CN104673809A - 一种苹果酸脱氢酶基因及其重组表达载体 - Google Patents
一种苹果酸脱氢酶基因及其重组表达载体 Download PDFInfo
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- CN104673809A CN104673809A CN201510033346.6A CN201510033346A CN104673809A CN 104673809 A CN104673809 A CN 104673809A CN 201510033346 A CN201510033346 A CN 201510033346A CN 104673809 A CN104673809 A CN 104673809A
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种苹果酸脱氢酶基因及其重组表达载体,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MIMDH2及其重组表达载体,具体涉及以深黄被孢霉(Mortierella isabellina)M6-22的总RNA反转录产物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MIMDH2,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1017bp(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MIMDH2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MIMDH2或上述重组表达载体的宿主细胞大肠杆菌(Escherichia coli)菌株BL21。
如SEQ ID NO:2所示的多肽或其片段。
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优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MIMDH2构建的大肠杆菌重组表达质粒pET32aMIMDH2质粒图谱
图2为本发明所构建重组表达质粒pET32aMIMDH2的酶切分析图;其中:1为pET32a(+)双酶切后条带;2为pET32aMIMDH2双酶切后条带;3为基因MIMDH2的PCR扩增产物;4为DNA Marker;
图3为本发明的苹果酸脱氢酶基因MIMDH2诱导表达并纯化后的SDS-PAGE分析图,其中:1为蛋白电泳Marker;2为转化了pET32a(+)并经IPTG诱导的大肠杆菌BL21总蛋白;3为转化了pET32aMIMDH2并经IPTG诱导的大肠杆菌BL21总蛋白;4为纯化的目的蛋白条带。
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MIMDH2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1ul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:MIMDH2F2: 5’-ATGTTTGCCGCTACCCGTG-3’ (SEQ ID NO:3)
引物P2:MIMDH2R2: 5’-TTAAGCCTTGACGAAAGACTTTCC-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
MIMDH2F2(10μmol/L) 2μL
MIMDH2R2(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、60℃ 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1017bp长的序列,命名为MIMDH2,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pET32aMIMDH2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P1:MIMDH2F3: 5’-CGCGGATCCATGTTTGCCGCTACCCGTG-3’ (SEQ ID NO:5)
引物P2:MIMDH2R4: 5’-CCGCTCGAGAGCCTTGACGAAAGACTTTCC-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
MIMDH2F3(10μmol/L) 1μL
MIMDH2R4(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、60℃ 45s、72℃ 2min进行30个循环,最后72℃ 10min;取纯化的PCR产物和质粒pET32a分别用BamHⅠ和XhoI酶切过夜,50μL PCR产物双酶切体系:PCR产物25μl,10×Tango Buffer 10ul,BamH I 2ul和Xho I 1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。50μL质粒pET-32a双酶切体系:质粒pET-32a 15μl,10×Tango Buffer 10μl, BamH I 2μL和Xho I1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。用凝胶回收试剂盒对酶切产物进行纯化和回收。再将回收片段进行连接,连接体系(10μL):纯化的PCR产物和表达载体pET-32a 按7:1的比例用0.5μl的T4 DNA连接酶,T4 Buffer 1μl,16℃连接过夜。连接产物转入大肠杆菌DH5α感受态细胞中。37℃振荡培养1h后,涂布在含氨苄的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pET32aMIMDH2,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2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3中该基因的PCR产物大小一致,大分子条带与泳道1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MIMDH2在大肠杆菌BL21中的诱导表达
1.苹果酸脱氢酶蛋白MIMDH2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pET32aMIMDH2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,然后加入950μL LB液体培养基于37℃、100rpm振荡孵育1h。孵育结束后,5000rpm离心10min,留下约80μL悬浮沉淀菌体并涂布于含有氨苄青霉素(Amp+)的LB固体平板,37℃倒置培养10h。挑取阳性转化子阳性克隆验证后,再接入100 mL LB(含100 μg/mL氨苄霉素)的液体培养基中,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80 rpm诱导培养8小时,12000 rpm离心15min收集菌体。SDS-PAGE分析显示,pET32aMIMDH2转化的大肠杆菌中表达出一条分子量约为53kD的蛋白(见图3泳道3),但在空载体pET32a(+)转化的大肠杆菌中没有(见图3泳道2)。
进一步将该菌体悬浮于适量(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,上清和沉淀分别用SDS-PAGE电泳检测,电泳检测发现目的蛋白pET32aMIMDH2是以包涵体的形式存在。首先对包涵体进行纯化:将破菌后的沉淀用PE缓冲液(20mmol/L磷酸钠,1mM EDTA pH7.2)重悬,用移液枪吹打混匀,于4℃,12000rpm离心15min收集沉淀,再用2mol/L的脲重悬沉淀振荡混匀静置20min后,于4℃,12000rpm离心30min收集沉淀,用30mL Triton X-100(0.5%) EDTA(10mmol/L)的溶液重悬沉淀,充分混匀后离心收集沉淀。最后再用PE缓冲液洗涤沉淀2-3次以出去残留的去污剂。对纯化后的包涵体进行变性:将纯化后的包涵体用变性缓冲液(100mmol/L Tris-HCl pH7.6, 8mol/L脲,10mmol/L DTT)溶解后,置于37℃恒温培养箱中变3-4h。将变性的包涵体溶液直接加入到1L的透析缓冲液中(100mmoL/L Tris-HCl 缓冲液pH7.6,5mmol/L β-巯基乙醇)。在4℃透析24h。中间换两次缓冲液以彻底透析复性。
将复性的蛋白质4℃、12000rpm离心15min,取上清液用0.2 μm的微型滤膜过滤,滤液上样于已用10mM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4)。
2.苹果酸脱氢酶MIMDH2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1nmolNADH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)
=[(0.207-0.194)×1.9×95]/(6.22×1×0.02×0.3445)
=54.748U/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MIMDH2的酶活为54.748U/mg,表明基因重组载体在大肠杆菌 BL21中诱导表达出来的蛋白MIMDH2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及其重组表达载体
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1017
<212> DNA
<213> 深黄被孢霉
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atgtttgccg ctacccgtgc cttcacctct ctcgctgcta agcgtacctt ctccacctct 60
gctgccaacc tctccaaggt tgctgtcctt ggtgctgccg gtggtatcgg tcaacccctt 120
tcccttttgc tcaagaacaa caccaaggtt accgacctct ccttgtacga tattgtcaac 180
tcccccggtg ttgctgctga tttgagccac atcaacacca actccaaggt cactggatac 240
ggccctgaga acgacggtct taagaaggct cttgaaggtt ctgagatcgt tgtcatcccc 300
gctggtgtcc ccagaaagcc cggtatgacc cgtgatgatc ttttcaacac taacgccagc 360
attgtccgtg accttgctga ggcctgcgcc aagcactgcc ccaaggctaa cttcctcatc 420
atctccaacc ccgtcaactc cactgttcct atcttcgctg aggtcctcaa gaaggctggt 480
gtctacaacc ccaagcgtct tttcggtgtc accacccttg atgttgtccg tgcctctcgt 540
ttcgtctctg aggtcaaggc taaggacccc aaggatgtta aggtcactgt cattggtggt 600
cactctggtg tcaccatcgt tccccttctc tcccaaactg gtgagaagtt ctccaaggag 660
gagctcgatg ctcttaccaa ccgtatccaa ttcggtggtg atgaagttgt caaggccaag 720
aacggtaccg gttccgctac cctctccatg gctttcgctg gtgcacgttt cgctgacagc 780
cttctcgatg ctcaacaagg caagaagggc gttgttgagc ctaccttcgt tgagtctccc 840
atctatgccg accaaggtgt tgagttcttc gctaccaacg ttgagcttgg ccccaacggt 900
gttgagaaga tccaccccat cggtgagacc tccgactacg agaaggatct catcaccaag 960
tgccttgagg acttgaagaa gaacattgcc aagggaaagt ctttcgtcaa ggcttaa 1017
<210> 2
<211> 338
<212> PRT
<213> 深黄被孢霉
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MET Phe Ala Ala Thr Arg Ala Phe Thr Ser Leu Ala Ala Lys Arg
10 15
Thr Phe Ser Thr Ser Ala Ala Asn Leu Ser Lys Val Ala Val Leu
20 30
Gly Ala Ala Gly Gly Ile Gly Gln Pro Leu Ser Leu Leu Leu Lys
40 45
Asn Asn Thr Lys Val Thr Asp Leu Ser Leu Tyr Asp Ile Val Asn
50 60
Ser Pro Gly Val Ala Ala Asp Leu Ser His Ile Asn Thr Asn Ser
70 75
Lys Val Thr Gly Tyr Gly Pro Glu Asn Asp Gly Leu Lys Lys Ala
80 90
Leu Glu Gly Ser Glu Ile Val Val Ile Pro Ala Gly Val Pro Arg
100 105
Lys Pro Gly MET Thr Arg Asp Asp Leu Phe Asn Thr Asn Ala Ser
110 120
Ile Val Arg Asp Leu Ala Glu Ala Cys Ala Lys His Cys Pro Lys
130 135
Ala Asn Phe Leu Ile Ile Ser Asn Pro Val Asn Ser Thr Val Pro
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Ile Phe Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asn Pro Lys
160 165
Arg Leu Phe Gly Val Thr Thr Leu Asp Val Val Arg Ala Ser Arg
170 180
Phe Val Ser Glu Val Lys Ala Lys Asp Pro Lys Asp Val Lys Val
190 195
Thr Val Ile Gly Gly His Ser Gly Val Thr Ile Val Pro Leu Leu
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Ser Gln Thr Gly Glu Lys Phe Ser Lys Glu Glu Leu Asp Ala Leu
220 225
Thr Asn Arg Ile Gln Phe Gly Gly Asp Glu Val Val Lys Ala Lys
230 240
Asn Gly Thr Gly Ser Ala Thr Leu Ser MET Ala Phe Ala Gly Ala
250 255
Arg Phe Ala Asp Ser Leu Leu Asp Ala Gln Gln Gly Lys Lys Gly
260 270
Val Val Glu Pro Thr Phe Val Glu Ser Pro Ile Tyr Ala Asp Gln
280 285
Gly Val Glu Phe Phe Ala Thr Asn Val Glu Leu Gly Pro Asn Gly
290 300
Val Glu Lys Ile His Pro Ile Gly Glu Thr Ser Asp Tyr Glu Lys
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Asp Leu Ile Thr Lys Cys Leu Glu Asp Leu Lys Lys Asn Ile Ala
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Lys Gly Lys Ser Phe Val Lys Ala ***
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<210> 3
<211> 19
<212> DNA
<213> 人工序列
<400> 3
atgtttgccg ctacccgtg 19
<210> 4
<211> 24
<212> DNA
<213> 人工序列
<400> 4
ttaagccttg acgaaagact ttcc 24
<210> 5
<211> 28
<212> DNA
<213> 人工序列
<400> 5
cgcggatcca tgtttgccgc tacccgtg 28
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<400> 6
ccgctcgaga gccttgacga aagactttcc 30
Claims (3)
1.一种苹果酸脱氢酶基因MIMDH2,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MIMDH2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MIMDH2或权利要求2所述的重组表达载体。
Priority Applications (1)
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