Transforming growth factor beta1 active polypeptide and application thereof

A transforming growth factor and active polypeptide technology, applied in the direction of transforming growth factor, growth factor/inducing factor, specific peptide, etc., can solve the problem that there is no transforming growth factor β1 active polypeptide gel, etc., to promote wound healing, improve Healing efficiency and effectiveness, inflammation-reducing effect

Pending Publication Date: 2022-04-29
PLASTIC SURGERY HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is no relevant report on the use of transf...

Method used

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  • Transforming growth factor beta1 active polypeptide and application thereof
  • Transforming growth factor beta1 active polypeptide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Screening of TGF-β1 active polypeptide sequence

[0032] Coat the 96-well plate with human TGF-β1 monoclonal antibody, add phage random dodecapeptide library (purchased from NEB Company, USA) and incubate overnight at 4°C, wash the plate 10 times with TBST, and wash the plate with elution buffer (0.2mol / L Glycine-HCl, pH 2.2, 0.1% BSA) to elute the bound phages, neutralization buffer (1mol / L Tris-HCl, pH 9.1) quickly neutralizes the eluate.

[0033] E.coli ER2738 host bacteria were used to amplify the eluate for the next round of screening. In the same way, three rounds of screening were performed on the eluate from each round. Titer determination was carried out on the eluate of the fourth round, phage coeruleus was randomly selected, amplified, and adjusted to 2.0×10 13 pfu / mL.

[0034] The ELISA method and the human TGF-β1 immunoassay kit (purchased from R&D Company, USA) were used to detect the binding ability of the monoclonal phage according to th...

Embodiment 2

[0044] Embodiment 2: Synthesis of TGF-β1 active polypeptide

[0045] The seven TGF-β1 polypeptide sequences screened in Example 1 were sent to Wuhan Minghao Biotechnology Co., Ltd. for the synthesis of TGF-β1 active polypeptides, purified to a purity of 98%, and then disulfide bonds were used to stabilize the peptides. Chain structure, at the same time, the C-terminus of the polypeptide is amidated and blocked to enhance the stability of the active polypeptide; the N-terminus of the polypeptide is labeled with a rhodamine laser dye to identify the affinity of the active polypeptide with fibroblasts, and to detect whether it can Associated with fibroblasts. Prepared into freeze-dried powder and stored at -20°C.

Embodiment 3

[0046] Embodiment 3: In vivo and in vitro experiments of TGF-β1 active polypeptide

[0047] The TGF-β1 active polypeptides in Example 2 are intervened in the cultured fibroblasts, because the TGF-β1 active polypeptides are labeled with rhodamine laser dye, so immunofluorescence detection can be used to detect their affinity with fibroblasts . The results show that only the TGF-β1 active polypeptide shown in SEQ ID NO.23 can bind to the specific receptor on fibroblasts.

[0048] Further, the TGF-β1 active polypeptide shown in SEQ ID NO.23 was co-cultured with human fibroblasts for in vitro experiments, and a blank control group, a TGF-β1 active polypeptide group and a TGF-β1 growth factor group were set. Using the CCK-8 method, it was found that compared with the negative control group, the TGF-β1 active polypeptide group could promote cell proliferation, and there was a significant difference (P<0.05), and it was dose-dependent. See Table 3 for details.

[0049] Table 3 CCK-...

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Abstract

The invention discloses a transforming growth factor beta 1 active polypeptide and application thereof, and belongs to the technical field of transforming growth factor beta 1 active polypeptides. The amino acid sequence of the transforming growth factor beta 1 active polypeptide is as shown in SEQ ID NO. 23. The invention also discloses application of the transforming growth factor beta1 active polypeptide in preparation of drugs for wound healing. According to the invention, a phage random dodecapeptide library is used for screening a TGF-beta1 key sequence, in-vitro synthesis of the TGF-beta1 active polypeptide is carried out, a large amount of TGF-beta1 active polypeptide can be synthesized in a short time, and the production cost is greatly reduced.

Description

technical field [0001] The invention relates to a transforming growth factor β1 active polypeptide and application thereof, belonging to the technical field of transforming growth factor β1 active polypeptide. Background technique [0002] Wound healing is a complex process of tissue defect repair that requires the coordinated and orderly participation of various cells, extracellular matrix and signaling pathways. At present, refractory wounds such as diabetic chronic wounds are clinical problems that need to be solved urgently. Topical application of growth factors can promote wound healing, but currently used growth factor products have complex production processes, high economic costs, short biological half-life, poor stability, and are difficult to achieve the expected therapeutic effect, which greatly limits their clinical application. [0003] At present, the research and application of peptides have reached an unprecedented level of prosperity. Compared with natural...

Claims

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Application Information

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IPC IPC(8): C07K14/495A61K38/18A61K9/06A61K47/32A61K47/10A61K47/14A61K47/36A61P17/02A61P29/00
CPCC07K14/495A61K9/06A61K9/0014A61K47/32A61K47/10A61K47/14A61K47/36A61P17/02A61P29/00A61K38/00
Inventor 宗宪磊杜宏靳小雷宋国栋
Owner PLASTIC SURGERY HOSPITAL CHINESE ACAD OF MEDICAL SCI
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