CN110295151A - 一类腺依赖病毒病毒重组质粒、重组病毒及构建方法 - Google Patents

一类腺依赖病毒病毒重组质粒、重组病毒及构建方法 Download PDF

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CN110295151A
CN110295151A CN201910647327.0A CN201910647327A CN110295151A CN 110295151 A CN110295151 A CN 110295151A CN 201910647327 A CN201910647327 A CN 201910647327A CN 110295151 A CN110295151 A CN 110295151A
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dependovirus
gland
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夏玉龙
刘玉竹
郝国静
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Boxin Instrument (tianjin) Co Ltd
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Abstract

本发明涉及一类5型腺依赖病毒重组病毒及生产方法，属于病毒改造及构建技术领域。本发明提供的5型腺依赖病毒重组病毒为：针对5型腺依赖病毒与腺依赖病毒受体(Adeno‑Associated Virus receptor，AAVR)的作用靶点区域氨基酸突变的5型腺依赖病毒(Adeno‑Associated Virus 5，AAV5)。本发明提供的腺依赖病毒重组病毒具有高纯度和高效价。(附图1)

Description

一类腺依赖病毒病毒重组质粒、重组病毒及构建方法
技术领域
本发明涉及病毒生产技术领域，具体涉及一类腺依赖病毒重组病毒的重组方法。
背景技术
1型血清型腺依赖病毒(Adeno-Associated Virus serotype 5，AAV5)，属于细小病毒家族属中的一员。由于其可以广泛地感染人的不同组织、具有低免疫原性、天然条件下不致病等优点，已逐渐成为国际上用于基因治疗的优选病毒载体。已有文献报道通过全基因组功能损失的筛选，发现腺依赖病毒受体(Adeno-Associated Virus Receptor,AAVR)蛋白，是包含1型腺依赖病毒在内的多种血清型的腺依赖病毒入侵宿主细胞的必要受体。AAV5与AAVR的相互作用直接且特殊，这种相互作用需要AAV5结合到靶细胞上，AAV5的衣壳蛋白直接识别细胞表面的AAVR受体蛋白，AAV5的衣壳蛋白与AAVR受体蛋白的亲和度将影响病毒的感染效率。
在腺依赖病毒的基因治疗临床使用中，病毒的感染效率不足增加了病毒的使用剂量，从而既增加了成本，又增强了病毒过量带来的致死风险。
因此，发现从何种角度去增强病毒的感染效率变得十分紧迫。本发明立足于结构生物学手段，发现了AAV5与受体蛋白AAVR的相互作用位点，以及能够提高病毒感染效率的突变位点，从而为重组病毒的设计提供了依据。
发明内容
本发明的目的在于提供一类腺依赖病毒病毒重组质粒、重组病毒及构建方法。本发明提供的1型腺依赖病毒重组病毒具有高病毒滴度和病毒增殖能力。
优选的是腺依赖病毒病毒衣壳VP1蛋白的第531位丝氨酸由Ser突变为Ala(S531A)。
本发明提供了一种腺依赖病毒重组病毒，所述腺依赖病毒重组病毒为：针对AAV5与AAVR的作用靶点区域氨基酸突变的腺依赖病毒。
本发明还提供了一类腺依赖病毒病毒重组质粒，所述腺依赖病毒重组质粒为负载上述技术方案所述腺依赖病毒重组病毒全长核苷酸序列的质粒。
优选的是，用于负载上述技术方案所述能够表达腺依赖病毒全长氨基酸序列的质粒包括pRC-AV5。
优选的是，所述能够表达腺依赖病毒全长氨基酸序列的未经突变的质粒，核苷酸全序列如SEQ ID NO.1,表达出的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了上述技术方案所述重组质粒pRC-S531A的构建方法，包括以下步骤：
1)设计突变引物；
2)构建突变质粒pRC-S531A，经聚合酶链式反应、DMT酶切消化后，进行转化和提取鉴定以完成定点突变，得到含5型腺依赖病毒突变基因的重组质粒；
优选的是，步骤2)所述定点突变采用全式金公司快速定点突变试剂盒进行。
本发明还提供了上述技术方案所述腺依赖病毒重组病毒的包装方法，包括以下步骤：
将上述方法所构建的突变重组质粒，连同辅助质粒和基因组质粒共转染细胞，裂解细胞并纯化得到1型腺依赖病毒突变重组病毒。
优选的是，所述细胞包括但不限定于293T细胞。
优选的是，所述辅助质粒包括但不限定于pHelper。
优选的是，所述基因组质粒包括但不限定于pGFP。
本发明提供了一类针对5型腺依赖病毒与病毒受体AAVR的作用靶点区域氨基酸突变的腺依赖病毒。所述腺依赖病毒重组病毒具有高病毒滴度和感染能力。本发明还提供了一类腺依赖病毒重组质粒。
附图说明
图1为本发明实施例1参考的AAV5与AAVR相互作用位点示意图；
图2为本发明实施例1提供的AAV5原始克隆图谱；
图3为本发明实施例1提供的细胞感染病毒流式细胞术检测结果图；
图4为本发明实施例1提供的病毒纯化后的负染电镜图；
具体实施方式
下面结合具体实施例对本发明所述的一类5型腺依赖病毒突变重组质粒、5型腺依赖病毒突变重组病毒的构建方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
(1)构建病毒衣壳蛋白突变质粒。
本实验进行突变的原始质粒是能够包装AAV5病毒颗粒的pRC-AV5质粒。该质粒由合作实验室提供，是一种商业化质粒。质粒结构图见图2。
首先根据全式金公司提供的突变试剂盒说明书，对第531位的突变位点进行突变引物的设计。
然后按照说明书上提供的引物、载体、预混物的配比，配制20ulPCR体系，并且根据说明书上的步骤进行聚合酶链式反应(PCR)。随后PCR产物中加入1ul DMT酶(突变试剂盒提供)，37度消化1小时后，产物置于冰上5分钟。
取5ul消化产物，加进50ul Trans10(全式金公司)大肠杆菌感受态细胞中
(2)包装突变病毒。
以三质粒包装系统在HEK293T细胞中包装生产，CsCl密度梯度离心浓缩纯化，得到纯度大于95％的AAV5病毒颗粒，其中溶液环境为磷酸缓冲液PBS，pH 7.4。纯化后的病毒浓缩至0.5mg/ml,经Western Blot和负染电镜检测无误后，用于细胞感染实验。
(3)突变病毒感染细胞
AAV1-GFP突变重组病毒单突变类型及生产纯化：S531A，如上所述。并经过qPCR法进行病毒定量，获得各突变型AAV5-S531A病毒。
野生型细胞中检测突变病毒转导效率：在24孔板中接种293T细胞并在细胞生长至70％汇合度时，将不同突变类型病毒与野生型病毒以10000v.g./cell感染细胞。AAV5-GFP各突变体的生物学功能在感染细胞24h后进行检测。各个24孔板内细胞以lysis buffer消化后用流式细胞术进行荧光值测定。测定结果经过各孔细胞数矫正统一。并以野生型AAV5-GFP病毒为标准比较突变型的转导效率变化，其结果如图3。可见相比于野生型病毒组，突变类型病毒转导效率明显提高。
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> 博鑫仪器（天津）有限公司
<120> 一类腺依赖病毒病毒重组质粒、重组病毒及构建方法
<130> 2019.7.17
<160> 2
<170> SIPOSequenceListing 1.0
<210> 3
<211> 2172
<212> DNA
<213> 人工序列(Artificial Sequence)
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tttttgggcc ttgaagcggg cccaccgaaa ccaaaaccca atcagcagca tcaagatcaa 120
gcccgtggtc ttgtgctgcc tggttataac tatctcggac ccggaaacgg tctcgatcga 180
ggagagcctg tcaacagggc agacgaggtc gcgcgagagc acgacatctc gtacaacgag 240
cagcttgagg cgggagacaa cccctacctc aagtacaacc acgcggacgc cgagtttcag 300
gagaagctcg ccgacgacac atccttcggg ggaaacctcg gaaaggcagt ctttcaggcc 360
aagaaaaggg ttctcgaacc ttttggcctg gttgaagagg gtgctaagac ggcccctacc 420
ggaaagcgga tagacgacca ctttccaaaa agaaagaagg cccggaccga agaggactcc 480
aagccttcca cctcgtcaga cgccgaagct ggacccagcg gatcccagca gctgcaaatc 540
ccagcccaac cagcctcaag tttgggagct gatacaatgt ctgcgggagg tggcggccca 600
ttgggcgaca ataaccaagg tgccgatgga gtgggcaatg cctcgggaga ttggcattgc 660
gattccacgt ggatggggga cagagtcgtc accaagtcca cccgaacctg ggtgctgccc 720
agctacaaca accaccagta ccgagagatc aaaagcggct ccgtcgacgg aagcaacgcc 780
aacgcctact ttggatacag caccccctgg gggtactttg actttaaccg cttccacagc 840
cactggagcc cccgagactg gcaaagactc atcaacaact actggggctt cagaccccgg 900
tccctcagag tcaaaatctt caacattcaa gtcaaagagg tcacggtgca ggactccacc 960
accaccatcg ccaacaacct cacctccacc gtccaagtgt ttacggacga cgactaccag 1020
ctgccctacg tcgtcggcaa cgggaccgag ggatgcctgc cggccttccc tccgcaggtc 1080
tttacgctgc cgcagtacgg ttacgcgacg ctgaaccgcg acaacacaga aaatcccacc 1140
gagaggagca gcttcttctg cctagagtac tttcccagca agatgctgag aacgggcaac 1200
aactttgagt ttacctacaa ctttgaggag gtgcccttcc actccagctt cgctcccagt 1260
cagaacctct tcaagctggc caacccgctg gtggaccagt acttgtaccg cttcgtgagc 1320
acaaataaca ctggcggagt ccagttcaac aagaacctgg ccgggagata cgccaacacc 1380
tacaaaaact ggttcccggg gcccatgggc cgaacccagg gctggaacct gggctccggg 1440
gtcaaccgcg ccagtgtcag cgccttcgcc acgaccaata ggatggagct cgagggcgcg 1500
agttaccagg tgcccccgca gccgaacggc atgaccaaca acctccaggg cagcaacacc 1560
tatgccctgg agaacactat gatcttcaac agccagccgg cgaacccggg caccaccgcc 1620
acgtacctcg agggcaacat gctcatcacc agcgagagcg agacgcagcc ggtgaaccgc 1680
gtggcgtaca acgtcggcgg gcagatggcc accaacaacc agagctccac cactgccccc 1740
gcgaccggca cgtacaacct ccaggaaatc gtgcccggca gcgtgtggat ggagagggac 1800
gtgtacctcc aaggacccat ctgggccaag atcccagaga cgggggcgca ctttcacccc 1860
tctccggcca tgggcggatt cggactcaaa cacccaccgc ccatgatgct catcaagaac 1920
acgcctgtgc ccggaaatat caccagcttc tcggacgtgc ccgtcagcag cttcatcacc 1980
cagtacagca ccgggcaggt caccgtggag atggagtggg agctcaagaa ggaaaactcc 2040
aagaggtgga acccagagat ccagtacaca aacaactaca acgaccccca gtttgtggac 2100
tttgccccgg acagcaccgg ggaatacaga accaccagac ctatcggaac ccgatacctt 2160
acccgacccc tt 2172
<210> 3
<211> 724
<212> PRT
<213> 人工序列(Artificial Sequence)
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Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu
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Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp
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Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn
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Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe
115 120 125
Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile
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Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser
145 150 155 160
Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln
165 170 175
Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr
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Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala
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Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp
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Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro
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Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp
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Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
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Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln
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Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val
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Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr
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Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp
325 330 335
Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys
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Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn
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Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser
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Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp
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Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly
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Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu
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Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr
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Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile
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Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu
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Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg
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Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser
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Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro
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Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp
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Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met
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Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn
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Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser
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Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu
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Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln
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Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp
690 695 700
Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu
705 710 715 720
Thr Arg Pro Leu

Claims (3)

1.一类5型腺依赖病毒重组病毒(AAV5)，其特征在于，所述5型腺依赖病毒重组病毒为：针对5型腺依赖病毒与受体的作用靶点区域氨基酸的一个或多个点突变的腺依赖病毒重组病毒。
2.一类5型腺依赖病毒重组病毒，其特征在于，所述点突变为：1型腺依赖病毒全长724个氨基酸中，第531位的丝氨酸突变为丙氨酸，以及包含此位点突变为丙氨酸的一组突变位点。
3.一类5型腺依赖病毒病毒重组质粒，其特征在于，所述腺依赖病毒重组质粒为负载权利要求1所述腺依赖病毒重组病毒全长氨基酸所对应核苷酸序列的质粒。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627617B1 (en) * 1999-10-01 2003-09-30 University Of North Carolina At Chapel Hill Temperature-sensitive regulation of viral vector production
CN106661591A (zh) * 2014-05-02 2017-05-10 建新公司 用于视网膜及cns基因疗法的aav载体
CN107532177A (zh) * 2015-03-24 2018-01-02 加利福尼亚大学董事会 腺相关病毒变体及其使用方法
CN108137655A (zh) * 2015-09-28 2018-06-08 北卡罗来纳-查佩尔山大学 逃避抗体的病毒载体的方法和组合物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627617B1 (en) * 1999-10-01 2003-09-30 University Of North Carolina At Chapel Hill Temperature-sensitive regulation of viral vector production
CN106661591A (zh) * 2014-05-02 2017-05-10 建新公司 用于视网膜及cns基因疗法的aav载体
CN107532177A (zh) * 2015-03-24 2018-01-02 加利福尼亚大学董事会 腺相关病毒变体及其使用方法
CN108137655A (zh) * 2015-09-28 2018-06-08 北卡罗来纳-查佩尔山大学 逃避抗体的病毒载体的方法和组合物
US20190048041A1 (en) * 2015-09-28 2019-02-14 The University Of North Carlolina At Chapel Hill Methods and compositions for antibody-evading virus vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAN ZHANG等: "Adeno-associated virus 2 bound to its cellular receptor AAVR", 《NATURE MICROBIOLOGY》 *

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