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Preparation method of recombinant sialic acid excision enzyme, expression gene, recombinant expression vector and construction method

A technique for expressing genes and exonucleases, which is applied in the field of preparation of recombinant exosialidases, which can solve the problems of stable expression of exosialidases and achieve the effects of enhanced soluble expression and simple construction

Pending Publication Date: 2021-05-14
SHANGHAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, there is no scheme for stably expressing exosialidase using prokaryotic expression system

Method used

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  • Preparation method of recombinant sialic acid excision enzyme, expression gene, recombinant expression vector and construction method
  • Preparation method of recombinant sialic acid excision enzyme, expression gene, recombinant expression vector and construction method
  • Preparation method of recombinant sialic acid excision enzyme, expression gene, recombinant expression vector and construction method

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Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Preparation method, expression gene, recombinant expression vector and vector construction method of recombinant exosialidase AMUC_0625

[0041] 1. Primer design

[0042] Query the Genbank database Amuc_0625 gene sequence (NM_053615) in the National Center for Biotechnology Information (National Center for Biotechnology Information), remove the C-terminal signal peptide through bioinformatics analysis, design PCR primers, and add NdeI digestion at the 5' end of Amuc_0625 site, a 6×HisTag sequence and a PstI restriction site were added to the 3' end to amplify the Amuc_0625 gene (SEQ ID NO: 1).

[0043] Upstream primer 5'-CATATGAGCGCCGGGGAGGGTAATCCC-3' (SEQ ID NO: 3)

[0044]Downstream primer 5'-CTGCAGCTACTTGAGAACAGGAGC-3' (SEQ ID NO: 4)

[0045] 2. The cultivation of Ekmanmyxobacteria and the acquisition of RNA

[0046] Inoculate Ekmanmyxobacterium in a special medium, and the medium composition is shown in Table 1 and Table 2 below, cultured to OD600=1 und...

Embodiment 2

[0115] Example 2 Detection of activity and biochemical indicators of recombinant protein AMUC_0625

[0116] Using 4MU-NANA (4-methylumbelliferyl-α-acetylneuraminic acid) as the substrate of exosialidase AMUC_0625 to detect its activity, AMUC_0625 can hydrolyze 4MU-NANA to generate 4MU and acetylneuraminic acid, wherein 4MU is a fluorescent dye with a maximum absorption wavelength of 365nm and a maximum emission wavelength of 450nm. After drawing the 4MU standard curve, detect the 4MU fluorescence value, which can reflect the hydrolysis rate of AMUC_0625 4MU-NANA.

[0117] The reaction system is as follows:

[0118] 1mM 4MU-NANA 20μl

[0119] 48 μg / ml AMUC_0625 1 μl

[0120] Reaction buffer 79μl;

[0121] After reacting at room temperature for 20 min, 200 μl of reaction termination solution (100 mM glycine / sodium hydroxide, pH 10.7) was added to terminate the reaction, and the fluorescence value was detected. Under the same reaction system, different reaction temperatures ...

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Abstract

The invention discloses a preparation method of recombinant sialic acid excision enzyme, an expression gene, a recombinant expression vector and a construction method. The expression gene comprises a nucleotide sequence as shown in SEQ ID No.1, a restriction enzyme cutting site NdeI, a restriction enzyme cutting site PstI and 6*HisTag located at the carboxyl terminal of recombinant protein. The recombinant sialic acid excision enzyme AMUC_0625 gene disclosed by the invention can realize massive soluble expression in escherichia coli, and the expressed sialic acid excision enzyme AMUC_0625 has specific biological activity on sialic acid, can be used for degrading alpha-sialic acid at the tail end of a glycoprotein carbohydrate chain, and has important significance on biological pharmacy and clinical treatment; and the recombinant protein AMUC_0625 provided by the invention is a novel sialic acid excision enzyme with an application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method, expression gene, recombinant expression vector and construction method of a recombinant exosialidase. Background technique [0002] Sialic acid is a general term for a class of acidic amino sugars containing 9 carbon atoms and a pyranose structure. It is a natural carbohydrate with more than 50 derivatives, mainly including three types: ① N-acetylneuraminic acid ( Neu5Ac), ②N-hydroxyneuraminic acid (Neu5Gc), ③keto-deoxynonylonic acid (KDN). The so-called sialic acid mostly refers to N-acetylneuraminic acid, usually in the form of α-glycoside at the non-reducing end of glycoprotein or glycolipid. Sialic acid is directly or indirectly involved in a variety of cellular activities, including 1) itself as a recognized receptor, 2) intercellular information transmission, hormones, lectins, antibodies and other molecules are ligands of sialic acid, 3) regulation of im...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/62C07K19/00C12N15/70C12N15/66C12R1/19
CPCC12N9/2402C12Y302/01018C12N15/70C12N15/66C07K2319/21C07K2319/24
Inventor 孙正龙顾依然关淼
Owner SHANGHAI UNIV
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