CN105838724A - Malate dehydrogenase gene RGMDH1 and recombinant expression vector containing same - Google Patents

Abstract

The invention discloses a nucleotide sequence which is separated from rhodotorula glutinis YM25079 and is used for coding malate dehydrogenase. The nucleotide sequence is shown in SEQ ID NO:1. An amino acid sequence coded by a gene is shown in SEQ ID NO:2. By constructing a recombinant vector and expressing the recombinant vector in escherichia coli, an expression product has the function of malate dehydrogenase and can catalytically convert malic acid to oxaloacetic acid.

Description

A kind of malate dehydrogenase gene RGMDH1 and recombinant expression carrier thereof

Technical field

The present invention relates to a kind of malate dehydrogenase gene RGMDH1 and recombinant expression carrier thereof, be specifically related to viscous red ferment The cDNA of female (Rhodotorula glutinis) YM25079 total serum IgE reverse transcription is template, and amplification obtains encoding malate dehydrogenation The gene of enzyme (MDH), is cloned into abduction delivering after coli expression carrier, and affinity chromatography obtains pure enzyme after purification, And this enzyme has been carried out the correlational study of enzyme activity determination and zymologic property, belong to genetic engineering and enzyme engineering field.

Background technology

Malic dehydrogenase (MDH) is distributed widely in organism, is the enzyme that a kind of activity is the strongest, and it is catalyzed oxalyl Acetate and the mutual conversion reaction of malate are relevant to the oxidoreduction of dinucleotide coenzyme.Oxaloacetate is in many Metabolic pathway all plays an important role, including tricarboxylic acid cycle, glyoxylate bypass, Amino acid synthesis, gluconeogenesis etc., and maintains Redox equilibrium, moreover it is possible to promote kytoplasm and the exchange of subcellular organelle metabolite.According to the function difference of organism, histological difference, Its expression kind of the difference of intracellular targeting is different, and MDH has multiple isozyme form.CyMD (cMDH) is deposited In being cell cytoplasm, it is responsible for and NADH is proceeded to mitochondrial important task, and also regulation and control tricarboxylic acid cycle is had effect, simultaneously CMDH or an ingredient of nucleic acid path (NACh) complex.

Malic dehydrogenase (MDH) is widely distributed in animal tissue, microorganism and plant.It is that a kind of activity is the strongest Enzyme, according to Subcellular Localization, malic dehydrogenase can be divided into 5 types, be present in glyoxalic acid body, mitochondrion, peroxidase precursor, In chloroplast, cytoplasm and trypanosomicide glycerol body.MDH is polymer enzyme, the dimer being made up of same or similar subunit or The tetramer, the molecular weight of subunit is that 30-35kDa, MDH also cause increasing concern such as to utilize genetic engineering in terms of medical science Vaccine prevention human body taeniasis has been the research direction received much concern, by the biology of Bovine luteinizing hormone MDH gene Bioinformatics analysis, it is predicted that endochylema type MDH is a potential diagnostic antigen, this grinds at diagnosis, medicine and vaccine for band cestode 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 inravascular coagulation), myocardial infarction, acute, chronic hepatitis etc..At field of food, malic dehydrogenase is used In the mensuration of organic acid content, the mensuration of the materials such as acid as bright in L-Herba Marsileae Quadrifoliae, acetic acid, citric acid, application prospect is extensive.Utilize at the bottom of MDH Thing specificity, also can use it for splitting D, L MALIC ACID enzyme.In a word, MDHs is as the key of organism central metabolism approach Enzyme, to it, oneself has carried out relatively broad research both at home and abroad, and MDHs isozyme is just being applied to biological classification, species differentiation, heredity The researchs such as variation, species hybridization and ontogeny.Therefore the physio-biochemical characteristics of MDHs, structure and function, catalysis are understood in depth Mechanism, for expression, purification and the Immunogenicity of enzyme recombiant protein, inquires into the metabolism and of MDHs in organism The molecule mechanism of causing a disease of a little diseases has great significance.The applied research of MDH also will promote MDHs transgenic plant simultaneously And the further development of chiral drug.

Summary of the invention

It is an object of the invention to provide what one separated from rhodotorula glutinis (Rhodotorula glutinis) YM25079 Malate dehydrogenase gene RGMDH1, this gene nucleotide series as shown in SEQ ID NO:1 or the fragment of this nucleotide sequence, Or the nucleotide sequence complementary with SEQ ID NO:1, this gene order a length of 978bp(base), the aminoacid of this gene code Sequence polypeptide as shown in SEQ ID NO:2 or its fragment.

Another object of the present invention is to provide the recombinant expressed of a kind of malate dehydrogenase gene RGMDH1 containing and having separated Carrier, by gene shown in SEQ ID NO:1 directly with different expression vectors (plasmid, virus or carrier) connection constructed by Recombinant vector.

Another object of the present invention be to provide a kind of recombinant expression carrier containing this malate dehydrogenase gene RGMDH1 or Host cell E. coli (Escherichia coli) the bacterial strain BL21 of above-mentioned recombinant expression carrier.

Can be with this with nucleotide sequence of the present invention or the recombinant vector transformed host cell containing nucleotide sequence Method known to the technical staff in field is carried out.When host is prokaryote such as escherichia coli, use CaCl₂, the side such as electroporation Method is carried out.When host is eukaryote, can be selected for the methods such as DNA infection protocol, microinjection, electroporation, liposome packaging.

The nucleotide sequence that the present invention provides is a kind of malate dehydrogenase gene efficient, specific, can by its with Carrier converts to microbial cell body production malic dehydrogenase after connecting, have product specificities high, with short production cycle, raw Produce and do not affected and utilize different strains and culture medium to be suitable for exploitation commercialization malic dehydrogenase by place, weather, season Etc. advantage；Present invention application technique for gene engineering builds specificity and produces the transgenic escherichia coli production Herba Marsileae Quadrifoliae of malic dehydrogenase Fruit acid dehydrogenase, has simple to operate, low cost, feasibility advantages of higher, produces for malate dehydrogenase gene through engineering approaches and establishes Fixed basis.

Accompanying drawing explanation

Fig. 1 is the escherichia coli utilizing the rhodotorula glutinis YM25079 malate dehydrogenase gene RGMDH1 of the present invention to build Recombinant expression plasmid pET32aRGMDH1 plasmid map；

Fig. 2 is malate dehydrogenase gene RGMDH1 abduction delivering the SDS-PAGE analysis chart after purification of the present invention, wherein: 1 is protein electrophoresis Marker；2 for converted pET32a (+) and through IPTG induction e. coli bl21 total protein；3 for converting PET32aRGMDH1 through the e. coli bl21 total protein of IPTG induction；4 is the destination protein band of purification.

Detailed description of the invention

With embodiment, the present invention is described in further detail below in conjunction with the accompanying drawings, but scope is not limited to Described content, the reagent used in embodiment and method, if no special instructions, all use conventional reagent and use conventional method.

Embodiment 1: the clone of rhodotorula glutinis malate dehydrogenase gene RGMDH1

Use OMEGA test kit E.Z.N.A Fungal RNA Kit from rhodotorula glutinis (Rhodotorula glutinis) YM25079 extracts total serum IgE, with Reverse Transcription 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 are 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 ol/L) 5 μ L

cDNA 1μL

RGMDHF1(10 μm ol/L) 2 μ L

RGMDHR1(10 μm ol/L) 2 μ L

Fast Pfu DNA polymerase(5U/ μ L) 2 μ L

Aseptic ddH₂O complements to 50 μ L；

Amplification condition: 94 DEG C of degeneration 4min, then carry out 30 circulations with 94 DEG C of 45s, 56 DEG C of 45s, 72 DEG C of 90s, last 72 DEG C 10min, takes product 1 μ L, then in the agarose gel that concentration is 1%, carries out electrophoretic analysis after having reacted.Through gel imaging After system imaging confirms that clip size is correct, purify with hundred Tyke Bioisystech Co., Ltd many kinetic energy DNA and reclaim test kit and return Receive purpose fragment, then the genes of interest that PCR amplification obtains is connected on pMD18-T, connect product and convert bacillus coli DH 5 α, screens with the LB solid plate containing ampicillin (Amp+), and the transformant on picking flat board carries out bacterium colony PCR sieve Select positive colony, be then sent for Shanghai raw work order-checking.Sequencing result result shows, it is thus achieved that the sequence of one section of 978bp length, named RGMDH1, sequence composition nucleotide sequence as shown in SEQ ID NO:1.

Embodiment 2: the structure of recombinant expression plasmid pET32aRGMDH1

Using the cDNA in embodiment 1 is that template carries out PCR amplification, reaction the primer combination, reactive component and amplification condition 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 L) composition is as follows:

5×Fast Pfu Buffer 10μL

DNTP(2.5 mol/L) 5 μ L

cDNA 1μL

RGMDHF2(10 mol/L) 1 μ L

RGMDHR2(10 mol/L) 1 μ L

Fast Pfu DNA polymerase(5U/ L) 1 μ L

Aseptic ddH₂O complements to 50 μ L；

Amplification condition: 94 DEG C of degeneration 4min, then carry out 30 circulations with 94 DEG C of 45s, 59 DEG C of 45s, 72 DEG C of 2min, last 72 ℃ 10min;The PCR primer and the plasmid pET-32a that take purification use BamH I and Xho I enzyme action overnight respectively, 50 μ l PCR primer Reaction system: PCR primer 25 μ L, 10 × Tango Buffer 10 μ l, BamH I 2 μ l and Xho I 1.5 μ L, double with sterilizing Steaming water polishing, 37 DEG C of enzyme action are overnight.50 μ l plasmid pET-32a reaction systems: plasmid pET-32a 15 μ l, 10 × Tango Buffer 10 μ l, BamH I 2 μ l and Xho I1.5 μ L, with the distilled water polishing of sterilizing, 37 DEG C of enzyme action are overnight.Electrophoresis inspection enzyme Cut product, and reclaim test kit with gel and digestion products is purified and reclaims.Linked system (10 μ L): the PCR of purification produces Thing and expression vector pET-32a press 7:1 sample-adding, T4DNA ligase 0.5 μ L, T4 Buffer 1 μ L, and 16 DEG C connect overnight.Take Connect product to proceed in bacillus coli DH 5.After 37 DEG C of shaken cultivation 1.5h, the culture fluid coating LB culture medium containing ammonia benzyl is put down Plate, cultivates 12h in 37 DEG C of incubators, the transformant on picking flat board carries out bacterium colony PCR, screening positive clone, builds and obtains weight The group named pET32aRGMDH1 of expression plasmid, this plasmid map is as shown in Figure 1.

Embodiment 3: malate dehydrogenase gene RGMDH1 abduction delivering in e. coli bl21

1, the abduction delivering of malate dehydrogenase pheron RGMDH1 and purification

In order to verify the activity of this gene coded protein, 1 μ g recombiant plasmid pET32aRGMDH1 is added 50 μ L escherichia coli In BL21 competent cell, by after whole system ice bath 30min in 42 DEG C of thermal shock 90s, again ice bath 2min, then will even Junctor system draws and adds to 950 μ L LB fluid mediums, 37 DEG C, 100rpm oscillation incubation 1h.Hatch after end in 5000rpm is centrifuged 10 min, coats the LB containing ampicillin (Amp+) solid after leaving about 80 μ L supernatant suspension precipitations Body flat board, is inverted for 37 DEG C and cultivates 10h.

Picking positive transformant in 100 mL LB (containing 100 μ g/mL ammonia benzyl mycins) culture medium, 37 DEG C of shaken cultivation mistakes At night, the bacterium solution of enrichment being inoculated in 1L LB fluid medium in 1% ratio, in 37 DEG C, 160rpm cultivates to OD600 value about 0.8.Taking 5ml bacterium solution as blank, remaining adds IPTG to final concentration of 1mmol/L, lures in 15 DEG C of constant-temperature table 80rpm Leading cultivation 8 hours, 12000 rpm are centrifuged 15min and collect thalline.SDS-PAGE analyzes display, and it is big that pET32aRGMDH1 converts Enterobacteria gives expression to the albumen (see Fig. 2 swimming lane 3) that a molecular weight is about 50kD, but at empty carrier pET32a(+) convert Escherichia coli do not have (see Fig. 2 swimming lane 2).

It is suspended in this thalline further and (makes the OD of bacteria suspension in right amount₆₀₀≈ 20) 30 mM imidazole buffer in, on ice Sonicated cells, 4 DEG C, 14000 rpm be centrifuged 15 min.The supernatant miniature membrane filtration of 0.2 μm after being centrifuged, filter Liquid is splined on the His Trap HP post (1 ml, GE Healthcare) balanced with 30mM imidazole buffer, uses 200mM miaow Azoles buffer carries out eluting, and eluent centrifuge tube is collected in order, elution samples SDS-PAGE electrophoresis detection, it is thus achieved that is pure 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, along with product SHENGCAO ethyl acetoacetic acid and NADH.Owing to the enzyme work of MDH concentration change with product NADH within certain response time is line Sexual relationship, so the activity of MDH can be measured by the concentration change of detection NADH.With malic acid and NAD(+) it is that substrate adds Malic dehydrogenase reacts, and measures enzyme with ultraviolet spectrophotometer and live at 340nm.MDH enzyme live calculating:

Unit definition: enzyme activity unit refers to enzyme amount when 25 DEG C needed for generation 1 molNADH per minute.

Malic dehydrogenase enzyme computing formula alive:

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-----reaction solution cumulative volume (ml)

The absorbance of the NADH that ε-----measures at 340nm is 6.42

D-----optical path length (1cm) (cuvette diameter)

Vs-----enzyme liquid amasss (ml)

C-----protein concentration (mg/ml)

The change of absorbance at 340nm in Δ e/ Δ t----1min

Df----dilution gfactor

Result shows, the enzyme work of purified malic dehydrogenase RGMDH1 is 318.93 U/mg, shows that gene recombined vector exists In e. coli bl21, abduction delivering malic dehydrogenase RGMDH1 out has the activity of malic dehydrogenase.

Sequence table

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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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Ser Asn Pro Val Asn Ser Thr Val Pro Ile Ala Ala Glu Val Leu Lys Ala Ala Gly Val

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Phe Asp Pro Lys Arg Val Phe Gly Val Thr Thr Leu Asp Val Val Arg Ala Ser Thr MET

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Claims (2)

1. a malate dehydrogenase gene RGMDH1, its nucleotide sequence as shown in SEQ ID NO:1, the ammonia of this gene code Base acid sequence is as shown in SEQ ID NO:2.

2. the recombinant expression carrier containing malate dehydrogenase gene RGMDH1 described in claim 1.