Multi-copy gene carrier of high efficient expression mannanase

Abstract

The present invention relates to gene engineering bacteria, and is especially one expression vector for high efficiency secreting expression on mannase gene and its multicopy body. Primer is designed according to the cloning site of Pichia yeast pAO815, the polyclonal site region of the target expression vector is replaced into Not I, XhoL I and EcoR I to form polyclonal sites for easy cloning, the alpha-factor signal peptide and the target gene are fused and connected to the expression vector for the expression vector containing the target gene to secrete and express mannase, and the restriction enzymes Bgl II and BamH I with complementary sticky ends are cleaved, linked and converted to obtain multicopy expression unit plasmid containing mannase gene. The multicopy body after transforming Pichia yeast can express mannase in high efficiency and is used to prepare mannase from konjaku meal.

Description

technical field

[0001] The invention relates to an expression carrier of genetically engineered bacteria, in particular to a multi-copy expression carrier capable of efficiently secreting and expressing the mannanase gene and the construction primers thereof. technical background

[0002] The main components of plants are cellulose, hemicellulose and lignin. Among them, mannan is the second largest component of hemicellulose, which is widely distributed. Studies have found that mannan has good physical and chemical properties such as low heat, stability, safety, and non-toxicity. It also has the functions of protecting the intestinal tract and improving immunity. It can be used as health food and fine chemical products, and has broad market prospects and commercial interests. . For this reason, in recent years, the production and application research of oligosaccharides have been carried out successively at home and abroad. At present, the functional oligosaccharide produ...

Examples

Embodiment 1

[0036] Example 1 Screening of mannanase-producing bacteria

[0037] Take the soil under the old haystacks, and carry out enrichment culture in: 2% konjac flour, 0.5% ammonium sulfate, 0.5% dipotassium hydrogen phosphate, 0.1% magnesium sulfate medium, 30°C, 180rpm. After 10 days, the supernatant was taken and diluted according to the ratio of 10 to 1000 to coat a plate, which was composed of LB medium, 0.3% konjac flour and 0.5% Trypon-blue. After static culture at 30°C for 16 hours, observe the transparent circle on the plate, and select the bacterium with a large transparent circle as the mannanase-producing strain man37.

Embodiment 2

[0038] Cloning of embodiment 2 mannanase gene

[0039] After the mannanase-producing bacteria were cultured in LB medium at 30°C overnight, the genomic DNA was extracted according to the following method: Centrifuge the cultured bacteria in a 1.5mL centrifuge tube at 12000rpm for 2-3min, Collect the cells; suspend the cells in 200 μL TE (pH 8.0), add 50 μL of 20 mg / mL lysozyme, and incubate at 37°C for 30 minutes; add 400 μL of lysis buffer, and break the cells by high-speed shaking; then add 300 μL of 5M NaCl liquid , mix well, and centrifuge at 12000rpm for 15min; transfer the supernatant to another new centrifuge tube, add an equal volume of chloroform, mix gently, and centrifuge at 12000rpm for 5min; then transfer the upper aqueous phase to another new centrifuge tube, add 2 times the volume DNA was precipitated with absolute ethanol; washed with 70% ethanol, dried and dissolved in TE for later use. The extracted DNA was partially digested with Sau3A I to obtain a series ...

Embodiment 3

[0043] Example 3 Directed Mutagenesis of Mannanase

[0044] Take known mannanase, design primers for its two ends, use mannanase as a template, add 5-bromouracil solution with a concentration of 1% to 10% in the PCR system, and use rTaq DNA polymerase to carry out PCR (conditions are 94°C for 4min; 94°C for 30s, 50°C for 30s, 72°C for 1min, 35cycles; 72°C for 5min; 4°C hold), using the mismatch generated by gene amplification in the 5-bromouracil system, the The mutation is introduced into the mannanase gene, and then screened on a plate made of LB medium, 100 μg / mL AP, 0.3% konjac flour and 0.4% Trypon-blue, and the gene with a large hydrolysis circle is selected and screened, which is the construction vector The desired expressed gene was named man37-1.

technical field

[0001] The invention relates to an expression carrier of genetically engineered bacteria, in particular to a multi-copy expression carrier capable of efficiently secreting and expressing the mannanase gene and the construction primers thereof. technical background

[0002] The main components of plants are cellulose, hemicellulose and lignin. Among them, mannan is the second largest component of hemicellulose, which is widely distributed. Studies have found that mannan has good physical and chemical properties such as low heat, stability, safety, and non-toxicity. It also has the functions of protecting the intestinal tract and improving immunity. It can be used as health food and fine chemical products, and has broad market prospects and commercial interests. . For this reason, in recent years, the production and application research of oligosaccharides have been carried out successively at home and abroad. At present, the functional oligosaccharide produ...

Examples

Embodiment 1

[0036] Example 1 Screening of mannanase-producing bacteria

[0037] Take the soil under the old haystacks, and carry out enrichment culture in: 2% konjac flour, 0.5% ammonium sulfate, 0.5% dipotassium hydrogen phosphate, 0.1% magnesium sulfate medium, 30°C, 180rpm. After 10 days, the supernatant was taken and diluted according to the ratio of 10 to 1000 to coat a plate, which was composed of LB medium, 0.3% konjac flour and 0.5% Trypon-blue. After static culture at 30°C for 16 hours, observe the transparent circle on the plate, and select the bacterium with a large transparent circle as the mannanase-producing strain man37.

Embodiment 2

[0038] Cloning of embodiment 2 mannanase gene

[0039] After the mannanase-producing bacteria were cultured in LB medium at 30°C overnight, the genomic DNA was extracted according to the following method: Centrifuge the cultured bacteria in a 1.5mL centrifuge tube at 12000rpm for 2-3min, Collect the cells; suspend the cells in 200 μL TE (pH 8.0), add 50 μL of 20 mg / mL lysozyme, and incubate at 37°C for 30 minutes; add 400 μL of lysis buffer, and break the cells by high-speed shaking; then add 300 μL of 5M NaCl liquid , mix well, and centrifuge at 12000rpm for 15min; transfer the supernatant to another new centrifuge tube, add an equal volume of chloroform, mix gently, and centrifuge at 12000rpm for 5min; then transfer the upper aqueous phase to another new centrifuge tube, add 2 times the volume DNA was precipitated with absolute ethanol; washed with 70% ethanol, dried and dissolved in TE for later use. The extracted DNA was partially digested with Sau3A I to obtain a series ...

Embodiment 3

[0043] Example 3 Directed Mutagenesis of Mannanase

[0044] Take known mannanase, design primers for its two ends, use mannanase as a template, add 5-bromouracil solution with a concentration of 1% to 10% in the PCR system, and use rTaq DNA polymerase to carry out PCR (conditions are 94°C for 4min; 94°C for 30s, 50°C for 30s, 72°C for 1min, 35cycles; 72°C for 5min; 4°C hold), using the mismatch generated by gene amplification in the 5-bromouracil system, the The mutation is introduced into the mannanase gene, and then screened on a plate made of LB medium, 100 μg / mL AP, 0.3% konjac flour and 0.4% Trypon-blue, and the gene with a large hydrolysis circle is selected and screened, which is the construction vector The desired expressed gene was named man37-1.

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