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Mutant of human hepatocyte growth factor (hHGF) and application of mutant

A technology of hepatocyte growth factor and mutant, applied in the field of host cells, can solve problems such as lack of serine protease activity

Active Publication Date: 2019-03-29
BEIJING NORTHLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is now known that the hairpin structure and the K1 region are the key sites for the binding of HGF to the receptor c-Met; the hairpin structure and the K2 region together constitute the necessary structure for the affinity of HGF with heparin and heparan sulfate; the β chain contains serine protease like fold region , but no serine protease activity

Method used

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  • Mutant of human hepatocyte growth factor (hHGF) and application of mutant
  • Mutant of human hepatocyte growth factor (hHGF) and application of mutant
  • Mutant of human hepatocyte growth factor (hHGF) and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] The preparation of embodiment 1.hHGF and its mutant

[0105] The amino acid sequence of native hHGF (SEQ ID NO: 1 ) can be found at NCBI Accession No. NP_000592.3. Using SEQ ID NO:1 as a template, the following three hHGF mutants were designed:

[0106] (1) Mutating Ser at position 130 of SEQ ID NO:1 to Arg to obtain hHGF mutant 130Arg-hHGF, the amino acid sequence of which is shown in SEQ ID NO:2;

[0107] (2) Mutating Ser at position 130 of SEQ ID NO:1 to His to obtain hHGF mutant 130His-hHGF, the amino acid sequence of which is shown in SEQ ID NO:3; and

[0108] (3) Mutating Ser at position 130 of SEQ ID NO:1 to Lys to obtain hHGF mutant 130Lys-hHGF, the amino acid sequence of which is shown in SEQ ID NO:4.

[0109] Using preferred codons in CHO cells, the whole gene synthesizes polynucleosides encoding native hHGF (SEQ ID NO:1) and the above three hHGF mutants (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4) acid, and introduce a restriction enzyme cutting site and a star...

Embodiment 2

[0112] Example 2. Evaluation of in vitro biological activity of hHGF mutants and native hHGF

[0113] In this example, the effects of hHGF mutant and native hHGF on endothelial cell migration were evaluated by in vitro endothelial cell migration experiment, so as to evaluate the in vitro biological activity of hHGF mutant and native hHGF.

[0114] 1. Materials and methods

[0115] 1.1 Protein sample

[0116] The hHGF mutants prepared above (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) and natural hHGF were prepared with physiological saline to the required concentration immediately before use.

[0117] 1.2 Cell lines

[0118] ECV304 cell line (umbilical vein endothelial cells), used to test the biological activity of HGF.

[0119] 1.3 Reagents

[0120] DMEM medium: provided by Hyclone. The preparation method is as follows: take 1 bag of DMEM medium powder (the specification is 1 L), add water to dissolve and dilute to 1000 ml, and then add 2.1 g of sodium bicarbonate. Then,...

Embodiment 3

[0143] Example 3. Evaluation of the therapeutic effect of hHGF mutants and natural hHGF on rabbit lower extremity arterial ischemia model

[0144] In this example, the rabbit lower limb arterial ischemia model was used to evaluate the effect of hHGF mutant and natural hHGF on the re-formation of blood vessels and collateral circulation in the rabbit lower limb ischemia model, so as to evaluate the therapeutic effect of hHGF mutant and natural hHGF.

[0145] 1. Materials and methods

[0146] 1.1 Protein sample

[0147] The hHGF mutants prepared above (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) and natural hHGF were prepared with physiological saline to the required concentration immediately before use.

[0148] 1.2 Animal model

[0149]New Zealand male white rabbits, 12-14 months old, weighing 3.5-4.0kg, were provided by Beijing Weitong Lihua Company. According to the method described by Takeshita et al. (Therapeutics angiogenesis. A single intraarterial bolus of vascular end...

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Abstract

The application of the invention relates to a mutant of a human hepatocyte growth factor (hHGF). The application also relates to a nucleic acid molecule coding the mutant, a vector containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule or the vector. The application also relates to a medical composition containing the hHGF mutant, and the use of the hHGF mutant.

Description

technical field [0001] The present application relates to mutants of human hepatocyte growth factor (hHGF). The present application also relates to nucleic acid molecules encoding said mutants, vectors containing said nucleic acid molecules, host cells containing said nucleic acid molecules or vectors. The present application also relates to the pharmaceutical composition containing the hHGF mutant and the application of the hHGF mutant. The hHGF mutants can be used, for example, to promote endothelial cell growth and / or migration, to promote angiogenesis, or to treat diseases that would benefit from the activity of native hHGF (such as the treatment of lower extremity arterial ischemia, myocardial infarction, and / or diabetic peripheral neuropathy) , and thus can be used in the preparation of medicines. Background technique [0002] Hepatocyte growth factor (Hepatocyte growth factor, HGF) was originally isolated from rat plasma and platelets, is a secreted heparin affinity...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K1/22C07K1/18C12N15/12C12N15/85A61K38/18A61P9/10A61P3/00A61P3/10A61P25/02A61P25/28A61P25/16A61P9/00
CPCA61P3/00A61P3/10A61P9/00A61P9/10A61P25/02A61P25/16A61P25/28C12N15/85C07K14/4753A61K38/00C12N2800/22A61K48/005
Inventor 聂李亚许松山马素永
Owner BEIJING NORTHLAND BIOTECH
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