Dehalogenase DhmB, encoding gene and application thereof
A technology for encoding genes and dehalogenases, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of not easy rapid degradation, threats to human and animal health, etc., and achieve broad industrial application prospects, easy purification, and good stability. Effect
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Embodiment 1
[0050] Embodiment 1, the acquisition of DhmB protein and its coding gene
[0051] Using the genomic DNA of Huangia manganoxydans (Huangia manganoxydans) 8047CGMCC No.6697 as a template, PCR amplification was performed with primers DhmBF and DhmBR to obtain PCR amplification products. The primer sequences are as follows (the underlined sequence is the restriction site):
[0052] DhmBF: 5′-G GAATTCCATATGATGCCCGCTGTCTACGTCTTC-3′;
[0053] DhmBR: 5′-CCC AAGCTT TCAGTCCGCGCGCAGGCTGATC-3'.
[0054] The PCR amplification product was recovered and cloned into pGEM-T vector to obtain the recombinant vector, which was transformed into Escherichia coli DH5α and then sent for sequencing.
[0055] Sequencing results show that: the nucleotide sequence of the PCR amplification product is shown in sequence 1 in the sequence listing, the gene shown in the sequence is named DhmB, the protein encoded by the gene is named DhmB, and the amino acid sequence of DhmB is shown in the sequence lis...
Embodiment 2
[0056] Example 2, the acquisition of dehalogenase DhmB and its functional verification
[0057] 1. Dehalogenase acquisition
[0058] 1. Obtaining the recombinant vector
[0059] Use restriction endonucleases to Nde I and Hind III to perform double enzyme digestion on the PCR amplification product obtained in the above example 1 and the pET-28a(+) vector, and connect to obtain the recombinant vector pET-DhmB, and submit it for sequencing .
[0060] Sequencing results show that: pET-DhmB replaces the DNA between the Nde I and Hind III sites of the pET-28a (+) vector with the DhmB gene shown in sequence 1 in the sequence table, and keeps the other elements of the pET-28a (+) vector Recombinant vector obtained with unchanged sequence. The amino acid sequence of the protein encoded by the DhmB gene is shown in sequence 2 in the sequence listing.
[0061] 2. Acquisition of recombinant bacteria
[0062] The recombinant vector pET-DhmB obtained in step 2 is transformed into Esche...
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