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

Abstract

The invention discloses a nucleotide sequence encoding malate dehydrogenase isolated from Rhodotorula viscosus YM25079, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO : As shown in 2, by constructing a recombinant vector and expressing it in Escherichia coli, the expression product has the function of malate dehydrogenase, which can catalyze the conversion of malic acid into oxaloacetic acid.

Description

A malate dehydrogenase gene RGMDH1 and its recombinant expression vector

technical field

The invention relates to a malate dehydrogenase gene RGMDH1 and its recombinant expression vector, in particular to cDNA reverse-transcribed from the total RNA of Rhodotorula glutinis (Rhodotorula glutinis) YM25079, amplified to obtain a gene encoding malate dehydrogenase (MDH ) gene, which was cloned into an Escherichia coli expression vector and induced to express, purified by affinity chromatography to obtain a pure enzyme, and the enzyme activity assay and enzymatic properties of the enzyme were carried out. It belongs to genetic engineering and enzyme engineering field.

Background technique

Malate dehydrogenase ( MDH ) is widely distributed in organisms and is a very active enzyme, which catalyzes the mutual conversion reaction of oxaloacetate and malate, and is related to the redox of dinucleotide coenzymes. Oxaloacetate plays an important role in many metabolic pathways, including tricarboxylic acid cycle, glyoxylate bypass, amino acid synthesis, gluconeogenesis, etc., and maintains redox balance, and can also promote cytoplasmic and subcellular organelle exchange of metabolites. According to the different functions of organisms, tissue differences, and different intracellular locations, its expression types are different, and MDH has a variety of isozyme forms. Cytoplasmic malate dehydrogenase (cMDH) exists in the cytoplasm of cells and is responsible for transferring NADH to mitochondria, and also plays a role in regulating the tricarboxylic acid cycle. At the same time, cMDH is also a member of the nucleic acid pathway (NACh) complex component.

Malate dehydrogenase (MDH) is widely distributed in animal tissues, microorganisms and plants. It is the most active enzyme. According to subcellular localization, malate dehydrogenase can be divided into 5 types, which exist in glyoxysomes, mitochondria, peroxisomes, chloroplasts, cytoplasm and trypanoglycerol. MDH is a multimeric enzyme, which is a dimer or tetramer composed of the same or similar subunits. The molecular weight of the subunit is 30-35kDa. MDH has also attracted more and more attention in medicine, such as the use of genetic engineering vaccines The prevention of human taeniasis has been a research direction that has attracted much attention. Through the bioinformatics analysis of the MDH gene of the Asian subspecies of Taenia saginata, it has been predicted that the cytoplasmic MDH is a potential diagnostic antigen, which provides a basis for the diagnosis and drug use of Taenia saginata. And the application prospects in vaccine research provide important clues. It is used in multi-enzyme analysis and early diagnosis of diseases in clinical diagnosis, such as for the diagnosis of DIC (disseminated intravascular coagulation), myocardial infarction, acute and chronic hepatitis, etc. In the field of food, malate dehydrogenase is used for the determination of organic acid content, such as the determination of L-malic acid, acetic acid, citric acid and other substances, and has a wide application prospect. Taking advantage of the substrate specificity of MDH, it can also be used to resolve D,L-malic enzyme. In short, MDHs, as the key enzymes of the central metabolic pathways of organisms, have been extensively studied at home and abroad. MDHs isoenzymes are being used in the studies of biological taxonomy, species differentiation, genetic variation, species hybridization and individual development. Therefore, an in-depth understanding of the physiological and biochemical characteristics, structure and function, and catalytic mechanism of MDHs is of great significance for the expression, purification, and analysis of immune characteristics of enzyme recombinant proteins, and for exploring the metabolism of MDHs in organisms and the molecular pathogenic mechanisms of some diseases. At the same time, the application research of MDH will also promote the further development of MDHs transgenic plants and chiral drugs.

Contents of the invention

The object of the present invention is to provide a malate dehydrogenase gene RGMDH1 isolated from Rhodotorula glutinis YM25079, the nucleotide sequence of which is shown in SEQ ID NO: 1 or a fragment of the nucleotide sequence , or a nucleotide sequence complementary to SEQ ID NO: 1, the gene sequence is 978 bp (base), and the amino acid sequence encoded by the gene is the polypeptide shown in SEQ ID NO: 2 or a fragment thereof.

Another object of the present invention is to provide a recombinant expression vector containing the isolated malate dehydrogenase gene RGMDH1, which is obtained by directly linking the gene shown in SEQ ID NO: 1 with different expression vectors (plasmids, viruses or carriers). The constructed recombinant vector.

Another object of the present invention is to provide a recombinant expression vector containing the malate dehydrogenase gene RGMDH1 or the host cell Escherichia coli strain BL21 of the recombinant expression vector.

Transformation of host cells with the nucleotide sequence of the present invention or a recombinant vector containing the nucleotide sequence can be carried out by methods well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, methods such as CaCl ₂ and electroporation are used. When the host is a eukaryote, methods such as DNA transfection, microinjection, electroporation, and liposome packaging can be used.

The nucleotide sequence provided by the present invention is a high-efficiency and specific malate dehydrogenase gene, which can be transformed into microbial cells to produce malate dehydrogenase after being connected with a carrier, and has high product specificity and short production cycle. Short, production is not affected by the site, climate, season, and the use of different strains and culture media is suitable for the development of commercial malate dehydrogenase and other advantages; the present invention uses genetic engineering technology to construct a transgene that specifically produces malate dehydrogenase The production of malate dehydrogenase by Escherichia coli has the advantages of simple operation, low cost and high feasibility, and lays the foundation for the genetically engineered production of malate dehydrogenase.

Description of drawings

Fig. 1 is the recombinant expression plasmid pET32aRGMDH1 plasmid map of Escherichia coli constructed by Rhodotorula viscosus YM25079 malate dehydrogenase gene RGMDH1 of the present invention;

Fig. 2 is the SDS-PAGE analysis graph after the induction expression of malate dehydrogenase gene RGMDH1 of the present invention and purification, wherein: 1 is protein electrophoresis Marker; Protein; 3 is the total protein of Escherichia coli BL21 transformed with pET32aRGMDH1 and induced by IPTG; 4 is the purified target protein band.

detailed description

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail, but protection scope of the present invention is not limited to described content, reagent and method used in the embodiment, if no special instructions, all adopt conventional reagent and use conventional method.

Example 1: Cloning of Rhodotorula viscosus malate dehydrogenase gene RGMDH1

Total RNA was extracted from Rhodotorula glutinis YM25079 using the OMEGA kit E.Z.N.A Fungal RNA Kit, cDNA was synthesized using the reverse transcription kit Thermo Scientific Maxima H Minus FirstStrand cDNA Synthesis Kit, and 1 μl was used as a template for polymerase chain reaction . Design primers (primer 1 and primer 2) for PCR amplification. The primers, components and amplification conditions used in the reaction are as follows:

Primer P1: RGMDHF1: 5`-ATGGGCCTCAAGACTGCTGTTCTC-3` (SEQ ID NO: 3)

Primer P2: RGMDHR1: 5`-TTACTGCTTCATGAAGTTCTGAC-3` (SEQ ID NO: 4)

The composition of the PCR amplification system (50 μL) is as follows:

5×Trans PFU Buffer 10μL

dNTP (2.5μmol/L) 5μL

cDNA 1 μL

RGMDHF1 (10 μmol/L) 2 μL

RGMDHR1 (10 μmol/L) 2 μL

Fast Pfu DNA polymerase (5U/μL) 2μL

Make up to 50 μL with sterile ddH ₂ O;

Amplification conditions: Denaturation at 94°C for 4 minutes, followed by 30 cycles at 94°C for 45s, 56°C for 45s, and 72°C for 90s, and finally at 72°C for 10 minutes. , for electrophoretic analysis. After confirming that the size of the fragment is correct by gel imaging system imaging, the target fragment was recovered with the multi-kinetic DNA purification and recovery kit of Biotech Biotechnology Co., Ltd., and then the target gene obtained by PCR amplification was connected to pMD18-T, and the ligated product was transformed into large intestine Bacillus DH5α was screened with LB solid plates containing ampicillin (Amp+), and the transformants on the plate were picked for colony PCR to screen positive clones, and then sent to Shanghai Sangon for sequencing. Sequencing results showed that a 978bp long sequence was obtained, which was named RGMDH1, and the sequence consisted of the nucleotide sequence shown in SEQ ID NO:1.

Example 2: Construction of recombinant expression plasmid pET32aRGMDH1

Adopt the cDNA among the embodiment 1 to carry out PCR amplification as template, the primer combination used in reaction, reaction component and amplification condition are as follows:

Primer P3: RGMDHF2: 5`-TACGGATCCATGGGCCTCAAGACTGCT-3` (SEQ ID NO: 5)

Primer P4: RGMDHR2: 5`-CGTCTCGAGCTGCTTCATGAAGTTCTG-3` (SEQ ID NO: 6)

The composition of the PCR amplification system (50 µL) is as follows:

5×Fast Pfu Buffer 10μL

dNTP (2.5 µmol/L) 5 µL

cDNA 1 μL

RGMDHF2 (10 µmol/L) 1 µL

RGMDHR2 (10 µmol/L) 1 µL

Fast Pfu DNA polymerase (5U/µL) 1µL

Make up to 50 μL with sterile ddH ₂ O;

Amplification conditions: Denaturation at 94°C for 4 minutes, followed by 30 cycles of 94°C for 45s, 59°C for 45s, and 72°C for 2 minutes, and finally 10 minutes at 72°C; the purified PCR product and plasmid pET-32a were purified with BamH I and Xho I enzymes, respectively. Cut overnight, 50μl PCR product reaction system: PCR product 25μL, 10×Tango Buffer 10μl, BamH I 2μl and Xho I 1.5μL, make up with sterilized double distilled water, digest overnight at 37°C. 50 μl plasmid pET-32a reaction system: plasmid pET-32a 15 μl, 10×TangoBuffer 10 μl, BamH I 2 μl and Xho I 1.5 μl, filled with sterilized double distilled water, digested overnight at 37°C. The digested products were checked by electrophoresis, and the digested products were purified and recovered with a gel recovery kit. Ligation system (10 μL): Purified PCR product and expression vector pET-32a were added at a ratio of 7:1, T4 DNA ligase 0.5 μL, T4 Buffer 1 μL, ligated overnight at 16°C. The ligated product was transferred into Escherichia coli DH5ɑ. After shaking culture at 37°C for 1.5h, the culture solution was coated on the LB medium plate containing ampicillin, cultured in the incubator at 37°C for 12h, the transformants on the plate were picked for colony PCR, positive clones were screened, and the recombinant expression plasmid was constructed and named as The map of pET32aRGMDH1 is shown in FIG. 1 .

Example 3: Induced expression of malate dehydrogenase gene RGMDH1 in Escherichia coli BL21

1. Induced expression and purification of malate dehydrogenase protein RGMDH1

In order to verify the activity of the protein encoded by the gene, 1 μg of the recombinant plasmid pET32aRGMDH1 was added to 50 μL of Escherichia coli BL21 competent cells, and the whole system was ice-bathed for 30 minutes, then heat-shocked at 42°C for 90 seconds, and ice-bathed for 2 minutes again, and then the connection system was absorbed and removed. Add it to 950 μL LB liquid medium, and incubate at 37° C. with shaking at 100 rpm for 1 h. After the incubation, centrifuge at 5000 rpm for 10 min, leave about 80 μL of the supernatant to suspend the precipitate, and spread it on the LB solid plate containing ampicillin (Amp+), and incubate it upside down at 37°C for 10 h.

Pick positive transformants in 100 mL LB (containing 100 μg/mL ampicillin) culture medium, shake culture at 37 °C overnight, inoculate the enriched bacterial liquid into 1L LB liquid medium at a ratio of 1%, and inoculate at 37 °C , cultivated at 160rpm until the OD600 value was about 0.8. Take 5ml of the bacterial solution as a blank control, and add IPTG to the rest to a final concentration of 1mmol/L, induce culture on a constant temperature shaker at 15°C at 80rpm for 8 hours, and centrifuge at 12000rpm for 15min to collect the bacteria. SDS-PAGE analysis showed that a protein with a molecular weight of about 50kD was expressed in Escherichia coli transformed with pET32aRGMDH1 (see lane 3 in Figure 2), but not in Escherichia coli transformed with empty vector pET32a (+) (see lane 2 in Figure 2) .

Further, the cells were suspended in an appropriate amount (the OD ₆₀₀ of the bacterial suspension ≈ 20) of 30 mM imidazole buffer, the cells were ultrasonically disrupted on ice, and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant after centrifugation was filtered with a 0.2 μm micro-filter, and the filtrate was loaded on a His Trap HP column (1 ml, GE Healthcare) that had been equilibrated with 30 mM imidazole buffer, and eluted with 200 mM imidazole buffer. The eluate was collected sequentially in a centrifuge tube, and the eluted sample was detected by SDS-PAGE electrophoresis to obtain a pure protein band (see lane 4 in Figure 2).

2. Enzyme activity determination of malate dehydrogenase RGMDH1

Malate dehydrogenase is a key enzyme regulating malate metabolism, which can catalyze the dehydrogenation and oxidation of malate, accompanied by the production of oxaloacetate and NADH. Since the enzymatic activity of MDH has a linear relationship with the concentration change of the reaction product NADH within a certain reaction time, the activity of MDH can be determined by detecting the concentration change of NADH. Malic acid and NAD (+) were used as substrates to add malate dehydrogenase for reaction, and the enzyme activity was measured at 340nm with a UV spectrophotometer. Calculation of MDH enzyme activity:

Unit definition: One unit of enzyme activity refers to the amount of enzyme required to generate 1 µmol NADH per minute at 25°C.

Calculation formula of malate dehydrogenase enzyme activity:

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 of NADH measured at ε-----340nm is 6.42

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

Vs-----enzyme solution volume (ml)

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

Δe/Δt----change of absorbance at 340nm within 1min

df----dilution factor

The results showed that the enzyme activity of the purified malate dehydrogenase RGMDH1 was 318.93 U/mg, indicating that the malate dehydrogenase RGMDH1 induced and expressed by the gene recombinant vector in Escherichia coli BL21 had malate dehydrogenase activity.

sequence listing

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Claims (2)

1. A malate dehydrogenase gene RGMDH1, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 2. 2. A recombinant expression vector containing the malate dehydrogenase gene RGMDH1 according to claim 1.