CN110295150A - 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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CN110295150A
CN110295150A CN201910647322.8A CN201910647322A CN110295150A CN 110295150 A CN110295150 A CN 110295150A CN 201910647322 A CN201910647322 A CN 201910647322A CN 110295150 A CN110295150 A CN 110295150A
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赵文渊
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Abstract

本发明涉及一类1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1型腺依赖病毒重组病毒为：针对1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1型腺依赖病毒(Adeno‑Associated Virus 1，AAV1)。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 1，AAV1)，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。AAV1与AAVR的相互作用直接且特殊，这种相互作用需要AAV1结合到靶细胞上，AAV1的衣壳蛋白直接识别细胞表面的AAVR受体蛋白，AAV1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AAV1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386位谷氨酰胺由Gln突变为Ala(Q386A)。
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AAV1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-AV1。
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-Q386A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-Q386A，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1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1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AAV1与AAVR相互作用位点示意图；
图2为本发明实施例1提供的AAV1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1型腺依赖病毒突变重组质粒、1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AAV1病毒颗粒的pRC-AV1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386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AAV1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：Q386A，如上所述。并经过qPCR法进行病毒定量，获得各突变型AAV1-Q386A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。AAV1-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AAV1-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> 天津润德生物科技有限公司 赵文渊
<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> 1
<211> 2208
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60
gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120
gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180
aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240
cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300
caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360
gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420
ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480
aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540
tcagtccccg atccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600
actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660
gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720
accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780
tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840
gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900
atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960
gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020
gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080
ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140
ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200
tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260
ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320
caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380
ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440
ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaat 1500
tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560
ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620
atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680
acagacgaag aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg 1740
gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca tgctatggga 1800
gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860
aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920
aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980
gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040
gtggaaattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100
tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160
tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctg 2208
<210> 2
<211> 736
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
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Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
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Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly
145 150 155 160
Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190
Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala
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Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile
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Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His
260 265 270
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe
275 280 285
His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn
290 295 300
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln
305 310 315 320
Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn
325 330 335
Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350
Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365
Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
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Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro
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Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
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Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp
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Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg
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Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser
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Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro
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Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn
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Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn
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Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys
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Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly
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Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile
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Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg
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Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala
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Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln
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Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
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Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu
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Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
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Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr
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Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
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Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn
690 695 700
Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu
705 710 715 720
Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu
725 730 735

Claims (3)

1.一类1型腺依赖病毒重组病毒(AAV1)，其特征在于，所述1型腺依赖病毒重组病毒为：针对1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1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736个氨基酸中，第386位的谷氨酰胺突变为丙氨酸，以及包含此位点突变为丙氨酸的一组突变位点。
3.一类1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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