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Sialidase gene recombinant expression vector and construction method thereof and sialidase and preparation method thereof

A technology of sialidase and gene recombination, which is applied in recombinant DNA technology, biochemical equipment and methods, and the introduction of foreign genetic material using vectors. question

Active Publication Date: 2017-06-30
INST OF DEEP SEA SCI & ENG CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been no reports on the research on sialidase derived from deep-sea invertebrate symbiotic microorganisms. Due to the special ecological environment of the deep sea (low temperature, high pressure and oligotrophic, etc.) and the lack of sampling techniques and tools, it is relatively difficult to capture organisms, prompting There are very limited reports of sialidases from deep-sea invertebrate commensal microorganisms, so little is known about sialidases from this source

Method used

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  • Sialidase gene recombinant expression vector and construction method thereof and sialidase and preparation method thereof
  • Sialidase gene recombinant expression vector and construction method thereof and sialidase and preparation method thereof
  • Sialidase gene recombinant expression vector and construction method thereof and sialidase and preparation method thereof

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preparation example Construction

[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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Abstract

A preparation method of sialidase comprises the following steps: preparation of a sialidase gene fragment; cloning of the sialidase gene fragment into pMD-18T to obtain a connection product, conversion of the connection product into E.coliJM109, and selection of a positive clone for sequencing to obtain a gene sequence as shown in SEQIDNO:1; connection of the positive clone and a prokaryotic expression vector to obtain a recombinant expression vector; conversion of the recombinant expression vector to obtain recombinant engineering bacteria; fermentation, induced expression and purification of the recombinant engineering bacteria to obtain the sialidase. The preparation method of the sialidase comprises bacterial genome DNA extraction, PCR amplification, construction of the recombinant expression vector, conversion of the recombinant expression vector into host cells for construction of recombinant expression cells, culture and induced expression of the recombinant expression cells, isolation and purification of protease from the cultured host cells, and analysis of protease enzymatic properties. The sialidase prepared by the preparation method has high temperature resistance and Ph resistance, and provides more basic materials and scientific bases for industrial and social application.

Description

technical field [0001] The invention relates to the technical field of expression vector construction methods, in particular to a sialidase gene recombinant expression vector and a construction method thereof, sialidase and a preparation method thereof. Background technique [0002] Sialic acid is a kind of acidic amino sugar with 9 carbon atoms and pyranose structure, widely exists in animal tissues and microorganisms, and is an important component of cell membrane glycoproteins and glycolipids. Sialic acid generally does not exist in a free form. It usually combines with biomacromolecules such as glycoproteins and glycolipids on the cell surface to form a conjugate, and exists at the end of the conjugate. It plays an important role in physiological and biochemical processes such as intermolecular interactions. More than 50 kinds of sialic acid with different structures have been found in nature, and the most important and most researched sialic acid is N-acetylneuraminic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N9/24
CPCC12N9/2402C12N15/66C12N15/70C12Y302/01018
Inventor 王少露贺丽生王勇
Owner INST OF DEEP SEA SCI & ENG CHINESE ACADEMY OF SCI
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