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Method for producing DSPA alpha 1 by yeast expression system

An expression system, the technology of Pichia pastoris, applied in the field of biomedicine, can solve the problems of unsuitability for industrial production and no successful expression of DSPAα1, and achieve the effect of low cost, suitable for industrial production, and easy technology

Inactive Publication Date: 2009-07-22
QILU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

For thrombolytic proteins, tPA was previously reported to be expressed in Pichia pastoris, but it was not suitable for industrial production due to its low enzyme activity; in 2007, it was reported that DSPAα2 was expressed in Pichia pastoris, and its expression level was only 25mg / L, but so far there is no report on the successful expression of DSPAα1 in Pichia pastoris

Method used

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  • Method for producing DSPA alpha 1 by yeast expression system
  • Method for producing DSPA alpha 1 by yeast expression system
  • Method for producing DSPA alpha 1 by yeast expression system

Examples

Experimental program
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Effect test

Embodiment 1

[0049] 1. Gene synthesis of DSPAα1 gene and Pichia pastoris preferred codon sequence

[0050] Synthesize the nucleotide sequence of the DSPAα1 gene or optimize the nucleotide sequence of the DSPAα1 gene according to the preferred codon sequence of Pichia pastoris.

[0051] On the basis of not changing the amino acid sequence of DSPAα1, the whole gene sequence of DSPAα1 was synthesized according to the preferred codons of Pichia pastoris, and the XhoI restriction site and the cleavage signal amino acid codon ( AAAAGA), while introducing EcoRI or NotI restriction sites at the 3' end, the nucleotide sequence of the synthetic Pichia pastoris preferred codon-optimized DSPAα1 gene is shown in SEQ ID NO.1 (synthetic work provided by Shanghai Sangon Bioengineering Technology Services Co., Ltd.).

[0052] 2. Construction of recombinant plasmids

[0053] The steps are as follows: use restriction endonuclease XhoI and EcoRI to double digest the DSPAα1 synthetic gene and pPIC9 expressio...

Embodiment 2

[0077] 1. Gene synthesis of DSPAα1 gene and Pichia pastoris preferred codon sequence

[0078] Step is with embodiment 1.

[0079] 2. Construction of recombinant plasmids

[0080] The constructed pPIC9K-DSPAα1 was digested with XhoI and NotI, and the fragment containing DSPAα1 was recovered and ligated with the expression plasmid pPICZαA digested with the same enzyme using T4 DNA ligase to construct the recombinant plasmid pPICZαA-DSPAα1.

[0081] 3. Pichia pastoris strain transformation and plate screening

[0082] SalI linearized the plasmid pPICZαA-DSPAα1 and the blank plasmid pPICZαA, and took 5 μg to 20 μg of the plasmid to transform Pichia pastoris GS115 competent cells by electroporation.

[0083] The parameters of the GenepulsterII electroporation instrument (Bio-Rad laboratories Inc., Hercules, CA) used were: voltage 1.5kV, capacitance 25μF, resistance 200Ω, and discharge time 4.0ms. Immediately after electric shock, add 1.0 mL of ice-cold 1.0 mol / L sorbitol, and in...

Embodiment 3

[0094] 1. Gene synthesis of DSPAα1 gene and Pichia pastoris preferred codon sequence

[0095] Step is with embodiment 1.

[0096] 2. Construction of recombinant plasmids

[0097] The constructed pPIC9K-DSPAα1 was digested with XhoI and NotI, and the fragment containing DSPAα1 was recovered and ligated with the expression plasmid pGAPαA digested with the same restriction enzyme using T4 DNA ligase to construct the recombinant plasmid pGAPαA-DSPAα1.

[0098] 3. Pichia pastoris strain transformation and plate screening

[0099] The SalI linearized plasmid pGAPαA-DSPAα1 and the blank plasmid pGAPαA, the transformation of Pichia pastoris strain GS115 and the plate screening are the same as in Example 2 for plate screening.

[0100] 4. Fermentation screening of Pichia pastoris recombinant strains

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Abstract

The invention discloses a DSPA-Alpha1 production method that utilizes a pichia pastoris expression system and comprises the steps as follows: (1) a nucleotide sequence of a DSPA-Alpha1 gene is synthesized or optimized according to a preferred codon sequence of pichia pastoris; (2) a recombinant expression plasmid X-DSPA-Alpha1 is constructed by utilizing the double-digestion synthesized or optimized DSPA-Alpha1 gene sequence; (3) a pichia pastoris strain cell and a positive flat filtering transformant are transformed; (4) a recombinant pichia pastoris strain is fermented and filtered; and (5) the separated and purified DSPA-Alpha-1 gene in a supernatant is expressed by the recombinant pichia pastoris strain. The DSPA-Alpha1 production method of the invention has low cost, short period and easy technical implementation, the shaking flask fermentation can reach the expression amount of 75mg / L, primary industrial production level is achieved, and the separated and purified DSPA-Alpha1 gene can reach the final purity of over 97 percent, and can be directly used in the reagent research and preparation.

Description

technical field [0001] The invention relates to a production method of a plasminogen activator, in particular to a method for producing a recombinant vampire bat plasminogen activator desmoteplase α1 (DSPAα1) using a yeast expression system, and belongs to the technical field of biomedicine. Background technique [0002] Desmoteplase (DSPA) is a plasminogen activator isolated from the saliva of a vampire bat in South America. It mainly includes four proteins: DSPAα1, DSPAα2, DSPAβ and DSPAγ. It was found that DSPAα1 has the best biochemical and pharmacological properties, so it was further studied. Studies have shown that: under the stimulation of fibrin, the activity of DSPAα1 increases by 10500 times, while that of tissue plasminogen activator (tPA) is only 550 times; in the presence of fibrin and fibrinogen, the ratio of DSPAα1 activity is 12900 times , while tPA is only 72-fold, DSPAα1 has a higher fibrin selectivity. Pharmacological studies have shown that in the mode...

Claims

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Application Information

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IPC IPC(8): C12N15/58C12N15/81C12N1/19C12N9/48C12R1/84
Inventor 李剑凤武国栋王庆民闫岩孙丽霞王晶翼
Owner QILU PHARMA
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