CN105838724B - A kind of malate dehydrogenase gene RGMDH1 and its recombinant expression carrier - Google Patents

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

A kind of malate dehydrogenase geneRGMDH1And its recombinant expression carrier

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

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gtgcgcgacc tcgcccaggg catcgccgac ttctgcccaa aggccttcgt cctcatcatc 360

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acctacgtcg agtcgcccct ctacgccgac aagggcgtca cgttcttttc gtcgcccgtg 840

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Lys Gly Val Ala Ala Asp Ile Gly His Val Asp Ser Pro Ala Val Thr Thr Gly Tyr Val

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Lys Asp Glu Asp Gly Leu Lys Gly Ala Leu Thr Gly Ala Asp Leu Val Val Ile Pro Ala

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Gly Val Pro Arg Lys Pro Gly MET Thr Arg Asp Asp Leu Phe Asn Ile Asn Ala Gly Ile

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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

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Asp Glu Val Val Lys Ala Lys Asp Gly Ala Gly Ser Ala Thr Leu Ser MET Ala Tyr Ala

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Gly Ala Asp Trp Ala Asp Ser Leu Leu Arg Ala MET Asn Gly Glu Gln Val Glu Ile Cys

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Thr Ile Ser Ser Glu Gly Thr Val Gly Glu Ile Lys Pro Val Gly Gln Leu His Glu Ser

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tacggatcca tgggcctcaa gactgct 27

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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.