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Mutant heparinase I, encoding nucleotide sequence thereof, recombinant carrier and host cell with nucleotide sequence and application

A technology of nucleotide sequence and recombinant vector, applied in the field of molecular biology, can solve the problems of poor stability of heparinase I, decrease, and the yield is difficult to exceed 10%, etc.

Pending Publication Date: 2019-10-18
宝锐生物科技泰州有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the stability of the existing heparanase I is very poor, and the existing heparanase I is stored in a liquid form at 4°C, and the activity is reduced to half of the original within a short period of time. After one freeze-thaw and one freeze-drying, its activity can only maintain 45% and 25% of the original
Moreover, the active loss of the enzyme in the current preparation method is very large, and the yield is difficult to exceed 10%. Therefore, it is very necessary and necessary to research and develop a mutant heparanase I with strong stability by means of molecular biology. urgent

Method used

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  • Mutant heparinase I, encoding nucleotide sequence thereof, recombinant carrier and host cell with nucleotide sequence and application
  • Mutant heparinase I, encoding nucleotide sequence thereof, recombinant carrier and host cell with nucleotide sequence and application
  • Mutant heparinase I, encoding nucleotide sequence thereof, recombinant carrier and host cell with nucleotide sequence and application

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preparation example Construction

[0050] According to one aspect of the present invention, a method for preparing mutant heparanase I, the preparation method comprises the following steps:

[0051] (a), first synthesizing the nucleotide sequence encoding the above-mentioned mutant heparanase I, and then combining the nucleotide sequence with the eukaryotic recombinant expression vector to obtain the recombinant vector;

[0052] (b) Transforming the recombinant vector into host cells, followed by inducing expression to obtain mutant heparanase I.

[0053] The preparation method of the mutant heparanase I provided by the present invention, the preparation method combines the synthesized nucleotide sequence encoding the above mutant heparanase I with the eukaryotic recombinant expression vector to obtain a recombinant vector by means of genetic engineering , and transfer the recombinant vector into host cells to induce expression, and obtain mutant heparanase Ⅰ. The preparation method can produce mutant heparana...

Embodiment 1

[0057] Example 1 Synthesis of Mutant Heparanase I Nucleotide Sequence and Construction of Expression Vector

[0058] (a), search the UniProt library to obtain the protein sequence of Flavobacterium heparinus heparanase I (AccessionNo.Q05819), and enter the sequence into the codon bias analysis tool Gene designer software;

[0059] (b) According to the codon usage preference of Pichia pastoris in the Pichia pastoris codon preference data table, the protein sequence is reverse-translated into a DNA sequence, so that the codons of the DNA sequence are all preferred by Pichia pastoris (using online codon optimization tool for verification). At the same time, we input the protein sequence from amino acid 22 to amino acid 384 of Q05819 into the online tool "PeptideCutter" to predict potential protease cleavage sites;

[0060] (c) Perform mutation transformation on potential protease cleavage sites, specifically, site-directed mutation of Gln at position 2 of the existing heparanase...

Embodiment 2

[0063] The electrotransformation of embodiment 2 expression vector

[0064] 1. First prepare GS115 competent cells:

[0065] (1) Pick a single colony of yeast GS115 from the YPD plate, inoculate it in 5ml of YPD liquid medium, and culture overnight at 29°C with shaking at 180rpm; inoculate 1mL of the overnight cultured GS115 bacterial liquid into 100mL of YPD medium at 29°C , Shake culture at 180rpm until the OD600 value is about 1.3-1.5;

[0066] (2) Take 50mL of GS115 bacteria solution, centrifuge at 4°C, 4000rpm for 5min to collect the bacteria, discard the supernatant, and absorb the residual liquid; add 40mL of ice-cold sterile water to wash the bacteria, and centrifuge at 4°C, 4000rpm for 5min , discard the supernatant; and repeat this step once;

[0067] (3) Add 10 mL of ice-cold sterile water to wash the cells, centrifuge at 4° C. 4000 rpm for 5 min, discard the supernatant; resuspend the cells with 2 mL of ice-pre-cooled 1 M sorbitol, and centrifuge at 4° C. 4000 rp...

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Abstract

The invention provides mutant heparinase I, an encoding nucleotide sequence thereof, a recombinant carrier and host cell with the nucleotide sequence and application, and relates to the technical field of molecular biology. By means of the mutant heparinase I, mutation is conducted on a protease restriction enzyme cutting site, probably influencing the stability of the heparinase I, of an amino acid sequence of existing heparinase I, and specifically, the Gln fixed point in the 2nd site of the amino acid sequence of the existing heparinase I is mutated into His, and the Glu fixed point in the78th site is mutated into Met. The mutant heparinase I obtained after mutation is higher in stability than existing heparinase I under the condition of not influencing the activity of the heparinase I.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a mutant heparanase I and its coding nucleotide sequence, a recombinant vector including the nucleotide sequence, a host cell and application. Background technique [0002] Heparinase I (heparinase I) is a kind of lyase that can degrade heparin substances. Heparanase I destroys the basic structure of the extracellular matrix and basement membrane by hydrolyzing the heparin side chain of heparan sulfate proteoglycan, releases and activates the active substances connected to the heparin side chain, and plays an important role in angiogenesis, tumor metastasis, and inflammation. and other pathological processes are closely related. It has important uses in preparing low molecular weight heparin, eliminating heparin anticoagulant in extracorporeal circulation, and determining the precise structure of heparin. However, the expression of existing natural heparinase I needs ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/81C12N1/19C12R1/84C12R1/865
CPCC12N9/88C12N15/81C12N15/815C12Y402/02007
Inventor 严俊刘涛卿小红
Owner 宝锐生物科技泰州有限公司
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