确认无气泡；
16.将Top滤纸覆盖在凝胶上，用滚轮滚动排气泡；
17.盖好转印槽盖子，放入转膜仪；
18.对于6％分离胶，使用11min进行转膜，对于12％分离胶，使用14min进行转膜；
19.转膜完成后，用镊子小心取出膜，在此后，膜应避免干燥；
20.用超纯水洗5min；
21.配制TBS：取100ml 10×TBS(生工，P/N C520002)，用水稀释至1000ml；
22.配制TBST：取1000ml TBS加入1ml Tween-20(sigma，P/N P1379)；
23.配制5％脱脂牛奶：取2.5g脱脂奶粉(伊利脱脂奶粉)溶解于50ml TBST；
24.去除超纯水，用TBS洗5min；
25.去除TBS，用TBST洗5min；
26.用5％脱脂牛奶在室温封闭1h
27.6％凝胶转的膜，用于检测S蛋白，12％凝胶转的膜，用于检测GAPDH蛋白；
28.目的蛋白一抗孵育：取3μl TGFβ3或FGF18一抗稀释到3ml 5％脱脂牛奶中(稀释比1:1000)，置于4℃摇床过夜孵育；
29.GAPDH蛋白一抗孵育：取0.25μL GAPDH antibody(proteintech，P/N 60004-1-Ig)稀释到5ml 5％脱脂牛奶中(稀释比1:20000)，置于4℃过夜孵育；
30.一抗孵育结束后，去除一抗溶液，用TBST洗三次，每次5min；
31.二抗孵育：取1μL HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)(proteintech，P/N SA00001-1)稀释到5ml 5％脱脂牛奶中(稀释比1:5000)，常温孵育1h；
32.二抗孵育结束后，去除二抗溶液，用TBST洗三次，每次5min；
33.最后将膜浸泡在TBS中；
34.准备好显影液(thermo，P/N 34580)，把成像仪(analytikjena，型号UVPChemstudio touch)的相机和软件打开，进行预冷；
35.预冷完成后，取500μL显影A液和500μL显影B液混合，把膜放在托盘中，向表面倒入500μL显影混合液，反应1min；
36.在成像仪中选择ECL程序进行成像，曝光时间根据情况而定；
37.保存图片；
6.15.6.1结果判定
结果如图3所示，供试品应呈现明显色带，GAPDH为内参蛋白，1-4分别代表seq1-4。
实施例2
根据本发明的编码软骨生长因子的mRNA，检测其免疫原性，评价标准为将所述mRNA对小鼠进行肌肉注射后血清中TNFα水平。
将6-8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养，将Seq1-1、Seq2-1、Seq3-1、Seq4-1所示的mRNA对balb/c小鼠进行肌肉注射，每只小鼠的注射剂量为100μg，24h后对小鼠进行眼眶取血，分离血清。用小鼠TNFa试剂盒(RayBio)进行酶联免疫吸附分析(ELISA)。实验结果如图4所示。
表3小鼠注射软骨生长因子mRNA制剂后小鼠TNFα表达水平
小鼠注射本发明提供的软骨生长因子mRNA制剂后，TNFα表达水平低于注射TGFβ3和FGF18蛋白的处理，可见本发明提供的mRNA剂型免疫原性低，安全性好。
实施例3
编码软骨生长因子的mRNA修复大鼠关节软骨损伤将6～8周龄的SD大鼠(购自北京维通利华)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养，膝关节注射单点乙酸(MIA)诱导关节软骨损伤(详细步骤参见参考文献Takahashi I,Matsuzaki T,Kuroki H,et al.Induction ofosteoarthritis by injecting monosodium iodoacetateinto the patellofemoral joint of an experimental rat model[J].PLoS One,2018,13(4):e0196625.)，待造模成功后(通过大鼠膝关节组织切片番红固绿染色和HE染色，观察到明显的软骨组织损伤；认为是造模成功)，将Seq1-1、Seq2-1、Seq3-1、Seq4-1所示的mRNA对大鼠进行关节腔注射，每只大鼠关节的注射剂量为50μl(15μg)，同时设置蛋白对照组，每只大鼠关节注射10μg相应蛋白药物(TGFβ3和FGF18蛋白)，注射频率为每周一次，持续4周，随后对大鼠关节处进行切片和染色，观察软骨修复状况。
OARSI评分标准如下：
0正常
1表面轻微纤维化，但没有软骨损失
2关节软骨表面出现轻微裂痕；
3关节软骨表面裂痕磨损<25％；
4关节软骨表面裂痕磨损25％～50％；
5关节软骨表面裂痕磨损50％～75％；
6关节软骨表面裂痕磨损>75％。
结果如图5和图6所示，蛋白药物对骨关节软骨修复没有明显效果，对应的mRNA药物对骨关节炎有明显的治疗效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 深圳市瑞吉生物科技有限公司
<120> 一种mRNA剂型的骨关节炎药物制剂及其制备方法和应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 135
<212> PRT
<213> Artificial Sequence
<400> 1
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Gly Ser Ala Leu Asp Thr Asn Tyr Cys Phe Arg
20 25 30
Asn Leu Glu Glu Asn Cys Cys Val Arg Pro Leu Tyr Ile Asp Phe Arg
35 40 45
Gln Asp Leu Gly Trp Lys Trp Val His Glu Pro Lys Gly Tyr Tyr Ala
50 55 60
Asn Phe Cys Ser Gly Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr
65 70 75 80
His Ser Thr Val Leu Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser
85 90 95
Ala Ser Pro Cys Cys Val Pro Gln Asp Leu Glu Pro Leu Thr Ile Leu
100 105 110
Tyr Tyr Val Gly Arg Thr Pro Lys Val Glu Gln Leu Ser Asn Met Val
115 120 125
Val Lys Ser Cys Lys Cys Ser
130 135
<210> 2
<211> 133
<212> PRT
<213> Artificial Sequence
<400> 2
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Asp Gly Ser Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu
20 25 30
Glu Glu Asn Cys Cys Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp
35 40 45
Leu Gly Trp Lys Trp Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe
50 55 60
Cys Ser Gly Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser
65 70 75 80
Thr Val Leu Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser
85 90 95
Pro Cys Cys Val Pro Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr
100 105 110
Val Gly Arg Thr Pro Lys Val Glu Gln Leu Ser Asn Met Val Val Lys
115 120 125
Ser Cys Lys Cys Ser
130
<210> 3
<211> 190
<212> PRT
<213> Artificial Sequence
<400> 3
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Glu Glu Asn Val Asp Phe Arg Ile His Val Glu
20 25 30
Asn Gln Thr Arg Ala Arg Asp Asp Val Ser Arg Lys Gln Leu Arg Leu
35 40 45
Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Ile Gln Val Leu Gly
50 55 60
Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln Leu
65 70 75 80
Leu Val Glu Thr Asp Thr Phe Gly Ser Gln Val Arg Ile Lys Gly Lys
85 90 95
Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg Lys Gly Lys Leu Val Gly
100 105 110
Lys Pro Asp Gly Thr Ser Lys Glu Cys Val Phe Ile Glu Lys Val Leu
115 120 125
Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala Lys Tyr Ser Gly Trp Tyr
130 135 140
Val Gly Phe Thr Lys Lys Gly Arg Pro Arg Lys Gly Pro Lys Thr Arg
145 150 155 160
Glu Asn Gln Gln Asp Val His Phe Met Lys Arg Tyr Pro Lys Gly Gln
165 170 175
Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr Thr Val Thr Lys
180 185 190
<210> 4
<211> 188
<212> PRT
<213> Artificial Sequence
<400> 4
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Asp Glu Glu Asn Val Asp Phe Arg Ile His Val Glu Asn Gln
20 25 30
Thr Arg Ala Arg Asp Asp Val Ser Arg Lys Gln Leu Arg Leu Tyr Gln
35 40 45
Leu Tyr Ser Arg Thr Ser Gly Lys His Ile Gln Val Leu Gly Arg Arg
50 55 60
Ile Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln Leu Leu Val
65 70 75 80
Glu Thr Asp Thr Phe Gly Ser Gln Val Arg Ile Lys Gly Lys Glu Thr
85 90 95
Glu Phe Tyr Leu Cys Met Asn Arg Lys Gly Lys Leu Val Gly Lys Pro
100 105 110
Asp Gly Thr Ser Lys Glu Cys Val Phe Ile Glu Lys Val Leu Glu Asn
115 120 125
Asn Tyr Thr Ala Leu Met Ser Ala Lys Tyr Ser Gly Trp Tyr Val Gly
130 135 140
Phe Thr Lys Lys Gly Arg Pro Arg Lys Gly Pro Lys Thr Arg Glu Asn
145 150 155 160
Gln Gln Asp Val His Phe Met Lys Arg Tyr Pro Lys Gly Gln Pro Glu
165 170 175
Leu Gln Lys Pro Phe Lys Tyr Thr Thr Val Thr Lys
180 185
<210> 5
<211> 408
<212> DNA
<213> Artificial Sequence
<400> 5
atggaaaccg atacattact tctctgggta ttgctgctct gggtccccgg gagtacgggt 60
gatgggagcg cattagatac caactattgc tttagaaacc tcgaagaaaa ttgctgtgtg 120
cgcccgcttt atattgactt tcgtcaagat cttggatgga aatgggtcca cgagcctaag 180
ggctattatg caaatttctg ttctggtcct tgcccttatt taaggtcggc tgacacgaca 240
cactcgaccg tattggggct ttataacaca ttgaaccctg aagcctctgc aagtccctgc 300
tgcgtccctc aagatttaga accccttacg atcttgtatt atgttggtcg aacgccgaaa 360
gttgaacagt tatccaacat ggtcgtgaag tcgtgcaaat gtagttga 408
<210> 6
<211> 408
<212> DNA
<213> Artificial Sequence
<400> 6
atagagaccg atacgttgct tttatgggtt cttctgctct gagttcctgg tagtacaggg 60
gacgggagtg ccttagatac gaactactgc ttccgcaacc tggaggaaaa ctgctgcgtg 120
cggccactgt acatcgactt ccgccaggat cttggatgaa agtgggtaca cgaacccaaa 180
ggttattatg ctaacttttg ctctgggcca tgtccatact tgcggtctgc cgatactact 240
cactctacag tgttaggact ttataacacg ttgaacccag aggcttctgc tagcccctgc 300
tgcgttcctc aggatcttga gcctctcacc attttatatt atgtaggacg cactcctaaa 360
gttgaacaat tatcaaacat ggtcgtgaaa tcgtgtaagt gttcctga 408
<210> 7
<211> 402
<212> DNA
<213> Artificial Sequence
<400> 7
atggactgga cctggatatt atttctggta gcagctgcga cacgagtcca ttctgatggc 60
tccgctctag ataccaatta ctgctttcgg aacttagagg agaattgttg cgtgcgaccc 120
ttatatattg actttcggca ggacttaggt tggaagtggg tccacgagcc caagggatat 180
tacgcgaact tctgtagtgg tccctgccca tacctgaggt ccgctgatac aactcatagt 240
accgttcttg ggctatataa tactctaaac ccagaggctt cagcttcgcc ttgttgtgtt 300
ccgcaggatc tagaaccatt aacaatacta tattatgtag ggcgaacccc taaagtcgag 360
caacttagta acatggtggt gaagtcgtgc aagtgcagtt ga 402
<210> 8
<211> 402
<212> DNA
<213> Artificial Sequence
<400> 8
atggactgaa catgaatcct tttcttagta gcagccgcta cgcgagtcca ctcggatggc 60
agtgcgctcg atactaacta ttgttttcgc aatttggaag aaaactgctg tgtacgtcct 120
ctatacattg actttcgcca ggatttgggt tgaaaatgag tgcatgagcc taaaggctac 180
tatgccaatt tctgttccgg cccttgtccc tatcttcgct ctgcagatac aactcactct 240
acggtactag ggctctataa cactcttaac cccgaagcgt cagcctcgcc atgttgtgtg 300
ccccaagatc tcgagcccct cactattctc tactatgttg gtcgcactcc taaagttgag 360
caactatcaa atatggtggt caaatcctgc aagtgttcgt ga 402
<210> 9
<211> 573
<212> DNA
<213> Artificial Sequence
<400> 9
atggagacag acactctcct gctttgggtt ttgcttttat gggtaccagg gtcgaccgga 60
gatgaagaaa acgtcgactt ccgtatccac gttgaaaatc agactagagc tagagacgac 120
gtgagtagga agcagcttag actctaccag ctttactcga ggactagcgg aaagcatata 180
caggttcttg gccgacgtat ctcggcccgg ggggaagatg gagacaagta cgcgcaacta 240
ttagtggaga cggacacatt tggttcgcaa gttcgaatca aggggaagga gactgaattc 300
tatttgtgca tgaaccgcaa gggaaagtta gtgggtaagc cggacggaac aagtaaagaa 360
tgcgtgttta tcgagaaggt cctagagaat aactacacag cccttatgag cgcaaaatac 420
tcaggctggt atgtcggttt cacaaaaaag ggtcgacccc ggaagggccc gaagactcgc 480
gagaatcaac aagatgtgca cttcatgaaa cggtacccca aaggacagcc cgagctacag 540
aagcctttta aatatacaac cgttacgaaa tga 573
<210> 10
<211> 573
<212> DNA
<213> Artificial Sequence
<400> 10
atggagacag acactctgct attatgagtg ctactgttat gagtacctgg atctactggg 60
gacgaggaga acgtcgattt tcgcatccac gttgaaaatc agacacgagc acgcgacgat 120
gtatcacgaa aacagctacg gctctaccaa ctgtactcgc gcactagcgg aaaacatatc 180
caggtccttg gccggcggat ctcagcccga ggtgaagatg gggacaaata cgcccagcta 240
ctggtcgaaa ccgacacgtt cggctcgcag gtacgaatca agggcaaaga aacagagttc 300
tacctttgca tgaatcggaa aggcaaactg gtcgggaagc cagatggtac gtctaaggag 360
tgcgtgttca ttgagaaagt tctagaaaat aattacaccg ccctgatatc cgctaaatat 420
agtgggtggt atgttggctt cacaaaaaag ggtcgtccgc ggaaggggcc aaaaacacga 480
gagaatcaac aggatgtcca ttttataaag cgatatccaa aggggcaacc cgagcttcag 540
aaacccttta agtacacaac cgtaactaaa tga 573
<210> 11
<211> 567
<212> DNA
<213> Artificial Sequence
<400> 11
atggactgga cttggatact attcttagtg gctgccgcga ctagggtgca tagtgacgag 60
gaaaatgtag actttcgtat ccacgtggaa aaccagaccc gggcgcgaga tgacgtttca 120
cggaaacagc tccggttata ccagttgtat tcacgcacaa gcggaaaaca cattcaggta 180
ttagggagac ggatttccgc tcgaggcgaa gacggagaca aatatgccca actactcgtt 240
gagaccgaca cctttggtag ccaagttcgt ataaaaggga aagagacgga gttctattta 300
tgcatgaata ggaagggtaa gcttgttgga aaacctgatg ggacctcgaa ggagtgcgta 360
tttatagaga aagtcctgga aaacaactat acggctctca tgagcgccaa atattccggt 420
tggtacgttg gcttcaccaa aaaagggaga ccccgaaaag gacctaagac gcgtgagaat 480
caacaagatg tgcactttat gaagcgttat ccaaagggcc aaccggagct gcagaaacca 540
ttcaaataca cgactgttac taagtga 567
<210> 12
<211> 567
<212> DNA
<213> Artificial Sequence
<400> 12
atggattgaa cttggatctt atttctagta gcagccgcta ctcgcgttca ctccgatgag 60
gaaaatgttg attttcgtat tcatgttgag aaccagactc gcgcccgaga cgatgtaagc 120
cgcaaacaac tgcggctcta tcaattgtac tcacgcactt caggcaagca tatccaagtc 180
ttgggtcggc gtatcagtgc acggggcgaa gacggtgaca agtatgccca actcttagtt 240
gaaacggata catttggatc gcaggttcga atcaagggca aagaaacaga attttaccta 300
tgcataaacc ggaaaggaaa attggtgggc aagccagacg gtacgtcaaa agaatgcgtt 360
tttatcgaga aagtgcttga aaacaactat acggcactga tatcagccaa gtattcgggt 420
tgatatgtgg gttttacaaa aaaagggcgt cctcgaaagg gtcccaagac tcgcgagaat 480
cagcaggatg tgcatttcat aaaacgttac cctaaaggcc aaccagagct ccaaaagccc 540
ttcaaataca cgacagtgac taaatga 567
Claims (10)
1.一种mRNA剂型的骨关节炎药物制剂，其特征在于，包括活性成分mRNA，所述mRNA包括编码如SEQ ID No.1～SEQ ID No.4所示的重组蛋白的mRNA序列中的一种或几种。
2.根据权利要求1所述的骨关节炎药物制剂，其特征在于，所述mRNA的5’端连接帽子结构和5’UTR；所述mRNA的3’端连接3’UTR和多聚A尾。
3.根据权利要求1或2所述的骨关节炎药物制剂，其特征在于，所述mRNA的序列如SEQID No.5～SEQ ID No.12所示；其中SEQ ID No.5或SEQ ID No.6所示的mRNA序列编码SEQID No.1所示的重组蛋白；SEQ ID No.7或SEQ ID No.8所示的mRNA序列编码SEQ ID No.2所示的重组蛋白；SEQ ID No.9或SEQ ID No.10所示的mRNA序列编码SEQ ID No.3所示的重组蛋白；SEQ ID No.11或SEQ ID No.12所示的mRNA序列编码SEQ ID No.4所示的重组蛋白。
4.根据权利要求1所述的骨关节炎药物制剂，其特征在于，所述骨关节炎药物制剂为液体制剂。
5.根据权利要求4所述的骨关节炎药物制剂，其特征在于，所述骨关节炎药物制剂为注射制剂。
6.根据权利要求5所述的骨关节炎药物制剂，其特征在于，所述活性成分mRNA在骨关节炎药物制剂中的浓度为200～2000μg/ml。
7.根据权利要求6所述的骨关节炎药物制剂，其特征在于，所述活性成分mRNA的溶剂为生理盐水。
8.权利要求1～7任意一项所述的骨关节炎药物制剂的制备方法，包括以下步骤：
1)合成转录权利要求1～7任意一项所述骨关节炎药物制剂中的活性成分mRNA的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒；
2)将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；
3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。
9.根据权利要求8所述的制备方法，其特征在于，所述RNA体外合成体系以1600μl计，包括以下组分：
10.根据权利要求8或9所述的制备方法，其特征在于，所述RNA体外合成的条件为36～38℃，8～12h。
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