Nerve growth factor combined with collagen specificity and its coading gene and application

A nerve growth factor, specific binding technology, applied in the directions of nerve growth factor, growth factor/inducing factor, application, etc., can solve the problems of safety concerns, large dose, high treatment cost, etc., and achieves broad clinical application prospects and improved Viability, effect of reducing dosage

Active Publication Date: 2006-11-29
独步吾奇生物医疗科技(江苏)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of exogenous β-NGF into the body through direct administration is affected by many factors. The maintenance of its activity and effect are related to the route, method, and dosage of the drug. The continuous effect of continuous administration requires a large amount of medicine, the cost of treatment is very

Method used

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  • Nerve growth factor combined with collagen specificity and its coading gene and application
  • Nerve growth factor combined with collagen specificity and its coading gene and application
  • Nerve growth factor combined with collagen specificity and its coading gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1. Cloning, Expression and Purification of Nerve Growth Factor Encoding Gene with Collagen-Specific Binding Ability

[0032] 1. Design primers

[0033] Three forward primers (CBDU, NGFU1 and NGFU2, whose sequences are shown in Table 1) and two reverse primers (CBDD and NGFD), whose sequences are shown in Table 1, were designed with the help of the software Primer premier 5.05, wherein, CBDU (restriction endonuclease Enzyme Nde I recognition site) and CBDD (introduction of restriction endonuclease Hind III recognition site) are used to amplify the coding sequence of collagen binding domain and connecting peptide (LINKER), wherein, the amino acid sequence of collagen binding domain conservation For TKKTLRT (SEQ ID №: 1 in the sequence listing), the aminoacid sequence of linking peptide is GSAGSAAGSGGK; NGFU1 (introducing restriction endonuclease Nde I recognition site) and NGFD (introducing stop codon and restriction endonuclease Xho I recognition site) is used t...

Embodiment 3

[0040] Example 3, Activity Detection of Nerve Growth Factor with Collagen-Specific Binding Ability

[0041] Rat adrenal chromaffin cells PC12 can differentiate and generate axons under the induction of NGF. In addition, NGF can promote the survival of neurons. Therefore, through PC12 (purchased from the cell bank of the Basic Medical College of Peking Union Medical College) axon induction experiments and MTT experiments Detect the activity of CBD-NGF and NAT-NGF obtained in Example 2 (Howe, C.L., Depolarization of PC12 cells induces neurite outgrowth and enhances nerve growth factor-induced neurite outgrowth in rats. Neurosci Lett, 2003.351 (1): p.41- 5.), the method is: stimulate PC12 cells with CBD-NGF and NAT-NGF at a concentration of 0-64nM, and use unstimulated PC12 cells as a control. After one day of action, PC12 cells have axon induction (see Figure 2A ); Calculate the proportion of long axon cells, the statistical results are as follows Figure 2B As shown, it can b...

Embodiment 4

[0042] Example 4, Detection of Collagen Binding Ability of CBD-NGF

[0043] Using genetic engineering technology and microbial fermentation to prepare CBD-NGF, the purpose is to enhance its binding ability to collagen three-dimensional scaffold materials for tissue engineering while ensuring its activity, so as to reduce the effective dosage of β-NGF. The improved ELISA experiments were used to detect the binding ability of NAT-NGF and CBD-NGF to collagen membrane (Finnis, M.L. and M.A. Gibson, Microfibril-associated glycoprotein-1 (MAGP-1) binds to the pepsin-resistant domain of the alpha3 (VI)chain of type VI collagen.J Biol Chem, 1997.272(36):p.22817-23.), and according to the method provided by Matsushita (Matsushita, O., et al., A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J Biol Chem, 1998.273 (6): p.3643-8.) Calculate the dissociation constant Kd of the two proteins for collagen. Load gradually increasing doses of NAT-NGF an...

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Abstract

A nerve growth factor with specific combination power to collagen features that a collagen combining structure domain is fused at the amino terminate of its beta subset. It can be used for treating the nerve injury. Its coding gene is also disclosed.

Description

technical field [0001] The present invention relates to nerve growth factor and its encoding gene and application, in particular to a nerve growth factor capable of specifically combining with collagen, its encoding gene and the expression method of the nerve growth factor. Background technique [0002] Nerve Growth Factor (NGF) is a complex composed of α, β, γ three different subunits, with a molecular weight of about 140Kd. Studies have shown that the β subunit of NGF (β-NGF) is a homodimeric molecule, and its single chain contains 118 amino acid residues (Greene, L.A. and E.M. Shooter, Thenerve growth factor: biochemistry, syn thesis, and mechanism of action.AnnuRev Neurosci, 1980.3: p.353-402.), it is a functional subunit of NGF, which is closely related to many aspects such as the survival, migration, growth, differentiation and establishment of functional connections with other cells of neurons. The dual role of neurotrophic and neurite-promoting is one of the most im...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/48C12N15/12C12N15/09C12N15/70A61P21/00
Inventor 孙文杰林航戴建武
Owner 独步吾奇生物医疗科技(江苏)有限公司
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