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Acetaldehyde dehydrogenase recombinant gene as well as lactic acid bacteria vector and application thereof

An acetaldehyde dehydrogenase and recombinant gene technology, which is applied in the field of acetaldehyde dehydrogenase recombinant gene and its lactic acid bacteria carrier, can solve problems such as unsuitable health food preparation, and achieve the effects of reducing acetaldehyde concentration and prolonging half-life

Pending Publication Date: 2020-07-31
广州辉园苑医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2005, Qiu Lizhen realized the heterologous high-efficiency expression of the acetaldehyde dehydrogenase gene in Escherichia coli BL21, and the specific activity of the recombinant acetaldehyde dehydrogenase reached 331.7U / mg (reference: Qiu Lizhen. Human acetaldehyde dehydrogenase 2 Gene cloning and expression. Zhejiang University 2005.), but in view of the food safety problems of Escherichia coli, it is not suitable for the preparation of health food
At present, there is no application research on the expression of acetaldehyde dehydrogenase using lactic acid bacteria engineering bacteria

Method used

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  • Acetaldehyde dehydrogenase recombinant gene as well as lactic acid bacteria vector and application thereof
  • Acetaldehyde dehydrogenase recombinant gene as well as lactic acid bacteria vector and application thereof
  • Acetaldehyde dehydrogenase recombinant gene as well as lactic acid bacteria vector and application thereof

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Effect test

Embodiment 1

[0042] This embodiment provides a recombinant gene encoding human recombinant acetaldehyde dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO:1. The artificial synthesis method of this recombinant gene is as follows:

[0043] 1) Obtain the ALDH2 gene sequence (No. CR45699) from the NCBI database, use Signal P 3.0Server online analysis website to analyze and according to the codon preference of lactic acid bacteria, replace the codons in the ALDH2 gene sequence that are used less frequently in the target lactic acid bacteria Synthesize optimized ALDH2-CO (Codonoptimized) enzyme gene for codons with high frequency usage.

[0044] 2) Introduce restriction endonuclease KpnI and EcoNI digestion sites at the 5' end and 3' end of the ALDH2-CO enzyme gene respectively, and obtain the ALDH2 target gene through PCR amplification, agarose gel electrophoresis and gel recovery in sequence fragment.

[0045] Among them, the specific operation of PCR amplification is as ...

Embodiment 2

[0050] This embodiment provides a lactic acid bacteria engineering bacterium pNZ8148-coALDH2, which is a lactic acid bacteria expression vector expressing the recombinant gene of Example 1. The establishment method of the vector is as follows: figure 2 As shown, specifically: NZ9000 lactic acid bacteria were bathed in ice water until they melted. In a 200 μL system, the OD 600nm of the bacterial solution was 0.4 and 10 μg of plasmid, and mixed by pipetting. After 15 minutes of ice-water bath, the mixture was transferred to an electric cup and continued to ice-bath for 15 minutes. Electroporation conditions were voltage 1400V / cm, resistance 400Ω, capacitance 25μF, ice-water bath immediately after electroporation for 5ms; electroporation product 200μL was added with a final concentration of 20mM MgCl 2 and 2mM CaCl 2 Incubate in 800 μl GM17 liquid medium at 30°C for 2 hours. Spread the bacteria solution on the MRS medium plate containing 10 μg / ml chloramphenicol and wait for t...

Embodiment 3

[0052] Fermentation of the starting strain (NZ3900), the control strain (pNZ8148-ALDH2), and the codon-optimized strain (pNZ8148-coALDH2) obtained in Example 2, and enzyme activities under different conditions.

[0053] The construction method of the control strain (pNZ8148-ALDH2) is basically the same as that of the above pNZ8148-coALDH2, the difference is that the pNZ8148-ALDH2 vector is used, and the codons of ALDH2 are not optimized.

[0054] The starting strain NZ3900, the control strain (pNZ8148-ALDH2), and the codon-optimized strain (pNZ8148-coALDH2) were inoculated in the MRS lactic acid bacteria medium respectively, and activated at 30°C, and then the three bacterial solutions were simultaneously transferred to fresh yeast Extract the peptone glucose medium for cultivation, and the specific operation steps are as follows:

[0055] (1) Activation of bacteria

[0056] Take 30 μL of the original bacteria and genetically engineered bacteria stored in glycerol tubes and a...

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Abstract

The invention provides an acetaldehyde dehydrogenase recombinant gene as well as a lactic acid bacteria vector and application thereof. The nucleotide sequence of the recombinant gene is as shown in SEQ ID NO: 1. The amino acid sequence of encoded human-derived acetaldehyde dehydrogenase is shown as SEQ ID NO: 2. The artificial synthesis method of the recombinant gene comprises the following steps: carrying out codon optimization on an ALDH2 gene sequence obtained by an NCBI database to obtain an ALDH2-CO (Codon optimized) enzyme gene; separately introducing restriction endonucleases cutting sites KpnI and EcoNI to the 5'end and the 3 'end of the ALDH2-CO enzyme gene, and performing PCR amplification, agarose gel electrophoresis and gel recovery in sequence to obtain an ALDH2 target gene segment; and connecting the ALDH2 target gene segment to a lactic acid bacteria expression vector, and converting the gene segment into lactic acid bacteria cells. The invention provides the novel recombinant acetaldehyde dehydrogenase gene and lactic acid bacteria expressing the recombinant gene, and provides a new way and theoretical basis for research and development of hangover alleviating products.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a recombinant gene of acetaldehyde dehydrogenase and its lactic acid bacteria carrier and application. Background technique [0002] In organisms, acetaldehyde is a highly reactive and highly toxic metabolite of ethanol, which has been shown to be carcinogenic in experimental animals. Ethanol and its metabolite acetaldehyde are mainly absorbed in the digestive tract, where the rich blood circulation The aerobic bacteria, facultative anaerobic bacteria and digestive tract endothelial cells colonized here can quickly oxidize and metabolize ethanol to acetaldehyde, but their ability to metabolize acetaldehyde is poor. Therefore, the digestive tract becomes an important accumulation site of acetaldehyde in the body. For example, in the colorectum, the intestinal bacteria are highly enriched and there are many types. The alcohol dehydrogenase activi...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/74C12N1/21C12R1/01
CPCC12N9/0008C12Y102/0101C12N15/74
Inventor 黄新珠邹翠云江福能张蓓钟惟德朱建国贺俊波
Owner 广州辉园苑医药科技有限公司
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