Preparation method of type I collagen-like fiber

A collagen fiber and collagen technology, applied in the field of genetic engineering, can solve the problems of limited application, inability to form collagen fibers, and inability to form nanofiber morphology, etc., and achieve the effect of simple preparation process and low cost

Active Publication Date: 2020-06-26
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant collagen has potential applications in the production of biomaterials, but lacks the driving force for self-assembly to form higher-order structures and cannot form collagen fibers, which limits its application in biomaterials and tissue engineering
Barbara Brodsky et al. expressed the full-length and double-length Streptococcus pyogenes collagen Scl2, and the collagen region after the globular-guided folding domain was excised to self-assemble into fibers, and the ability to self-assemble could be promoted by lengthening the sequence length, but these recombinations Collagen sequences cannot form nanofiber morphology with regular light and dark bands similar to natural collagen [2]

Method used

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  • Preparation method of type I collagen-like fiber
  • Preparation method of type I collagen-like fiber
  • Preparation method of type I collagen-like fiber

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Sequence Design and Collagen Preparation

[0059] according to The structure shown is designed, and the specific steps are:

[0060] (1) With N and C terminals (GPP) 10 As a fixed sequence motif, a variable collagen region is inserted in the middle to obtain a three-segment chimeric sequence (abbreviated as P 10 CLP 10 ). In this example, the CL-domain adopts the amino acid sequence (abbreviation For H) as bacterial collagen, wherein the Scl2 collagen region is divided into three equal-length regions A, B and C, in the following examples, the designed CL domains are respectively A, B, C, and BB (repeated 2 a B region) and ABC (equivalent to the complete Scl2 collagen region).

[0061] (2) A globular domain derived from Scl2 (shown in SEQ ID NO.1) is inserted at the N-terminus of the sequence to guide the correct folding of the collagen triple helix, and a protease cleavage site is inserted between the globular domain and the fixed sequence unit of the...

Embodiment 2

[0078] Example 2 Determination of the secondary structure of collagen

[0079] The collagen prepared in Example 1 was made into a concentration of 1 mg / mL. Then stand at 4°C for more than 24h, use a 1mm cuvette, scan the full wavelength of circular dichroism at 4°C, the wavelength is from 190nm to 260nm, the wavelength interval is 1nm, and stay at each wavelength for 5s. The thermal change experiment was measured at 220nm, the temperature was from 4°C to 80°C, each temperature was equilibrated for 8s, and the temperature increment rate was 1°C / 6min. The typical CD spectrum of collagen triple helix structure shows a positive absorption peak at 220nm.

[0080] like Figure 4 As shown, under full-wavelength scanning, the protein designed in Example 1 has a characteristic absorption peak around 220nm; the thermal variation experiment results show that as the temperature increases, the characteristic absorption value at 220nm changes suddenly at around 50°C, It is manifested by ...

Embodiment 3

[0081] Example 3 Effects of Substitution of Collagen Domain Sequence on Fiber Structure

[0082] The lyophilized collagen P prepared in Example 1 10 AP 10 ,P 10 BP 10 ,P 10 CP 10 , P 10 HP 10 Use 10mM PB to prepare a solution with a final concentration of 0.5mM, place it at 4°C for 3.5 days, take a small amount and drop it on the copper grid, absorb it for 45s, then blot it dry with filter paper, then negatively stain it with 0.75% phosphotungstic acid for 20s, and blot it dry on the filter paper , observed with a Hitachi H-7650 transmission electron microscope, as Figure 5 According to the transmission electron microscopy results shown, the designed collagen can self-assemble to form ribbon fibers with periodic light and dark stripes, and the periodic light and dark stripes formed by sequences A, B and C have the same length. by negatively staining P 10 BP 10 The light and dark lines of the fiber are measured, and the lengths of the light and dark lines are respect...

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Abstract

The invention discloses a preparation method of a type I collagen-like fibers, belonging to the technical field of gene engineering. On the basis of a (GPP)<n> sequence of N and C terminals, a continuous collagen sequence of a Gly-Xaa-Yaa triplet is inserted into the middle of the (GPP)<n> sequence to form a three-section type chimeric collagen P-CL-P mode. Self-assembly is driven through interaction among triple helixes of the (GPP)<n> of the N and C terminals, so the strip-shaped fiber with periodically bright and dark stripes is formed. The method provided by the invention can be used for preparing the clean-source fiber which can be self-assembled to form the periodically bright and dark stripes, and the structure of the fiber is similar to the structure of type I collagen; the preparation method is simple in process; and the collagen fiber with low cost can be produced on a large scale and has wide application prospects in the field of biological materials.

Description

technical field [0001] The invention relates to a method for preparing type I collagen fibers, belonging to the technical field of genetic engineering. Background technique [0002] Collagen is a biological polymer, which is a triple helix structure formed by three chains intertwined with each other. Collagen can be divided into 28 types according to its gene sequence and function, the most important of which is type I collagen. In higher biological cells, after the original protein of type I collagen undergoes a series of maturation processes such as post-translational modification, folding, and cleavage, multiple collagen triple helices (collagen for short) are staggered to form light and dark stripes with uniform spacing. collagen fibers ( figure 1 ), whose morphology plays a key role in cell adhesion and growth [1] , is also a biomaterial that promotes the repair and regeneration of human tissues and organs. [0003] Type I collagen is mainly used in biomedical mater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/70C12N1/21C12P21/06C12R1/19
CPCC07K14/78C12N15/70C12P21/06
Inventor 许菲胡金远维卡斯·南达戴维·史瑞伯张萌锁娜儿·格拉瓦特
Owner JIANGNAN UNIV
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