CN108728491B - 一种广谱抗凋亡杆状病毒表达载体 - Google Patents

一种广谱抗凋亡杆状病毒表达载体 Download PDF

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CN108728491B
CN108728491B CN201810640684.XA CN201810640684A CN108728491B CN 108728491 B CN108728491 B CN 108728491B CN 201810640684 A CN201810640684 A CN 201810640684A CN 108728491 B CN108728491 B CN 108728491B
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Abstract

本发明公开了一种广谱抗凋亡杆状病毒表达载体，通过载体表达靶向Spodoptera frugiperda和Trichoplusia ni昆虫细胞caspase‑1共有序列的siRNA，实现广谱抗细胞凋亡的效果。该杆状病毒载体上含有一段特定的DNA序列，这段DNA序列包含RNA聚合酶III启动子转录的靶向Sf‑caspase‑1和Tn‑caspase‑1共有序列的siRNA序列；该载体的重组病毒可以在宿主细胞中表达双链小RNA，通过RNA干扰途径沉默宿主细胞编码的caspase‑1，从而抑制宿主细胞的凋亡，使外源蛋白表达水平显著提高。本发明可用蛋白制剂和疫苗的工业化生产。

Description

一种广谱抗凋亡杆状病毒表达载体
技术领域
本发明属于重组蛋白质表达技术领域，尤其涉及一种在多种昆虫细胞中抗凋亡的杆状病毒表达载体。
背景技术
杆状病毒是特异感染节肢动物的双链DNA病毒，苜蓿银纹夜蛾多核型多角体病毒（Autographa californica nucleopolyhedrovirus, AcMNPV）是杆状病毒的模式种。自从1983年Smith GE等首次用杆状病毒在昆虫细胞中表达人β-干扰素基因以来，由于具有低成本、高产量，而且具备各种翻译后修饰系统等优点，杆状病毒表达载体系统在科研和生产中被广泛应用。
杆状病毒表达系统是一种瞬时表达系统，昆虫细胞被病毒感染3-4天后就会凋亡。基于晚期启动子的杆状病毒表达系统的表达时限是从感染后20小时开始至宿主细胞死亡。如果能延长感染细胞的存活时间，就能增加外源重组蛋白的产量。
细胞凋亡是宿主抵御病毒侵染的策略之一，其中细胞编码的caspase活性对细胞凋亡非常重要。而草地贪夜蛾（Spodoptera frugiperda, Sf）细胞中目前研究最多的caspase是执行caspase Sf-caspase-1。2007年，中国台湾一个课题组用RNA干扰载体在Sf9细胞中表达Sf-caspase-1的dsRNA，成功沉默了细胞中的Sf-caspase-1，并筛选出抑制Sf9细胞凋亡的细胞系（Lin CC, Hsu JTA, Huang KL, et al. Sf-Caspase-1-repressedstable cells: resistance to apoptosis and augmentation of recombinant proteinproduction. Biotechnology and applied biochemistry. 2007, 48(1): 11-19.）。
凋亡是生物体清理衰退细胞的积极途径。如果昆虫细胞系自身的抗凋亡途径被阻断，势必会使细胞系退化。为了解决这个问题，张潇月等将靶向Sf-caspase-1的双链小RNA编码序列直接克隆到杆状病毒基因组上，使表达的荧光素酶活性提高10倍，但蛋白质表达水平提高不多，推测抗凋亡可能更多的是影响了宿主蛋白质质量控制系统的活性（ZhangX, Xu K, Ou Y, Xu X, Chen H. Development of a baculovirus vector carrying asmall hairpin RNA for suppression of sf-caspase-1 expression and improvementof recombinant protein production. BMC Biotechnol. 2018, 18(1):24.）。
来源于粉纹夜蛾（Trichoplusia ni, Tn）的商业化细胞系High Five因其产量高于Sf细胞系，在工业上已获得了巨大成功。有研究表明，人工合成的Tn-caspase-1 dsRNA能抑制High Five细胞凋亡（Hebert CG, Valdes JJ, Bentley WE. Investigatingapoptosis: characterization and analysis of Trichoplusia ni-caspase-1 throughoverexpression and RNAi mediated silencing. Insect Biochem Mol Biol. 2009, 39(2):113-24.）。进一步把Tn-caspase-1 dsRNA克隆到High Five细胞中，获得的抗凋亡细胞系能显著提高外源基因的表达（Hebert CG, Valdes JJ, Bentley WE. In vitro and invivo RNA interference mediated suppression of Tn-caspase-1 for improvedrecombinant protein production in High Five cell culture with the baculovirusexpression vector system. Biotechnol Bioeng. 2009, 104(2):390-9.）。虽然HighFive细胞的表达量高于Sf细胞系，但它不适合用于杆状病毒的转染。工业生产上通常都是两种细胞交叉使用，但遗憾的是，目前还没有能同时在Sf细胞和Tn细胞中抗凋亡的杆状病毒表达载体。如果杆状病毒表达载体能同时在Sf和Tn细胞系中表现出抗凋亡特性，这将极大地推进抗凋亡杆状病毒表达载体在工业上的应用。
发明内容
本发明的目的在于提供一种广谱抗宿主凋亡的重组杆状病毒载体，旨在解决背景技术提及的问题。
本发明通过Sf-caspase-1和Tn-caspase-1编码序列对比，获得唯一一段大于21nt的共有序列SEQ ID NO:1。根据此序列，本发明设计了两段RNAi的靶位点：
gccgcactgagacagatggct（互补序列为SEQ ID NO:2）
gcactgagacagatggctcac（互补序列为SEQ ID NO:3）
本发明是这样实现的，将靶向上述序列的小RNA表达盒克隆到杆状病毒载体基因组中。
进一步，该表达盒序列包括人U6启动子序列、21nt引导链互补序列、9nt loop序列、21nt引导链编码序列、作为转录终止信号的TTTTT序列；
所述21nt引导链互补序列分别为SEQ ID NO:4或SEQ ID NO:5；
所述21nt引导链编码序列分别为SEQ ID NO:2或SEQ ID NO:3。
进一步，根据RNA中存在G-U配对现象，在与引导链互补序列中引进若干C到T的点突变，在不影响siRNA加工的前提下，降低DNA链形成发夹结构的倾向。
本发明另一目的在于用上述的抗凋亡重组杆状病毒载体制备蛋白制品。
本发明另一目的在于用上述的抗凋亡重组杆状病毒载体制备疫苗。
本发明把干扰两种工业上常用昆虫细胞Sf-caspase-1和Tn-caspase-1的siRNA序列和调控序列克隆到杆状病毒基因组上，构建了能延长感染细胞存活期的杆状病毒表达载体。该载体可用于构建表达外源基因的重组杆状病毒。因为病毒载体上携带抑制昆虫细胞凋亡的siRNA序列，所获得的重组杆状病毒感染昆虫细胞后，蛋白表达水平显著提高。经测试，以荧光素酶作为报告基因，本发明的抗凋亡载体荧光素酶活性以及蛋白量在Sf9细胞和High Five细胞中提高大约1倍（见附图3、4），说明本发明确实能在两种细胞系中发挥预期作用。本发明获得的杆状病毒载体可用于蛋白药物和疫苗的工业化生产。
附图说明
图1 本发明提供的重组杆状病毒载体的抗凋亡机制示意图。
图2 使用本发明的不同实施例表达GFP荧光强度对比。Ctrl为不编码siRNA的对照；563-4T, 563-5T, 566, 566-4T为4种抗凋亡载体的实施例；外源基因GFP由p10启动子转录。
图3 使用本发明提供的重组杆状病毒载体制备的表达荧光素酶的SDS-PAGE检测结果。
图4 使用本发明提供的重组杆状病毒载体制备的表达荧光素酶的酶活检测结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
下面结合附图对本发明的应用原理作详细的描述。
本发明根据Sf-caspase-1和Tn-caspase-1共有序列SEQ ID NO:1设计了两段RNAi的靶位点：
gccgcactgagacagatggct（563-583）
gcactgagacagatggctcac（566-586）
根据这两个靶位点设计四个siRNA编码序列（DNA序列分别为：SEQ ID NO:4 +loop + SEQ ID NO:2 + TTTTT 和 SEQ ID NO:5 + loop + SEQ ID NO:3 + TTTTT）：
563-4T: gctgtattgagatagatggctgcttattaaagccatctgtctcagtgcggcttttt
563-5T: gctgtattgagatagatggttgcttattaaagccatctgtctcagtgcggcttttt
566: gcactgagacagatggctcacgcttattaagtgagccatctgtctcagtgcttttt
566-4T: gcattgagatagatggtttacgcttattaagtgagccatctgtctcagtgcttttt
进一步，通过重叠PCR，将上述序列分别连接在人U6启动子下游，使转录从上述序列的第一个碱基“G”开始；
进一步，将带有U6启动子的siRNA序列敲入到杆状病毒表达载体上（如图1所示，敲入方法参见Zhang X, Xu K, Ou Y, Xu X, Chen H. Development of a baculovirusvector carrying a small hairpin RNA for suppression of sf-caspase-1expression and improvement of recombinant protein production. BMC Biotechnol.2018, 18(1):24.），获得抗凋亡杆状病毒表达载体。
下面结合实施例对本发明的应用效果进一步说明。
（1）表达GFP评估上述四种抗凋亡杆状病毒表达载体
通过同源重组，获得5株表达GFP的重组杆状病毒，其中一株没有siRNA序列的作为对照（Ctrl）。如附图2所示，在感染Sf9细胞4天后，用流式细仪检测细胞中绿色荧光的强度。除了566-4T外，其余三个重组病毒感染的细胞中，绿色荧光的强度显著高于对照。说明本发明的shRNA确实能提高外源基因的表达量。其中563-5T作为候选抗凋亡载体，进一步检测其在不同细胞系中的表达效果。
（2）荧光素酶表达水平的电泳检测
通过同源重组，获得带有荧光素酶基因的重组杆状病毒。以MOI 4分别感染Sf9细胞和High Five细胞，在感染后2-5天收集细胞，SDS-PAGE后，考马斯亮蓝染色分析。如附图3所示，无论在两种细胞中的任何一种中，任意时间的表达量，抗凋亡载体的表达量均明显高于对照。
（3）荧光素酶表达水平的酶活检测
如图4所示：用上述病毒感染Sf9细胞和High Five细胞2-5天后，直接用荧光素酶底物定量检测荧光素酶的表达水平。结果显示，带有siRNA的重组病毒的荧光素酶的表达水平在两种细胞中都有提高，在感染后第4-5天表达量可以达到对照的2倍。
以上所述仅为本发明的实施例而已，并不用以限制本发明。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 陕西杆粒生物科技有限公司
<120> 一种广谱抗凋亡杆状病毒表达载体
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 1
agccgcactg agacagatgg ctcacc 26
<210> 2
<211> 21
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 2
agccatctgt ctcagtgcgg c 21
<210> 3
<211> 21
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 3
gtgagccatc tgtctcagtg c 21
<210> 4
<211> 21
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 4
gcygyaytga gayagatggy t 21
<210> 5
<211> 21
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 5
gcaytgagay agatggytya c 21

Claims (6)

1.一种shRNA，其特征在于，所述shRNA为563-5T，其中，563-5T的引导链互补序列为gctgtattgagatagatggtt，563-5T的引导链编码序列如SEQ.ID.NO.2所示。
2.如权利要求1所述的shRNA，其特征在于，所述563-5T的核苷酸序列为：gctgtattgagatagatggttgcttattaaagccatctgtctcagtgcggcttttt。
3.一种表达载体，其特征在于，其包含如权利要求1或2所述的shRNA。
4.如权利要求3所述的表达载体，其特征在于，所述表达载体为杆状病毒。
5.一种如权利要求3或4所述的表达载体在制备重组蛋白中的应用。
6.一种如权利要求3或4所述的表达载体在制备疫苗中的应用。
CN201810640684.XA 2018-06-21 2018-06-21 一种广谱抗凋亡杆状病毒表达载体 Active CN108728491B (zh)

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