Recombinant human-like collagen protein-human cell growth factor fusion protein and preparation method and application thereof

A kind of human-like collagen, human collagen technology, applied in the field of genetic engineering, can solve the problem of collagen easily carrying viruses, infecting health and other problems

Active Publication Date: 2014-10-15
浙江肽源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Collagen sponge loaded with growth factors has the functions of tissue repair, residual cavity filling, hemostasis, drug carrier and anti-adhesion, and has been widely used in various operations, but the mature process of obtaining collagen is to use animal tissue Collagen extracted from mammalian bovine tissue and bird animal chicken tissue is easy to carry virus, and then infects and endangers the risk of human health

Method used

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  • Recombinant human-like collagen protein-human cell growth factor fusion protein and preparation method and application thereof
  • Recombinant human-like collagen protein-human cell growth factor fusion protein and preparation method and application thereof
  • Recombinant human-like collagen protein-human cell growth factor fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Expression of recombinant human-like collagen-human epidermal growth factor fusion protein in Escherichia coli

[0050] 1. Acquisition of gene encoding recombinant human-like collagen-human epidermal growth factor fusion protein

[0051] Send the DNA fragment (SEQ ID NO.2) encoding human-like collagen to Guangzhou Jetway Biotech Co. Ltd. (China) for whole gene synthesis, and use the DNA sequence and the whole gene The synthesized human epidermal growth factor (EGF) sequence (SEQ ID NO.3) was used as a template, and the two primers Ecoll F (SEQ ID NO.4) Ecoll R (SEQ ID NO.5) and the intermediate primer Ecoll-IN were added F (SEQ ID NO.6), Ecoll-IN R (SEQ ID NO.7) (primers synthesized by BGI) (primer concentration 10μM) each 1μL, then add dNTP (each 2.5μM) 10μL, 10x PCR pfu buffer Solution 10μL and pfu Taq DNA polymerase 1μL (5U / μL), add water to a total volume of 100μL, according to the touch-down PCR reaction conditions (94°C denaturation for 4min, enter the cycle, the...

Embodiment 2

[0057] Expression of Recombinant Human Collagen-Epidermal Growth Factor Fusion Protein in CHO Cells

[0058] First construct transfection CHO-dhfr according to the method described in Example 1 - Cell expression vector pSV2-dhfr-Ecoll. Using the Ecoll described in Example 1 as a template to utilize the upstream primer Ecoll F2 (SEQ ID NO.8) and the downstream primer Ecoll R2 (SEQ ID NO.9) to amplify the coding fusion protein sequence Ecoll, with TOPO TA Cloning Kit (purchased from Invitrogen Enzyme amplification in ), ligated to T vector (pCR TM 2.1-TOPO vector), verified by sequencing, obtained the pT-Ecoll recombinant plasmid of the Ecoll gene fragment containing the HindIII (aagctt) restriction site at both ends. For the construction of recombinant fusion proteins in CHO-dhfr - The expression vector pSV2-dhfr in the cell was single cut by HindIII, the vector was dephosphorylated with CIAP alkaline phosphatase (TAKARA), and then ligated with pT-Ecoll, the small fragment r...

Embodiment 3

[0062] Expression of recombinant human-like collagen-growth factor fusion protein in yeast cells

[0063] First, the recombinant expression vector pPICZαA-Ecoll for transforming yeast cell GS115 was constructed according to the method described in Example 1. Using the Ecoll described in Example 1 as a template, use the upstream primer Ecoll F3 (SEQ ID NO.10) and the downstream primer Ecoll R3 (SEQ ID NO.11) to amplify the Ecoll containing the α-factor secretion signal peptide recognition site sequence at the N-terminus The gene was digested with Xho I and Xba I, connected with the large fragment pPICZαA of the vector recovered after the same double digestion, transformed into Top10 competent cells, and the transformants were screened on bleomycin-resistant YPD solid medium and identified by sequencing After the plasmid is correct, extract the plasmid, linearize the recombinant plasmid pPICZαA-Ecoll with Sac I, purify the plasmid by ethanol precipitation, transform GS115 compet...

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Abstract

The invention discloses a recombinant human-like collagen protein-human original cell growth factor fusion protein and a preparation method and application thereof. The prepared fusion protein is provided with a special fibrous structure and good biological tissue compatibility, and can promote proliferation and differentiation of epidermis cells, endothelial cells and fibroblast cells. The preparation method comprises the following steps: obtaining encoded DNA sequence of the fusion protein, reconstructing and recombining fusion protein expressed by an expression vectors in escherichia coli, pichia pastoris or chinese hamster ovary cells. The fusion protein comprises a first zone and a second zone, wherein the first zone has at least 85% of sequence homology with human collagen protein and has the function of the human collagen protein, and the second zone has at least 85% of human original cell growth factor and has the function of the human original cell growth factor; connecting peptide is arranged between the first zone and the second zone; the general formula of the connecting peptide is (GGGS)n; preferably, the amino acid sequence of the fusion protein is SEQ ID NO. 15.

Description

technical field [0001] The invention relates to the field of genetic engineering, and obtains recombinant human-like collagen-human cell growth factor fusion protein through gene recombination. Specifically, the present invention relates to a preparation method and application of recombinant human-like collagen-human cell growth factor fusion protein. Background technique [0002] In recent years, with the advancement of biotechnology and material science, great progress has been made in the treatment of severe bleeding wounds that were difficult to suture and skin grafting in the past. Leave less scars and pursue the height of perfect repair of structure and function (Fu Xiaobing. New issues in wound repair and tissue regeneration [J]. Journal of Clinical Surgery, 2007, 15(11): 739-740). The existing clinical growth factors have significant advantages in wound healing, but when applied to wounds that are difficult to treat by conventional methods, they have the disadvantag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61L27/22A61L27/54A61K38/17A61K8/64A61P7/04A61P17/02A61Q19/00
Inventor 黄亚东项琪崔宇张卉张齐肖巧学郑青
Owner 浙江肽源生物科技有限公司
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