Tissue-type plasminogen activator mutant and application thereof

A plasminogen and mutant technology, applied in the field of biomedicine, can solve the problems of reduced tPA drug titer, insufficient resistance, and no ability to enhance plasminogen activation, and achieve enhanced plasminogen activation. effect of ability

Active Publication Date: 2018-03-06
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the stimulation of thrombin, platelets are continuously activated, releasing a large amount of PAI-1, which will also greatly reduce the potency of tPA drugs in the blood
Although a TNK-tPA mutant with the ability to resist PAI-1 has been developed. However, the KHRR contained in the TNK-tPA mutant 296-299 Mutations to AAAA were not sufficiently resistant to PAI-1 and did not appear to enhance plasminogen activation

Method used

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  • Tissue-type plasminogen activator mutant and application thereof
  • Tissue-type plasminogen activator mutant and application thereof
  • Tissue-type plasminogen activator mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Construction, expression and purification of embodiment 1 tPA (A146Y) mutant

[0021] The tPA(A146Y) here is based on the hydrolase domain (tPA-SPD) of tPA for A146Y mutation, also referred to as tPA(A146Y), or tPA-SPD(A146Y) (the amino acid naming method is Chymotrypsinogen numbering):

[0022] (1) Construction of tPA-SPD-pPICZαA plasmid.

[0023] Using human hepatocyte cDNA as a template, the tPA-SPD gene fragment was amplified by PCR. Cut the tPA-SPD fragment with restriction endonuclease XhoI and SacI, and cut the pPICZαA plasmid (pPICZαA plasmid was purchased from Invitrogen) with the same endonuclease XhoI and SacI, and connect the tPA-SPD fragment to the pPICZαA plasmid with T4 ligase middle. The enzyme-linked product was transformed into Escherichia coli DH5α after heat stimulation at 42°C, plated, picked a single colony, and carried out gene sequencing. The DH5α strain containing the correct tPA-SPD sequence was expanded and cultivated, and the ZDNA plasmid s...

Embodiment 2

[0037] Example 2 Detection of tPA(A146Y) Mutant Enzyme Activity

[0038] 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 tPA protein is added. Then add the luminescent substrate S2288 (Chromogenix), tPA can specifically recognize the enzyme cutting site and cut off its chromophore-p-nitroaniline (pNA), and finally detect the absorbance value at 405nm by a microplate reader. The activity of the tPA enzyme can be assayed.

[0039] Specific measurement process:

[0040] (a) Materials

[0041] tPA, tPA(A146Y), obtained in Example 1 above, tPA substrate S-2288.

[0042] Buffer: 20mM Tris-HCl pH7.4, 150mM NaCl, 0.2% BSA. 0.22μm pore size membrane filter.

[0043] (b) steps

[0044] The mas...

Embodiment 3

[0049] Example 3 Detection of tPA(A146Y) Activation Ability to Natural Substrate Plasminogen

[0050] Measured with 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 tPA and plasminogen (PLG for short) are added, tPA activates PLG to generate plasmin (Pn for short), and then Pn-specific As for the luminescent substrate S-2403, Pn can specifically recognize the cleavage site and cut off its chromophore-p-nitroaniline (pNA), but tPA will not digest the luminescent substrate S2403. Finally, the activating ability of tPA to PLG can be determined by detecting the absorbance value at 405 nm with a microplate reader.

[0051] Specific measurement process:

[0052] (a) Materials

[0053] tPA obtained in Example 1 above, tPA (A146...

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Abstract

The invention provides a tissue-type plasminogen activator mutant and application thereof. The mutant comprises an A146 locus which is a brand new mutation site, referring to substitution, deletion oraddition of at least one or more amino acid residues comprising the A146. The mutant disclosed by the invention has the effects of obviously resisting ability inhibited by an endogenous inhibitor (PAI-1) (Plasminogen Activator Inhibitor 1) and enhanced ability of activating plasminogen. The mutant can be applied to preparing medicines for treating thrombotic diseases comprising acute myocardial infarction, acute pulmonary embolism, cerebral apoplexy, phlebothrombosis and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a tissue-type plasminogen activator mutant and application thereof. Background technique [0002] With the rapid development of society and economy, people's quality of life is improving day by day. Among them, high-sugar, high-protein, and high-fat foods account for an increasing proportion of people's daily nutritional intake, which leads to the formation of diseases such as hypertension, hyperlipidemia, coronary heart disease, myocardial infarction, and cerebral infarction, and affects the entire human body. Health poses a huge threat. At present, cardiovascular disease has become the number one killer threatening human health, especially thromboembolic disease. It mainly includes three categories: (1) coronary artery thrombosis, mainly acute myocardial infarction (AMI); (2) cerebrovascular thrombosis, namely acute ischemic stroke; (3) venous thrombosis, such as acute ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48A61K38/49A61P7/02A61P9/10A61P11/00
CPCA61K38/00C12N9/6459C12Y304/21068
Inventor 黄明东彭双周袁彩雪光浦李金宇
Owner FUZHOU UNIV
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