CN104673810A - 一种苹果酸脱氢酶基因mimdh1及其重组表达载体 - Google Patents
一种苹果酸脱氢酶基因mimdh1及其重组表达载体 Download PDFInfo
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- CN104673810A CN104673810A CN201510033347.0A CN201510033347A CN104673810A CN 104673810 A CN104673810 A CN 104673810A CN 201510033347 A CN201510033347 A CN 201510033347A CN 104673810 A CN104673810 A CN 104673810A
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- mimdh1
- malate dehydrogenase
- gly
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Abstract
本发明公开了一种苹果酸脱氢酶基因MIMDH1及其重组表达载体,其是从深黄被孢霉M6-22中分离的编码苹果酸脱氢酶的核苷酸序列,其核苷酸序列如SEQ ID NO:1所示,该基因编码的氨基酸序列如SEQ ID NO:2所示,通过构建重组载体并在大肠杆菌中表达,表达产物具有苹果酸脱氢酶功能,能催化苹果酸转化成草酰乙酸。
Description
技术领域
本发明涉及一种苹果酸脱氢酶基因MIMDH1及其重组表达载体,具体涉及以深黄被孢霉(Mortierella isabellina)M6-22总RNA反转录的cDNA为模板,扩增得到编码苹果酸脱氢酶(MDH)的基因,将其克隆到大肠杆菌表达载体后诱导表达,亲和层析法纯化后得到纯酶,并对该酶进行了酶活测定及酶学性质的相关研究,属于基因工程和酶工程领域。
背景技术
苹果酸脱氢酶( MDH)广泛分布于生物体内,是一种活性非常强的酶,它催化草酰乙酸盐和苹果酸盐的相互转化反应,与二核苷酸辅酶的氧化还原相关。草酰乙酸盐在许多代谢途径中都有重要作用,包括三羧酸循环、乙醛酸旁路、氨基酸合成、糖原异生等,并维持氧化还原平衡,还能促进胞质和亚细胞器代谢物的交换。依生物体的功能不同、组织差异、细胞内定位的不同其表达种类不同,MDH具有多种同工酶形式。胞质苹果酸脱氢酶(cMDH)存在于细胞胞质内,担负着将NADH转入线粒体的重任,并且还对调控三羧酸循环有作用,同时cMDH还是核酸通路(NACh)复合体的一个组成部分。
苹果酸脱氢酶(MDH)广泛分布在动物组织、微生物和植物中。它是一种活性最强的酶,根据亚细胞定位,苹果酸脱氢酶可分为5种类型,存在于乙醛酸体、线粒体、过氧化物体、叶绿体、细胞浆以及锥虫甘油体内。MDH为多聚体酶,是由相同或相似亚基组成的二聚体或四聚体,亚基的分子量为30-35kDa,MDH在医学方面也引起越来越多的关注如利用基因工程疫苗预防人体带绦虫病已是备受关注的研究方向,通过牛带绦虫亚洲亚种MDH基因的生物信息学分析,预测到胞浆型MDH是一个潜在的诊断抗原,这为带绦虫在诊断、药物及疫苗研究中的应用前景提供了重要的线索,在临床诊断中用于多酶分析及疾病的早期诊断,例如用于诊断DIC(弥散性血管内凝血),心肌梗塞,急慢性肝炎等。在食品领域,苹果酸脱氢酶用于有机酸含量的测定,如L-苹杲酸、醋酸、柠檬酸等物质的测定,应用前景广泛。利用MDH底物专一性,还可将其用于拆分D,L-苹果酸酶。总之,MDHs作为生物体中枢代谢途径的关键酶,国内外对其己进行了较为广泛的研究,MDHs同工酶正应用于生物分类、物种分化、遗传变异、物种杂交和个体发育等研究。因此深入了解MDHs的生理生化特性、结构及功能、催化机制,对于酶重组蛋白的表达、纯化及免疫特性分析,探讨生物体中MDHs的代谢作用以及一些疾病的分子致病机制有着重要的意义。同时MDH的应用研究也将会推动MDHs转基因植物及手性药物的进一步发展。
发明内容
本发明的目的是提供一种从深黄被孢霉(Mortierella isabellina)M6-22中分离的苹果酸脱氢酶基因MIMDH1,该基因核苷酸序列如SEQ ID NO:1所示或该核苷酸序列的片段,或与SEQ ID NO:1互补的核苷酸序列,该基因序列长为990bp(碱基),该基因编码的氨基酸序列如SEQ ID NO:2所示的多肽或其片段。
本发明另一目的是提供一种含有所分离的苹果酸脱氢酶基因MIMDH1的重组表达载体,是将SEQ ID NO:1所示基因直接与不同表达载体(质粒、病毒或运载体)连接所构建的重组载体。
本发明另一目的是提供一种含有该苹果酸脱氢酶基因MIMDH1的重组表达载体或上述重组表达载体的宿主细胞大肠杆菌(Escherichia coli)菌株BL21。
用本发明所述的核苷酸序列或含有核苷酸序列的重组载体转化宿主细胞可用本领域的技术人员熟知的方法进行。当宿主为原核生物如大肠杆菌时,用CaCl2、电穿孔等方法进行。当宿主是真核生物,可选用DNA转染法、显微注射、电穿孔、脂质体包装等方法。
本发明提供的核苷酸序列是一种高效、特异性的苹果酸脱氢酶基因,可以将其与载体连接后转化至微生物细胞体内生产苹果酸脱氢酶,具有产物特异性高、生产周期短、生产不受场地、气候、季节的影响及利用不同的菌种和培养基适合开发商业化苹果酸脱氢酶等优点;本发明应用基因工程技术构建特异性生产苹果酸脱氢酶的转基因大肠杆菌生产苹果酸脱氢酶,具有操作简单、成本低、可行性高等优点,为苹果酸脱氢酶基因工程化生产奠定基础。
附图说明
图1为利用本发明的深黄被孢霉M6-22苹果酸脱氢酶基因MIMDH1构建的大肠杆菌重组表达质粒pET32aMIMDH1质粒图谱;
图2为本发明所构建重组表达质粒pET32aMIMDH1的酶切分析图;其中:1为DNA Marker;2为pET32a(+)双酶切后条带;3为pET32aMIMDH1双酶切后条带;4为基因MIMDH1的PCR扩增产物;
图3为本发明的苹果酸脱氢酶基因MIMDH1诱导表达并纯化后的SDS-PAGE分析图,其中:1为转化了pET32a(+)并经IPTG诱导的大肠杆菌BL21总蛋白;2为转化了pET32aMIMDH1并经IPTG诱导的大肠杆菌BL21总蛋白;3为纯化的目的蛋白条带;4为蛋白电泳Marker。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法。
实施例1:深黄被孢霉苹果酸脱氢酶基因MIMDH1的克隆
采用OMEGA试剂盒E.Z.N.A Fungal RNA Kit从深黄被孢霉(Mortierella isabellina)中提取总RNA,用反转录试剂盒Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit合成cDNA,取1μl为模板进行聚合酶链式反应。设计引物(引物1和引物2)进行PCR 扩增,反应所用引物、组分和扩增条件如下:
引物P1:MIMDHF1: 5`-ATGGTCAAAGTTACAGTTTGCGG-3` (SEQ ID NO:3)
引物P2:MIMDHR1: 5`-TTACAACTTGGCATCAGTGATGAAAG-3` (SEQ ID NO:4)
PCR扩增体系(50 μL)组成如下:
5×Trans PFU Buffer 10μL
dNTP(2.5μmol/L) 5μL
cDNA 1μL
MIMDHF1(10μmol/L) 2μL
MIMDHR1(10μmol/L) 2μL
Fast Pfu DNA polymerase(5U/μL) 2μL
无菌ddH2O 补足至50μL
扩增条件:94℃变性4min,再用94℃ 45s、64℃ 45s、72℃ 2min进行30个循环,最后72℃ 10min,反应完后取产物1μL,然后在浓度为1%的琼脂糖凝胶中,进行电泳分析。经凝胶成像系统成像确认片段大小正确后,用百泰克生物技术有限公司多动能DNA纯化回收试剂盒回收目的片段,然后将PCR扩增得到的目的基因连接到pMD18-T上,连接产物转化大肠杆菌DH5α,用含有氨苄青霉素(Amp+)的LB固体平板进行筛选,挑取平板上的转化子进行菌落PCR筛选阳性克隆,然后送去上海生工测序。测序结果结果显示,获得一段990bp长的序列,命名为MIMDH1,序列组成如SEQ ID NO:1所示的核苷酸序列。
实施例2:重组表达质粒pET32aMIMDH1的构建
采用实施例1中的cDNA为模板进行PCR 扩增,反应所用引物组合、反应组分和扩增条件如下:
引物P3:MIMDHF2: 5`-CGCGGATCCATGGTCAAAGTTACAGTTTGCGG-3` (SEQ ID NO:5)
引物P4:MIMDHR2: 5`-CCGCTCGAGCAACTTGGCATCAGTGATGAAAG-3` (SEQ ID NO:6)
PCR扩增体系(50μL)组成如下:
5×Fast Pfu Buffer 10μL
dNTP(2.5μmol/L) 5μL
cDNA 1μL
MIMDHF2(10μmol/L) 1μL
MIMDHR2(10μmol/L) 1μL
Fast Pfu DNA polymerase(5U/μL) 1μL
无菌ddH2O 补足至50μL;
扩增条件:94℃变性4min,再用94℃ 45s、64℃ 45s、72℃ 2min进行30个循环,最后72℃ 10min;取纯化的PCR产物和质粒pET-32a分别用BamHⅠ和Xho I酶切过夜,50μl PCR产物反应体系:PCR产物25μL,10×Tango Buffer 10μl,BamHⅠ2μl和Xho I 1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。50μl质粒pET-32a反应体系:质粒pET-32a 15μl,10×Tango Buffer 10μl,BamHⅠ2μl和Xho I1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。电泳检验酶切产物,并用凝胶回收试剂盒对酶切产物进行纯化和回收。连接体系(10μL):纯化的PCR产物和表达载体pET-32a 按7:1加样,T4DNA连接酶0.5μL,T4 Buffer 1μL,16℃连接过夜。取连接产物转入大肠杆菌DH5ɑ 中。37℃振荡培养1.5h后,培养液涂布含氨苄的LB培养基平板,37℃培养箱中培养12h,挑取平板上的转化子进行菌落PCR,筛选阳性克隆,构建获得重组表达质粒命名为pET32aMIMDH1,该质粒图谱如图1所示。进一步进行双酶切分析鉴定,如图2第3泳道所示,用BamH I和Xho I双酶切,重组质粒产生两条带,小分子条带与泳道4中该基因的PCR产物大小一致,大分子条带与泳道2中用相同两个内切酶酶切pET32a(+)产生的条带大小一致,表明所构建的重组表达质粒正确,进一步测序分析也证明这一点。此外,通过核苷酸序列所编码的氨基酸相似性搜索表明,该基因编码的蛋白与真菌来源的苹果酸脱氢酶相似,但不完全相同。
实施例3:苹果酸脱氢酶基因MIMDH1在大肠杆菌BL21中的诱导表达
1、苹果酸脱氢酶蛋白MIMDH1的诱导表达及纯化
为了验证该基因编码蛋白的活性,将1μg重组质粒pET32aMIMDH1加入50μL大肠杆菌BL21感受态细胞中,,将整个体系冰浴30min之后于42℃热击90s,再次冰浴2min,然后将连接体系吸取并加入至950μL LB液体培养基中,37℃、100rpm振荡孵育1h。孵育结束后于5000rpm离心10 min,留下约80μL 上清液悬浮沉淀后涂布于含有氨苄青霉素(Amp+)的LB固体平板,37℃倒置培养10h。
挑取阳性转化子于100 mL LB(含100μg/mL氨苄霉素)培养基中,37 ℃振荡培养过夜,将富集的菌液按1%比例接种到1L LB液体培养基中,于37℃,160rpm培养至OD600值约为0.8。取5ml菌液作为空白对照,其余加入IPTG至终浓度为1mmol/L,于15℃恒温摇床80rpm诱导培养8小时,12000 rpm离心15min收集菌体。SDS-PAGE分析显示,pET32aMIMDH1转化的大肠杆菌中表达出一条分子量约为50kD的蛋白(见图3泳道2),但在空载体pET32a(+)转化的大肠杆菌中没有(见图3泳道1)。
进一步用该菌体悬浮于适量(使菌悬液的OD600≈20)30 mM的咪唑缓冲液中,冰上超声破碎细胞,4℃、14000 rpm离心15 min。将离心后的上清液用0.2μm的微型滤膜过滤,滤液上样于已用30mM咪唑缓冲液平衡好的His Trap HP柱(1 ml,GE Healthcare),用200mM咪唑缓冲液进行洗脱,洗脱液用离心管按顺序收集,洗脱样品用SDS-PAGE电泳检测,获得一纯蛋白条带(见图3泳道3)。
2、苹果酸脱氢酶MIMDH1的酶活测定
苹果酸脱氢酶是调控苹果酸代谢的关键酶,可以催化苹果酸进行脱氢氧化,伴随着产生草酰乙酸和NADH。由于MDH的酶活在一定的反应时间内与反应产物NADH的浓度变化呈线性关系,所以MDH的活性可通过检测NADH的浓度变化来测定。以苹果酸和NAD(+)为底物加入苹果酸脱氢酶进行反应,用紫外分光光度计在340nm处测定酶活。MDH酶活的的计算:
单位定义:一个酶活力单位是指25℃时每分钟生成1μmolNADH所需的酶量。
苹果酸脱氢酶酶活计算公式:
E=[(Δe/Δt) ×Vt×df]/(ε×D×Vs×C)
=[(0.305-0.276)×1.9×95]/(6.22×1×0.02×0.2482)
=168.85U/mg
Vt-----反应溶液总体积(ml)
ε-----340nm处测定的NADH的吸光度为6.22
D-----光路长(1cm)(比色皿直径)
Vs-----酶液体积(ml)
C-----蛋白质浓度(mg/ml)
Δe/Δt----1min内340nm处吸光度的变化
df----稀释因子
结果显示,所纯化的苹果酸脱氢酶MIMDH1的酶活为168.85 U/mg,表明基因重组载体在大肠杆菌 BL21中诱导表达出来的苹果酸脱氢酶MIMDH1具有苹果酸脱氢酶的活性。
序列表
<110> 昆明理工大学
<120> 一种苹果酸脱氢酶基因MIMDH1及其重组表达载体
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<170> PatentIn version 3.5
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ttgaagcaat ccgccaacat cactcacttg agcttgttcg atattgttaa cacccctggc 120
gttgctgtcg acttgagcca catcaacacc gtctccaagg ttactggcca tgttggcaac 180
gaacagatgg aagaggctct caaggactct actattgtcg tcatccctgc tggtgttcct 240
cgcaagcctg gtatgacccg tgatgatctt ttcaagatca acgctggtat catccgtgac 300
cttgctgttg gtgttgctaa gcacgctccc aaggcgtttg tcttggttat ttccaacccc 360
gttaactcta ccgtccccat cgttactgag gtgttcaaga agcacggtgt ttatgaccct 420
aagagaatct tcggtgtcac cactcttgac attgttcgcg cttccacttt cgtttctgag 480
ttgattggca ctgatcccgc tcagcttaag gttcctgttg ttggtggaca cagcggtgtc 540
accatccttc cccttctctc tcaagtctct ggcactgaga agctttccca agagcaagtc 600
gagcaagtca ctaaccgcat tcaattcggt ggtgatgagg ttgtcaaggc caaggctggt 660
actggttctg caaccttgtc catggccttt gctggtgctc gtttcacctt ggctgttttg 720
gacgctatcc aaggcaagac cggtgttgtt gaatgcactt atgtcaactt ggagtccgat 780
cctgagggtg gtaaggctat caagcaggtt gttggtgatg atgtcactta cttctctgct 840
caagttgagc tcggtaagga cggtgtttcc aagatcttgt ctgttggcaa gctttccgac 900
tacgagaaga agcttttcga ggctgctgtc cccgaattga agggcaacat cgtcaagggt 960
gcttctttca tcactgatgc caagttgtaa 990
<210> 2
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<213> 深黄被孢霉
<400> 2
MET Val Lys Val Thr Val Cys Gly Ala Ala Gly Gly Ile Gly Gln
1 10 15
Pro Leu Ser Leu Leu Leu Lys Gln Ser Ala Asn Ile Thr His Leu
20 30
Ser Leu Phe Asp Ile Val Asn Thr Pro Gly Val Ala Val Asp Leu
40 45
Ser His Ile Asn Thr Val Ser Lys Val Thr Gly His Val Gly Asn
50 60
Glu Gln MET Glu Glu Ala Leu Lys Asp Ser Thr Ile Val Val Ile
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Pro Ala Gly Val Pro Arg Lys Pro Gly MET Thr Arg Asp Asp Leu
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Phe Lys Ile Asn Ala Gly Ile Ile Arg Asp Leu Ala Val Gly Val
100 105
Ala Lys His Ala Pro Lys Ala Phe Val Leu Val Ile Ser Asn Pro
110 120 Val Asn Ser Thr Val Pro Ile Val Thr Glu Val Phe Lys Lys His
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Gly Val Tyr Asp Pro Lys Arg Ile Phe Gly Val Thr Thr Leu Asp
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Ile Val Arg Ala Ser Thr Phe Val Ser Glu Leu Ile Gly Thr Asp
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Pro Ala Gln Leu Lys Val Pro Val Val Gly Gly His Ser Gly Val
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Thr Ile Leu Pro Leu Leu Ser Gln Val Ser Gly Thr Glu Lys Leu
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Ser Gln Glu Gln Val Glu Gln Val Thr Asn Arg Ile Gln Phe Gly
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Gly Asp Glu Val Val Lys Ala Lys Ala Gly Thr Gly Ser Ala Thr
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Leu Ser MET Ala Phe Ala Gly Ala Arg Phe Thr Leu Ala Val Leu
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Asp Ala Ile Gln Gly Lys Thr Gly Val Val Glu Cys Thr Tyr Val
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Asn Leu Glu Ser Asp Pro Glu Gly Gly Lys Ala Ile Lys Gln Val
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Val Gly Asp Asp Val Thr Tyr Phe Ser Ala Gln Val Glu Leu Gly
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Lys Asp Gly Val Ser Lys Ile Leu Ser Val Gly Lys Leu Ser Asp
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Tyr Glu Lys Lys Leu Phe Glu Ala Ala Val Pro Glu Leu Lys Gly
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Asn Ile Val Lys Gly Ala Ser Phe Ile Thr Asp Ala Lys Leu ***
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atggtcaaag ttacagtttg cgg 23
<210> 4
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<213> 人工序列
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ttacaacttg gcatcagtga tgaaag 26
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cgcggatcca tggtcaaagt tacagtttgc gg 32
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<211> 32
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ccgctcgagc aacttggcat cagtgatgaa ag 32
Claims (3)
1.一种苹果酸脱氢酶基因MIMDH1,其核苷酸序列如SEQ ID NO:1所示,该基因编码的氨基酸序列如SEQ ID NO:2所示。
2.一种含有权利要求1所述苹果酸脱氢酶基因MIMDH1的重组表达载体。
3.一种宿主细胞,所述宿主细胞含有权利要求1所述的苹果酸脱氢酶基因MIMDH1或权利要求2所述的重组表达载体。
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