Sialidase gene recombinant expression vector and its construction method, sialidase and its preparation method
A sialidase and gene recombination technology, which is applied in recombinant DNA technology, biochemical equipment and methods, and the introduction of foreign genetic material using vectors. question
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[0034] The preparation method of the sialidase of one embodiment, comprises the steps:
[0035] S10. Prepare a recombinant expression vector for the sialidase gene, that is, a recombinant engineered bacterium.
[0036] In S10, the construction method of the sialidase gene recombinant expression vector comprises the following steps:
[0037] S110. Prepare a sialidase gene fragment.
[0038] The method for preparing the sialidase gene fragment is as follows: the nucleotide sequence of the sialidase gene (NanA) is obtained by cloning from the stomach of the sialidase gene. Using PowerLyzer R The Powersoil DNA isolation kit extracted the genomic DNA of the gastrointestinal microbes of the giant king worm as a template for polymerase chain reaction to obtain the sialidase gene fragment.
[0039] Wherein, the primers used in polymerase chain reaction are as follows:
[0040] SEQ ID NO: 2
[0041] Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3';
[0042] SEQ ID NO: 3
[0043] ...
Embodiment 1
[0079] Example 1: The nucleotide sequence of the sialidase gene (NanA) was cloned from the gut of the phalaenopeda. Using PowerLyzer R The Powersoil DNA isolation kit extracts the genomic DNA of the gastrointestinal microbes of the phalaenopsis, takes 1 μL as a template for polymerase chain reaction (PCR), and designs primers (primer F and primer R) in combination with the carrier used. The primers, group The separation and amplification conditions were as follows:
[0080] Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3'
[0081] Primer R: 5'-GTCGACTTATGAAAGATATTTATCAATTATGTT-3'
[0082] PCR reaction system: 10 μL 5×Buffer, 4 μL dNTP, 0.5 μL PrimeSTAR HS DNA Polymerase, 1 μL primer F, 1 μL primer R, 1 μL template, 32.5 μL H 2 O, the total volume is 50 μL.
[0083] The PCR amplification conditions are as follows:
[0084] Pre-denaturation at 98°C for 10 seconds, denaturation at 98°C for 10 seconds, annealing at 50°C for 15 seconds, extension at 72°C for 1 minute, three ste...
Embodiment 2
[0085] Embodiment 2: Construction of recombinant expression vector
[0086]For the fragment amplified in Example 1, the 5' end of primer F contains a BamHI restriction site (base 1 to base 6), and the 5' end of primer R contains a SalI restriction site (from the 1st base to the 6th base), so use these two enzymes to double-digest the fragment, and pET-30a is double-digested with the same enzyme, and the digested fragment is recovered by electrophoresis, and connected with T4 Enzymes were ligated at 16°C for 2 hours to construct a recombinant expression vector. The ligation product was transformed into Escherichia coli BL21(DE3), cultured on LB solid plates containing ampicillin (100 μg / mL), and the white colonies growing on the plate were picked, and screened by colony PCR and extracted plasmid single and double enzyme digestion A positive clone was obtained and identified by sequencing, and an E. coli host cell containing the sialidase gene was successfully constructed.
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