Preparation of type I collagen-like fiber and method for regulating and controlling the D-periodic of fiber thereof

Abstract

The disclosure discloses a preparation of type I collagen-like fibers and a method for regulating and controlling the periodic length of fiber stripes thereof, belonging to the technical field of genetic engineering. The disclosure produces a three-segment chimeric collagen P-CL-P pattern by inserting a continuous Gly-Xaa-Yaa triplet collagen sequence in the middle based on the N- and C-terminal (GPP).sub.n sequences. The self-assembly is driven by the interaction between the N- and C-terminal (GPP).sub.n triple helixes to form banded fibers with periodic bright and dark stripes. According to the method of the disclosure, a fiber from a clean source, which can self-assemble to form periodic bright and dark stripes can be prepared, the structure of which is similar to type I collagen, the preparation process is simple, the collagen fiber with low cost can be produced on a large scale, and the method has broad application prospects in the field of biological materials.

Claims

1. A single-chain protein for expressing a type I-like collagen, wherein: the single-chain protein consists of an amino acid sequence as set forth in: ##STR00006## V-domain has an amino acid sequence as set forth in SEQ ID NO:1; (GPP)n is (glycine-proline-proline)n; n=10; the CL-domain has an amino acid sequence as set forth in any sequence of SEQ ID NOS: 2 to 6; HIS is a 6×His tag; the V-domain and (GPP).sub.n are ligated by LVPRGSP (SEQ ID NO:33); and the single-chain protein self-assembles with additional single-chain proteins to form the type I-like collagen fibers with D-periodic band structure comprising uniform spacing of bright and dark alternating stripes when viewed with electron microscope.

2. The single-chain protein according to claim 1, wherein the bright and dark stripes have a length of p×1 nm, wherein p is an integer greater than 5.

3. A gene encoding the single-chain protein according to claim 1.

4. A plasmid or a cell, wherein the plasmid comprises the gene according to claim 3, and wherein the cell comprises the gene according to claim 3.

5. The plasmid or the cell according to claim 4, wherein the plasmid is a pColdIII series plasmid or a pET series plasmid.

6. The plasmid or the cell according to claim 4, wherein the cell is an E. coli cell selected from the group consisting of: E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, and E. coli TOP10.

7. A method for preparing type I collagen-like fiber, comprising the following steps: (1) culturing the cell of claim 4 under conditions that induce expression of the gene; (2) adding a trypsin to a purified gene product of step (1) and incubating at 20° C. to 25° C. for at least 6 hours to obtain the single-chain protein of the type I-like collagen; and (3) adding the single-chain protein of a type I-like collagen obtained in step (2) to a solution at a final concentration of 0.1 mmol/L to 1 mmol/L, and storing the solution at 2° C. to 37° C.

8. The method according to claim 7, wherein the gene in step (1) has a nucleotide sequence as set forth in any one of SEQ ID NOS: 13 to 15, 17, and 18.

9. The method according to claim 7, wherein the storing time in step (3) is 24 hours or more.

10. The method according to claim 7, wherein in step (3), the collagen is added to the solution at a final concentration of 0.5 mmol/L, and storing the solution at 4° C. to 37° C. for at least 2 days.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows the morphology of type I collagen fibers;

(2) FIG. 2 A is a schematic diagram of the three-segment chimeric sequence, x is sequence A, B, C or H; FIG. 2B is collagen SDS-PAGE after purification and enzyme cleavage;

(3) FIG. 3 shows MALDI-TOF molecular weight identification of the designed collagen;

(4) FIG. 4A is the full-wavelength scan spectrum of a circular dichroism; FIG. 4B is the thermo transition curve of the circular dichroism.

(5) FIG. 5A are transmission electron micrographs of P.sub.10CLP.sub.10 self-assembled fibers;

(6) FIG. 5B are statistics of bright and dark stripes in length of the TEM results.

(7) FIG. 6A are transmission electron micrographs of P.sub.10BP.sub.10, P.sub.10BBP.sub.10 and P.sub.10ABCP.sub.10 self-assembled fibers; FIG. 6B are statistics of bright and dark stripes in length of the TEM results.

(8) FIG. 7A is the full-wavelength scan spectrum of P.sub.5BP.sub.5 and P.sub.5B.sub.2P.sub.5; FIG. 7B is thermo transition curve of P.sub.5BP.sub.5 and P.sub.5B.sub.2P.sub.5; FIG. 7C is the transmission electron micrograph of P.sub.5BP.sub.5 and P.sub.5B.sub.2P.sub.5 self-assembled fibers.

(9) FIG. 8A is the adhesion ability of smooth muscle cells under different collagen concentrations; FIG. 8B is the adhesion diagram of smooth muscle cells when the collagen concentration is 0.02 mg/mL; FIG. 8C is the relative percentage of the number of 3T3 mouse fibroblasts growing on the collagen substrate; FIG. 8D is the fluorescence staining image of 3T3 mouse fibroblasts growing on the collagen substrate.

(10) FIG. 9A is the full-wavelength scan spectrum of B, P.sub.10B, BP.sub.10 and BP.sub.10B; FIG. 9B is the thermo transition curve of the circular dichroism; FIG. 9C are transmission electron micrographs of B, P.sub.10B, BP.sub.10 and BP.sub.10B.

DETAILED DESCRIPTION

1. Technical Terms

(11) Unless otherwise specified, the “type I collagen-like” in this application refers to a triple helix structure formed by three single protein chains with periodic repetition (Gly-Xaa-Yaa).sub.n coiling around a common central axis. The “type I collagen-like fiber” refers to a biological macromolecule with uniform spacing and a morphology of bright and dark stripes formed by the staggered arrangement, spontaneous aggregation or assembly of type I collagen-like.

2. Materials and Methods Used in the Disclosure

(12) 1) Media:

(13) LB solid medium: 15 g/L agar, 10 g/L tryptone, 5 g/L yeast extract powder, 10 g/L NaCl, pH 7.0.

(14) LB liquid medium: 10 g/L tryptone, 5 g/L yeast extract powder, 10 g/L NaCl, pH 7.0.

(15) TB liquid medium: 12 g/L tryptone, 24 g of yeast extract powder, 4 mL glycerol, 2.31 g of KH.sub.2PO.sub.4, 12.54 g of K.sub.2HPO.sub.4, pH 7.5, diluting to 1 L.

(16) 2) Bacterial Culture Methods:

(17) E. coli seed culture conditions: The LB liquid medium was inoculated with a single colony grown via a streak plate method, the medium loading volume was 10%, a 250 mL shake flask was used for culture, the culture temperature was 37° C., the culture time was 10 h, and the rotate speed was 200 rpm.

(18) Fermentation and culture conditions of pET28a recombinant strains: A TB medium was used, the medium loading volume was 20%, the inoculum size was 1%, a 500 mL shake flask was used for culture, the culture temperature was 25° C., when OD.sub.600 reached 2.5, IPTG with a final concentration of 1 mM was used for induction, the induction temperature was 35° C., the induction time was 24 h, and the rotate speed was 200 rpm.

(19) Fermentation and culture conditions of pCold recombinant strains: A TB medium was used, the medium loading volume was 20%, the inoculum size was 1%, a 500 mL shake flask was used for culture, after culturing at 37° C. for 24 h, IPTG with a final concentration of 1 mM was used for induction, the induction was carried out at 25° C. for 10 h, then at 15° C. for 14 h, and the rotate speed was 200 rpm.

Example 1 Sequence Design and Collagen Preparation

(20) A sequence was designed according to the structure as shown in

(21) ##STR00004##
and the specific steps comprised:

(22) (1) taking N- and C-terminal (GPP).sub.10 as fixed sequence motifs, inserting a variable collagen region in the middle to obtain a three-segment chimeric sequence

(23) ##STR00005##
(abbreviated as P.sub.10CLP.sub.10). In this example, collagen Scl2 derived from Streptococcus pyogenes (Genbank ID: AAL50184.1) or an amino acid sequence (abbreviated as H) truncated from a human type I collagen α1 chain (UniProt ID: P02452.5) was used as a bacterial collagen for CL-domain, wherein the Scl2 collagen region was divided into three regions A, B, and C of equal length, and in the following examples, the designed CL domains were A, B, C, BB (two repeated B regions) and ABC (equivalent to the complete Scl2 collagen region), respectively; and

(24) (2) inserting the globular domain derived from Scl2 (as shown in SEQ ID NO. 1) at the N-terminus of the sequence to induce the correct folding of the collagen triple helix, and inserting a protease cleavage site LVPRGSP (SEQ ID NO:33) between the globular domain and the fixed sequence unit of the collagen region, and inserting 6×His at the N-terminus of the sequence for purification.

(25) Amino acid sequences were designed as follows:

(26) TABLE-US-00001 V-P.sub.10AP.sub.10 (as shown in SEQ ID NO. 7): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGQDGRNGERGEQGPTGPTGPAGPRGLQGLQGFP GERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGP PGPPGPPGPPGPPGPPGPPGPPGPPGPPG; V-P.sub.10BP.sub.10 (as shown in SEQ ID NO. 8): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFP GERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGP PGPPGPPGPPGPPGPPGPPGPPGPPGPPG; V-P.sub.10CP.sub.10 (as shown in SEQ ID NO. 9): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKD GQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGPPGP PGPPGPPGPPGPPGPPGPPGPPGPPG; V-P.sub.10B.sub.2P.sub.10 (as shown in SEQ ID NO. 10): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFP GERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGP RGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRG ETGPVGPRGERGEAGPAGKDGERGPVGPAGPPGPPGPPGPPGPPGPPGPP GPPGPPGPPG; V-P.sub.10ABCP.sub.10 (as shown in SEQ ID NO. 11): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQ GERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGP RGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETG PVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKD GLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQ PGKPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG; V-P.sub.10HP.sub.10 (as shown in SEQ ID NO. 12): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGERGPPGPQGARGLPGAPGQMGPRGLPGERGRP GAPGPAGARGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGE PGPTGPAGPKGSPGEAGRPGEAGLPGPPGPPGPPGPPGPPGPPGPPGPPG PPGPPG.

(27) Genes encoding the above amino acid sequences were synthesized, wherein the nucleotide sequence encoding V-P.sub.10AP.sub.10 was shown in SEQ ID NO. 13; the gene sequence encoding V-P.sub.10BP.sub.10 was shown in SEQ ID NO. 14; the gene sequence encoding V-P.sub.10CP.sub.10 was shown in SEQ ID NO. 15; the gene sequence encoding V-P.sub.10B.sub.2P.sub.10 was shown in SEQ ID NO. 16; the gene sequence encoding V-P.sub.10ABCP.sub.10 was shown in SEQ ID NO. 17; the nucleotide sequence encoding V-P.sub.10HP.sub.10 was shown in SEQ ID NO. 18; the nucleotide sequences shown above contained a 5′ Ncol enzyme cleavage site, a 5′ flanking sequence GC and 3′ BamHI enzyme cleavage site, respectively. The above genes as synthesized were respectively inserted between the Ncol and BamHI of the pET28a and pCOLD III-Tu plasmids to obtain corresponding recombinant collagen plasmids, and then the recombinant plasmids were respectively transformed into E. coli BL21 (DE3) competent cells by a CaCl.sub.2 method, plated on LB plates containing antibiotics, and cultured at 37° C., 200 rpm for 10 h. After screening, recombinant strains for preparing hybrid collagen were obtained; the pCOLD III-Tu plasmid was constructed by mutating the pCold Ill plasmid with the primers shown in SEQ ID NO. 23 and SEQ ID NO. 24 to introduce the Nco I site.

(28) The recombinant strains were induced and fermented. The specific steps were as follows: a TB medium with a medium loading volume of 20% and an inoculum size of 1% was used and a 500 mL shake flask was used for culture, after culturing at 37° C. and 200 rpm for 24 h, IPTG with a final concentration of 1 mM was used for induction, the induction was carried out at 25° C. for 10 h, and then at 15° C. for 14 h. The induced cell culture solution was centrifuged at 8,000 rpm for 5 min to collect cells. The cells were resuspended in a phosphate buffer solution, the cells were lysed with an ultrasonic cell disruptor under ice bath conditions, then centrifuged at 10,000 rpm for 20 min at 4° C. to remove cell debris, and then the supernatant was filtered with a microporous filter membrane (0.45 μm) to remove impurities. The sample was injected into a 5 mL His-trap hp affinity chromatography column installed on a protein purifier, and then washed with a washing solution for 8 column volumes. The protein was eluted with an elution buffer in which the imidazole content increased stepwise (140 mM, 400 mM). The protein fractions were collected, and analyzed by SDS-PAGE electrophoresis. Then, the protein was digested with trypsin at a final concentration of 0.05 mg/mL at 25° C. for 6 h to excise the globular guide folding domain, and then desalted with a desalting column and freeze-dried to obtain freeze-dried collagen powder.

(29) A small amount of freeze-dried powder was dissolved in water and identified by SDS-PAGE and Maldi-tof. FIG. 2 B shows that all of the digested protein has a single band detected by SDS-PAGE. Since collagen is a rod-shaped protein, the protein Marker used is a globular molecule, and the molecular weight shown by SDS-PAGE is greater than an expected molecular weight. As shown in FIGS. 3 A-(H), the molecular weight obtained by mass spectrometry is consistent with a theoretical molecular weight, and the collagen with a correct molecular weight is obtained.

Example 2 Determination of Secondary Structure of Collagen

(30) The collagen prepared in Example 1 was formulated to a concentration of 1 mg/mL, and then allowed to stand at 4° C. for 24 h or more. A 1 mm cuvette was used to carry out the full-wavelength scan of the circular dichroism at 4° C., the wavelength range was from 190 nm to 260 nm, the wavelength interval was 1 nm, and retention time was 5 s at each wavelength. The thermo transition experiment was determined at 220 nm, the temperature range was from 4° C. to 80° C., the balance time was 8 s at each temperature, and the temperature increasing speed was 1° C./6 min. The typical CD spectrum of the triple helix structure of collagen shows a positive absorption peak at 220 nm.

(31) As shown in FIG. 4A and FIG. 4B, under full-wavelength scan, the protein designed in Example 1 has a characteristic absorption peak near 220 nm; the thermo transition experiment results show that the characteristic absorption value at 220 nm changes suddenly at about 50° C. with the increase of temperature, manifested by the destruction of the secondary structure of collagen and the unwinding of the triple helix. The CD full spectrum and the thermo transition experiment results show that the three-segment chimeric collagen designed in Example 1 can be correctly folded to form a collagen triple helix structure, and has a high thermal stability.

Example 3 the Regulation and Control of Fibrous Structure by Replacing Collagen Region Sequence

(32) The freeze-dried collagen P.sub.10AP.sub.10, P.sub.10BP.sub.10, P.sub.10CP.sub.10, and P.sub.10HP.sub.10 prepared in Example 1 were formulated into a solution with a final concentration of 0.5 mM with 10 mM PB and placed at 4° C. for 3.5 days. After that, a small amount of the solution was dropped on copper grids, after adsorption for 45 s, blot-dried with filter paper, then negatively stained with 0.75% phosphotungstic acid for 20 s, blot-dried with filter paper, and observed with a Hitachi H-7650 transmission electron microscope.

(33) The transmission electron microscopy results shown in FIG. 5A show that all of the collagen designed in Example 1 can self-assemble to form banded fibers with periodic bright and dark stripes, and the periodic bright and dark stripes formed by sequences A, B and C have the same length. Through the measurement of the bright and dark stripes of the negatively stained P.sub.10BP.sub.10 fibers, and the statistics of at least 5 different TEM images and 200 or more sets of data, the lengths of the bright and dark stripes are found to be 10.4 nm and 24.0 nm, respectively, which are consistent with the theoretical lengths of (PPG).sub.10 and sequences A, B and C. The length of each Gly-Xaa-Yaa triplet is about 0.9 nm. The lengths of sequences A, B and C are all 81 amino acids, that is, 27 triplets, and the theoretical length is 24.3 nm. The collagen derived from human sequences can also self-assemble to form fibers with periodic bright and dark stripes in this mode. The length of the bright stripes is consistent with the theoretical (PPG).sub.10 length, and the dark stripes are 32.6 nm in length, which is consistent with the theoretical length of the sequence H (36 Gly-Xaa-Yaa triplets), proving that the three-segment chimeric design model can form a stable periodic fiber under the drive of N- and C-terminal (PPG).sub.10 and is not affected by the sequence replacement of collagen regions.

Example 4 Control of Periodic Length of Fibers by Length of Collagen Region

(34) The freeze-dried collagen P.sub.10BP.sub.10, P.sub.10B.sub.2P.sub.10, and P.sub.10ABCP.sub.10 prepared in Example 1 were formulated as a collagen solution according to the method of Example 3, and the fiber morphology was observed. The transmission electron microscope results as shown in FIG. 6A show that the dark stripes of the fibers change with the length of the sequence, which are 24.0 nm, 47.4 nm, and 72.3 nm and are consistent with the theoretical lengths of collagen regions B, 2B, and ABC. The dark stripes of P.sub.10B.sub.2P.sub.10 are about twice that of P.sub.10BP.sub.10, the dark stripes of P.sub.10ABCP.sub.10 are about 3 times that of P.sub.10BP.sub.10, and the length of the bright stripes are all about 10 nm. The results show that the length of the dark stripes of collagen fibers can be controlled by adjusting the length of the collagen region under the three-segment chimeric sequence mode.

Example 5 Verification of Function of Collagen Fibers

(35) The self-assembled fibers in Example 3 were diluted to concentrations of 0.02, 0.04, 0.08, and 0.1 mg/mL. After that, 200 μL of the collagen fiber solution prepared in Example 1, 200 μL of 5% bovine serum albumin (BSA) as a negative control, and 200 μL of 0.04 mg/mL type I collagen as a positive control were added to a 48-well plate, performed in triplicate for each group, allowed to stand at 4° C., and adsorbed for 24 h. After that, the solution was aspirated, 200 μL of DMEM medium containing 5% BSA was added, and allowed to stand at room temperature for 2 h. The mixture was washed 3 times with PBS buffer, then smooth muscle cells were resuspended in DMEM containing 10% FBS at a density of 20,000 cells per well and a cell culture plate is inoculated with 200 μL. After 2 h, the cell suspension was aspirated and the cells were washed with PBS 3 times and then stained with crystal violet. The absorbance was measured at 590 nm and the cell adhesion was observed.

(36) As shown in FIG. 8 A and FIGS. 8B, compared with BSA, P.sub.10BP.sub.10 and P.sub.10B.sub.2P.sub.10 can promote cell adhesion very well, and different concentrations have no great effect on cell adhesion. At a concentration of 0.04 mg/mL, their adhesion abilities are about 0.58 times and 0.57 times that of natural type I collagen, respectively.

(37) In the same way, the collagen fibers were adsorbed to a 96-well plate, and then mouse 3T3 cells were resuspended in DMEM containing 4% FBS at a density of 5000 cells per well, and a cell culture plate was inoculated with 100 μL. After culturing for 24 h, the cells were stained with Dapi and phalloidin, the number of cells was counted and the cell morphology was observed. As shown in FIG. 8 C and FIG. 8 D, the adhesion abilities of P.sub.10BP.sub.10 and P.sub.10B.sub.2P.sub.10 to 3T3 cells are comparable to that of natural type I collagen, and are 0.94 times and 1.31 times that of type I collagen. The observation of cell morphology shows that 3T3 cells based on P.sub.10BP.sub.10 and P.sub.10B.sub.2P.sub.10 grow well and have a higher cell extension.

(38) The function of other collagen fibers prepared in Example 1 was verified according to the above method. The results show that the adhesion ability and cell extension of other collagen fibers were equivalent to the effects of P.sub.10BP.sub.10 and P.sub.10B.sub.2P.sub.10.

Comparative Example 1

(39) The specific implementation mode was the same as that in Example 1, except that (PPG).sub.10 was replaced with (PPG).sub.5.

(40) TABLE-US-00002 The amino acid sequence of V-P.sub.5BP.sub.5 (as shown in  SEQ ID NO. 19): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAK GDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPPGPPGPPGPPGPPG,  and the nucleotide sequence encoding the amino  acid sequence is shown in SEQ ID NO. 20. The amino acid sequence of V-P.sub.5B.sub.2P.sub.5 (as shown in  SEQ ID NO. 21): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAK GDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGEQGPQGLPGKDGE AGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAG PAGKDGERGPVGPAGPPGPPGPPGPPGPPG, and the nucleotide  sequence encoding the amino acid sequence is shown  in SEQ ID NO. 22.

(41) As shown in FIG. 7A˜ FIG. 7 C, the results of full-wavelength scan and thermo transition experiment show that both of the chimeric collagen P.sub.5BP.sub.5 and P.sub.5B.sub.2P.sub.5 designed in this patent can be folded correctly to form a collagen triple helix structure, and have a high thermal stability, but the transmission electron microscopy results show that although the designed collagen P.sub.5BP.sub.5 and P.sub.5BP.sub.5 can self-assemble to form fibers, the fibers do not have periodic bright and dark stripes.

Comparative Example 2

(42) The specific implementation mode was the same as in Example 1, except that (GPP).sub.10 was inserted only at the C-terminus, N-terminus, or middle portion of the CL-domain (using the B collagen sequence of Scl2 here), or no (GPP).sub.10 was added.

(43) TABLE-US-00003 (1) The amino acid sequence of V-B (as shown in  SEQ ID NO. 25): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPRGEQGPQGLPG KDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGER GEAGPAGKDGERGPVGPAG. The nucleotide sequence encoding V-B (as shown in SEQ ID NO. 26): CCATGGGCCATCATCATCATCACCACGCCGATGAACAAGAAGAGAAAGCA AAGGTGCGCACCGAACTGATTCAAGAACTGGCACAAGGTCTGGGCGGTAT CGAAAAGAAGAACTTCCCGACTTTAGGTGATGAGGATTTAGATCACACCT ACATGACCAAACTGCTGACCTATTTACAAGAACGCGAACAAGCTGAAAAT AGCTGGCGCAAACGTCTGCTGAAAGGCATCCAAGATCATGCACTGGATCT GGTTCCGCGTGGTAGCCCCGGTCCTCGCGGTGAACAAGGTCCGCAAGGTC TGCCGGGTAAAGATGGTGAAGCCGGTGCACAAGGTCCGGCTGGTCCTATG GGCCCGGCCGGCTTTCCGGGCGAACGTGGTGAAAAAGGCGAACCGGGTAC CCAAGGTGCCAAAGGTGATCGTGGCGAAACCGGTCCGGTTGGCCCTCGTG GCGAACGCGGTGAAGCTGGTCCGGCTGGCAAAGACGGTGAACGTGGTCCC GTTGGTCCGGCCGGTTAAGGATCC. (2) The amino acid sequence of V-P.sub.10B (as shown in SEQ ID NO. 27): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPG PPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFP GERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAG. The nucleotide sequence encoding V-P.sub.10B (as shown in SEQ ID NO. 28): CCATGGGCCATCATCATCATCACCACGCCGATGAGCAAGAAGAAAAGGCC AAGGTTCGCACCGAACTGATTCAAGAACTGGCCCAAGGTCTGGGTGGCAT CGAGAAAAAGAACTTCCCGACTTTAGGCGACGAAGATTTAGACCACACCT ATATGACCAAGCTGCTGACCTATTTACAAGAACGCGAACAAGCTGAAAAC AGTTGGCGTAAACGTTTACTGAAGGGTATCCAAGATCACGCACTGGATCT GGTTCCGCGTGGTTCTCCCGGTCCCCCCGGCCCCCCCGGTCCCCCCGGTC CCCCCGGTCCTCCCGGCCCCCCCGGTCCCCCCGGTCCTCCGGGTCCCCCC GGTCCGCCCGGTCCCCGTGGTGAACAAGGCCCGCAAGGTTTACCGGGCAA AGACGGTGAAGCTGGTGCACAAGGTCCGGCTGGTCCTATGGGCCCGGCCG GTTTTCCGGGTGAGCGTGGTGAAAAAGGCGAACCGGGCACACAAGGCGCA AAAGGTGATCGCGGTGAAACCGGCCCCGTTGGCCCTCGTGGCGAACGTGG CGAAGCTGGTCCGGCCGGCAAAGATGGTGAGCGTGGCCCCGTTGGCCCCG CTGGCTAAGGATCC. (3) The amino acid sequence of V-BP.sub.10 (as shown in SEQ ID NO. 29): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPRGEQGPQGLPG KDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGER GEAGPAGKDGERGPVGPAGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG. The nucleotide sequence encoding V-BP.sub.10 (as shown in SEQ ID NO. 30): CCATGGGCCATCATCATCATCACCACGCCGATGAGCAAGAAGAAAAGGCC AAGGTTCGCACCGAACTGATTCAAGAACTGGCCCAAGGTCTGGGTGGCAT CGAGAAAAAGAACTTCCCGACTTTAGGCGACGAAGATTTAGACCACACCT ATATGACCAAGCTGCTGACCTATTTACAAGAACGCGAACAAGCTGAAAAC AGTTGGCGTAAACGTTTACTGAAGGGTATCCAAGATCACGCACTGGATCT GGTTCCGCGTGGTTCTCCCGGTCCGCGTGGCGAACAAGGTCCTCAAGGTT TACCGGGTAAAGATGGCGAAGCCGGTGCACAAGGTCCCGCTGGTCCTATG GGTCCCGCTGGTTTTCCCGGTGAACGCGGCGAAAAAGGTGAACCCGGTAC CCAAGGTGCAAAGGGTGACCGTGGTGAGACCGGTCCCGTTGGCCCTCGTG GTGAACGTGGTGAAGCCGGTCCGGCTGGTAAAGACGGCGAGCGCGGCCCG GTTGGCCCCGCTGGCCCCCCCGGTCCCCCCGGTCCCCCCGGTCCTCCCGG TCCCCCCGGTCCGCCCGGTCCCCCCGGTCCCCCCGGTCCCCCCGGTCCTC CGGGCTAAGGATCC. (3) The amino acid sequence of V-BP.sub.10B (as shown in SEQ ID NO. 31): HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPRGEQGPQGLPG KDGEAGAQGPAGPMGPAGFPGERGEKGEPGPPGPPGPPGPPGPPGPPGPP GPPGPPGPPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAG. The nucleotide sequence encoding V-BP.sub.10B (as shown in SEQ ID NO. 32): CCATGGGCCATCATCACCATCACCATGCCGATGAGCAAGAAGAAAAAGCC AAAGTGCGCACCGAACTGATCCAAGAACTGGCACAAGGTCTGGGTGGCAT CGAGAAGAAAAACTTCCCGACTTTAGGCGATGAAGATTTAGACCACACCT ACATGACCAAACTGCTGACCTATTTACAAGAACGTGAGCAAGCTGAGAAT AGCTGGCGCAAGCGTTTACTGAAAGGCATTCAAGATCATGCTTTAGATTT AGTTCCGCGTGGTAGTCCGGGTCCGCGTGGTGAACAAGGTCCTCAAGGTC TGCCGGGTAAAGACGGTGAAGCTGGTGCCCAAGGCCCGGCTGGTCCGATG GGTCCCGCTGGTTTTCCGGGCGAACGTGGTGAAAAAGGTGAACCCGGTCC CCCGGGTCCTCCCGGTCCGCCGGGCCCGCCCGGTCCCCCCGGTCCGCCCG GTCCCCCGGGCCCCCCCGGTCCTCCCGGCCCTCCGGGTACCCAAGGTGCC AAAGGTGATCGTGGTGAAACTGGTCCGGTTGGTCCTCGCGGTGAACGCGG CGAAGCTGGTCCCGCTGGTAAAGATGGTGAGCGCGGTCCCGTTGGTCCGG CTGGTTAAGGATCC.

(44) As shown in FIG. 8A˜D, the results show that the collagen sequences designed in this example can all be folded correctly to form a collagen triple helix structure, but the transmission electron microscope results show that none of the designed collagens can self-assemble to form fibers.