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Marine low-temperature MDH (malate dehydrogenase) gene and recombinant expression vector thereof

A malate dehydrogenase and expression vector technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology and other directions, can solve the problems of cumbersome extraction process and low enzyme activity, and achieve high product specificity and short production cycle. Effect

Inactive Publication Date: 2019-01-08
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of raw materials is low and the source is wide, but the extraction process is cumbersome and the enzyme activity of the product is low

Method used

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  • Marine low-temperature MDH (malate dehydrogenase) gene and recombinant expression vector thereof
  • Marine low-temperature MDH (malate dehydrogenase) gene and recombinant expression vector thereof
  • Marine low-temperature MDH (malate dehydrogenase) gene and recombinant expression vector thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Construction of MDH expression vector for malate dehydrogenase gene of Oceanobacillus picturae strain R-5321

[0024] (1) Amplification of marine cryogenic EMDH

[0025] First, using the genomic DNA of Oceanobacillus picturae strain R-5321 as a template, primers were designed at a certain length from both ends of the marine low-temperature malate dehydrogenase gene (MDH), and PCP amplification was performed to obtain the gene. Named EMDH. The gene fragment contains the complete sequence of the MDH gene, with a sequence length of 939 bp. The designed primer sequences are as follows:

[0026] EMDH-F:5'-ATGGCTATTAAAAGAAGTAAAATATCAGTAATCGGATCAGG-3'

[0027] EMDH-R:5'-GCAGATTCAGTTAAAAATGTCCTAAGTATATTGGGA-3'

[0028] The composition of PCR amplification system (50 μL) is as follows:

[0029]

[0030] Make up to 50μL of sterile ddH2O;

[0031] Amplification conditions: Stage 1: 95°C (5min); Stage 2: 94°C (30s), 52°C (1min), 72°C (3min), 30Reps;

[0032] Stage 3: 72°C...

Embodiment 2

[0051] Inducible expression and purification of marine low temperature malate dehydrogenase gene MDH in host cell C43(DE3)

[0052] 1. Inducible expression and purification of marine low temperature malate dehydrogenase protein MDH

[0053] (1) Preparation with kanamycin 32Y medium: [0.8% (w / v) peptone, 3.2% (w / v) yeast extract, 100 mmol / L NaCl, 10 mmol / L Tris-HCl, pH 7.4] .

[0054] (2) E. coli C43 (DE3) carrying the pET-28a-MDH plasmid was inoculated into the medium. Culture on a constant temperature shaker (30°C, 220 r / min). Take the bacterial solution to measure the OD600, when it reaches 0.25-0.35, add 0.1 mmol / LIPTG (isopropyl-β-d-thiogalactoside), and place it on a constant temperature shaker (20°C, 220r / min, 24h) to induce expression.

[0055] (3) The cultured cells were collected by centrifugation at 8000×g, suspended with PBS buffer, and centrifuged at 8000×g again. Repeat 2 times to wash the cells. Weigh the cells, resuspend each 2g of wet cells with 10-15ml PB...

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Abstract

The invention belongs to the field of bioengineering and relates to a marine low-temperature MDH (malate dehydrogenase) gene and a recombinant expression vector thereof. The low-temperature MDH gene is isolated from Oceanobacillus picturae strain R-5321 and encodes low-temperature MDH, the nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the gene isshown in SEQ ID NO: 2. A marine low-temperature MDH expression vector pET-28a-MDH is successfully constructed with an RF (restriction-free) cloning method. The expression vector is electrically transferred into host cell C43 (DE3) and induced to express marine low-temperature MDH protein by IPTG. An expressed product has a marine low-temperature MDH function and can catalyze conversion of malic acid into oxaloacetic acid.

Description

technical field [0001] The invention relates to the field of biological engineering, in particular to a marine low-temperature malate dehydrogenase gene and a recombinant expression vector thereof. Background technique [0002] MDH (Marine Low Temperature Malate Dehydrogenase), as a key enzyme in the central metabolic pathway of organisms, has important research value in genetic variation and ontogeny, and is widely used in clinical diagnosis and industrial testing. The market demand is increasing day by day. huge. Since the 1950s, some scholars have isolated MDH from animal myocardium. At this stage, the commercial sources of MDH on the market are mainly extracted from the cardiac muscle, liver, and skeletal muscle of pigs, rabbits, and cattle. The raw material cost is low and the source is wide, but the extraction process is cumbersome and the product enzyme activity is low. Therefore, it is particularly important to expand new sources. And marine microorganisms are a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12Y101/01037
Inventor 张庆芳肖景惠王梦雨迟乃玉
Owner DALIAN UNIV
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