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Coding gene of swainsonine degrading enzyme and application thereof

A technology encoding gene and swainsonine, applied in the fields of biotechnology and enzyme genetic engineering, can solve the problem of not being a long-term solution

Inactive Publication Date: 2015-08-19
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is still no specific cure for locopoisoning. The only and most effective way to prevent it is to avoid direct contact between animals and locoweeds. However, this method of prevention and control is not a long-term solution.

Method used

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  • Coding gene of swainsonine degrading enzyme and application thereof
  • Coding gene of swainsonine degrading enzyme and application thereof
  • Coding gene of swainsonine degrading enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Arthrobacter sp. HW 08 Extraction of total DNA (Genomic DNA).

[0020] 1.1 Arthrobacter Expansion of sp. HW 08

[0021] Stored at -70°C under sterile conditions Arthrobacter sp.HW 08 was picked up with an inoculation needle and streaked on Luria-Bertani (LB) solid medium, cultured at 30°C for 48 h. A single colony was picked and transferred to LB medium for enrichment at 200 r / min and 30°C for 16 h. Then transfer 10% of the volume into inorganic salt culture solution containing 5% SW at 200 r / min, 30°C for 24 h, and take 500 μL of the supernatant to detect SW content by thin-layer chromatography (to verify whether HW08 bacteria can degrade SW ). The LB solid / liquid medium described here are common terms, media, techniques and methods for microbial research, for details, please refer to "Molecular Cloning Experiment Guide" (J. Sambrook, et al.). Unless otherwise specified, the techniques, methods, culture media, reagents, and medicines used in microbi...

Embodiment 2

[0025] Example 2 Obtaining of SW degrading enzyme coding gene AAur_2040

[0026] After analyzing the differential proteins obtained by Label Free proteomics, some differential protein genes were selected for analysis experiments, and a gene capable of degrading SW was screened out.

[0027] The primer sequence of gene AAur_2040 is:

[0028] F 5'-CCG GAATTC ATGCCTACCGCTAATGCTT-3'

[0029] (underlined as EcoR I restriction site, the gene sequence is SEQ ID No. 3);

[0030] R 5'- CCC AAGCTT CTAGCCGATAGTGGAGACGT-3'

[0031] (underlined as Hind III restriction site, the gene sequence is SEQ ID No. 4)

[0032] 50 μL amplification system: 10×LA PCR Buffer II (Mg 2+ Plus) 5 μL; dNTP Mixture (2.5mM) 8 μL; primers (20 μM) 2 μL each; total DNA obtained in Example 1 2 μL; LA Taq enzyme 0.5 μL; ddH 2 O 30.5 μL. Reaction parameters: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 49.5°C for 30 sec, extension at 72°C for 1 min, 35 cycles of ...

Embodiment 3

[0036] Example 3 Expression of SW degrading enzyme AAur_2040 gene in Escherichia coli

[0037] 3.1 Construction of expression vector

[0038] The carrier pET32α (+) containing AAur_2040 gene in embodiment 2 is transformed into competent E.coilBL 21(DE3) expression bacteria were spread on the selective plate medium containing 50 μg / mL ampicillin for overnight culture, and 3 single colonies were randomly picked and inoculated in 3 LB liquid medium (containing 50 μg / mL ampicillin) in a test tube, cultivate until the bacterial density reaches OD 600 When it is 0.6, extract the plasmid and use EcoR I and hand Ⅲ double enzyme digestion ( figure 2 ) and sequencing methods to detect transformation results. Escherichia coli successfully transformed into the pET32α(+)-AAur_2040 plasmid is a positive transformant, which is E.coil BL 21(DE3) / pET32α(+) / AAur_2040.

[0039] 3.2 Induced expression

[0040] Will E.coil BL 21(DE3) / pET32α(+)-AAur_2040 transformants were cultured i...

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Abstract

The invention relates to a coding gene of swainsonine degrading enzyme and application thereof, belonging to the fields of biotechnology and enzyme gene engineering. The coding gene of swainsonine degrading enzyme (gene AAur_2040) has a nucleotide sequence as shown in SEQ ID No. 1. A protein coded by the coding gene has an amino acid sequence as shown in SEQ ID No. 2. According to invention, the gene segment-- AAur_2040 responsible for degradation of swainsonine is cloned from Arthrobacter sp. HW 08 capable of effectively degrading swainsonine. The coding gene of swainsonine degrading enzyme and application thereof can provide effective biotechnological means for degradation of swainsonine and are of important significance to restoration of poisoning of animals caused by intake of locoweed.

Description

technical field [0001] The invention belongs to the field of biotechnology and enzyme gene engineering, and relates to a degrading enzyme coding gene capable of degrading swainsonine (Swainsonine, SW for short) and its application. Background technique [0002] Locoweed (Locoweed) is a legume of the genus Oxytropis ( Oxytropis ) and Astragalus ( Astragalus ) is a general term for poisonous plants, which are the most serious poisonous weeds in the world that endanger the sustainable development of grassland animal husbandry production. In my country, locoweeds are mainly distributed in western regions such as Xinjiang, Tibet, Qinghai, Gansu, Ningxia, Shaanxi, Inner Mongolia, Shanxi and Sichuan. Cattle, horses, sheep, goats, antelopes, camels, red deer, donkeys, mules, pigs and other animals can cause poisoning after eating locoweed. Animals eating locoweed can cause symptoms of poisoning in varying degrees. The clinical manifestations are characterized by nervous system ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/11C12N15/70C12N1/21A23L1/015A23L5/20
Inventor 王妍李勤凡翟阿官王建华
Owner NORTHWEST A & F UNIV
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