CN116254237B - 一种降低眼压的表达nrp1的重组腺相关病毒的构建方法及其应用 - Google Patents
一种降低眼压的表达nrp1的重组腺相关病毒的构建方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种降低眼压的表达NRP1的重组腺相关病毒的构建方法及其应用。本发明利用AAV为载体携带重组Neuropilin1基因,采用ICAM2血管内皮特异性启动子,构建得到AAV9‑ICAM2‑hNeuropilin1,该AAV9‑ICAM2‑hNeuropilin1作为基因治疗药物,对小鼠进行单次前房注射,可实现AAV载体介导的眼部Neuropilin1基因转导,促进Schlemm’scanal的形成,改善房水外排和降低眼压,可持续时间长且无明显毒副作用,适用于慢性青光眼的治疗。
Description
技术领域
本发明属于生物技术领域,具体涉及一种降低眼压的表达NRP1的重组腺相关病毒的构建方法及其应用。
背景技术
青光眼是由于视网膜神经节细胞受损而导致失明的一类视神经疾病。高眼压是青光眼的主要致病因素,降低眼压是治疗青光眼的关键,但现有的降眼压药物存在许多局限性,如拟副交感神经药、β-肾上腺能受体阻滞剂、肾上腺能受体激动剂和前列腺素衍生物等需要患者每日滴眼1~3次,不仅用药频繁,而且容易导致副作用。对于慢性青光眼患者,药物治疗存在局限性,通常需要结合手术治疗,如小梁切除术或巩膜分层切除等,但这些手段存在一定的手术风险,可能带来严重的后遗症。高眼压主要是由于房水循环系统发生异常而导致。房水由睫状突上皮细胞产生,通过瞳孔进入前房,再由前房角经过小梁网进入施累姆氏管(Schlemm’s canal),最终汇入血液循环。Schlemm’s canal位于角膜缘处巩膜实质中,介导人眼中70-90%的房水外排。其功能障碍会导致房水外排阻滞,诱发眼压升高。
Schlemm’s canal由内皮细胞构成。在2014年,科学家首次证实小鼠Schlemm’scanal的发育始于角膜缘巩膜表层的血管内皮细胞以出芽方式迁移进入巩膜实质中,于出生后5天转分化成为Schlemm’s canal内皮细胞,随后Schlemm’s canal内皮细胞在巩膜中围绕角膜缘进行增殖延伸,至3周龄时结构发育完整。Neuropilin1在内皮细胞中高表达,与VEGF受体共同介导VEGF信号在内皮细胞中的传导。我们及其他许多学者的研究发现,Neuropilin1可通过促进内皮细胞迁移和细胞间连接而在内皮细胞所参与的众多生理进程中发挥重要作用。我们首次发现,内皮细胞Neuropilin1对促进Schlemm’s canal的形成、房水外排和降低眼压起重要作用。通过文献检索,尚未发现有关于Neuropilin1可通过促进Schlemm’s canal的形成以改善房水外排和降低眼压的文献报道。
发明内容
本发明所要解决的技术问题是如何在降眼压的同时减少用药频率,延长药物作用的时间。为了解决以上技术问题,本发明提供了一种有效的降眼压基因治疗药物,以腺相关病毒(AAV)为载体携带重组Neuropilin1(NRP1)基因的制备方法及运用。
本发明的第一个目的是提供一种降低眼压的表达NRP1的重组腺相关病毒的构建方法,包括以下步骤:
1)获取如SEQ ID NO.1所示的人源性hNeuropilin1基因片段;
2)获取如SEQ ID NO.2所示的人源性ICAM2启动子序列;
3)构建如SEQ ID NO.3所示的ICAM2-hNeuropilin1片段;
4)构建pAAV-ICAM2-hNeuropilin1重组质粒;
5)AAV9-ICAM2-hNeuropilin1的包装、纯化和浓缩。
优选的,步骤1)中获取人源性hNeuropilin1基因片段的扩增引物为:hNeuropilin1 F:5'-TGGAGACTGCCAGAGATGGAGAGGGGGCTG-3'和hNeuropilin1 R:5'-TCATGCCTCCGAATAAGTACTCTGTGTATTC-3'。
优选的,步骤2)中获取人源性ICAM2启动子序列的扩增引物为:ICAM2 F:
5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3'和ICAM2 R:5'-CAGCCCCCTCTCCATCTCTGGCAGTCTCCA-3'。
优选的,步骤3)中构建ICAM2-hNeuropilin1片段的扩增引物为:ICAM2-hNeuropilin1F:5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3'和ICAM2-hNeuropilin1 R:5'-ATCGGGATCCTCATGCCTCCGAATAAGTAC-3'。
本发明的第二个目的是提供一种降低眼压的表达NRP1的重组腺相关病毒,该重组腺相关病毒是根据上述的构建方法制备获得的。
本发明的第三个目的是提供上述的降低眼压的表达NRP1的重组腺相关病毒在制备防治青光眼的药物中的用途。
本发明的第四个目的是提供一种防治青光眼的药物,所述的药物包含作为活性成分的降低眼压的表达NRP1的重组腺相关病毒。
优选的,所述的药物还包含药学上可接受的载体。
优选的,所述的药物的剂型为注射剂。
优选的,所述的注射剂包括注射液和冻干粉针剂。
我们首次发现,内皮细胞Neuropilin1对促进Schlemm’s canal的形成、房水外排和降低眼压起重要作用。Neuropilin1的表达有助于增加内皮细胞迁移和细胞间连接,促进Schlemm’s canal的形成,改善房水外排和降低眼压;ICAM2是细胞间粘附分子2启动子,是血管内皮特异性启动子,有助于Neuropilin1的组织特异性表达;AAV9血清型在眼部基因治疗中应用广泛,具有良好的靶向性表达,且表达时间可长达一年以上。本发明以AAV9-ICAM2-hNeuropilin1作为基因治疗药物,对小鼠进行单次前房注射,可实现AAV载体介导的眼部Neuropilin1基因转导,促进Schlemm’s canal的形成,改善房水外排和降低眼压,可持续时间长且无明显毒副作用。
本发明利用AAV为载体携带重组Neuropilin1基因,采用ICAM2血管内皮特异性启动子,实现细胞特异性治疗,解决传统降眼压药物副作用多的问题。本发明采用AAV9血清型,对眼部组织感染效率高,且具有降眼压效果持久的优点,适用于慢性青光眼的治疗。
附图说明
图1为pAAV-ICAM2-hNeuropilin1重组质粒图谱。
图2为病毒制剂小鼠眼内注射3周后的眼内压值。AAV9-Ctr:AAV9-对照病毒;AAV9-Nrp1:AAV9-ICAM2-hNeuropilin1。
图3为病毒制剂小鼠眼内注射3周后Schlemm’s canal的面积统计。AAV9-Ctr:AAV9-对照病毒;AAV9-Nrp1:AAV9-ICAM2-hNeuropilin1。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1
1、获取人源性Neuropilin1(hNeuropilin1)基因片段A
1)依据NM_003873.7序列确定蛋白表达区域,设计并合成扩增hNeuropilin1蛋白表达区域的引物。引物序列如下:
hNeuropilin1 F:5'-TGGAGACTGCCAGAGATGGAGAGGGGGCTG-3';
hNeuropilin1 R:5'-TCATGCCTCCGAATAAGTACTCTGTGTATTC-3'。
2)扩增目的基因片段:10×Pfu Buffer(with Mg2+)5μL;dNTP(2.5mM each)4μL;引物混合物(0.8μM)4μL;Pfu DNA聚合酶(5U/μL)0.25μL;模板:人基因组DNA(10ng/μL)1μL;双蒸水35.75μL。轻轻吹打混匀,短暂离心,置于PCR仪中进行反应。反应条件为:94℃3min;94℃30s,55℃30s,72℃3min(30Cycles);72℃10min;4℃∞。扩增获得hNeuropilin1片段,其大小为:2772bp,具体核苷酸序列如SEO ID NO.1所示,具体如下:
ATGGAGAGGGGGCTGCCGCTCCTCTGCGCCGTGCTCGCCCTCGTCCTCGCCCCGGCCGGCGCTTTT
CGCAACGATAAATGTGGCGATACTATAAAAATTGAAAGCCCCGGGTACCTTACATCTCCTGGTTATCC
TCATTCTTATCACCCAAGTGAAAAATGCGAATGGCTGATTCAGGCTCCGGACCCATACCAGAGAATT
ATGATCAACTTCAACCCTCACTTCGATTTGGAGGACAGAGACTGCAAGTATGACTACGTGGAAGTC
TTCGATGGAGAAAATGAAAATGGACATTTTAGGGGAAAGTTCTGTGGAAAGATAGCCCCTCCTCCT
GTTGTGTCTTCAGGGCCATTTCTTTTTATCAAATTTGTCTCTGACTACGAAACACATGGTGCAGGATT
TTCCATACGTTATGAAATTTTCAAGAGAGGTCCTGAATGTTCCCAGAACTACACAACACCTAGTGGA
GTGATAAAGTCCCCCGGATTCCCTGAAAAATATCCCAACAGCCTTGAATGCACTTATATTGTCTTTGT
GCCAAAGATGTCAGAGATTATCCTGGAATTTGAAAGCTTTGACCTGGAGCCTGACTCAAATCCTCCA
GGGGGGATGTTCTGTCGCTACGACCGGCTAGAAATCTGGGATGGATTCCCTGATGTTGGCCCTCACA
TTGGGCGTTACTGTGGACAGAAAACACCAGGTCGAATCCGATCCTCATCGGGCATTCTCTCCATGGT
TTTTTACACCGACAGCGCGATAGCAAAAGAAGGTTTCTCAGCAAACTACAGTGTCTTGCAGAGCAG
TGTCTCAGAAGATTTCAAATGTATGGAAGCTCTGGGCATGGAATCAGGAGAAATTCATTCTGACCAG
ATCACAGCTTCTTCCCAGTATAGCACCAACTGGTCTGCAGAGCGCTCCCGCCTGAACTACCCTGAG
AATGGGTGGACTCCCGGAGAGGATTCCTACCGAGAGTGGATACAGGTAGACTTGGGCCTTCTGCGC
TTTGTCACGGCTGTCGGGACACAGGGCGCCATTTCAAAAGAAACCAAGAAGAAATATTATGTCAAG
ACTTACAAGATCGACGTTAGCTCCAACGGGGAAGACTGGATCACCATAAAAGAAGGAAACAAACC
TGTTCTCTTTCAGGGAAACACCAACCCCACAGATGTTGTGGTTGCAGTATTCCCCAAACCACTGATA
ACTCGATTTGTCCGAATCAAGCCTGCAACTTGGGAAACTGGCATATCTATGAGATTTGAAGTATACG
GTTGCAAGATAACAGATTATCCTTGCTCTGGAATGTTGGGTATGGTGTCTGGACTTATTTCTGACTCC
CAGATCACATCATCCAACCAAGGGGACAGAAACTGGATGCCTGAAAACATCCGCCTGGTAACCAGT
CGCTCTGGCTGGGCACTTCCACCCGCACCTCATTCCTACATCAATGAGTGGCTCCAAATAGACCTGG
GGGAGGAGAAGATCGTGAGGGGCATCATCATTCAGGGTGGGAAGCACCGAGAGAACAAGGTGTTC
ATGAGGAAGTTCAAGATCGGGTACAGCAACAACGGCTCGGACTGGAAGATGATCATGGATGACAG
CAAACGCAAGGCGAAGTCTTTTGAGGGCAACAACAACTATGATACACCTGAGCTGCGGACTTTTCC
AGCTCTCTCCACGCGATTCATCAGGATCTACCCCGAGAGAGCCACTCATGGCGGACTGGGGCTCAG
AATGGAGCTGCTGGGCTGTGAAGTGGAAGCCCCTACAGCTGGACCGACCACTCCCAACGGGAACT
TGGTGGATGAATGTGATGACGACCAGGCCAACTGCCACAGTGGAACAGGTGATGACTTCCAGCTCA
CAGGTGGCACCACTGTGCTGGCCACAGAAAAGCCCACGGTCATAGACAGCACCATACAATCAGAG
TTTCCAACATATGGTTTTAACTGTGAATTTGGCTGGGGCTCTCACAAGACCTTCTGCCACTGGGAAC
ATGACAATCACGTGCAGCTCAAGTGGAGTGTGTTGACCAGCAAGACGGGACCCATTCAGGATCACA
CAGGAGATGGCAACTTCATCTATTCCCAAGCTGACGAAAATCAGAAGGGCAAAGTGGCTCGCCTGG
TGAGCCCTGTGGTTTATTCCCAGAACTCTGCCCACTGCATGACCTTCTGGTATCACATGTCTGGGTC
CCACGTCGGCACACTCAGGGTCAAACTGCGCTACCAGAAGCCAGAGGAGTACGATCAGCTGGTCT
GGATGGCCATTGGACACCAAGGTGACCACTGGAAGGAAGGGCGTGTCTTGCTCCACAAGTCTCTG
AAACTTTATCAGGTGATTTTCGAGGGCGAAATCGGAAAAGGAAACCTTGGTGGGATTGCTGTGGAT
GACATTAGTATTAATAACCACATTTCACAAGAAGATTGTGCAAAACCAGCAGACCTGGATAAAAAG
AACCCAGAAATTAAAATTGATGAAACAGGGAGCACGCCAGGATACGAAGGTGAAGGAGAAGGTGA
CAAGAACATCTCCAGGAAGCCAGGCAATGTGTTGAAGACCTTAGACCCCATCCTCATCACCATCATA
GCCATGAGTGCCCTGGGGGTCCTCCTGGGGGCTGTCTGTGGGGTCGTGCTGTACTGTGCCTGTTGG
CATAATGGGATGTCAGAAAGAAACTTGTCTGCCCTGGAGAACTATAACTTTGAACTTGTGGATGGTGTGAAGTTGAAAAAAGACAAACTGAATACACAGAGTACTTATTCGGAGGCATGA。
2、获取人源性ICAM2启动子序列B
1)依据NM_001099786.2启动子序列,设计并合成扩增ICAM2启动子区域的引物。引物序列如下:
ICAM2 F:5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3';
ICAM2 R:5'-CAGCCCCCTCTCCATCTCTGGCAGTCTCCA-3'。
2)扩增目的基因片段:10×Pfu Buffer(with Mg2+)5μL;dNTP(2.5mM each)4μL;引物混合物(0.8μM)4μL;Pfu DNA聚合酶(5U/μL)0.25μL;模板:人基因组DNA(10ng/μL)1μL;双蒸水35.75μL。轻轻吹打混匀,短暂离心,置于PCR仪中进行反应。反应条件为:94℃3min;94℃30s,55℃30s,72℃1min(30Cycles);72℃10min;4℃∞。扩增获得ICAM2启动子片段,其大小为:354bp,具体核苷酸序列如SEQ ID NO.2所示,具体如下:
GAGAAGACTGAATAAGCCGTGAACTGGGCAGCTGTGACACGCGCACCTTGTCACCTTGTCACCAAT
TTCCTGCCCGGGATCCCAGAGCTACCCTTCTTGGAGCCCACAGGGGTAAGGTTGAGCTTCTCACTTT
CCCTCCAATGAAGCGTGGCAGCATGGGAAGGATTTTGGCAGTGTCGAGGTCTGAGTTTTAGTCCGT
TCCCCTCTCTGGGTCTCAGTTTCTCGCTGGAATAAAGTGAAACACCTCAGGCTCCAACCCTCCCAA
CCCAAGGCTCCCTCCTGGAGGGAGAGAGAGGATGGAGGACATCAGGCAGCCCTTGGCTGGTCCCTGCGAGCCCGTGGAGACTGCCAGAG。
3、构建ICAM2-hNeuropilin1片段C
两种产物混合,经变性及退火处理,A片段和B片段部分碱基互补配对,成杂交链。设计并合成扩增,同时引入保护碱基和酶切位点,扩增引物为:ICAM2-hNeuropilin1 F:5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3';ICAM2-hNeuropilin1 R:5'-ATCGGGATCCTCATGCCTCCGAATAAGTAC-3';PCR反应体系:10×Pfu Buffer(with Mg2+)5μL;dNTP(2.5mM each)4μL;引物混合物(0.8μM)4μL;Pfu DNA聚合酶(5U/μL)0.25μL;模板:人基因组DNA(10ng/μL)1μL;双蒸水35.75μL。轻轻吹打混匀,短暂离心,置于PCR仪中进行反应。设置温度梯度PCR,反应程序为:94℃3min;94℃30s,50-65℃30s,72℃3.5min(30Cycles);72℃10min;4℃∞。扩增获得ICAM2-hNeuropilin1片段大小为:3126bp,具体核苷酸序列如SEQID NO.3所示,具体如下:
GAGAAGACTGAATAAGCCGTGAACTGGGCAGCTGTGACACGCGCACCTTGTCACCTTGTCACCAAT
TTCCTGCCCGGGATCCCAGAGCTACCCTTCTTGGAGCCCACAGGGGTAAGGTTGAGCTTCTCACTTT
CCCTCCAATGAAGCGTGGCAGCATGGGAAGGATTTTGGCAGTGTCGAGGTCTGAGTTTTAGTCCGT
TCCCCTCTCTGGGTCTCAGTTTCTCGCTGGAATAAAGTGAAACACCTCAGGCTCCAACCCTCCCAA
CCCAAGGCTCCCTCCTGGAGGGAGAGAGAGGATGGAGGACATCAGGCAGCCCTTGGCTGGTCCCT
GCGAGCCCGTGGAGACTGCCAGAGATGGAGAGGGGGCTGCCGCTCCTCTGCGCCGTGCTCGCCCT
CGTCCTCGCCCCGGCCGGCGCTTTTCGCAACGATAAATGTGGCGATACTATAAAAATTGAAAGCCCC
GGGTACCTTACATCTCCTGGTTATCCTCATTCTTATCACCCAAGTGAAAAATGCGAATGGCTGATTCA
GGCTCCGGACCCATACCAGAGAATTATGATCAACTTCAACCCTCACTTCGATTTGGAGGACAGAGA
CTGCAAGTATGACTACGTGGAAGTCTTCGATGGAGAAAATGAAAATGGACATTTTAGGGGAAAGTT
CTGTGGAAAGATAGCCCCTCCTCCTGTTGTGTCTTCAGGGCCATTTCTTTTTATCAAATTTGTCTCTG
ACTACGAAACACATGGTGCAGGATTTTCCATACGTTATGAAATTTTCAAGAGAGGTCCTGAATGTTC
CCAGAACTACACAACACCTAGTGGAGTGATAAAGTCCCCCGGATTCCCTGAAAAATATCCCAACAG
CCTTGAATGCACTTATATTGTCTTTGTGCCAAAGATGTCAGAGATTATCCTGGAATTTGAAAGCTTTG
ACCTGGAGCCTGACTCAAATCCTCCAGGGGGGATGTTCTGTCGCTACGACCGGCTAGAAATCTGGG
ATGGATTCCCTGATGTTGGCCCTCACATTGGGCGTTACTGTGGACAGAAAACACCAGGTCGAATCC
GATCCTCATCGGGCATTCTCTCCATGGTTTTTTACACCGACAGCGCGATAGCAAAAGAAGGTTTCTC
AGCAAACTACAGTGTCTTGCAGAGCAGTGTCTCAGAAGATTTCAAATGTATGGAAGCTCTGGGCAT
GGAATCAGGAGAAATTCATTCTGACCAGATCACAGCTTCTTCCCAGTATAGCACCAACTGGTCTGCA
GAGCGCTCCCGCCTGAACTACCCTGAGAATGGGTGGACTCCCGGAGAGGATTCCTACCGAGAGTG
GATACAGGTAGACTTGGGCCTTCTGCGCTTTGTCACGGCTGTCGGGACACAGGGCGCCATTTCAAA
AGAAACCAAGAAGAAATATTATGTCAAGACTTACAAGATCGACGTTAGCTCCAACGGGGAAGACTG
GATCACCATAAAAGAAGGAAACAAACCTGTTCTCTTTCAGGGAAACACCAACCCCACAGATGTTGT
GGTTGCAGTATTCCCCAAACCACTGATAACTCGATTTGTCCGAATCAAGCCTGCAACTTGGGAAACT
GGCATATCTATGAGATTTGAAGTATACGGTTGCAAGATAACAGATTATCCTTGCTCTGGAATGTTGGG
TATGGTGTCTGGACTTATTTCTGACTCCCAGATCACATCATCCAACCAAGGGGACAGAAACTGGATG
CCTGAAAACATCCGCCTGGTAACCAGTCGCTCTGGCTGGGCACTTCCACCCGCACCTCATTCCTACA
TCAATGAGTGGCTCCAAATAGACCTGGGGGAGGAGAAGATCGTGAGGGGCATCATCATTCAGGGTG
GGAAGCACCGAGAGAACAAGGTGTTCATGAGGAAGTTCAAGATCGGGTACAGCAACAACGGCTCG
GACTGGAAGATGATCATGGATGACAGCAAACGCAAGGCGAAGTCTTTTGAGGGCAACAACAACTA
TGATACACCTGAGCTGCGGACTTTTCCAGCTCTCTCCACGCGATTCATCAGGATCTACCCCGAGAGA
GCCACTCATGGCGGACTGGGGCTCAGAATGGAGCTGCTGGGCTGTGAAGTGGAAGCCCCTACAGC
TGGACCGACCACTCCCAACGGGAACTTGGTGGATGAATGTGATGACGACCAGGCCAACTGCCACA
GTGGAACAGGTGATGACTTCCAGCTCACAGGTGGCACCACTGTGCTGGCCACAGAAAAGCCCACG
GTCATAGACAGCACCATACAATCAGAGTTTCCAACATATGGTTTTAACTGTGAATTTGGCTGGGGCT
CTCACAAGACCTTCTGCCACTGGGAACATGACAATCACGTGCAGCTCAAGTGGAGTGTGTTGACCA
GCAAGACGGGACCCATTCAGGATCACACAGGAGATGGCAACTTCATCTATTCCCAAGCTGACGAAA
ATCAGAAGGGCAAAGTGGCTCGCCTGGTGAGCCCTGTGGTTTATTCCCAGAACTCTGCCCACTGCA
TGACCTTCTGGTATCACATGTCTGGGTCCCACGTCGGCACACTCAGGGTCAAACTGCGCTACCAGA
AGCCAGAGGAGTACGATCAGCTGGTCTGGATGGCCATTGGACACCAAGGTGACCACTGGAAGGAA
GGGCGTGTCTTGCTCCACAAGTCTCTGAAACTTTATCAGGTGATTTTCGAGGGCGAAATCGGAAAA
GGAAACCTTGGTGGGATTGCTGTGGATGACATTAGTATTAATAACCACATTTCACAAGAAGATTGTG
CAAAACCAGCAGACCTGGATAAAAAGAACCCAGAAATTAAAATTGATGAAACAGGGAGCACGCCA
GGATACGAAGGTGAAGGAGAAGGTGACAAGAACATCTCCAGGAAGCCAGGCAATGTGTTGAAGAC
CTTAGACCCCATCCTCATCACCATCATAGCCATGAGTGCCCTGGGGGTCCTCCTGGGGGCTGTCTGT
GGGGTCGTGCTGTACTGTGCCTGTTGGCATAATGGGATGTCAGAAAGAAACTTGTCTGCCCTGGAG
AACTATAACTTTGAACTTGTGGATGGTGTGAAGTTGAAAAAAGACAAACTGAATACACAGAGTACTTATTCGGAGGCATGA。
4、构建pAAV-ICAM2-hNeuropilin1重组质粒
纯化片段C。配制50μL片段C酶切体系:包括双蒸水23μL,10×酶切缓冲液5μL,纯化目的片段C(200ng/μL)20μL,BamHI(20U/μL)1μL,EcoRI(20U/μL)1μL。按顺序依次加入体系中,轻轻吹打混匀,短暂离心,置于37℃反应2h。配制50μL AAV载体转移质粒pAAV酶切体系:包括双蒸水41μL,10×酶切缓冲液5μL,AAV载体质粒(1μg/μL)2μL,BamHI(20U/μL)1μL,EcoRI(20U/μL)1μL。按顺序依次加入体系中,轻轻吹打混匀,短暂离心,置于37℃反应2h。对片段和载体酶切产物进行琼脂糖凝胶电泳,回收目的条带,回收片段大小分别为3126bp和3199bp。连接片段C和载体酶切回收产物:配制10μL连接体系,包括双蒸水(补充至10μL),10×DNA连接酶缓冲液1μL,片段C酶切回收产物与载体酶切回收产物摩尔比2:1,置于16℃连接过夜。转化质粒:将1管感受态细胞放在冰上融化;取100μL感受态细胞加入约20ng质粒DNA,用移液器轻轻混匀,在冰上放置30min;将离心管放置于42℃水浴,热击45秒,过程中稳定离心管不晃动;迅速将离心管转移至冰上放置2min;加入300μL LB培养基或SOC培养基,在37℃温和摇动培养1小时;取适当体积均匀涂布于含有抗生素的LB平板;倒置培养皿,于37℃培养过夜。挑选单菌落,扩增并提取质粒DNA,使用BamHI和EcoRI双酶切,跑琼脂糖凝胶电泳确定插入片段大小为3126bp,构建得到pAAV-ICAM2-hNeuropilin1重组质粒(图1)。设计测序引物鉴定重组质粒插入片段序列:1-GAGAAGACTGAA;2-TGTCTTCAGGGC;3-TCGGGACACA;4-CCAAATAGACCTG;5-GGAGATGGCAACTT;6-CCTGGAGAACTAT。
5、AAV9-ICAM2-hNeuropilin1的包装、纯化和浓缩
质粒准备:将构建好的AAV载体转移质粒(pAAV-ICAM2-hNeuropilin1)、重组衣壳包装质粒(pAAV9)和腺病毒Helper质粒(pAdDeltaF6)扩增并提纯,OD260/280需在1.8左右。pAAV9含Rep和Cap,两侧是两个145碱基倒置末端重复序列(ITR);pAdDeltaF6含E4,E2a andVA,介导AAV的复制。AAV病毒的包装:复苏AAV-293细胞,大量培养于15cm培养皿中,待细胞汇合度达到70~80%时,采用脂质体转染法,将上述质粒一同转染AAV-293细胞。质粒使用量:pAAV-ICAM2-hNeuropilin1 6μg,pAAV9 10μg,辅助质粒pAdDeltaF6 12μg进行转染。转染结束后,将皿中的培养基替换为新鲜的细胞培养基(含10%胎牛血清的高糖DMEM培养基),常规继续培养66~72小时。AAV病毒的浓缩:将上清培养基和细胞一起收集于15mL的离心管中,500×g 3min离心分离细胞和上清。将上清另外存放,细胞用1mL PBS重悬。细胞悬浮液在液氮和37℃水浴中反复融3~4次,每次冻融过程约10min。高速离心去除细胞碎片,将离心上清转移到一个新离心管中并使用0.22μm滤膜过滤。AAV病毒的纯化和浓缩:在超速离心管中依次添加5%(1.031g/cm3),15%(1.085g/cm3),25%(1.085g/cm3),40%(1.215g/cm3),54%(1.291g/cm3)的碘克沙醇溶液。将病毒上清液缓慢加入到密度梯度离心介质中,使用超速离心机36,0000×g,16℃,离心2h。回收40%层溶液。使用Millipore超滤管进行浓缩,3500rpm离心30min,病毒颗粒使用500μL PBS溶液反复吹打后,于-80℃冰箱分装保存。病毒滴度的测定:采用定量PCR方法检测AAV载体的基因组拷贝数来测定AAV的病毒颗粒数,测定滴度>1013vg/mL。
6、AAV9-ICAM2-hNeuropilin1在降低眼压研究中的运用
重组AAV(AAV9-ICAM2-hNeuropilin1)眼内注射:取6月龄小鼠5只(雄鼠3只,雌鼠2只),用1.5%戊巴比妥钠,按50mg/kg予腹腔注射麻醉,确保整个实验过程在麻醉状态下进行。小鼠充分麻醉后,将小鼠置于体视显微操作平台上,用丙美卡因滴眼液局麻小鼠眼表,用干棉棒拭去结膜囊多余的液体。充分暴露小鼠眼球后。使用31G一次性胰岛素注射器于角膜周边部做前房穿刺,穿刺口尽量平行虹膜、确保穿刺口自闭;放出约2~3μL房水后,将显微注射器由穿刺口进入前房,一侧眼球注射AAV9-ICAM2-hNeuropilin1,另一侧眼球注射AAV9-对照病毒(1.5μL,约1.5*1010vg),末端注入少许无菌空气,尽量确保注射期间无明显房水流出;维持显微注射针头停留在前房6~8min,调整眼位使无菌小气泡位于穿刺口内口,缓慢、平稳地取出显微注射器,眼表涂抹抗生素眼凝胶,全程操作在手术室等级无菌层流条件下完成。术后每日观察,确定无明显眼内感染表现。
眼压检测:AAV9-ICAM2-hNeuropilin1或AAV9-对照病毒感染小鼠3周后检测眼压值,结果见图2。由图2可知,相较于眼内注射AAV9-对照病毒,眼内注射AAV9-ICAM2-hNeuropilin1可显著降低小鼠眼压值。
Schlemm’s canal面积测量:体视显微镜下分离角膜组织,PBS清洗组织,将样本浸没于1% Triton X-100通透50min,PBS洗2次。封闭液室温封闭样本30min,一抗4℃摇床孵育过夜(12~16h),抗体选择不同物种来源。第二天样品复温后,0.1%PBST洗5次,每次15min,二抗4℃摇床孵育8~10h,抗体选择避开一抗物种来源。第三天样品复温后,0.1%PBST洗5次,每次15min,1%多聚甲醛后固定2min,PBS洗3次,每次2min。将角膜展平于载玻片,抗淬灭剂封片。基于CD31免疫荧光染色图片,使用Image J软件测量Schlemm’s canal面积,结果见图3。由图3可知,相较于眼内注射AAV9-对照病毒,眼内注射AAV9-ICAM2-hNeuropilin1可显著增加Schlemm’s canal面积。
以上结果表明,AAV9-ICAM2-hNeuropilin1通过促进Schlemm’s canal的形成,改善房水外排和降低眼压。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,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。
Claims (7)
1.一种改善房水外排和降低眼压的表达hNeuropilin1的重组腺相关病毒的构建方法,其特征在于,包括以下步骤:
1)获取如SEQ ID NO.1所示的人源性hNeuropilin1基因片段;
2)获取如SEQ ID NO.2所示的人源性ICAM2启动子序列;
3)构建如SEQ ID NO.3所示的ICAM2-hNeuropilin1片段;
4)构建pAAV-ICAM2-hNeuropilin1重组质粒;
5)AAV9-ICAM2-hNeuropilin1的包装、纯化和浓缩;
步骤1)中获取人源性hNeuropilin1基因片段的扩增引物为:hNeuropilin1 F: 5'-TGGAGACTGCCAGAGATGGAGAGGGGGCTG-3'和hNeuropilin1R: 5'-TCATGCCTCCGAATAAGTACTCTGTGTATTC-3';
步骤2)中获取人源性ICAM2启动子序列的扩增引物为:ICAM2 F: 5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3'和ICAM2R: 5'-CAGCCCCCTCTCCATCTCTGGCAGTCTCCA-3';
步骤3)中构建ICAM2-hNeuropilin1片段的扩增引物为:ICAM2-hNeuropilin1 F: 5'-CCGCGAATTCGAGAAGACTGAATAAGCCGTG-3'和ICAM2-hNeuropilin1R:
5'- ATCGGGATCCTCATGCCTCCGAATAAGTAC-3'。
2.根据权利要求1所述的构建方法制备获得的改善房水外排和降低眼压的表达hNeuropilin1的重组腺相关病毒。
3.权利要求2所述的改善房水外排和降低眼压的表达hNeuropilin1的重组腺相关病毒在制备治疗青光眼的药物中的用途。
4.一种治疗青光眼的药物,其特征在于,所述的药物包含作为活性成分的权利要求2所述的改善房水外排和降低眼压的表达hNeuropilin1的重组腺相关病毒。
5.根据权利要求4所述的药物,其特征在于,所述的药物还包含药学上可接受的载体。
6.根据权利要求4所述的药物,其特征在于,所述的药物的剂型为注射剂。
7.根据权利要求6所述的药物,其特征在于,所述的注射剂包括注射液和冻干粉针剂。
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