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Design and application of reteplase mutant

A technology of reteplase and mutants, applied to medical preparations containing active ingredients, enzymes, peptidases, etc., can solve problems such as insufficient resistance, enhanced plasminogen activation ability, and lack of expression, and achieve The effect of excellent thrombolytic effect

Active Publication Date: 2019-12-31
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tenecteplase resistance was not sufficient and did not appear to enhance plasminogen activation

Method used

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  • Design and application of reteplase mutant
  • Design and application of reteplase mutant
  • Design and application of reteplase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction, expression and purification of reteplase mutants

[0023] (1) pPICZα-K 2 Construction of SPD plasmid

[0024] Using human hepatocyte cDNA as a template (NCBI Reference Sequence: NC_000008.11), PCR was used to amplify K 2 SPD gene fragment. Cut K with restriction endonucleases XhoI and AgeI 2 SPD fragment, and pPICZαA plasmid was cut with the same endonuclease XhoI and AgeI (pPICZαA plasmid was from Invotrogen Company), K 2 The SPD fragment was ligated into pPICZαA plasmid. Transform the enzyme-linked product into Escherichia coli DH5α after heat shock at 42°C, smear a plate, pick a single colony, and perform gene sequencing. 2 The DH5α strain of SPD sequence was expanded and cultivated, and pPiczαA-K was extracted by EZNA plasmid mini-extraction kit (OMEGA) 2 SPD plasmid for use in the following experiments.

[0025] K 2 SPD gene fragment sequence is as follows:

[0026] tcttaccaagggaattgctactttgggaatgggtcagcctaccgtggcacgcacagcctcaccgagt...

Embodiment 2

[0042] Example 2 Activity Detection of Reteplase Mutants

[0043] Enzyme activity was determined by the reported chromogenic assay [Gorlatova NV (2003). Mapping of aconformational epitope on plasminogen activator inhibitor-1 by randommutagenesis. Implications for serpin function. J Biol Chem.]. The reaction principle is as follows, in a reaction system with a volume of 200 µL, a certain concentration of reteplase mutant is added. Then add the luminescent substrate S2288 (Chromogenix), reteplase and its mutants can specifically recognize the enzyme cutting site and cut off its chromophore-p-nitroaniline (pNA). The activity of reteplase can be determined by detecting the absorbance value at 405 nm with a microplate reader.

[0044] Specific measurement process:

[0045] (a) Materials

[0046] Reteplase, reteplase mutant, and tPA substrate S2288 obtained in Example 1 above.

[0047] Buffer: 20 mM Tris-HCl pH7.4, 150 mM NaCl, 0.2% BSA. 0.22 μm pore size membrane filter.

[00...

Embodiment 3

[0054] Example 3 Detection of reteplase mutants' ability to activate natural substrate plasminogen

[0055] Measured by the reported chromogenic assay [Gorlatova NV (2003). Mapping of aconformational epitope on plasminogen activator inhibitor-1 by randommutagenesis. Implications for serpin function. J Biol Chem.]. The reaction principle is as follows. In a reaction system with a volume of 200 µL, a certain concentration of reteplase and its mutants and plasminogen (Plasminogen, PLG for short), K 2 SPD activates PLG to generate plasmin (plasmin, referred to as Pn), and then adds Pn-specific luminescent substrate S2403, Pn can specifically recognize the enzyme cutting site and convert its chromophore-p-nitroaniline (pNA ), but reteplase itself will not digest the luminescent substrate S2403. Finally, the activating ability of reteplase and its mutants to PLG can be determined by detecting the absorbance value at 405 nm with a microplate reader.

[0056] Specific measurement pr...

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Abstract

The invention provides a reteplase mutant and application thereof. The mutant comprises brand-new mutation sites of two amino acids in a self-shearing ring region, and substitution, deletion or addition of at least one of amino acid residues including alanine and proline is included. The mutant is named as reteplase mutant. The mutant has the abilities of remarkably resisting inhibition of an endogenous inhibitor (PAI-1) of the mutant and enhancing plasminogen activation, can be used as a thrombotic disease medicine and is used for treating various diseases including acute myocardial infarction, acute pulmonary embolism, cerebral apoplexy and venous thrombosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a reteplase mutant and application thereof. Background technique [0002] With the improvement of our people's daily life, the incidence of stroke has increased significantly. According to statistics, there are 2 million new stroke patients in China every year, and the incidence rate is as high as 1.20 / 100,000. In 2012, there were 7 million surviving stroke patients, 4.5 million of whom lost their labor force and were unable to take care of themselves to varying degrees, and the disability rate was as high as 75%. For many years, stroke has been a disease that has been paid much attention to in clinical practice. Whether it is research or clinical treatment, the attention has been focused on reducing the mortality rate, reducing complications and promoting the recovery of physical functions. In my country, according to statistics, 3 million people need thrombolytic drug t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72A61K38/48A61P7/02
CPCA61K38/00A61P7/02C12N9/6459C12Y304/21068
Inventor 黄明东陈珊莉袁彩程媛许燕艳江龙光
Owner FUZHOU UNIV
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