High-temperature recombinant xylanase as well as coding gene and application thereof
A technology of xylanase and xylan, which is applied in the field of genetic engineering, can solve the problems of lack of high-yield strains and achieve the effect of strong heat resistance
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Embodiment 1
[0022] Example 1 Xylanase gene xyn10A-01 preparation of
[0023] It can be prepared artificially as follows: xyn10A-01 genes, can also be Thermotoga thermarum The total DNA was obtained as a template by means of PCR. xylanase gene in this study xyn10A-01 Synthesized by Shanghai Jierui Bioengineering Co., Ltd., the gene sequence is shown in SEQ ID NO.2
Embodiment 2
[0024] Example 2 Xylanase gene xyn10A-01 subclones of
[0025] Use the following primers to PCR amplify the xylanase gene in embodiment 1:
[0026] Primer 1: 5'-GGAATTCCATATGGCAGTTGTGGCAAACTACGATTTTGA-3';
[0027] Primer 2: 5'-CCGCTCGAGCTCCGGCTTAACCAGAGCCCAATATGCCA-3.
[0028] When the above primers were synthesized, primer 1 was introduced Nde I restriction site, primer 2 introduction xho I restriction site.
[0029] PCR reaction system: 1 μL template DNA, 1 μL primer 1, 1 μL primer 2, 25 μL Premix ExTaq, 22 μL ultrapure water.
[0030] PCR reaction conditions: Denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.
[0031] The yield and specificity of the PCR products were detected by 1% agarose gel electrophoresis, and purified with a PCR product recovery kit (BIOMIGA, Shanghai).
Embodiment 3
[0032] Example 3 Recombinant cloning, expression vector pET-20b- xyn10A-01 build and verify
[0033] The purified PCR product and pET-20b were used respectively Nde I and xho I double enzyme digestion, agarose electrophoresis recovery restriction enzyme digestion PCR and large vector fragments. After the gel-tapping recovery, the target fragment and the carrier were concentrated and resuspended in 8 μL sterile water, 1 μL 10× Ligase Buffer and 1 μL Ligase were added, and ligated overnight at 16°C. Transform Escherichia coli pET-20b with the ligation reaction product, and then spread it on a petri dish containing 100 μg / mL Amp (ampicillin), and incubate at 37°C for 10-12h.
[0034] Pick multiple single colonies from the transformation plate, and use BIOMIGA's plasmid mini-extraction kit to extract the plasmid. The obtained plasmid was verified by double enzyme digestion and the obtained recombinant plasmid was sequenced. The sequencing results showed that the cloned ta...
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