CN105838724B - A kind of malate dehydrogenase gene RGMDH1 and its recombinant expression carrier - Google Patents
A kind of malate dehydrogenase gene RGMDH1 and its recombinant expression carrier Download PDFInfo
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Abstract
The invention discloses a kind of nucleotide sequences of encoding malate dehydrogenase separated from rhodotorula glutinis YM25079, its nucleotide sequence is as shown in SEQ ID NO:1, the amino acid sequence of gene coding is as shown in SEQ ID NO:2, by building recombinant vector and in expression in escherichia coli, expression product has the function of malic dehydrogenase, can be catalyzed malic acid and be converted to oxaloacetic acid.
Description
Technical field
The present invention relates to a kind of malate dehydrogenase genesRGMDH1And its recombinant expression carrier, and in particular to glue red ferment
Female (Rhodotorula glutinis) YM25079 total serum IgE reverse transcription cDNA be template, amplification obtain encoding malate dehydrogenation
The gene of enzyme (MDH) is cloned into inducing expression after coli expression carrier, and affinity chromatography obtains pure enzyme after purification,
And the correlative study of enzyme activity determination and zymologic property has been carried out to the enzyme, belong to genetic engineering and enzyme engineering field.
Background technique
Malic dehydrogenase (MDH) is distributed widely in organism, is a kind of enzyme that activity is very strong, it is catalyzed oxalyl
Acetate and malate mutually convert reaction, related to the redox of dinucleotides coenzyme.Oxaloacetate is in many
It all plays an important role, including tricarboxylic acid cycle, glyoxylate bypass, Amino acid synthesis, gluconeogenesis etc., and maintains in metabolic pathway
Redox equilibrium, moreover it is possible to promote the exchange of cytoplasm and subcellular organelle metabolin.According to the function difference of organism, histological difference,
Different its of intracellular targeting expresses type difference, and MDH has a variety of isodynamic enzyme forms.CyMD (cMDH) is deposited
It is in cell cytoplasm, is responsible for the important task that NADH is transferred to mitochondria, and also have effect to regulation tricarboxylic acid cycle, simultaneously
A component part of cMDH or nucleic acid access (NACh) complex.
Malic dehydrogenase (MDH) is widely distributed in animal tissue, microorganism and plant.It is that a kind of activity is strongest
Enzyme, according to subcellular localization, malic dehydrogenase can be divided into 5 seed types, be present in glyoxalic acid body, mitochondria, peroxidase precursor,
In chloroplaset, cytoplasm and trypanosome glycerol body.MDH be polymer enzyme, the dimer be made of same or similar subunit or
The tetramer, the molecular weight of subunit are 30-35kDa, and MDH also causes more and more to pay close attention to as utilized genetic engineering in terms of medicine
Vaccine prevention human body taeniasiss have been the research directions being concerned, and pass through the biology of Bovine luteinizing hormone MDH gene
Bioinformatics analysis, it is predicted that endochylema type MDH is a potential diagnostic antigen, this is that band tapeworm is ground in diagnosis, drug and vaccine
Application prospect in studying carefully provides important clue, for multienzyme analysis and the early diagnosis of disease in clinical diagnosis, such as
For diagnosing DIC(disseminated intravascular coagulation), myocardial infarction, acute, chronic hepatitis etc..In field of food, malic dehydrogenase is used
It is extensive in the measurement of organic acid content, such as the measurement of L- apple bright acid, acetic acid, citric acid substance, application prospect.Utilize the bottom MDH
Object specificity can also be used for splitting D, L MALIC ACID enzyme.In short, key of the MDHs as organism central metabolism approach
Enzyme, to it, oneself has carried out relatively broad research both at home and abroad, and MDHs isodynamic enzyme is just being applied to biological classification, species differentiation, heredity
The research such as variation, species hybridization and ontogeny.Therefore physio-biochemical characteristics, structure and function, the catalysis of MDHs are understood in depth
The metabolism and one of MDHs in organism is inquired into mechanism, expression, purifying and Immunogenicity for enzyme recombinant protein
The molecule pathogenic mechanism of a little diseases has great significance.The application study of MDH will also will push MDHs genetically modified plants simultaneously
And the further development of chiral drug.
Summary of the invention
The object of the present invention is to provide one kind from rhodotorula glutinis (Rhodotorula glutinis) separate in YM25079
Malate dehydrogenase geneRGMDH1, the gene nucleotide series as shown in SEQ ID NO:1 or the segment of the nucleotide sequence,
Or the nucleotide sequence complementary with SEQ ID NO:1, a length of 978bp(base of the gene order), the amino acid of gene coding
Sequence polypeptide as shown in SEQ ID NO:2 or its segment.
Another object of the present invention is to provide a kind of containing separated malate dehydrogenase geneRGMDH1Recombinant expression
Carrier is that gene shown in SEQ ID NO:1 is directly constructed from different expression vectors (plasmid, virus or carrier) connection
Recombinant vector.
Another object of the present invention is to provide one kind and contains the malate dehydrogenase geneRGMDH1Recombinant expression carrier or
Above-mentioned recombinant expression carrier host cell E. coli (Escherichia coli) bacterial strain BL21.
With nucleotide sequence of the present invention or the conversion of the recombinant vector containing nucleotide sequence host cell can use this
Method known to the technical staff in field carries out.When host is prokaryotes such as Escherichia coli, CaCl is used2, the side such as electroporation
Method carries out.When host is eucaryote, the methods of DNA infection protocol, microinjection, electroporation, liposome packaging can be selected.
Nucleotide sequence provided by the invention be it is a kind of efficiently, the malate dehydrogenase gene of specificity, can by its with
Carrier is ligated and transformed into malic dehydrogenase is produced in microbial cell body, has product specificities high, with short production cycle, raw
Producing is not influenced by place, weather, season and is suitble to exploitation commercialization malic dehydrogenase using different strain and culture medium
The advantages that;The transgenic escherichia coli of present invention application technique for gene engineering building specificity production malic dehydrogenase produces apple
Tartaric acid dehydrogenase has many advantages, such as that easy to operate, at low cost, feasibility is high, establishes for malate dehydrogenase gene engineering production
Fixed basis.
Detailed description of the invention
Fig. 1 is to utilize rhodotorula glutinis YM25079 malate dehydrogenase gene of the inventionRGMDH1The Escherichia coli of building
Recombinant expression plasmid pET32aRGMDH1 plasmid map;
Fig. 2 is malate dehydrogenase gene of the inventionRGMDH1Inducing expression and SDS-PAGE analysis chart after purification,
Wherein: 1 is protein electrophoresis Marker;2 be the e. coli bl21 total protein for having converted pET32a (+) and having induced through IPTG;3 are
The e. coli bl21 total protein for having converted pET32aRGMDH1 and having been induced through IPTG;4 be the destination protein band of purifying.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to
The content, reagent and method used in embodiment are all made of conventional reagent and use conventional method unless otherwise specified.
Embodiment 1: rhodotorula glutinis malate dehydrogenase geneRGMDH1Clone
Using OMEGA kit E.Z.N.A Fungal RNA Kit from rhodotorula glutinis (Rhodotorula glutinis)
Total serum IgE is extracted in YM25079, with reverse transcription reagent box Thermo Scientific Maxima H Minus First
Strand cDNA Synthesis Kit synthesizes cDNA, and taking 1 μ l is that template carries out polymerase chain reaction.Design primer (primer
1 and primer 2) carry out PCR amplification, reaction the primer, component and amplification condition it is as follows:
Primer P1:RGMDHF1:5`-ATGGGCCTCAAGACTGCTGTTCTC-3` (SEQ ID NO:3)
Primer P2:RGMDHR1:5`-TTACTGCTTCATGAAGTTCTGAC-3` (SEQ ID NO:4)
PCR amplification system (50 μ L) composition is as follows:
5×Trans PFU Buffer 10μL
DNTP(2.5 μm of ol/L) 5 μ L
cDNA 1μL
RGMDHF1(10 μm of ol/L) 2 μ L
RGMDHR1(10 μm of ol/L) 2 μ L
Fast Pfu DNA polymerase(5U/ μ L) 2 μ L
Sterile ddH2O complements to 50 μ L;
Amplification condition: 94 DEG C of denaturation 4min, then 30 circulations are carried out with 94 DEG C of 45s, 56 DEG C of 45s, 72 DEG C of 90s, finally
72 DEG C of 10min take 1 μ L of product after having reacted, then in the Ago-Gel that concentration is 1%, carry out electrophoretic analysis.Through gel
After imaging system images confirm that clip size is correct, with the more kinetic energy DNA purification and recovery reagents of hundred Tyke Bioisystech Co., Ltd
Box recycles target fragment, and the target gene for then obtaining PCR amplification is connected on pMD18-T, and connection product converts large intestine bar
Bacterium DH5 α is screened with the LB solid plate containing ampicillin (Amp+), and the transformant on picking plate carries out bacterium colony
PCR screening positive clone is then sent for the raw work sequencing in Shanghai.Sequencing result the results show that obtain one section of 978bp long sequence,
It is named asRGMDH1, sequence composition nucleotide sequence as shown in SEQ ID NO:1.
Embodiment 2: the building of recombinant expression plasmid pET32aRGMDH1
The cDNA in embodiment 1 is used to carry out PCR amplification, the combination of reaction the primer, reactive component and amplification for template
Condition is as follows:
Primer P3:RGMDHF2:5`-TACGGATCCATGGGCCTCAAGACTGCT -3` (SEQ ID NO:5)
Primer P4:RGMDHR2:5`-CGTCTCGAGCTGCTTCATGAAGTTCTG-3` (SEQ ID NO:6)
PCR amplification system (50 μ L) composition is as follows:
5×Fast Pfu Buffer 10μL
DNTP(2.5 μm of ol/L) 5 μ L
cDNA 1μL
RGMDHF2(10 μm of ol/L) 1 μ L
RGMDHR2(10 μm of ol/L) 1 μ L
Fast Pfu DNA polymerase(5U/ μ L) 1 μ L
Sterile ddH2O complements to 50 μ L;
Amplification condition: 94 DEG C of denaturation 4min, then 30 circulations are carried out with 94 DEG C of 45s, 59 DEG C of 45s, 72 DEG C of 2min, most
72 DEG C of 10min afterwards;The PCR product and plasmid pET-32a for taking purifying are used respectivelyBamH I andXhoI digestion is stayed overnight, 50 μ l PCR
Product reaction system: 25 μ L, 10 × Tango Buffer of PCR product, 10 μ l,Bam2 μ l of H I andXho I1.5 μ L, with sterilizing
Distilled water polishing, 37 DEG C of digestions stay overnight.50 μ l plasmid pET-32a reaction systems: plasmid pET-32a 15 μ l, 10 × Tango
10 μ l of Buffer,Bam2 μ l of H I andXhoI1.5 μ L, with the distilled water polishing of sterilizing, 37 DEG C of digestions are stayed overnight.Electrophoresis examines enzyme
Product is cut, and purification and recovery are carried out to digestion products with gel reclaims kit.Linked system (10 μ L): the PCR of purifying is produced
Object and expression vector pET-32a are loaded by 7:1, and 0.5 μ L, T4 Buffer of T4DNA ligase, 1 μ L, 16 DEG C of connections are overnight.It takes
Connection product is transferred in bacillus coli DH 5 ɑ.After 37 DEG C of shaken cultivation 1.5h, the LB culture medium that culture solution is coated with the benzyl containing ammonia is flat
Plate cultivates 12h in 37 DEG C of incubators, and the transformant on picking plate carries out bacterium colony PCR, screening positive clone, and building is weighed
Group expression plasmid is named as pET32aRGMDH1, and the plasmid map is as shown in Figure 1.
Embodiment 3: malate dehydrogenase geneRGMDH1Inducing expression in e. coli bl21
1, the inducing expression and purifying of malate dehydrogenase zymoprotein RGMDH1
In order to verify the activity of the gene coded protein, 50 μ L large intestine bars are added in 1 μ g recombinant plasmid pET32aRGMDH1
In bacterium BL21 competent cell, by, in 42 DEG C of thermal shock 90s, ice bath 2min, then will again after whole system ice bath 30min
Linked system is drawn and is added into 950 μ L LB liquid mediums, 37 DEG C, 100rpm oscillation incubation 1h.After incubation in
5000rpm is centrifuged 10 min, leaves about 80 μ L supernatants and suspends that be coated on the LB containing ampicillin (Amp+) after precipitating solid
10h is cultivated in body plate, 37 DEG C of inversions.
Picking positive transformant is in 100 mL LB (containing 100 μ g/mL ammonia benzyl mycins) culture medium, 37 DEG C of shaken cultivation mistakes
The bacterium solution of enrichment is inoculated into 1L LB liquid medium by night in 1% ratio, and in 37 DEG C, 160rpm, which is cultivated to OD600 value, is about
0.8.Take 5ml bacterium solution as blank control, remaining is added IPTG to final concentration of 1mmol/L, lures in 15 DEG C of constant-temperature table 80rpm
Culture 8 hours is led, 12000 rpm are centrifuged 15min and collect thallus.SDS-PAGE analysis shows that, pET32aRGMDH1 conversion it is big
The albumen (see Fig. 2 swimming lane 3) that a molecular weight is about 50kD is given expression in enterobacteria, but in empty carrier pET32a(+) conversion
Do not have in Escherichia coli (see Fig. 2 swimming lane 2).
Further being suspended in the thallus (makes the OD of bacteria suspension in right amount600≈ 20) 30 mM imidazole buffer in, on ice
Sonicated cells, 4 DEG C, 14000 rpm centrifugation, 15 min.By 0.2 μm of miniature membrane filtration of the supernatant after centrifugation, filter
Liquid is splined on the His Trap HP column (1 ml, GE Healthcare) balanced with 30mM imidazole buffer, with 200mM miaow
Azoles buffer is eluted, and eluent is collected in order with centrifuge tube, elution samples SDS-PAGE electrophoresis detection, and it is pure to obtain one
Protein band (see Fig. 2 swimming lane 4).
2, the enzyme activity determination of malic dehydrogenase RGMDH1
Malic dehydrogenase is the key enzyme of regulating apple acid metabolic, can be catalyzed malic acid and carry out dehydrogenation oxidation, adjoint
Generation oxaloacetic acid and NADH.Since the enzyme activity of MDH changes within certain reaction time with the concentration of reaction product NADH
It is in a linear relationship, so the activity of MDH can be measured by detecting the concentration variation of NADH.With malic acid and NAD(+) it is substrate
Malic dehydrogenase is added to be reacted, measures enzyme activity at 340nm with ultraviolet specrophotometer.The calculating of MDH enzyme activity:
Unit definition: an enzyme amount enzyme activity unit generates 1 μm of olNADH per minute when referring to 25 DEG C needed for.
Malic dehydrogenase enzyme activity calculation formula:
E=[(Δe/Δt) ×Vt×df]/(ε×D×Vs×C)
=[(0.315-0.236)×1.9×95]/(6.42×1×0.02×0.3482)
=318.93U/mg
Vt----- total volume of reaction solution (ml)
The absorbance for the NADH that ε --- -- is measured at 340nm is 6.42
D----- optical path length (1cm) (cuvette diameter)
Vs----- enzyme solution volume (ml)
C----- protein concentration (mg/ml)
In Δ e/ Δ t----1min at 340nm absorbance variation
Df---- dilution gfactor
The results show that the enzyme activity of purified malic dehydrogenase RGMDH1 is 318.93 U/mg, show that genetic recombination carries
The body malic dehydrogenase RGMDH1 that inducing expression comes out in e. coli bl21 has the activity of malic dehydrogenase.
Sequence table
<110>Kunming University of Science and Technology
<120>a kind of malate dehydrogenase geneRGMDH1And its recombinant expression carrier
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 978
<212> DNA
<213>rhodotorula glutinis
<400> 1
atgggcctca agactgctgt tctcggcgct gctggtggca tcggccagcc cctcgccctt 60
ctcctcaagc agaacccggc catcaccgag ctcgccctgt tcgatgtcgt ccccgtcgtc 120
aagggcgtcg ccgccgacat cggccacgtc gactcgcccg ccgtcacgac gggctacgtc 180
aaggacgagg acggccttaa gggcgccctc accggcgccg acctcgtcgt catccccgcc 240
ggcgtcccgc gtaagcccgg catgacgcgc gacgacctgt tcaacatcaa cgccggcatc 300
gtgcgcgacc tcgcccaggg catcgccgac ttctgcccaa aggccttcgt cctcatcatc 360
tcgaacccgg tcaactcgac cgtgcccatc gccgccgagg tcctcaaggc cgccggcgtc 420
tttgacccca agcgcgtgtt cggcgtcacc accctcgacg tcgtccgtgc gtcgaccatg 480
tcggcgcagg ccatcggcaa gcccaactcg gcgcccgagt acacgatccc ggtcgtcggc 540
ggccactctg gcgtgacgat cctcccgctc ctgtcgcagg cccagccggc gctccccaag 600
tcgctgttcg acgacgaggc caagctcaag gagctcgtca agcgcatcca gtttggcggt 660
gacgaggtcg tcaaggccaa ggacggcgct gggtcggcca ccctctcgat ggcgtacgcc 720
ggtgccgact gggccgactc gctcctccgc gccatgaacg gcgagcaggt cgagatctgc 780
acctacgtcg agtcgcccct ctacgccgac aagggcgtca cgttcttttc gtcgcccgtg 840
acgatctcct cggagggcac ggtcggcgag atcaagcccg tcggccagct gcacgagtcg 900
gagcagaagc tcctcgacgc gtgcctcccg gacctcaaga aaaacatcga ggccggtcag 960
aacttcatga agcagtaa 978
<210> 2
<211> 325
<212> PRT
<213>rhodotorula glutinis
<400> 2
MET Gly Leu Lys Thr Ala Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Pro Leu Ala Leu
5 15
Leu Leu Lys Gln Asn Pro Ala Ile Thr Glu Leu Ala Leu Phe Asp Val Val Pro Val Val
25 35
Lys Gly Val Ala Ala Asp Ile Gly His Val Asp Ser Pro Ala Val Thr Thr Gly Tyr Val
45 55
Lys Asp Glu Asp Gly Leu Lys Gly Ala Leu Thr Gly Ala Asp Leu Val Val Ile Pro Ala
65 75
Gly Val Pro Arg Lys Pro Gly MET Thr Arg Asp Asp Leu Phe Asn Ile Asn Ala Gly Ile
85 95
Val Arg Asp Leu Ala Gln Gly Ile Ala Asp Phe Cys Pro Lys Ala Phe Val Leu Ile Ile
105 115
Ser Asn Pro Val Asn Ser Thr Val Pro Ile Ala Ala Glu Val Leu Lys Ala Ala Gly Val
125 135
Phe Asp Pro Lys Arg Val Phe Gly Val Thr Thr Leu Asp Val Val Arg Ala Ser Thr MET
145 155
Ser Ala Gln Ala Ile Gly Lys Pro Asn Ser Ala Pro Glu Tyr Thr Ile Pro Val Val Gly
165 175
Gly His Ser Gly Val Thr Ile Leu Pro Leu Leu Ser Gln Ala Gln Pro Ala Leu Pro Lys
185 195
Ser Leu Phe Asp Asp Glu Ala Lys Leu Lys Glu Leu Val Lys Arg Ile Gln Phe Gly Gly
205 215
Asp Glu Val Val Lys Ala Lys Asp Gly Ala Gly Ser Ala Thr Leu Ser MET Ala Tyr Ala
225 235
Gly Ala Asp Trp Ala Asp Ser Leu Leu Arg Ala MET Asn Gly Glu Gln Val Glu Ile Cys
245 255
Thr Tyr Val Glu Ser Pro Leu Tyr Ala Asp Lys Gly Val Thr Phe Phe Ser Ser Pro Val
265 275
Thr Ile Ser Ser Glu Gly Thr Val Gly Glu Ile Lys Pro Val Gly Gln Leu His Glu Ser
285 295
Glu Gln Lys Leu Leu Asp Ala Cys Leu Pro Asp Leu Lys Lys Asn Ile Glu Ala Gly Gln
305 315
Asn Phe MET Lys Gln ***
325
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
atgggcctca agactgctgt tctc 24
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
ttactgcttc atgaagttct gac 23
<210> 5
<211> 27
<212> DNA
<213>artificial sequence
<400> 5
tacggatcca tgggcctcaa gactgct 27
<210> 6
<211> 27
<212> DNA
<213>artificial sequence
<400> 6
cgtctcgagc tgcttcatga agttctg 27
Claims (2)
1. a kind of malate dehydrogenase geneRGMDH1, nucleotide sequence is as shown in SEQ ID NO:1, the ammonia of gene coding
Base acid sequence is as shown in SEQ ID NO:2.
2. one kind contains malate dehydrogenase gene described in claim 1RGMDH1Recombinant expression carrier.
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CN108753802B (en) * | 2018-05-22 | 2021-07-16 | 昆明理工大学 | Malic dehydrogenase gene CIMDH1 and recombinant expression vector thereof |
CN109337879B (en) * | 2018-12-21 | 2021-01-26 | 厦门大学 | Malate dehydrogenase PbMDH and coding sequence and application thereof |
CN109777815B (en) * | 2019-03-28 | 2021-10-29 | 昆明理工大学 | HMG-CoA synthetase gene RKHMGCS and application thereof |
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CN104673810A (en) * | 2015-01-23 | 2015-06-03 | 昆明理工大学 | Malic dehydrogenase gene MIMDH1 and recombinant expression vector thereof |
CN105296509A (en) * | 2015-11-16 | 2016-02-03 | 昆明理工大学 | Malate dehydrogenase gene RKMDH2 and recombinant expression vector thereof |
CN105400711A (en) * | 2015-12-30 | 2016-03-16 | 江南大学 | Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid |
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CN104673810A (en) * | 2015-01-23 | 2015-06-03 | 昆明理工大学 | Malic dehydrogenase gene MIMDH1 and recombinant expression vector thereof |
CN105296509A (en) * | 2015-11-16 | 2016-02-03 | 昆明理工大学 | Malate dehydrogenase gene RKMDH2 and recombinant expression vector thereof |
CN105400711A (en) * | 2015-12-30 | 2016-03-16 | 江南大学 | Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid |
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