A low immunogenicity recombinant I & III fusion collagen with high stability, a preparation method and application thereof

Abstract

The application belongs to the technical field of synthetic biology, and particularly relates to a recombinant I&III fusion type collagen with high stability and low immunogenicity, and a preparation method and application thereof. The amino acid sequence of the recombinant I&III fusion type collagen and the nucleotide sequence of the coding gene thereof are disclosed. The recombinant I&III fusion type collagen with high stability and low immunogenicity is prepared by a fermentation method of an engineering bacterium. The results of examples show that the recombinant I&III fusion type collagen colI&III-1 provided by the application has good thermal stability, long-term stability and anti-degradation capacity. The animal experiment proves that the recombinant I&III fusion type collagen colI&III-1 has low immunogenicity, and does not cause immune balance disorder of animals. In addition, the recombinant I&III fusion type collagen has the characteristics of high biocompatibility and good safety, and has no influence on the overall health condition of animals.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a highly stable, low-immunogenic recombinant I&III fusion collagen, its preparation method, and its application. Background Technology

[0002] Collagen, a key component of the extracellular matrix, accounts for 70%–80% of the dermis. It not only provides structural support and maintains the elasticity of skin and muscles but also plays a significant role in regulating various biological processes such as cell communication and signal transduction. In recent years, with continuous advancements in collagen extraction and preparation technologies, the related market has continued to expand. Currently, the market is dominated by natural collagen, primarily derived from animal tissues and skin extracts. However, natural collagen faces limitations such as immunogenicity risks, potential viral contamination, and poor processing performance, restricting its further development and application.

[0003] In contrast, recombinant collagen, prepared through expression in genetically engineered bacteria, effectively avoids the risk of viral transmission and possesses excellent biocompatibility and water solubility, thus finding wide application in tissue engineering, biomedicine, and drug delivery. To date, 28 types of collagen with different genetic backgrounds have been discovered. Previous industry research has largely focused on type I, II, III, and XVII collagen. Existing studies indicate that the triple helix structure of recombinant collagen is sensitive to external factors such as temperature, and is prone to degradation during storage, transportation, and end-use, leading to a decrease in its bioactivity. Furthermore, achieving large-scale production with high purity and low immunogenicity still faces significant technical challenges, making it difficult to meet the growing market demand.

[0004] With the continuous iteration and maturation of technology, fusion-type collagen has gradually shown its application potential, becoming a type of functional biomolecule with broad development prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a highly stable, low-immunogenic recombinant I&III fusion collagen, its preparation method, and its applications. The recombinant I&III fusion collagen of this invention exhibits good thermal stability and anti-degradation ability, low immunogenicity, and high safety, and can be widely used in high-end functional skincare products and medical device filler materials. To achieve the above objective, this invention provides the following technical solution:

[0006] The present invention provides a collagen, wherein the collagen is a recombinant I&III fusion collagen, and the recombinant I&III fusion collagen includes type I collagen and type III collagen.

[0007] Preferably, the type I collagen includes any one or more of the proteins encoded by the sequences shown in SEQ ID NO. 3-5; the type III collagen includes any one or more of the proteins encoded by the sequences shown in SEQ ID NO. 6-8.

[0008] Preferably, the amino acid sequence of the collagen is shown in SEQ ID NO.9.

[0009] A nucleotide molecule encoding the aforementioned collagen, the sequence of which is shown in SEQ ID NO.10.

[0010] A recombinant plasmid comprising the aforementioned nucleotide molecules.

[0011] Preferably, the recombinant plasmid is obtained by linking pET28a with the above-mentioned nucleotide molecule.

[0012] An engineered bacterium containing the aforementioned recombinant plasmid.

[0013] Preferably, the engineered bacteria is Escherichia coli.

[0014] This invention also provides a method for preparing collagen, the method comprising the following steps:

[0015] The engineered bacteria were cultured, then induced to obtain a bacterial culture, and the bacterial cells in the culture were collected.

[0016] The bacterial cells were dissolved in water and then ruptured to obtain the supernatant. The supernatant was then purified to obtain collagen.

[0017] Preferably, the culture is a fermentation culture, and the specific steps include: inoculating the engineered bacteria into a fermenter after seed culture, and culturing to OD... 600 Greater than 60.

[0018] Preferably, the induction time is 15-16 hours.

[0019] Preferably, the rupture is achieved by dissolving the bacterial cells in water and then homogenizing them under high pressure.

[0020] Preferably, after dissolving the bacterial cells in water and rupturing them to obtain the supernatant, the method further includes adding saturated ammonium sulfate to the supernatant.

[0021] Preferably, the purification is performed using a chromatography system.

[0022] Preferably, the preparation method further includes lyophilization after purification.

[0023] The present invention also provides an application of the collagen, wherein the application is as follows:

[0024] The application of collagen in the preparation of skin care products; or, the application of collagen in the preparation of filler materials for medical devices. Beneficial effects

[0025] This invention provides a highly stable, low-immunogenic recombinant I&III fusion collagen, along with the gene encoding this recombinant I&III fusion collagen and a recombinant expression vector. The constructed recombinant expression vector was introduced into *E. coli* BL21(DE3), and the engineered *E. coli* successfully expressed the recombinant I&III fusion collagen with a theoretical molecular weight of 22.9 kDa. The recombinant I&III fusion collagen of this invention has a purity of over 99%, good thermal stability and anti-degradation ability, low immunogenicity, and high safety, showing broad application prospects in the high-end functional skincare industry and the biomedical materials industry. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is an SDS-PAGE electrophoresis image of colI&III-1 in Example 4 of the present invention;

[0028] Where M is the marker; 1 is the protein band induced by the empty vector BL21-pET28a(+); 2 is the protein band induced by BL21-pET28a(+)-colI&III-1 for 5 h; and 3 is the protein band induced by BL21-pET28a(+)-colI&III-1 for 20 h.

[0029] Figure 2 This is an SDS-PAGE electrophoresis image of colI&III-2 in Example 4 of the present invention;

[0030] Where M is the marker; 1 is the protein band induced by the empty vector BL21-pET28a(+); 2 is the protein band induced by BL21-pET28a(+)-colI&III-2 for 5 h; and 3 is the protein band induced by BL21-pET28a(+)-colI&III-2 for 20 h.

[0031] Figure 3 This is an SDS-PAGE electrophoresis image of the thermal stability of colI&III-1 in Example 8 of the invention;

[0032] Where M is Marker; 1 is unsterilized colI&III-1; 2 is colI&III-1 sterilized at 121℃ for 30 min; 3 is colI&III-1 sterilized at 115℃ for 30 min; 4 is colI&III-1 sterilized at 105℃ for 30 min.

[0033] Figure 4 This is an SDS-PAGE electrophoresis image of the thermal stability of colI&III-2 in Example 8 of the invention;

[0034] Where M is Marker; 1 is unsterilized colI&III-2; 2 is colI&III-2 sterilized at 121℃ for 30 min; 3 is colI&III-2 sterilized at 115℃ for 30 min; 4 is colI&III-2 sterilized at 105℃ for 30 min.

[0035] Figure 5 This is the HPLC chromatogram of colI&III-1 in Example 9 of the present invention after being stored at 4°C for 1 month;

[0036] Figure 6 This is the HPLC chromatogram of colI&III-1 in Example 9 of the present invention after being stored at 4°C for 3 months;

[0037] Figure 7 This is the HPLC chromatogram of colI&III-1 in Example 9 of the present invention after being stored at 4°C for 6 months;

[0038] Figure 8 This is the HPLC chromatogram of colI&III-1 in Example 9 of the present invention after being stored at 4°C for 12 months;

[0039] Figure 9 This is the HPLC chromatogram of colI&III-2 in Example 9 of the present invention after being stored at 4°C for 1 month;

[0040] Figure 10 HPLC chromatogram of colI&III-2 after being stored at 4°C for 3 months in Example 9 of this invention;

[0041] Figure 11 HPLC chromatogram of colI&III-2 after being stored at 4°C for 6 months in Example 9 of this invention;

[0042] Figure 12 The HPLC chromatogram of colI&III-2 after being stored at 4°C for 12 months in Example 9 of this invention. Detailed Implementation

[0043] This invention provides a recombinant I&III fusion collagen protein with high stability and low immunogenicity. This invention also provides a method for preparing this recombinant I&III fusion collagen protein.

[0044] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0045] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0046] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0047] Example 1: Sequence design of recombinant I & III fusion collagen

[0048] Human type I and type III collagen sequences were selected for screening and optimization. The sequences of human type I collagen α1 chain and type III collagen α1 chain are NCBI reference sequences, and their amino acid sequences are shown below (SEQ ID NO. 1~2):

[0049] MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPP GPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGE NGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAA GEPGKAGERGV PGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGAR GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKG ADGSP GKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEV GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSG EPGKQGPSGASGERGPP GPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGET GPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL(SEQ ID NO.1)MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSHLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPNGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAK GEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFR GPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPG GKGDAGAPGERGPPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQN GEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGA AGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAP GPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESPGPAGPAGAPGPAGSRGAP GPQGPRGDKGETGE RGAAGIKGHRGFP GNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIK VFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL (SEQ ID NO.2)

[0050] Six sequences (underlined and bolded portions) from the above-mentioned sequence were selected (amino acid sequences shown below as SEQ ID NO. 3~8) that possess strong cell adhesion, high biocompatibility, and good hydrophilicity, making them suitable for use in biomaterials, cosmetics, and other fields. No tags were introduced, avoiding the risk of immunogenicity.

[0051] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSP (SEQ ID NO.3)

[0052] GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP (SEQ ID NO.4)

[0053] GPAGPAGPVGPVGARGPAGPQGPRGDKGET (SEQ ID NO.5)

[0054] GEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRD (SEQ ID NO.6)

[0055] GEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAP (SEQ ID NO. 7)

[0056] GPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.8)

[0057] Combining portions of the above sequences yields the amino acid sequence of this invention:

[0058] GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.9) Its optimized nucleotide sequence:

[0059] CCATGGGGTCCTCCTGGTCCTCCAGGACCGGCGGGTGAGAAAGGTAGTCCTGGTGCTGATGGTCCTGCTGGTGCACCTGGAACCCCTGGACCACAAGGTATCGCCGGACAGAGAGGTGTTGTGGGGTTACCAGGACAGCGTGGAGAGCGTGGCTTCCCTGGTCTGCCTGGTCCTTCTGGAGAACCAGGAAAACAGGGTCCCTCGGGAGCATCCGGTGAACGAGGTCCGCCGGGACCGGCAGGCCCGGCCGGCCCAGTCGGACCTGTGGGAGCCCGTGGACCAGCAGGTCCACAGGGTCCTAGAGGTGACAAAGGTGAGACTGGCGAACCGGGTGGTAAAGGTGAGCGGGGTGCGCCAGGTGAAAAAGGAGAAGGTGGACCACCAGGTGTTGCTGGACCACCAGGTGGAAGCGGACCTGCTGGTCCACCTGGCCCGCAGGGCGTCAAGGGCGAACGCGGTAGCCCCGGCGGGCCGGGTGCCGCGGGCTTTCCGGGCGCGCGCGGCCTGCCGGGCCCGCCAGGTTCAAACGGCAATCCGGGGCCCCCGGGGCCGAGCGGATCACCGGGTAAAGACGGACCACCAGGACCTGCGGGTAACACCGGAGCTCCAGGTAGTCCTGGAGTATCTGGTCCGAAAGGCGATGCAGGTCAGCCGGGTGAAAAGGGTTCTCCTGGTGCGCAAGGTCCACCAGGTGCACCAGGTCCTCAAGGTCCTCGTGGGGATAAAGGTGAAACGGGAGAACGCGGCGCTGCCGGTATTAAAGGCCATCGTGGTTTTCCATAAAAGCTT (SEQ ID NO.10)

[0060] It should be noted that in the gene sequence shown in SEQ ID No.10, CCATGG is the NcoI restriction site, AAGCTT is the HindIII restriction site, and TAA is the stop codon.

[0061] The amino acid sequence of Comparative Example 1:

[0062] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.11)

[0063] Its optimized nucleotide sequence:

[0064]

[0065] It should be noted that in the gene sequence shown in SEQ ID No. 12, CCATGG is the NcoI restriction site, AAGCTT is the HindIII restriction site, and TAA is the stop codon.

[0066] The amino acid sequence of Comparative Example 2:

[0067] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAP GNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSPGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEP GKQGPSGASGERGPPGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGEPGGKGERGAP GEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPP (SEQ ID NO.13)

[0068]

[0069] It should be noted that in the gene sequence shown in SEQ ID No. 14, CCATGG is the NcoI restriction site, AAGCTT is the HindIII restriction site, and TAA is the stop codon.

[0070] Example 2: Construction of a vector for recombinant I & III fusion collagen

[0071] The nucleotide sequences of recombinant I&III fusion collagen shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14 were entrusted to Suzhou Genewise Biotechnology Co., Ltd. for gene synthesis, and plasmids PUC57-colI&III-1, PUC57-colI&III-2, and PUC57-colI&III-3 were obtained respectively.

[0072] Plasmid pET28a(+) was digested with plasmids PUC57-colI&III-1, PUC57-colI&III-2, and PUC57-colI&III-3 using NcoI and HindIII, respectively. The digested products were recovered, ligated, and transformed into E. coli DH5α competent cells. After antibiotic selection, positive transformants were selected and cultured. After PCR identification, sequencing was performed. The sequencing results were correct. Recombinant plasmids pET28a(+)-colI&III-1, pET28a(+)-colI&III-2, and pET28a(+)-colI&III-3 were extracted using a plasmid miniprep kit.

[0073] Example 3: Construction of genetically engineered bacteria for recombinant I&III fusion collagen

[0074] Take 1 μL of recombinant plasmids pET28a(+)-colI&III-1, pET28a(+)-colI&III-2, and pET28a(+)-colI&III-3 (concentration of 1 μg / μL) and mix them with 100 μL of thawed Escherichia coli BL21(DE3) competent cell suspension. After incubating on ice for 30 min, heat shock them in a 42℃ water bath for 90 s, then incubate on ice again for 5 min. Then add 400 μL of antibiotic-free LB medium and culture on a shaker at 37℃ and 200 rpm for 30 min. Take 200 μL of the bacterial suspension and spread it evenly on an LB agar plate containing 50 μg / mL kanamycin. Incubate upside down at 37℃ overnight. The following day, single colonies of the transformed bacteria were picked and inoculated into test tubes containing 5 mL of LB liquid medium (Kana concentration was 50 μg / mL). After overnight incubation at 37°C and 200 rpm, the plasmids of the bacterial culture were extracted. Nucleotide sequencing confirmed the presence of the plasmids, thus obtaining the genetically engineered bacteria BL21-pET28a(+)-colI&III-1, BL21-pET28a(+)-colI&III-2, and BL21-pET28a(+)-colI&III-3 expressing recombinant I&III fusion collagen. The empty vector genetically engineered bacteria BL21-pET28a(+) was then constructed using the same method.

[0075] Example 4: Induction of Recombinant I&III Fusion Collagen Genetically Engineered Bacteria for Expression

[0076] Single colonies of genetically engineered bacteria were selected and inoculated into 5 mL of LB broth (with a final Kana concentration of 50 μg / mL). The culture was incubated overnight at 37°C with shaking at 200 rpm. 300 μL of the overnight culture was then transferred to an Erlenmeyer flask containing 30 mL of LB broth (with a final Kana concentration of 50 μg / mL) and incubated for 2 hours at 37°C with shaking at 200 rpm to allow OD to develop. 600 When the concentration reaches 0.6-0.8, add IPTG as an inducer to a final concentration of 0.5 mM. The induction temperature is 20℃ and the induction time is 20 h. After induction, take 1 mL of bacterial culture, centrifuge at 12000 rpm and 4℃ for 3 min, discard the supernatant, add 500 μL of ddH2O to resuspend, take 40 μL of the resuspended liquid, add 10 μL of 5× loading buffer, mix well, boil in water for 10 minutes, briefly centrifuge, and take 20 μL of the liquid for 12% separating gel SDS-PAGE protein electrophoresis to detect the expression level.

[0077] The protein electrophoresis results of genetically engineered bacteria BL21-pET28a(+)-colI&III-1 and BL21-pET28a(+)-colI&III-2 are as follows: Figure 1 and Figure 2As shown, the genetically engineered bacterium BL21-pET28a(+)-colI&III-3 did not express recombinant I&III fusion collagen. Despite multiple adjustments to experimental conditions, including codon optimization, induction OD, induction temperature, inducer concentration, and induction time, the target protein was still not expressed.

[0078] Recombinant I&III fusion collagen colI&III-1 exhibits a specific band at 25-35 KD (theoretical molecular weight is 22.9 KD, but the apparent molecular weight is larger than the theoretical molecular weight). Figure 1 It can be seen that after induction, the supernatant obtained by cell disruption of the empty vector genetically engineered bacterium BL21-pET28a(+) showed no specific bands. The genetically engineered bacterium BL21-pET28a(+)-colI&III-1 showed protein expression after 5h and 20h of induction, and the expression level at 20h was much higher than that at 5h.

[0079] The recombinant I&III fusion collagen colI&III-2 exhibits a specific band at 55-75 KD (theoretical molecular weight is 45.8 KD, but the apparent molecular weight is larger than the theoretical molecular weight). Figure 2 It can be seen that after induction, the supernatant obtained by cell disruption of the empty vector genetically engineered bacterium BL21-pET28a(+) showed no specific bands. The genetically engineered bacterium BL21-pET28a(+)-colI&III-2 showed protein expression after 5h and 20h of induction, and the expression level at 20h was much higher than that at 5h.

[0080] Example 5 Fermentation culture of recombinant I&III fusion-type collagen-producing genetically engineered bacteria

[0081] In Example 3, 200 μL of the correctly identified genetically engineered bacteria BL21-pET28a(+)-colI&III-1 and BL21-pET28a(+)-colI&III-2 were inoculated into Erlenmeyer flasks containing 65 mL of LB medium and cultured at 200 rpm and 37°C for 16 h to obtain primary seed culture. 1.5 mL of the primary seed culture was inoculated into three Erlenmeyer flasks containing 65 mL of LB medium each, and cultured at 200 rpm and 37°C for 3 h. The remaining 195 mL of the bacterial culture was then inoculated into a fermenter containing 2.5 L of TB medium and cultured at 37°C and 1000 rpm until OD (dose retardation). 600 When the concentration reaches 60, add IPTG to a final concentration of 0.4 mM and induce fermentation for 15-16 hours to end the fermentation, thus obtaining the fermentation broth of the genetically engineered bacteria.

[0082] Example 6 Purification of recombinant I&III fusion collagen

[0083] The fermentation broths of each genetically engineered bacterium in Example 5 were centrifuged at 8000 rpm for 30 min, and the precipitated bacterial cells were collected. 7 mL of purified water was added to 1 g of bacterial cells to dissolve the cells. The cells were homogenized three times using a Nanoton high-pressure homogenizer (800 bar). After centrifugation at 8000 rpm for 30 min, the supernatant was collected. 40% saturated ammonium sulfate was slowly added to the supernatant until it was completely dissolved. After centrifugation at 8000 rpm for 30 min, the supernatant was collected for the next experiment.

[0084] The samples were purified using the Bio-Lab laboratory chromatography system from Hanbang Technology Co., Ltd., and a Tofflon Rigose MMC cationic mixed-mode high-strength agarose medium self-packed column. The purified protein stock solution was labeled as colI&III-1 and colI&III-2, both with a purity higher than 99%.

[0085] Example 7 Lyophilization of Recombinant I&III Fusion Collagen

[0086] The purified protein stock solution was freeze-dried using a vacuum freeze dryer from Haier Biomedical to obtain recombinant I&III fusion collagen freeze-dried powder. The freeze-drying process is shown in Table 1.

[0087] Table 1 Vacuum freeze-drying process

[0088]

[0089] Example 8: Thermal stability test of recombinant I&III fusion collagen

[0090] Lyophilized recombinant I&III fusion collagen colI&III-1 and colI&III-2 were prepared into 1 mg / ml solutions with pure water and sterilized in an autoclave at 121℃ for 30 min, 115℃ for 30 min, and 105℃ for 30 min, respectively. 20 μL of each solution was taken, 5 μL of 5× loading buffer was added, mixed well, boiled in water for 10 minutes, and then briefly centrifuged. 10 μL of the liquid was taken for 12% separating gel SDS-PAGE protein electrophoresis to detect its thermal stability.

[0091] The results are as follows Figure 3 and Figure 4 As shown, compared with the pre-sterilization state, both colI&III-1 lyophilized powder and colI&III-2 lyophilized powder exhibited varying degrees of degradation when sterilized at 121℃ for 30 min, 115℃ for 30 min, and 105℃ for 30 min. However, the degradation degree of colI&III-1 lyophilized powder was much lower than that of colI&III-2 lyophilized powder, indicating that the recombinant I&III fusion collagen colI&III-1 of the present invention has good thermal stability.

[0092] Example 9: Long-term stability experiment of recombinant I&III fusion collagen

[0093] Lyophilized powders of recombinant I&III fusion collagen colI&III-1 and colI&III-2 were stored at 4℃ for 1 month, 3 months, 6 months, and 12 months, respectively. The solutions were then prepared with pure water to a concentration of 10 mg / mL, and their purity was determined by reversed-phase high-performance liquid chromatography. The results are shown in Table 2. Figure 5 and Figure 6 .

[0094] Table 2. Purity test results of recombinant I&III fusion collagen

[0095]

[0096] Table 2 shows that the purity of recombinant I&III fusion collagen colI&III-2 decreased rapidly after being stored at 4℃ for 1 month, 3 months, 6 months, and 12 months, while the purity of colI&III-1 remained relatively stable, consistently above 99%. These results indicate that the recombinant I&III fusion collagen colI&III-1 of this invention possesses good long-term stability and anti-degradation ability.

[0097] Example 10 Animal Immunological Experiment of Recombinant I&III Fusion Collagen

[0098] SPF-grade Hartley guinea pigs, weighing 280-320g, were used and kept in a clean environment with a temperature maintained at 23±1℃ and humidity at 55±5%, maintaining a 12-hour day / night cycle. After one week of acclimatization feeding, an immunity-related test was conducted.

[0099] 10.1 Experimental Grouping

[0100] colI&III-1 group: colI&III-1 protein purity ≥99%, concentration 20mg / mL, sterile, endotoxin <0.5EU / mL; colI&III-2 group: colI&III-2 protein purity ≥99%, concentration 20mg / mL, sterile, endotoxin <0.5EU / mL.

[0101] Negative control group: physiological saline;

[0102] Positive control group: ovalbumin (OVA, concentration 10 mg / mL).

[0103] 10.2 Guinea Pig Grouping

[0104] Forty-eight guinea pigs were divided into four groups: colI&III-1, colI&III-2, negative control group, and positive control group, with 12 guinea pigs in each group, half male and half female (6 males and 6 females).

[0105] 10.3 Rearing conditions

[0106] Environment: Temperature 23±1℃ (Guinea pigs have a narrow suitable temperature range, and fluctuations must be strictly controlled), humidity 55±5%, light 12h / 12h (fixed light cycle to avoid affecting immune rhythm).

[0107] Feed: SPF grade feed specifically for guinea pigs (containing sufficient vitamin C to prevent scurvy in guinea pigs and avoid affecting their immune function).

[0108] Drinking water: Sterile distilled water + 0.2% vitamin C solution (free to drink, change daily).

[0109] Cage: Two animals are kept in a single cage (to avoid fighting and stress caused by group living, which can affect immune indicators).

[0110] 10.4 Administration

[0111] Dosage control: negative control group (0.1 mL / 100 g body weight); positive control group (1 mg / kg body weight); colI&III-1 group (20 mg / kg body weight); colI&III-1 group (20 mg / kg body weight). The injection volume was calculated precisely according to body weight, and the single injection volume for each guinea pig did not exceed 0.5 mL (to avoid excessive injection volume leading to local swelling).

[0112] Injection site: Subcutaneous tissue on both sides of the guinea pig's back (rotate sites to avoid repeated stimulation of the same site, which may interfere with the local immune response).

[0113] Injection frequency: One injection each on day 0, day 2, and day 4, for a total of 3 injections (multiple injections to induce a potential immune response are better than a single injection).

[0114] 10.5 Sample Collection and Processing

[0115] Six guinea pigs (3 males and 3 females) were anesthetized intraperitoneally (1% sodium pentobarbital, 30 mg / kg body weight) at 48 h and 72 h after the last injection, and 5-6 ml of blood was collected from the heart of each guinea pig.

[0116] Serum separation: 3 mL of blood was injected into a centrifuge tube without anticoagulant, allowed to stand at room temperature for 30 min, centrifuged at 3000 rpm for 15 min at 4℃, and the supernatant serum was collected and stored at -80℃ for the detection of immunoglobulins and cytokines.

[0117] Peripheral blood mononuclear cell (PBMC) isolation: 2 mL of blood was injected into an EDTA anticoagulant tube, diluted with an equal volume of physiological saline, and slowly layered onto the upper layer of lymphocyte separation medium (density 1.077 g / mL). The mixture was centrifuged at 800 rpm for 20 min at 4°C. The intermediate white membrane layer (PBMC) was aspirated, washed three times with physiological saline, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL, used to detect immune cell subsets.

[0118] 10.6 Immunological marker detection

[0119] 10.6.1 Immunoglobulin Detection

[0120] Serum total IgE and specific IgG were detected in each experimental group using a commercially available ELISA kit. Following the kit instructions, 100 μL of standard and serum samples from each group were added, incubated at 37°C for 1 hour, washed 5 times, and then incubated with enzyme-labeled secondary antibody at 37°C for 30 minutes. Substrate was added for color development for 15 minutes, and the incubation was terminated. OD was measured using an ELISA reader. 450 The nm value was used to construct a standard curve and calculate the concentration. The results are shown in Table 3. The serum total IgE content of guinea pigs in the colI&III-1 group was 5.65 ng / mL and the specific IgG content was 3.79 mg / mL, which was close to that of the negative control group, indicating that the recombinant I&III fusion collagen colI&III-1 of the present invention has low immunogenicity.

[0121] Table 3 Immunoglobulin detection results in each experimental group

[0122]

[0123] 10.6.2 Cytokine Detection

[0124] Serum IL-4, IFN-γ, and TNF-α (pro-inflammatory factors) in each experimental group were detected using a commercially available ELISA kit. Following the kit instructions, 100 μL of standards and serum samples from each group were added, incubated at 37°C for 1 hour, washed 5 times, and then incubated with enzyme-labeled secondary antibody at 37°C for 30 minutes. Substrate was added for color development for 15 minutes, and the incubation was terminated. OD levels were measured using an ELISA reader. 450 The nm value was used to construct a standard curve, and the concentrations of IL-4, IFN-γ, and TNF-α, as well as the IL-4 / IFN-γ ratio, were calculated. The results are shown in Table 4. The IL-4 content of guinea pigs in the colI&III-1 group was 4.58 pg / mL, the IFN-γ content was 3.82 pg / mL, the TNF-α content was 3.96 pg / mL, and the IL-4 / IFN-γ ratio was 1.2, which was close to that of the negative control group. This indicates that the recombinant I&III fusion collagen colI&III-1 of the present invention has low immunogenicity.

[0125] Table 4. Cytokine detection results for each experimental group

[0126]

[0127] 10.6.3 Detection of Immune Cell Subpopulations

[0128] Take 100 μL of PBMC suspension from each experimental group, add 5 μL each of CD4-PE and CD8-Cy5 antibodies, incubate at 4℃ in the dark for 30 min, wash twice with physiological saline, detect by flow cytometry, and calculate the proportion of immune cell subsets in each group. The results are shown in Table 5. The CD4+ / CD8+ ratio of guinea pigs in the colI&III-1 group was 1.43, which is within the normal range (1.2-1.8), indicating that the recombinant I&III fusion collagen colI&III-1 of the present invention did not cause immune balance disorder in guinea pigs.

[0129] Table 5. Proportion of immune cell subsets in each experimental group

[0130]

[0131] 10.7 Simultaneous observation of clinical symptoms

[0132] 10.7.1 Local reaction

[0133] The injection site was observed 24 hours after each injection (redness, swelling, induration, and exudation). The results were scored as "none (0 points), mild (1 point), moderate (2 points), and severe (3 points)" and are shown in Table 6. Compared with the colI&III-2 group, no redness, swelling, induration, or exudation was observed at the injection site in the colI&III-1 group, indicating that the recombinant I&III fusion collagen colI&III-1 of the present invention has high biocompatibility and good safety.

[0134] Table 6 Local reaction scoring table for each experimental group

[0135]

[0136] 10.7.2 Systemic Reactions

[0137] The mental state (active / sleepy), food intake (a decrease of >20% from baseline was considered abnormal), and scratching behavior (>5 times per day suggested possible allergic itching) of guinea pigs were observed daily. The incidence of abnormalities was recorded. The results are shown in Table 7. Compared with the colI&III-2 group, the colI&III-1 group showed more active mental state, stable food intake, and no scratching behavior. The overall condition of the guinea pigs was good, indicating that the recombinant I&III fusion collagen colI&III-1 of the present invention has no effect on the overall health of guinea pigs.

[0138] Table 7. Results of abnormality incidence in each experimental group

[0139]

[0140] As demonstrated by the above embodiments, the recombinant I&III fusion collagen colI&III-1 provided by this invention exhibits good thermal stability, long-term stability, and anti-degradation ability. Animal experiments have shown that the recombinant I&III fusion collagen colI&III-1 of this invention has low immunogenicity and does not cause immune imbalance in animals. Furthermore, it possesses high biocompatibility and good safety profile, and has no impact on the overall health of animals.

[0141] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A collagen protein, characterized in that, The amino acid sequence of the collagen is shown as SEQ ID NO.

9.

2. A nucleotide molecule encoding the collagen of claim 1, characterized in that, The sequence of the nucleotide molecule is shown as SEQ ID NO.

10.

3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleotide molecule of claim 2.

4. An engineered bacterium, characterized in that, The engineering bacteria contain the recombinant plasmid of claim 3.

5. A method for preparing collagen, characterized by, The preparation method comprises the following steps: The engineering bacteria are cultured, and then induced to obtain bacterial liquid; the bacterial bodies in the bacterial liquid are collected, dissolved with water, broken to obtain supernatant, and then the supernatant is purified to obtain the collagen; the engineering bacteria are the engineering bacteria of claim 4.

6. The method of claim 5, wherein, The culture is a fermentation culture, and the specific steps include: inoculating the engineering bacteria into a fermenter after seed culture, culturing to OD 600 greater than 60.

7. The method of claim 5, wherein, After the bacterial bodies are dissolved with water and then broken to obtain the supernatant, the method further comprises the following step: adding saturated ammonium sulfate into the supernatant.

8. Use of the collagen of claim 1 or the collagen prepared by the method of any one of claims 5-7 in the preparation of skin care products.

9. Use of the collagen of claim 1 or the collagen prepared by the method of any one of claims 5-7 in the preparation of filling materials for medical devices.

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