CN106834312B - 一种超氧化物歧化酶Cu,Zn-SOD基因及其应用 - Google Patents
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Abstract
本发明涉及基因工程技术领域,特别涉及一种超氧化物歧化酶Cu,Zn‑SOD基因，核苷酸序列如序列表中序列1所示，GLN49(CAG)突变为TRP(TGG)；GLY90(GCT)突变为ILE(ATA)。超氧化物歧化酶Cu,Zn‑SOD基因在氧自由基清除中的应用。突变前后蛋白稳定性的比较，SOD突变后，分别在37℃，55℃水浴保温一定时间后测酶活，稳定性都提高5‑10倍，耐酸碱性能明显提高，对蛋白酶和胰蛋白酶耐受性也明显提高。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及一种超氧化物歧化酶Cu,Zn-SOD基因,还涉及此基因的应用。
背景技术
超氧化物歧化酶(SOD)是1938年首次从牛红血球中分离得到超氧化物歧化酶开始算起，人们对SOD的研究已经有70多年的历史，1969年由Fridovich和McCord发现其发生作用的机制是发生歧化反应，故正式命名为超超氧化物歧化酶(Superoxide Dismutase，SOD)。SOD是一种自由基清除剂，广泛存在于自然界一切生物体内，通过催化超氧阴离子自由基
发生歧化反应，减轻或者消除超氧阴离子对机体的氧化或过氧化损害。SOD是体内一种重要的氧自由基清除剂，还是一种应用广泛的药用酶。目前，SOD主要集中在治疗关节炎、类风湿性关节炎、缺血再灌注综合症，具有显著疗效，此外在抗辐射、抗肿瘤和预防衰老起到了重要作用。因此，对人体不断补充SOD具有抗衰老的特殊效果，已被广泛应用于生物医药、保健品和化妆品等行业。
根据所含金属离子的不同可以主要将SOD分为三类：①Cu,Zn-SOD:呈蓝绿色，由两个亚基组成，每个亚基含有Cu、Zn两种金属离子，每个亚基的分子量为16kD,主要存在于真核细胞的细胞质中,同时也是目前研究最多的一种SOD；②Mn-SOD:呈粉红色，存在与哺乳动物的线粒体内以及原核生物中，哺乳动物的Mn-SOD主要由四个亚基组成；原核生物中则是由两个亚基组成，每个亚基含有一个Mn离子，每个亚基的分子量为24Kd；③Fe-SOD：呈黄色，由两个亚基组成，每个亚基含有一个Fe，主要存在于原核生物和少数植物细胞内。此外，还包括含有金属Ni的Ni-SOD等。
但由于天然SOD作为一种酶用于临床存在体内停滞时间短、半衰期只有6-8min,易于被蛋白酶水解，并且具有免疫原性等不利因素。因此，对SOD进行分子改造以改变分子的理化性质，增加对高温和极端pH等的稳定性，克服上述不利因素就显得十分必要。目前对SOD进行分子改造和化学修饰主要有两条途径。第一种采用聚乙二醇、聚蔗糖、右旋糖酐、肝素、壳聚糖等对SOD的氨基酸残基进行化学修饰，主要是对其非活性部位进行修饰以提高其稳定性并仍能保留最大活性。第二种采用对SOD进行酶切改造以达到降低分子量和抗原性的目的。但是鉴于化学修饰不能实现定点修饰，而酶切改造的SOD在体内半衰期仍然很短。
CN101525600A公开了一种提高重组人Cu,Zn-SOD活性蛋白产量的方法，突变位点为人Cu,Zn-SOD蛋白的第6位和111位半胱氨酸残基定点突变为亲水性氨基酸残基，使得裂菌上清中的重组蛋白占重组蛋白总体表达的比例由40％提高到80％，并且从上清中纯化的突变体rhCu,Zn-SOD的比活性与上清中纯化的未突变rhCu,Zn-SOD的相当。此方法只是提高了Cu,Zn-SOD产量，对活性及稳定性并没有影响。
能够提高rhCu,Zn-SOD的稳定性的突变，至今仍无相关记载。
发明内容
为了解决以上现有技术中Cu,Zn-SOD的稳定性差的问题，本发明提供了一种体内半衰期长、稳定性高的超氧化物歧化酶Cu,Zn-SOD基因。
本发明还提供了上述基因的应用。
本研究拟通过对SOD基因进行突变来改造SOD分子，提高SOD稳定性。传统意义上的氨基酸定点突变，是通过基因突变的方法对某个氨基酸位点的碱基进行突变，借此改变该位点的氨基酸类型。尽管方法比较成熟，但对于实际操作而言，通常需要将某个位点突变为其余19种氨基酸，即单位点全突变扫描或多位点全突变扫描，突变结果往往难以估算，给实验带来困难。随着生物信息学技术的发展，计算机软件在对蛋白结构分析方面发挥着日益重要的作用。最近受到业内广泛重视的Discovery Studio4.0(DS)软件是面向生命科学领域新一代分子建模和模拟软件，应用于蛋白质的结构功能研究，以及药物的发现，为生命科学领域提供易用的蛋白模拟、优化工具。本研究采用Discovery Studio4.0(DS)软件对昆明鼠Cu,Zn-SOD分子通过突变能及分子动力学分析确定突变位点，进而进行试验验证。
本发明是通过以下步骤得到的：
一种超氧化物歧化酶Cu,Zn-SOD基因，核苷酸序列如序列表中序列1所示，GLN49(CAG)突变为TRP(TGG)；GLY90(GCT)突变为ILE(ATA)。
所述的超氧化物歧化酶Cu,Zn-SOD基因，氨基酸序列如序列表中序列2所示。
所述的超氧化物歧化酶Cu,Zn-SOD基因的载体的构建方法，包括以下步骤：
(1)涉及引物，核苷酸序列如下：
扩增引物为：上游引物P1:5’-ATAGGATCCATGGCGATGAAAGCGGTGT-3’，
下游引物P2:5’-GCAGTCGACTTACTGCGCAATCCCAATCACT-3’，
单点突变引物：上游引物P3:5’-GGGTTCCACGTCCATTGGTATGGGGACAATAC-3’，
下游引物P4:5’-GTATTGTCCCCATACCAATGGACGTGGAACCC-3’，
双点突变引物：上游引物P5:5’-GTGACTGCTATAAAGGACGGTG-3’，
下游引物P6:5’-CACCGTCCTTTATAGCAGTCAC-3’，
(2)由小鼠血液中提取RNA，反转为cDNA并经上下游引物P1、P2进行PCR得到目的基因序列，然后以目的基因为模板，以单点突变引物P3、P4及双点突变引物P5、P6为引物，通过巢式PCR扩增以得到所需突变基因，切胶回收所需目的片段，酶切转入表达载体。
所述的超氧化物歧化酶Cu,Zn-SOD基因在氧自由基清除中的应用。
本发明的有益效果：
突变前后蛋白稳定性的比较，SOD突变后，分别在37℃，55℃水浴保温一定时间后测酶活，稳定性都提高5-10倍，耐酸碱性能明显提高，对蛋白酶和胰蛋白酶耐受性也明显提高。
对双突变位点Top10进行10ns的分子动力学模拟，得到最优的突变位点组合为：GLN49突变为TRP；GLY90突变为ILE。
附图说明
图1为野生型和突变蛋白的RMSD对比图；
图2为野生型和突变蛋白的RMSF对比图；
图3为野生型和突变蛋白的势能对比图；
图4为野生型和突变蛋白的H-Bond对比图。
具体实施方式
下面结合具体实施例对本发明进行进一步说明：
实施例1
由NCBI中获得来自于猩猩、人、Mus Musculus等的Cu,Zn-SOD氨基酸序列；于ClustalX2软件中将来自牛、猩猩、人等的Cu,Zn-SOD氨基酸序列进行比对，并由DNAman对比对图进行处理，确定非保守氨基酸序列为27个。
在PDB数据库中由登录号3GTT得到Mus Musculus的Cu,Zn-SOD的三维晶体结构，将三维晶体结构导入Discovery Studio4.0软件中；本实验使用Discovery Studio 4.0的以下几个模块对蛋白进行突变：Build Mutants；②Calculate Mutation Energy(Stability)；③Predict Stabilizing Mutations。将非保守序列的27个氨基酸分别突变为其他19种氨基酸，根据突变前后突变能△△Gmut(△△Gmut＝△Gfolding(mut)-△Gfolding(wt)；△Gfolding＝△Gfolded-△Gunfolded)变化得到双点突变最优top10即突变状态为Stabilizing的top10如表1；
表1双点突变的突变能预测(top10)
具体步骤为：
①模型准备：启动Discovery Studio4.0，载入步骤2)模型，使用Prepare Protein模块对各模型进行初始化准备后，应用CHARMm力场；
②选择待突变氨基酸
使用组合键Ctrl+H，切换出Hierarchy窗口，Ctrl+鼠标左键选择非保守区氨基酸残基；
③启动突变预测模块Protocol
Macromolecules/Design Protein Tools/Predict Stabilizing Mutations
④参数设置
选择Cu,Zn-SOD作为Input Typed Molecule，Residuces to Mutate选择非保守氨基酸残基；设定要突变成的氨基酸类型为20中天然氨基酸；设定Maximum Mutations类型为Twofold mutations Parallel Processing为True，Processors为4，其他参数默认。
⑤点击Run，开始进行Protocol
对双点突变的top10序列进行10ns的分子动力学分析，具体步骤为：
①蛋白质体系准备：将步骤2的蛋白模型导入Molecule window，Prepare蛋白模型，然后Clean Protein删除多余原子或分子，应用隐水式模型，添加CHARMm力场；
②优化体系能量：约束蛋白分子，对水分子进行5000步的smart minimize；去除蛋白质约束，对整个体系进行5000步的smart minimize，并以此作为分子动力学模拟的初始结构；
③升温模拟：模拟迭代步长为1fs，采用SHAKE模型约束体系，进行10ps的升温模拟，设定最终温度为310K；
④平衡模拟：模拟迭代步长为1fs，采用SHAKE模型约束体系，进行10ps的平衡模拟；
⑤取样模拟：模拟迭代步长为1fs，采用SHAKE模型约束体系，进行10ns的取样模拟；
以上模拟过程均在等温等压条件下进行，其他参数为默认值。每一步均进行评估，符合标准再往下操作。
对野生型(WT-SOD)和突变(MSOD)蛋白的RMSD、RMSF、势能、H-Bond等进行比较见图1-4：
图1RMSD可知MSOD的蛋白质较WT-SOD能够更快的达到稳定，说明MSOD比WT-SOD蛋白质的构象更加稳定；
图2RMSF展示的是蛋白质中每个氨基酸的柔性变化由图可知MSOD蛋白质氨基酸的柔性明显小于WT-SOD突变位点的氨基酸，所以MSOD的蛋白质更加稳定；
图3Potential Eneygy中突变前后蛋白都能在1ns后达到平衡状态，表明分子动力学模拟体系进入平衡状态，可以对轨迹文件进行取样分析，而对于势能数值大小的比较无实际意义；
图4 H-Bond显示MSOD蛋白内部的键能明显高于WT-SOD的键能，表明蛋白质分子能够结合的更加紧密，也更加稳定。
由以上动力学分析最终确定最优双点突变为GLN49突变为TRP，GLY90突变为ILE。
实施例2
(1)设计引物
扩增引物为：上游引物P1:5’-ATAGGATCCATGGCGATGAAAGCGGTGT-3’，
下游引物P2:5’-GCAGTCGACTTACTGCGCAATCCCAATCACT-3’，
单点突变引物：上游引物P3:5’-GGGTTCCACGTCCATTGGTATGGGGACAATAC-3’，
下游引物P4:5’-GTATTGTCCCCATACCAATGGACGTGGAACCC-3’，
双点突变引物：上游引物P5:5’-GTGACTGCTATAAAGGACGGTG-3’，
下游引物P6:5’-CACCGTCCTTTATAGCAGTCAC-3’。
(2)由昆明鼠血液中提取RNA，反转为cDNA并经普通PCR得到目的基因序列；然后以目的基因为模板，以步骤5中的单点及双点突变引物为引物，通过巢式PCR扩增以得到所需突变基因；然后通过切胶回收所需目的片段；
选择表达载体为pGEX6p-1，在37℃条件下以BamHI和SalI对载体和目的基因双酶切8h，再次胶回收酶切目的片段，并以T4 DNA连接酶将载体与目的基因4℃连接过夜，得到连接体系；
通过氯化钙法制备DH5α感受态细胞，并将连接体系加入到感受态细胞中，冰上静置
30min，然后将离心管置于42℃水浴锅中热激45-90s,取出后立即冰上放置2-3min,加入预热至37℃培养基中，摇床震荡培养45-60min,后将培养的适量菌液涂布至含有氨苄的平板中，于37℃培养箱中培养12h；于步骤8的平板上挑取单菌落接种至含有氨苄的5ml菌液的试管中摇12h,取菌液进行菌液PCR，筛选出阳性克隆。
由碱裂解的方法从阳性菌中提取重组质粒，制备BL21感受态细胞，并将提取的重组质粒转入到BL21感受态细胞中，根据菌液PCR筛选出正确转入重组质粒的菌株。
正确转入重组质粒的BL21在28℃,1mM IPTG条件下诱导12h,并将诱导菌液超声破碎后将全菌液、上清、沉淀进行SDS-PAGE电泳检测蛋白表达情况。表达蛋白，可以通过谷胱甘肽-琼脂糖亲和层析法进行纯化，纯化出的带有GST标签的融合蛋白可经由Prescission蛋白酶切割，释放靶蛋白。
对纯化出的突变及未突变蛋白进行温度稳定性的比较。突变前后的蛋白，分别在37℃，55℃水浴保温一定时间后测酶活，稳定性都提高5-10倍(表2)。
表2突变-SOD和SOD的热稳定性(pH7.0,37℃,55℃)
突变前后耐酸碱性和胃胰蛋白酶耐受性也显著提高，结果见下表3、4、5、6。
表3突变-SOD和SOD的耐酸性(pH5.20,25℃)
表4突变-SOD和SOD的耐碱性(pH10.80,25℃)
表3、4结果表明，SOD经突变后，其耐酸碱性能明显提高。
表5突变-SOD和SOD对胃蛋白酶耐受性(pH1.34，25℃)
表6突变-SOD和SOD对胰蛋白酶耐受性(pH7.84，25℃)
表5、6结果表明，SOD经突变后，对蛋白酶和胰蛋白酶耐受性明显提高。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。
<110>曲阜师范大学
<120>一种超氧化物歧化酶Cu,Zn-SOD基因及其应用
<160> 7
<210> 1
<211> 465
<212> DNA
<213>小鼠（Mus musculus）
<400> 1
atggcgatga aagcggtgtg cgtgctgaag ggcgacggtc cggtgcaggg aaccatccac 60
ttcgagcaga aggcaagcgg tgaaccagtt gtgttgtcag gacaaattac aggattaact 120
gaaggccagc atgggttcca cgtccatcag tatggggaca atacacaagg ctgtaccagt 180
gcaggacctc attttaatcc tcactctaag aaacatggtg gcccggcgga tgaagagagg 240
catgttggag acctgggcaa tgtgactgct ataaaggacg gtgtggccaa tgtgtccatt 300
gaagatcgtg tgatctcact ctcaggagag cattccatca ttggccgtac aatggtggtc 360
catgagaaac aagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gactggaaat 420
gctgggagcc gcttggcctg tggagtgatt gggattgcgc agtaa 465
<210> 2
<211>28
<212> DNA
<213>人工序列
<400> 2
ATAGGATCCA TGGCGATGAA AGCGGTGT 28
<210>3
<211>31
<212> DNA
<213>人工序列
<400> 3
GCAGTCGACT TACTGCGCAA TCCCAATCAC T 31
<210>4
<211>32
<212> DNA
<213>人工序列
<400> 4
GGGTTCCACG TCCATTGGTA TGGGGACAAT AC 32
<210>5
<211>32
<212> DNA
<213>人工序列
<400> 5
GTATTGTCCC CATACCAATG GACGTGGAAC CC 32
<210>6
<211>22
<212> DNA
<213>人工序列
<400> 6
GTGACTGCTA TAAAGGACGG TG 22
<210>7
<211>22
<212> DNA
<213>人工序列
<400> 7
CACCGTCCTT TATAGCAGTC AC 22
Claims (3)
1.一种超氧化物歧化酶Cu,Zn-SOD基因，其特征在于核苷酸序列如序列表中序列1所示。
2.一种含有权利要求1中所述的超氧化物歧化酶Cu,Zn-SOD基因的载体的构建方法，其特征在于包括以下步骤：
（1）涉及引物，核苷酸序列如下：
扩增引物为：上游引物P1:5’- ATAGGATCCATGGCGATGAAAGCGGTGT-3’，
下游引物P2:5’- GCAGTCGACTTACTGCGCAATCCCAATCACT-3’，
单点突变引物：上游引物P3:5’-GGGTTCCACGTCCATTGGTATGGGGACAATAC-3’，
下游引物P4:5’-GTATTGTCCCCATACCAATGGACGTGGAACCC- 3’，
双点突变引物：上游引物P5:5’-GTGACTGCTATAAAGGACGGTG- 3’，
下游引物P6: 5’-CACCGTCCTTTATAGCAGTCAC- 3’，
（2）由小鼠血液中提取RNA，反转为cDNA并经上下游引物P1、P2进行PCR得到目的基因序列，然后以目的基因为模板，以单点突变引物P3、P4及双点突变引物P5、P6为引物，通过巢式PCR扩增以得到所需突变基因，切胶回收所需目的片段，酶切转入表达载体。
3.一种权利要求1所述的超氧化物歧化酶Cu,Zn-SOD基因在制备氧自由基清除剂中的应用。
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