CN101603048A - 一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法 - Google Patents
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Abstract
本发明提供了一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,它包括以下步骤:BbSod1基因的克隆;酿酒酵母Lys7基因的克隆;原核表达载体pET-BbSod1、pET-BbSod1-Lys7及pET-BbSod1-Mut的构建;重组质粒的扩增及蛋白表达及蛋白纯化。本发明巧妙地利用基因融合及定点突变技术,实现了BbSod1在技术体系成熟的原核表达体系中的高效表达。尤其所表达的BbSod1定点突变体BbSod1-Mut,工程菌株粗提物的SOD酶活相当于带分子伴侣的融合酶BbSod1-Lys7的2.8倍和无分子伴侣BbSod1的21.2倍;纯化BbSod1-Mut的SOD酶活相当于BbSod1-Lys7S的5.3倍。本发明有效克服了真核生物Cu/Zn-SOD对专一性分子伴侣CCS的普遍依赖性,为解决工业上从动植物中提取SOD的原料限制问题提供了基于原核工程菌株的新技术,具有重要的工业应用前景。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法。
背景技术
超氧化物歧化酶(Superoxide Dismutase,简称SOD;EC 1.15.1.1)系一类金属抗氧化酶,广泛存在于生物体中,它能够催化超氧阴离子(O2-)发生歧化反应,从而清除自由基,在生物体防御氧化损伤的过程中发挥着重要作用(Fridovich I.1995.Superoxide radical and superoxide dismutases.Annu Rev Biochem 64:97-112.)。根据酶分子中所含金属辅基的不同,SOD主要可分为Cu/Zn-SOD(多存在于真核生物)、Mn-SOD(多存在于原核细胞和真核细胞线粒体)和Fe-SOD(存在于原核细胞和高等植物叶绿体)等类型。SOD作为自由基清除剂,已被广泛应用于医药、食品和日用化妆品等各个领域(方允中,李文杰。自由基与酶。科学出版社,1989)。在医药上,SOD可用于治疗氧中毒、糖尿病、轻度烧伤及多种炎症等,在临床上都有较好的效果。在食品工业上,由于SOD无毒,无副作用,可作为食品添加剂用于饮料、口香糖及保健食品的生产。此外,在化妆品中添加SOD,可起到防止皮肤衰老和治疗皮肤疾病的作用。
现在市售的SOD多数为从哺乳动物牛、猪等动物肝脏中提取,随着疯牛病、口蹄疫、禽流感、猪流感等流行,安全性问题日渐凸显。另外,传统的提取方法还存在原料有限、分离纯化困难、产量低等缺陷。因此,利用基因工程技术构建工程菌株以实现SOD的高效、廉价化生产和制备无疑成为SOD规模化生产的有效途径。相对于酵母、昆虫细胞等众多外源表达系统,大肠杆菌由于具有生长速度快、培养基廉价、技术方案简单可行、载体及受体菌株多元化等优势而成为理想的基因工程受体菌。
真核生物一般具有Cu/Zn-型和Mn-型两类SOD,其中多数的Cu/Zn-SOD都需要专一的分子伴侣CCS来传递金属辅基Cu2+并催化分子间二硫键的形成而实现酶的转录后激活。研究表明酿酒酵母CCS基因Lys7缺失突变株的Cu/Zn-SOD表达量不变,但不具备清除氧自由基的活性(Culotta VC,Klomp LWJ,Strain J,Casareno RLB,Krems B,Gitlin JD.1997.The copper chaperone for superoxide dismutase.J Biol Chem272:23469-23472)。真核生物Cu/Zn-SOD对专一分子伴侣的普遍依赖性无疑成为此类活性蛋白在缺乏CCS的原核表达体系中实现高效表达的最大瓶颈。但是据报道,在少数真核生物中仍存在不依赖于专一性分子伴侣CCS的Cu/Zn-SOD激活途径。如人源的Cu/Zn-SOD(hSOD)在在缺失分子伴侣的情况下仍具有一定活性(CarrollMC,Girouard JB,Ulloa JL,Subramaniam JR,Wong PC,Valentine JS,Culotta VC.2004.Mechanisms for activating Cu and Zn-containing superoxide dismutase in the absence ofthe CCS Cu chaperone.Proc Natl Acad Sci USA,101:5964-5969);秀丽广杆线虫Caenorhabditis elegans的SOD(CeSOD)激活则完全不需要伴侣(Jensen LT,CulottaVC.2005.Activation of CuZn superoxide dismutases from Caenorhabditis elegans doesnot require the copper chaperone CCS.J Biol Chem 280:41373-41379)。通过三者序列比对推测,酿酒酵母SOD对CCS的完全依赖性可能与其C末端存在的两个脯氨酸有关。将hSOD(Carroll et al.,2004)和CeSOD(Jensen et al.,2005)中相应位点的氨基酸突变为脯氨酸后,这两种酶在CCS缺失条件下亦失活。这一现象为我们改造真核生物Cu/Zn-SOD,克服其对专一分子伴侣的依赖性,实现此类SOD在E.Coli体系中的规模化生产提供了思路,但迄今这一技术路线未见在真菌中成功实施的案例。
发明内容
本发明所要解决的技术问题是提供一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,它通过基因融合及定点突变技术,克服真核Cu/Zn-SOD对分子伴侣的普遍依赖性,实现真核Cu/Zn-SOD在缺乏伴侣的原核表达体系中的的简单化和规模化生产。
为此,本发明采用以下技术方案,它包括以下步骤:
(1)BbSod1基因的克隆
①BbSod1保守区序列的克隆:通过Clustal比对GenBank数据库中多种丝状真菌的Cu/Zn-SOD氨基酸序列(http://www.ncbi.nlm.nih/gov/genbank/index.htlm),针对保守区域设计简并引物;以球孢白僵菌Bb2860基因组为模板,扩增得到BbSod1的部分片段(282bp)。②BbSod1全长基因克隆及结构分析:以①所得的保守序列为基础设计引物,利用DNA Walking SpeedUpTM Premix Kit-II试剂盒(Seegene,Seoul,Korea)扩增获得保守区段的侧翼序列。对所得的序列进行拼接,并分析BbSod1基因的结构(内含子-外显子排列),获得该基因的编码区序列。③BbSod1 cDNA编码区序列的克隆:根据BbSod1编码区的5′端和3′端序列设计引物;TRIZOL法提取Bb2860的总RNA,并反转录,以此为模板扩增得到BbSod1的ORF序列。
(2)酿酒酵母Lys7基因的克隆
①酿酒酵母Y187基因组提取:挑取Y187单克隆于2ml YPD培养液(2%胰蛋白胨,2%葡萄糖,1%酵母提取物)中28℃培养过夜,利用真菌基因组提取试剂盒(Bioflux fungal genomic DNA extraction kit,Bioer,China)提取基因组。②Lys7基因的克隆:根据Lys7基因编码区序列(GenBank Accession number:AY558398)设计引物,扩增得到750bp的Lys7基因序列。
(3)原核表达载体pET-BbSod1、pET-BbSod1-Lys7及pET-BbSod1-Mut的构建
①构建pET-BbSod1:BbSod1编码区序列经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建原核表达载体pET-BbSod1。②构建pET-BbSod1-Lys7:应用基因重叠延伸技术(splicing-by-overlap extension,SOE),融合BbSod1基因与Lys7基因,经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建融合基因的原核表达载体pET-BbSod1-Lys7。③构建pET-BbSod1-Mut:利用定点突变技术,将BbSod1编码蛋白C末端的两个两个脯氨酸分别突变为丝氨酸和亮氨酸(P143S与P145L),突变后的基因BbSod1-Mut经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建原核表达载体pET-BbSod1-Mut。
(4)重组质粒的扩增及蛋白表达
将重组质粒pET-BbSod1、pET-BbSod1-Lys7及pET-BbSod1-Mut经CaCl2化学转化法分别转入到大肠杆菌(Escherichia coli)DH5α菌株中,经菌落PCR鉴定、酶切鉴定及测序确认后,提取阳性克隆质粒,然后将各质粒转入E.coli BL21(DE3)菌株中,培养至对数生长期时加入Isopropyl-D-thiogalactopyranoside(IPTG)、Cu2+和Zn2+进行诱导,获得超表达的可溶性目的蛋白。
(5)蛋白纯化
诱导表达目的蛋白的工程菌细胞培养物,经超声波破碎和离心后,获得可溶蛋白的粗提物,再通过Ni2+亲和层析进行纯化,收集电泳纯的活性洗脱液,脱盐后于-20℃下长期保存。
步骤一,(1)-①所述的扩增BbSod1保守区的简并引物序列为:
正向引物:5′-GACAAYACCAACGGCTGCAC-3′
反向引物:5′-GGYACYGAYGAYCTYGG-3′
步骤二,(1)-③所述的扩增BbSod1编码区的引物序列为:
正向引物:5′-GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′
反向引物:5′-CCCAAGCTTGTTGGCAACGCCAATGACACC-3′
步骤三,(3)-③所述的用于定点突变BbSod1的引物为:
正向引物:5′-GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′
反向引物:
5′-CCCAAGCTTGTTGGCAACGCCAATGACACCGCAGGCGAGGCGAGA
TCCAGCG-3′
步骤四,(4)所述的诱导目的蛋白表达加入的Cu2+和Zn2+离子浓度分别为2.5mM和0.5mM。
由于采用本发明的技术方案,本发明巧妙地利用基因融合及定点突变技术,实现了BbSod1在技术体系成熟的原核表达体系中的高效表达。尤其所表达的BbSod1定点突变体BbSod1-Mut,工程菌株粗提物的SOD酶活相当于带分子伴侣的融合酶BbSod1-Lys7的2.8倍和无分子伴侣BbSod1的21.2倍;纯化BbSod1-Mut的SOD酶活相当于BbSod1-Lys7S的5.3倍。本发明有效克服了真核生物Cu/Zn-SOD对专一性分子伴侣CCS的普遍依赖性,为解决工业上从动植物中提取SOD的原料限制问题提供了基于原核工程菌株的新技术,具有重要的工业应用前景。
附图说明
图1是本发明中重组球孢白僵菌BbSod1、BbSod1-Lys7和BbSod1-Mut蛋白的可溶性分析及纯化图谱。
具体实施方式
本发明所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法依次包括以下步骤:
1.BbSod1基因的克隆
(1)BbSod1保守区序列的克隆
①球孢子白僵菌(Beauveria bassiana)2860菌株总DNA的提取:接种孢子悬浮液于SDAY(4%葡萄糖,1%胰蛋白胨,1%酵母提取物,1.5%琼脂粉)平板上,在25℃下培养3天后,通过液氮研磨法提取总DNA。②简并引物设计:通过Clustal比对GenBank数据库中6种已报道的丝状真菌Cu/Zn-SOD氨基酸序列(登录号分别为AF305546、DQ493760、AJ344050、DQ530599、AY176061及M58687),针对保守区域设计简并引物。简并引物序列为:
正向引物(序列表中第1个序列):5′-GACAAYACCAACGGCTGCAC-3′
反向引物(序列表中第2个序列):5′-GGYACYGAYGAYCTYGG-3′
③保守片段的扩增:以2860菌株基因组为模板,扩增得BbSod1的部分片段(282bp)。PCR采用50μl体系,包括50ng真菌DNA、0.2μM引物、0.2mM dNTP、2.5mMMgCl2、1×taq聚合酶缓冲液和2.5U taq聚合酶。混匀后先94℃变性5min,此后经35个循环:94℃变性30s、60℃退火30s和72℃延伸30s;循环后72℃再延伸7min。扩增得到接近300bp的片段,割胶回收,与pGEMT-easy载体连接,经转化及鉴定后送样测序,确定得到的序列为BbSod1的部分序列(BbSod1 partial)如下(序列表中第3个序列):
gacaacaccaacggctgcacctcggctggccctcacttcaacccgcacggcaagacccacggcgctccctctgacgccgtcc
gccacgttggtgatctcggcaacattaagacggacgcccagggcaacgccaagggctccgttcaggacagccacgtcaagct
gattggccctcacagcgttgttggcgtacgtgcatccctggtaattcgagtctcgttgcctttgctaacatgtgtttagcgtaccgtc
gttgtccacgctggcaccgacgacctcggc
(2)BbSod1全长基因克隆及结构分析
以(1)所得的保守序列为基础设计引物,利用DNA Walking SpeedUpTM PremixKit-II试剂盒(Seegene,Seoul,Korea)扩增获得保守区段的侧翼序列。对所得的序列进行拼接,并分析BbSod1基因的结构(内含子-外显子排列),获得该基因的编码区序列。
上游侧翼序列Walking用引物序列:
Sod-upR1(序列表中第4个序列);TACGCCAACAACGCTGTGAGG
Sod-upR2(序列表中第5个序列);GAGCGCCGTGGGTCTTGC
下游侧翼序列Walking用引物:
Sod-dnF1(序列表中第6个序列):CAACCCGCACGGCAAGAC
Sod-dnF2(序列表中第7个序列):CCTCACAGCGTTGTTGGCGT
所获BbSod1基因全序列如下(序列表中第8个序列):
gcttggttcgtagcgctttttttattgcaaagcttggcccctccaacggagccgtagcttttccaggaccggccagcgtctggctga
gcgttttcgtcgggcagaagaaaagccgcccacgaccagacagctttattaagctcccctcttcccctccccctccactgcatcat
cacctcctccacttcgaatcatcaccatcccaactcaaaagcaaaacaaacaaaatcaatcaaa
ATGGTCAAAGCAGgtaagaatcgatcgagaataccatcaccgcatcgcgatatacctgtccccagatgatgctagtg
gcagaactgctagggtggcacccgggacatttataggtcgtggcggctgcgtgtgaacgcgctggtttgcgctggtttttagccc
gcccggttgcatcatggctcattcgataccttgatggactgggccgcgctgcacctttgatcaagggccttgatggcaccgcatct
gcattgtctcagcagctgtttactcattattacccaaaaaaggaaaaaaaaaactgccaacaagctccgagctaacctctgccgtt
ccgtctggttctagTCTGTGTCCTTCGTGGCGACGCCAGGGTTGCCGGTACTGTCACCT
TTGAGCAAGAGTCCGAGTCGGCTGCCACCACCATCACCTGGGACATCTCGGG
CAACGACCCCAACGCGGAGCGCGGCTTCCACATCCACACCTTTGGTGACAAC
ACCAACGGCTGCACCTCGGCTGGCCCTCACTTCAACCCGCACGGCAAGACCC
ACGGCGCTCCCTCTGACGCCGTCCGCCACGTTGGTGATCTCGGCAACATTAAG
ACGGACGCCCAGGGCAACGCCAAGGGCTCCGTTCAGGACAGCCACGTCAAG
CTGATTGGCCCTCACAGCGTTGTTGGCgtacgtgcatccctggtaattcgagtctcgttgcctttgctaac
atgtgtttagCGTACCGTCGTTGTCCACGCTGGCACCGACGACCTCGGCAAGGGCGG
CAACGAAGAGTCCCTCAAGACTGGCAACGCTGGACCTCGCCCGGCCTGCGgta
agacttacatctggcaacagacattgcgtgaatactaaccgtttatcagGTGTCATTGGCGTTGCCAACTAG
gatgcgcaactccgaaaaaacgaaaatctggtatgacaactgattgatacaaaattatgtagctcaaaatacgggtccggatccg
gacttggagctttgtagcttgatgttaggtagagataatttcatccaacgaagaatcacgtcatcactgcttcgcttgatagtatcatc
tgtgctgcagtcgtttacttttttccgtctcggcgcacttagacgctcaattttacatatagc
(3)BbSod1 cDNA编码区序列的克隆
①球孢子白僵菌总RNA的提取:接种球孢白僵菌孢子悬浮液于SDAY平板上,在25℃条件下培养3天后,称取100mg菌丝于研钵中,液氮研磨至粉末,而后加入1mlReagent,再研磨,而后采用酚-氯仿法抽提总RNA。②mRNA的逆转录:取2μl高纯水和1μl 0.66mg/ml Oligo(dt)引物[5′-gactcgagtcgacatcga(t)17-3′]与白僵菌RNA样品2μl混匀,用于反转录。反应体系在70℃下保温5min后,将PCR管转入冰浴50min以上,再加入dNTP、RNase抑制剂、RT enzyme及缓冲液,混匀后25℃下5min、42℃下60min、70℃下15min进行处理,获得cDNA模板用于扩增。③BbSod1编码区的扩增:据BbSod1编码区的5’端和3’端序列设计引物,扩增BbSod1编码区,PCR扩增体系及循环条件同(1)-③。
正向引物(序列表中第9个序列):
5′-GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′
反向引物(序列表中第10个序列):
5′-CCCAAGCTTGTTGGCAACGCCAATGACACC-3′
上下游引物的5′端分别加上酶切位点NdeI和HindIII。扩增获得450bp左右的目的片段,割胶回收后连接到pGEM-T并转化,经PCR及酶切鉴定后的阳性克隆作测序鉴定。
2.酿酒酵母Lys7基因的克隆
(1)酿酒酵母Y187基因组提取
挑取Y187单克隆于2ml YPD培养液(2%胰蛋白胨,2%葡萄糖,1%酵母提取物)中培养过夜(28℃,150r/m),利用真菌基因组提取试剂盒(Bioflux fungal genomicDNA extraction kit,Bioer,China)提取基因组。
(2)Lys7基因的克隆
根据Lys7基因编码区序列(GenBank Accession number:AY558398)设计引物,扩增Lys7基因序列,PCR扩增体系及循环条件同(1)③,延伸时间为45sec。上下游引物序列:
正向引物(序列表中第9个序列):
5′-GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′
反向引物(序列表中第10个序列):
5′-CCCAAGCTTGTTGGCAACGCCAATGACACC-3′
扩增获得750bp左右的目的片段,割胶回收后连接到pGEM-T并转化,经PCR及酶切鉴定后的阳性克隆送Invitrogen上海公司测序。
3.原核表达载体pET-BbSod1、pET-BbSod1-Lys7及pET-BbSod1-Mut的构建
①pET-BbSod1构建:BbSod1编码区序列经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建原核表达载体pET-BbSod1。②pET-BbSod1-Lys7构建:应用基因重叠延伸技术(splicing-by-overlap extension,SOE),融合BbSod1基因与Lys7基因,经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建融合基因的原核表达载体pET-BbSod1-Lys7。③pET-BbSod1-Mut构建:利用定点突变技术,将BbSod1蛋白C末端的两个两个脯氨酸分别突变为丝氨酸和亮氨酸(即P143S和P145L),突变后的基因BbSod1-Mut经限制性内切酶NdeI和HindIII消化后插入原核表达载体pET-29b+中,构建原核表达载体pET-BbSod1-Mut。上下游引物序列:
正向引物(序列表中第11个序列):
5′-GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′
反向引物(序列表中第12个序列):
5′-CCCAAGCTTGTTGGCAACGCCAATGACACCGCAGGCGAGGCGAGA
TCCAGCG-3′
4.重组质粒的扩增及目的蛋白表达
将重组质粒pET-BbSod1、pET-BbSod1-Lys7及pET-BbSod1-Mut经CaCl2化学转化法分别转化大肠杆菌E.coli DH5α,经过菌落PCR鉴定及酶切鉴定及测序后,提取阳性克隆质粒转化E.coli BL21(DE3),于37℃、150r/min条件下培养至对数生长期(OD600=0.8),而后加入0.5mM IPTG、2.5mM Cu2+和0.5mM Zn2+在20℃和150r/min条件下诱导培养过夜。
5.重组蛋白的可溶性分析
通过离心收集步骤4中诱导后的菌液,进行细胞超声破碎,运行程序为为超声时间15s、间隙时间15s及全程时间20min。4℃下10000g离心收集上清后,用不连续的十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳缓冲液系统进行分析。取20μL破碎上清液,加入5.0μL5×上样缓冲液[60mM Tris-HCl,pH6.8;25%(w/v)甘油;2%(w/v)SDS,14.4mM β-巯基乙醇和0.1%溴酚蓝]。不连续凝胶中浓缩胶和分离胶浓度分别为5%和12.5%。各泳道的上样量为15μL,在Hoefer miniVE垂直电泳系统(Amersham Pharmacia Biotech)上进行电泳。样品在浓缩胶中的电流保持12mA,在分离胶中的电流保持18mA。电泳结束后。凝胶在染色液(1.20g考马撕亮蓝R250、250mL乙醇、80mL乙酸和670mL dd-H2O)中染色过夜,然后在脱色液(250mL乙醇、80mL乙酸和670mL dd-H2O)中脱色,直至背景无色。
图1中SDS-PAGE结果显示,三种重组蛋白菌在受体菌中均以可溶性形式大量表达。图1中M代表蛋白分子量标记。泳道1~3分别为大肠杆菌BL21工程菌株培养液粗提物中可溶性的BbSod1、BbSod1-Mut和BbSod1-Lys7图谱(箭头),泳道4~6分别为纯化后的BbSod1、BbSod1-Mut及BbSod1-Lys7图谱。
6.蛋白纯化
由于重组蛋白带有6×His标签,可采用Ni2+亲和层析进行纯化。
(1)取诱导后菌液50ml,4℃下5000r/min离心10min收集菌体,沉淀悬浮于25ml Binding Buffer(50mM Tris-HCl,pH7.8,500mM NaCl和5mM咪唑)中,冰浴下超声波破碎菌体,4℃下12000r/min离心25min,取上清4℃保存备用。
(2)采用purifier(GE Healthcare,Uppsala,Sweden)纯化系统对目的蛋白进行纯化。上样前,使用10倍柱体积的Binding Buffer以5ml/min的流速平衡镍柱;流速为1ml/min上样,再次以Binding Buffer以5ml/min的流速平衡镍柱5-10个体积;梯度洗脱,依次以10%、20%、30%和100%的Elution Buffer洗脱5个柱体积,流速为5ml/min,进行OD280蛋白吸收峰收集洗脱液。收集洗脱峰通过SDS-PAGE分析检测纯度。
(3)SDS-PAGE鉴定获得的纯化部分用SephadexTM G-25脱盐。
7.粗酶液及纯化蛋白的酶活及蛋白浓度测定
(1)酶活测定。通过测定样品对邻苯三酚自氧化速度的抑制率来反应样品中SOD的含量。酶活性单位的定义:每分钟抑制邻苯三酚自氧化速率达50%时的酶量定义为一个酶活单位。由此,每毫升酶液的SOD酶活单位数(U/ml)S计算为:
式中,A0为邻苯三酚自氧化速率(OD325的变化速率控制在0.060/min),A1为SOD抑制邻苯三酚后的自氧化速率,N样品稀释倍数,V0反应液总体积(0.9ml),V1加入的待测样品体积。
(2)蛋白浓度测定。用Folin-酚法测定SOD酶液中的总蛋白浓度(mg/ml)。
(3)绝对SOD酶活的换算。根据(1)和(2)的测定结果,将目的蛋白的SOD酶活换算为每毫克蛋白的酶活单位数(U/mg蛋白),列于下表:
表1球孢白僵菌BbSod1、BbSod1-Mut和BbSod1-Lys7在大肠杆菌BL21菌株中重组表达的的粗提物与纯化品的SOD酶活比较
*同一列中相同字母的均值得表示差异不显著(LSD,P>0.05)
序列表
<110>浙江大学
<120>一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法
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<400>8
gcttggttcg tagcgctttt tttattgcaa agcttggccc ctccaacgga gccgtagctt 60
ttccaggacc ggccagcgtc tggctgagcg ttttcgtcgg gcagaagaaa agccgcccac 120
gaccagacag ctttattaag ctcccctctt cccctccccc tccactgcat catcacctcc 180
tccacttcga atcatcacca tcccaactca aaagcaaaac aaacaaaatc aatcaaaatg 240
gtcaaagcag gtaagaatcg atcgagaata ccatcaccgc atcgcgatat acctgtcccc 300
agatgatgct agtggcagaa ctgctagggt ggcacccggg acatttatag gtcgtggcgg 360
ctgcgtgtga acgcgctggt ttgcgctggt ttttagcccg cccggttgca tcatggctca 420
ttcgatacct tgatggactg ggccgcgctg cacctttgat caagggcctt gatggcaccg 480
catctgcatt gtctcagcag ctgtttactc attattaccc aaaaaaggaa aaaaaaaact 540
gccaacaagc tccgagctaa cctctgccgt tccgtctggt tctagtctgt gtccttcgtg 600
gcgacgccag ggttgccggt actgtcacct ttgagcaaga gtccgagtcg gctgccacca 660
ccatcacctg ggacatctcg ggcaacgacc ccaacgcgga gcgcggcttc cacatccaca 720
cctttggtga caacaccaac ggctgcacct cggctggccc tcacttcaac ccgcacggca 780
agacccacgg cgctccctct gacgccgtcc gccacgttgg tgatctcggc aacattaaga 840
cggacgccca gggcaacgcc aagggctccg ttcaggacag ccacgtcaag ctgattggcc 900
ctcacagcgt tgttggcgta cgtgcatccc tggtaattcg agtctcgttg cctttgctaa 960
catgtgttta gcgtaccgtc gttgtccacg ctggcaccga cgacctcggc aagggcggca 1020
acgaagagtc cctcaagact ggcaacgctg gacctcgccc ggcctgcggt aagacttaca 1080
tctggcaaca gacattgcgt gaatactaac cgtttatcag gtgtcattgg cgttgccaac 1140
taggatgcgc aactccgaaa aaacgaaaat ctggtatgac aactgattga tacaaaatta 1200
tgtagctcaa aatacgggtc cggatccgga cttggagctt tgtagcttga tgttaggtag 1260
agataatttc atccaacgaa gaatcacgtc atcactgctt cgcttgatag tatcatctgt 1320
gctgcagtcg tttacttttt tccgtctcgg cgcacttaga cgctcaattt tacatatagc 1380
<210>9
<211>32
<212>DNA
<213>人工序列
<400>9
ggaattccat atggtcaaag cagtctgtgt cc 32
<210>10
<211>30
<212>DNA
<213>人工序列
<400>10
cccaagcttg ttggcaacgc caatgacacc 30
<210>11
<211>32
<212>DNA
<213>人工序列
<400>11
ggaattccat atggtcaaag cagtctgtgt cc 32
<210>12
<211>52
<212>DNA
<213>人工序列
<400>12
cccaagcttg ttggcaacgc caatgacacc gcaggcgagg cgagatccag cg 52
Claims (10)
1.一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于它包括对BbSod1基因的克隆,在获得BbSod1基因的基础上构建重组质粒pET-BbSod1-Mut,然后对重组质粒进行扩增及蛋白表达;
所述BbSod1基因的克隆包括对BbSod1保守区序列的克隆,在BbSod1保守区序列的基础上获得BbSod1编码区序列,然后在BbSod1编码区序列的基础上再获得BbSod1 cDNA编码区序列;
所述原核表达载体pET-BbSod1-Mut的构建是将BbSod1编码蛋白C末端的两个脯氨酸分别突变为丝氨酸和亮氨酸(P143S与P145L),突变后的基因BbSod1-Mut经限制性酶酶切后插入原核表达载体中得到。
2.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述原核表达方法还包括酿酒酵母Lys7基因的克隆,所述酿酒酵母Lys7基因的克隆包括酿酒酵母Y187基因组的提取和Lys7基因的克隆。
3.如权利要求2所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于在构建所述重组质粒pET-BbSod1-Mut的同时,还构建重组质粒pET-BbSod1和pET-BbSod1-Lys7。
4.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述BbSod1保守区序列克隆的简并引物序列为:
正向引物:5′-GACAAYACCAACGGCTGCAC-3′,
反向引物:5′-GGYACYGAYGAYCTYGG-3′。
5.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述BbSod1保守区序列以球孢白僵菌Bb2860(Beauveria bassiana(Balsamo)Vuellemin)基因组为模板扩增得到。
6.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述BbSod1编码区的引物序列为:
正向引物:5′GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′,
反向引物:5′-CCCAAGCTTGTTGGCAACGCCAATGACACC-3′。
7.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述重组质粒pET-BbSod1-Mut的基因BbSod1-Mut经限制性内切酶NdeI和HindIII消化后插入到原核表达载体。
8.如权利要求6所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述用于定点突变BbSod1的引物为:
正向引物:5′GGAATTCCATATGGTCAAAGCAGTCTGTGTCC-3′,
反向引物:
5’-CCCAAGCTTGTTGGCAACGCCAATGACACCGCAGGCGAGGCGAGATCCAGCG-3’。
9.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述所述蛋白表达的诱导条件为20℃和150r/min振荡诱导过夜。
10.如权利要求1所述的一种真菌Cu/Zn-SOD的免分子伴侣的原核表达方法,其特征在于所述所述诱导目的蛋白表达时加入的IPTG、Cu2+和Zn2+离子浓度分别为0.5mM、2.5mM和0.5mM。
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