Construction method of bombyx mori for producing recombinant human III-type collagen and application of bombyx mori

Abstract

The invention discloses a construction method of bombyx mori for producing recombinant human III-type collagen and application of the bombyx mori, and belongs to the technical field of biology. On the basis of a bombyx mori silk gland bioreactor for efficiently synthesizing foreign proteins, specific expression of partial or complete full-length recombinant human III-type collagen in bombyx mori posterior silk glands is realized through a piggyBac mediated transgenic technology, and the recombinant human III-type collagen is successfully secreted into silkworm cocoons. The recombinant human III-type collagen which is single in component and high in safety is obtained through subsequent extraction and purification of posterior silk glands and silkworm cocoons, a novel biological manufacturing method of high-added-value protein is constructed, and meanwhile, the economic value of silkworms is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing silkworms to produce recombinant human type III collagen and its application. Background Technology

[0002] Collagen, a major component of the extracellular matrix, is the most widely distributed and abundant functional protein in the human body, accounting for approximately 25%–30% of total body protein. It plays a crucial role in maintaining the normal physiological functions of cells, tissues, and organs, as well as in damage repair. Due to its excellent bioactivity, biocompatibility, and biodegradability, it has wide applications in biomedicine, food, and cosmetics (Collagen and Leather, 2023, 5, 20). Based on differences in primary structure and amino acid chain combination, collagen in the human body is classified into 29 subtypes. Type III collagen, mainly found in blood vessels and skin, is a rebuilding collagen that strengthens blood vessel strength and elasticity, provides sufficient nutrients to cells, and is an important element in maintaining plump, smooth, and lustrous cells (International Journal of Molecular Sciences, 2021, 22, 13329; Tissue Engineering Part C: Methods, 2024, 30, 53–62).

[0003] Currently, the traditional source of collagen is mainly animal-derived collagen, mostly from terrestrial and marine animals. However, the utilization of animal-derived collagen involves risks related to animal-borne diseases, batch-to-batch inconsistencies, environmental problems during extraction, and complications related to eliminating protein impurities. Given these shortcomings, with advancements in bioengineering, there is a growing preference for recombinant gene technology and microbial expression systems to produce collagen. Recombinant collagen prepared using this method has advantages such as single-component composition, absence of viral infection, stable production process, low immunogenicity, good processability, and good water solubility, making it the best alternative to animal-derived collagen in biomedicine and tissue engineering (Regenerative Biomaterials, 2024, 11, rbad106). However, traditional microbial expression systems (such as E. coli and yeast) have inherent limitations, lacking post-translational modifications for complex proteins, making it difficult to produce proteins with complete natural activity and correct structure. The silkworm, as a higher eukaryotic expression host, possesses a complete protein processing mechanism within its cells, capable of efficiently performing various complex and precise post-translational modifications, thereby significantly improving the bioactivity and stability of recombinant proteins. In addition, silkworms are not pathogen carriers and pose no risk of carrying zoonotic diseases, giving them a unique advantage in terms of biosafety.

[0004] The silkworm, one of the most important economic insects and a model organism of Lepidoptera, undergoes four metamorphic developmental stages: egg, larva, pupa, and adult. Silkworms are docile, small in size, grow quickly, are easy to raise, and have a high silk conversion rate, making them an important source of income for people in some parts of my country. With the development of life sciences, scientists have gained a deeper understanding of silkworms, leading to greater development and utilization of this species. Silkworms possess an open circulatory system, abundant fat bodies, and silk glands capable of synthesizing, secreting, and storing large amounts of silk proteins; these characteristics endow the silkworm with the characteristics of a natural bioreactor (Frontiers in Genetics, 2021, 12, 816075). Therefore, based on the silkworm's efficient protein synthesis capabilities and the increasingly mature gene recombination technology, the silk gland bioreactor has become an ideal bioreactor for producing exogenous proteins.

[0005] In 2003, Tomita et al. successfully constructed transgenic silkworms expressing recombinant human type III procollagen in silk fibroin light chains using transgenic technology. However, the recombinant protein expressed by this system only contained 1 / 5 of the mature type III collagen peptide sequence, which was insufficient to meet the production requirements of high homology and high yield (Nature Biotechnology. 2003, 21, 52-56). In contrast, the silk fibroin heavy chain expression system exhibits superior protein expression capabilities. Its large molecular weight and stable structure allow it to accommodate longer exogenous gene sequences, providing a unique foundation for expressing complex, full-length recombinant proteins (Journal of Biological Chemistry, 275, 40517-40528). It is noteworthy that, to date, there have been no reports of successfully expressing partial or complete full-length recombinant human type III collagen using the silk fibroin heavy chain expression system.

[0006] Therefore, this invention utilizes a silkworm fibroin heavy chain expression system to construct transgenic silkworm lines expressing both partial and full-length recombinant human type III collagen. The recombinant protein is specifically expressed in the posterior silk gland and successfully secreted into the cocoon. Cell proliferation experiments confirmed that the expressed full-length recombinant human type III collagen possesses bioactivity that promotes the proliferation of mouse fibroblasts NIH / 3T3, providing a new strategy for applications in biomedical materials and medical aesthetics. Summary of the Invention

[0007] The first objective of this invention is to provide a method for establishing a silkworm strain that produces partial recombinant human type III collagen and its application in producing recombinant human type III collagen. The second objective of this invention is to provide a method for establishing a silkworm strain that produces complete, full-length recombinant human type III collagen and its application in producing recombinant human type III collagen. The third objective of this invention is to provide a method for producing recombinant human type III collagen using silkworms.

[0008] To address the problems of the prior art, the present invention provides the following solution:

[0009] A recombinant human type III collagen fragment, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] A gene encoding a recombinant human type III collagen fragment, optimized according to the codon preference of silkworm, has the nucleotide sequence shown in SEQ ID NO.3, used to encode the recombinant human type III collagen fragment of claim 1.

[0011] A full-length recombinant human type III collagen containing the above-mentioned recombinant human type III collagen fragment, wherein the amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.2.

[0012] A gene encoding a full-length recombinant human type III collagen, optimized according to the codon preference of silkworm, has the nucleotide sequence shown in SEQ ID NO.4, and is used to encode the full-length recombinant human type III collagen as described in claim 3.

[0013] A transgenic plasmid comprising the coding gene of the above-mentioned recombinant human type III collagen fragment or the coding gene of the above-mentioned full-length recombinant human type III collagen.

[0014] A method for constructing a silkworm that produces recombinant human type III collagen, obtained by introducing the above-mentioned transgenic plasmid into silkworm cells.

[0015] The above construction method includes the following steps:

[0016] Step 1, constructing transgenic plasmids

[0017] Using the transgenic vector pXL-BacII-IE1-DsRed2-FibH-P, which is the promoter of the silkworm fibroin heavy chain gene, as the original plasmid, the gene encoding the recombinant human type III collagen fragment described in claim 2 and the gene encoding enhanced green fluorescent protein EGFP or the full-length recombinant human type III collagen described in claim 4 were ligated into the original plasmid through homologous recombination to obtain the transgenic plasmid.

[0018] Step 2, genetic transformation of silkworms

[0019] The transgenic plasmid constructed in step 1 is mixed with the helper plasmid and injected into silkworm embryos. After induction, hatching, rearing, and moth emergence, G0 generation silkworm moths are obtained. Male and female moths of the G0 generation mate, and the extra moths mate with wild-type silkworm moths. Then, the female moths are placed on oviposition paper to lay eggs, and the eggs laid are the G1 generation. The G1 offspring are selected by fluorescent selection to obtain transgenic positive silkworm individuals, that is, silkworms that produce recombinant human type III collagen. The helper plasmid is a recombinant plasmid expressing transposase. The mixing mass ratio of the transgenic plasmid to the helper plasmid is 0.5-10:1.

[0020] As an improvement, the original transgenic vector in step 1 is pXL-BacII-IE1-DsRed2-Fib HP; the linking elements of the transgenic vector from the 5' end to the 3' end are, in sequence, the silk fibroin heavy chain promoter FibH-P, the silk fibroin heavy chain N-terminal domain FibH-NTD, and the silk fibroin heavy chain C-terminal domain FibH-CTD.

[0021] As an improvement, the amplification primers used in step 1 for amplifying the recombinant human type III collagen fragment are pp_F and pp_R, with nucleotide sequences as shown in SEQ ID NO. 6-7; the amplification primers used for amplifying the full-length recombinant human type III collagen are mp_F and mp_R, with nucleotide sequences as shown in SEQ ID NO. 8-9; the amplification primers used for amplifying enhanced green fluorescent protein (EGFP) are EGFP_F and EGFP_R, with nucleotide sequences as shown in SEQ ID NO. 10-11; and the nucleotide sequence of enhanced green fluorescent protein (EGFP) is shown in SEQ ID NO. 5.

[0022] A method for producing recombinant human type III collagen using silkworms, comprising collecting the posterior silk glands or cocoons of silkworms constructed by any of the methods described above, and extracting recombinant human type III collagen.

[0023] Specifically, when the collected raw material is silkworm silk glands, RIPA lysis buffer is added, and extraction is performed by centrifugation. When the collected raw material is silkworm cocoons, urea solution is added, and extraction is performed by centrifugation.

[0024] Beneficial effects:

[0025] Compared with traditional technologies, the present invention provides a method for constructing silkworms to produce recombinant human type III collagen and its application, which has the following advantages:

[0026] First, this invention constructs a silkworm strain for producing recombinant human type III collagen. By specifically expressing a partial or complete full-length recombinant human type III collagen gene in the silk glands of the silkworm, heterologous expression of recombinant human type III collagen is achieved. Recombinant human type III collagen can be spontaneously secreted into the cocoon by the silkworm, providing a new pathway for the production of recombinant human type III collagen.

[0027] Secondly, the full-length recombinant human type III collagen significantly improved the cell proliferation activity of mouse fibroblasts NIH / 3T3, providing strong evidence for the wide application of full-length recombinant human type III collagen in biomedicine, cosmetics and other fields.

[0028] Finally, this invention enables recombinant human type III collagen to become a byproduct of sericulture, greatly improving economic efficiency. Attached Figure Description

[0029] Figure 1 A schematic diagram of a transgenic plasmid expressing a recombinant human type III collagen fragment in silkworms;

[0030] Figure 2 A schematic diagram of a transgenic plasmid expressing full-length recombinant human type III collagen in silkworms;

[0031] Figure 3 Fluorescence observation of the posterior silk gland of FH-pp transgenic silkworm;

[0032] Figure 4 Fluorescence observation of FH-pp transgenic silkworm cocoons;

[0033] Figure 5 The coverage of COL3A1 peptide in the posterior silk gland of FH-mp transgenic silkworm;

[0034] Figure 6 Analysis of COL3A1 peptide coverage and N / C-terminal sequence in FH-mp transgenic silkworm cocoons; the red arrow indicates the N-terminus.

[0035] Figure 7 The image shows the effect of FH-mp transgenic silkworm cocoon extract on the proliferation of NIH / 3T3 cells. Detailed Implementation

[0036] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention. It should be noted that the reagents and biological materials used in the present invention are all conventional commercially available products.

[0037] This invention, based on piggyBac-mediated transgenic technology, achieves the specific expression of human type III collagen in the posterior silk gland of silkworms, thus realizing the goal of using silkworms to produce partial and complete full-length recombinant human type III collagen.

[0038] Example 1: Construction of a complete full-length human type III collagen open reading frame

[0039] The complete human-derived COL3A1 gene sequence of the type III collagen α1 chain (COL3A1-mature peptide, COL3A1-mp) was downloaded from the NCBI database. Simultaneously, bases 577 to 996 and bases 3463 to 3780 of this gene were extracted and spliced ​​into a human type III collagen fragment (COL3A1-partial peptide, COL3A1-pp). Based on the codon bias of the silkworm gene, the sequence of this open reading frame was optimized and then synthesized by Nanjing Sangon Biotech Co., Ltd. The sequence was cloned into the pUC57 plasmid, retaining Nru I restriction sites at both ends.

[0040] The amino acid sequence encoding the recombinant human type III collagen fragment is shown in SEQ ID NO.1 (5'-3'): MQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYG

[0041] The nucleotide sequence encoding the recombinant human type III collagen fragment after codon optimization is shown in SEQ ID NO.3 (5'-3'):

[0042] ATGCAATATGATTCTTATGATGTTAAATCTGGAGTTGCTGTTGGAGGATTAGC

[0043] TGGATATCCTGGACCTGCGGGACCACCAGGACCTCCTGGACCTCCTGGAAC

[0044] TTCTGGACATCCTGGATCTCCTGGATCTCCTGGATATCAAGGACCTCCAGGA

[0045] GAACCTGGACAAGCTGGACCTTCTGGTCCTCCTCTGGAGCTATA

[0046] GGGCCTAGCGGACCAGCTGGTAAAGATGGTGAGTCGGGTAGACCCGGTAG

[0047] GCCAGGTGAAAGAGGACTTCCTGGACCACCTGGAATTAAAGGACCTGCTG

[0048] GAATTCCTGGATTTCCTGGTATGAAAGGACATAGGGATTTGATGGAAGAA

[0049] ATGGTGAAAAGGAGAAACTGGAGCTCCTGGATTAAAGGTGAAAATGGT

[0050] TTACCTGGTGAAAATGGTGCTCCTGGTAGTCCAGGACCTGCCGGGCAACA

[0051] AGGTGCTATAGGTAGTCCTGGTCCTGCAGGACCTAGGGGACCGGTAGGTCC

[0052] TAGTGGACCTCCAGGTAAAGATGGAACTTCAGGTCATCCTGGACCTATCGG

[0053] GCCTCCTGGACCAAGGGGTAATAGAGGTGAACGTGGTAGTGAGGGATCAC

[0054] CTGGACATCCTGGACAACCTGGTCCGCCTGGTCCTCCGGGAGCTCCTGGAC

[0055] CTTGTTGTGGAGGAGTTGGTGCTGCTGCTATAGCTGGAATTGGAGGAGAAA

[0056] AGGCTGGAGGATTTGCTCCTTATTATGGA

[0057] The amino acid sequence of the full-length recombinant human type III collagen containing the above-mentioned recombinant human type III collagen fragment is shown in SEQ ID NO.2 (5'-3'):

[0058] MQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPG

[0059] EPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGF

[0060] PGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERG

[0061] RPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGS

[0062] NGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGAR

[0063] GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGED

[0064] GKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAG

[0065] PRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGP

[0066] SGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGP

[0067] PGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAP

[0068] GGKGDAGAPGERGPPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTP

[0069] GLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAG

[0070] QPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGE

[0071] RGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGAR

[0072] GLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGE

[0073] KGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKP

[0074] GANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDR

[0075] GENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGP

[0076] QGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPR

[0077] GPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGA

[0078] PGPCCGGVGAAAIAGIGGEKAGGFAPYYG

[0079] The nucleotide sequence encoding full-length recombinant human type III collagen after codon optimization is shown as SEQ ID NO.4 (5'-3'):

[0080] ATGCAATATGATTCATATGATGTAAAAAGTGGTGTAGCTGTAGGAGGTTTAG

[0081] CTGGTTATCCTGGTCCAGCAGGACCACCGGGTCCTCCAGGACCACCTGGA

[0082] ACTAGTGGTCACCCTGGTAGTCCAGGTAGTCCAGGCTACCAAGGACCCCCT

[0083] GGTGAACCCGGACAAGCGGGCCCTAGCGGCCCTCCGGGTCCCCCAGGTGC

[0084] TATAGGACCATCCGGGCCTGCCGGTAAAGACGGTGAATCTGGACGACCTGG

[0085] TAGACCTGGTGAACGAGGCCTACCGGGACCGCCTGGGATTAAGGGACCGG

[0086] CCGGGATTCCTGGGTTCCCCGGAATGAAGGGGCATAGAGGATTTGACGGTC

[0087] GTAACGGTGAGAAGGGTGAGACCGGTGCGCCTGGATTAAAGGGTGAGAAT

[0088] GGACTACAGGTGAAAATGGGGCCCCCGGACCGATGGGACCTAGAGGAGC

[0089] ACCCGGTGAGCGGGGTAGGCCTGGCTTGCCTGGTGCGGCCGGCGCTAGGG

[0090] GTAATGATGGAGCTAGAGGGTCTGACGGCCAGCCGGGCCCCCCTGGACCG

[0091] CCTGGTACAGCAGGGTTCCCCGGTAGCCCCGGTGCTAAGGGTGAGGTAGG

[0092] ACCCGCCGGGTCACCGGGTAGTAATGGAGCGCCGGGACAGCGAGGTGAGC

[0093] CGGGGCCGCAGGGGCATGCCGGTGCTCAGGGGCCACCGGGACCACCTGG

[0094] AATTAACGGTAGCCCAGGAGGGAAGGGTGAGATGGGGCCCGCCGGCATCC

[0095] CAGGAGCACCCGATTGATGGGCGCTAGAGGACCTCCTGGACCTGCAGGG

[0096] GCTAACGGAGCCCCTGGGTTAAGAGGCGGAGCCGGTGAACCTGGTAAAAA

[0097] CGGGGCTAAGGGTGAACCTGGCCCTAGAGGTGAACGCGGTGAAGCAGGA

[0098] ATTCCTGGAGTTCCAGGAGCTAAGGGTGAGGATGGTAAAGACGGCTCACC

[0099] AGGTGAACCGGGCGCTAATGGCCTGCCGGAGCGGCAGGTGAGAGAGGC

[0100] GCCCCAGGGTTTAGAGGTCCCGCCGGACCTAATGGTACCCTGGTGAAAAG

[0101] GGACCTGCTGGTGAGCGTGGCGCCACCGGGACCGGCGGGTCCAAGGGGAG

[0102] CTGCTGGTGAGCCCGGACGGGACGGAGTGCCGGGTGGCCCAGGAATGAG

[0103] AGGAATGCCAGGTAGTCCTGGAGGCCCCGGATCTGATGGTAAACCTGGTCC

[0104] ACCCGGTAGCCAAGGTGAGTCAGGTAGGCCGGGTCCACCTGGTCCTAGCG

[0105] GACCTCGAGGACAACCTGGAGTTATGGGCTTCCCAGGCCCTAAAGGCAAC

[0106] GATGGTGCTCCAGGAAAAAATGGTGAGAGGGGCGTCCTGGTGGCCCCGG

[0107] CCCCCAGGGGCCTCCTGGAAAAAATGGAGAAACAGGTCCACAGGGACCTC

[0108] CCGGTCCCACTGGCCCTGGTGGGGATAAGGGTGACACGGGACCGCCGGGC

[0109] CCACAAGGATTGCAGGGATTGCCAGGTACTGGGGGGCCGCCCGGTGAGAA

[0110] CGGTAAACCAGGTGAACCAGGTCCGAAAGGCGACGCTGGCGCTCCCGGA

[0111] GCTCCAGGAGGAAAAGGGGATGCAGGCGCTCCTGGTGAGCGCGGCCCGC

[0112] CCGGCCTGGCGGGTGCTCCCGGACTTCGAGGAGGTGCCGGCCCACCAGGC

[0113] CCCGAAGGCGGTAAAGGAGCCGCCGGCCCGCCTGGTCCGCCGGGGGCTGC

[0114] TGGTACTCCTGGATTGCAAGGAATGCCTGGTGAACGGGGGGGGCTTGGGT

[0115] CCCCAGGACCTAAAGGGGACAAAGGTGAACCAGGCGGACCTGGGGCTGA

[0116] TGGCGTCCCAGGTAAAGACGGCCCCCGTGGTCCCACCGGTCCGATTGGTCC

[0117] ACCTGGACCCGCGGGCCAGCCTGGTGACAAAGGCGAAGGAGGTGCACCT

[0118] GGCTTACCTGGAATCGCAGGCCCTCGAGGCTCGCCTGGAGAACGGGGTGA

[0119] AACGGGCCCCCCTGGGCCAGCCGGTTTTCCGGGCGCACCGGGTCAAAACG

[0120] GGGAACCAGGCGGAAAGGGTGAGCGAGGAGCCCCTGGTGAAAAGGGCGA

[0121] GGGAGGCCCTCCCGGCGTTGCGGGACCGCCTGGCGGATCGGGCCCAGCTG

[0122] GCCCTCCCGGACCACAAGGCGTAAAAGGCGAAAGGGGGTCCCCCGGAGG

[0123] TCCCGGCGCAGCTGGCTTCCCAGGTGCCAGAGGTCTGCCAGGGCCTCCAG

[0124] GCAGTAATGGAAACCCTGGTCCACCGGGTCCGAGCGGCTCTCCAGGCAAG

[0125] GATGGACCGCCCGGTCCAGCTGGTAATACGGGTGCGCCAGGGTCCCCTGGT

[0126] GTCTCTGGCCCTAAGGGAGATGCCGGGCAACCAGGCGAAAAGGGTAGCCC

[0127] AGGCGCCCAAGGTCCCCCAGGCGCTCCAGGCCCGCTTGGAATCGCCGGAA

[0128] TAACAGGAGCTAGGGGTTTAGCCGGTCCACCTGGTATGCCGGGTCCTAGAG

[0129] GTTCACCTGGACCACAAGGAGTCAAAGGAGAAAGCGGTAAACCTGGTGCT

[0130] AACGGACTGTCTGGAGAAAGAGGTCCCCCAGGACCGCAGGGTCTCCCTGG

[0131] TTTGGCTGGTACTGCAGGAGAACCTGGACGTGACGGTAATCCAGGTAGCG

[0132] ACGGACTGCCAGGTAGAGATGGTAGCCCTGGTGGTAAAGGAGATAGAGGA

[0133] GAAAATGGTTCACCCGGTGCTCCAGGTGCCCCTGGTCACCCTGGTCCACCA

[0134] GGACCTGTTGGTCCAGCTGGTAAATCAGGTGACAGAGGCGAATCCGGACC

[0135] AGCTGGTCCTGCTGGAGCTCCAGGACCAGCTGGATCACGTGGTGCTCCGG

[0136] GACCTCAAGGACCTAGAGGAGATAAAGGTGAAACTGGTGAAAGAGGTGCT

[0137] GCCGGTATTAAAGGTCACAGAGGATTTCCCGGAAACCCTGGAGCTCCAGGT

[0138] AGCCCTGGACCTGCTGGTCAACAAGGTGCTATCGGATCTCCGGGTCCTGCT

[0139] GGACCAAGAGGACCTGTTGGCCCTTCTGGACCACCTGGTAAAGATGGTAC

[0140] TTCAGGACACCCTGGACCTATCGGACCACCTGGCCCTCGTGGTAACAGAG

[0141] GTGAAAGAGGTTCCGAAGGTTCACCAGGTCATCCGGGTCAACCAGGTCCT

[0142] CCGGGTCCACCAGGTGCCCCAGGTCCATGTTGCGGTGGTGTGGGTGCTGCT

[0143] GCTATAGCTGGTATTGGTGGTGAAAAAGCTGGTGGTTTTCGCTCCATACTACG

[0144] GA

[0145] Example 2 Construction of transgenic plasmid

[0146] The original transgenic vector used in this embodiment is pXL-BacII-IE1-DsRed2-FibH-P (which can be constructed using conventional techniques), derived from the piggyBac transposon.

[0147] Specific primers with Nru I restriction sites were designed to amplify partial and complete full-length recombinant human type III collagen from the pUC57 plasmid containing the gene sequence synthesized in Example 1. The primer pairs used were:

[0148] pp_F:ctagtgctgcagtctcgcgaATGCAATATGATTCTTATGATGTTAAATCT(SEQ ID NO.6)

[0149] pp_R:tcctcgcccttgctcaccatTCCATAATAAGGAGCAAATCCTCC(SEQ ID NO.7)mp_F:ctagtgctgcagtctcgcgaATGCAATATGATTCATATGATGTAAAAAG(SEQ ID NO.8)

[0150] mp_R:ccgtaactgacgcttcgcgaATGATGGTGATGGTGGTGTCCGTAGTATGGAGCGAAAACCA(SEQID NO.9)

[0151] Specific primers with homologous arms of p were designed. Enhanced green fluorescent protein (EGFP) was obtained by cloning and purifying the original vector pXL-BacII-IE1-EGFP (which can be constructed using conventional techniques) using high-fidelity PCR polymerase. The primer pairs used were:

[0152] EGFP_F: GATTTGCTCCTTATTATGGAatggtgagcaagggcgagg (SEQ ID NO.10) EGFP_R:ccgtaactgacgcttcgcgaCTTGTACAGCTCGTCCATGCC (SEQ ID NO.11)

[0153] The nucleotide sequence of the enhanced green fluorescent protein is shown as (SEQ ID NO.5): ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0154] After amplifying and purifying the target fragment using high-fidelity PCR polymerase, the original transgenic vector pXL-BacII-IE1-DsRed2-FibH-P was linearized using the restriction endonuclease Nru I. The corresponding target fragment was then ligated into the original transgenic vector via homologous recombination, transformed into DH5α competent cells, and single colonies were picked for sequencing verification. This resulted in the construction of the transgenic plasmids pXL-BacII-IE1-DsRed2-FibH-P-COL3A1-pp-EGFP and pXL-BacII-IE1-DsRed2-FibH-P-COL3A1-mp. Schematic diagrams of these transgenic plasmids are shown below. Figure 1 and Figure 2 As shown.

[0155] Example 3: Construction of transgenic silkworm strains

[0156] In this embodiment, PHA3PIG was used as a helper plasmid to produce transposase (see Tamura et al., Nature Biotechnology, 2000). Commercially available silkworm embryos (less than 8 hours after birth) from the non-diapause line Nistrali were selected for microinjection. The transgenic plasmid obtained in Example 2 (at a working concentration of 200–500 ng / μL) and the PHA3PIG helper plasmid (at a mixed solution concentration of 100–300 ng / μL) were mixed and injected into the silkworm embryos. The injection site was sealed with non-toxic glue after injection. The injected silkworm eggs underwent priming, hatching, rearing, and moth emergence to obtain the G0 generation silkworm moths. The male and female moths of the G0 generation mate, and the extra moths mate with wild-type silkworm moths. When the embryos turn green, fluorescent markers are screened using a fluorescence microscope to obtain positive G1 generation silkworm individuals, namely silkworm strains expressing recombinant human type III collagen fragments (FH-pp) or silkworm strains expressing complete full-length recombinant human type III collagen (FH-mp).

[0157] Because the silkworm strain FH-pp, which expresses a recombinant human type III collagen fragment, fused with enhanced green fluorescent protein, the specific expression of the recombinant human type III collagen fragment in the posterior silk gland can be observed using fluorescence microscopy at a specific excitation wavelength. Figure 3 ), and successfully secreted into the silkworm cocoon ( Figure 4 Mass spectrometry analysis revealed that the COL3A1 peptide coverage in the posterior silk gland of FH-mp transgenic silkworms was 7%, consistent with the GXY structural characteristics of collagen. Figure 5 Simultaneously, mass spectrometry analysis revealed that the COL3A1 peptide coverage in the cocoons of FH-mp transgenic silkworms was 55%, with the N-terminus completely matching the theoretical sequence. Figure 6This indicates that the complete, full-length recombinant human type III collagen is specifically expressed in the posterior silk gland and successfully secreted into the silkworm cocoon.

[0158] Example 4: Cell proliferation experiment

[0159] Silk material containing complete, full-length recombinant human type III collagen COL3A1-mp was extracted from FH-mp transgenic silkworm cocoons using urea, and its effect on NIH / 3T3 cell proliferation was analyzed. After digestion with 0.25% trypsin, the material was diluted to 1×10⁻⁶ with complete culture medium. 4 Cell suspension was prepared at 100 μL / mL. 100 μL of the cell suspension was seeded into 96-well plates and incubated for 24 h. The culture medium was then removed and aspirated. In the experimental groups, extracts of FH-mp transgenic silkworm cocoons and wild-type silkworm cocoons diluted with DMEM medium were added. Simultaneously, commercial recombinant human type III collagen (purchased from Nanjing Bakaxing Biotechnology Co., Ltd.) at the same concentration was used as a positive control. The control group consisted of cells cultured in DMEM medium, and the blank group consisted of cell-free DMEM medium. After further incubation for 24 h, the culture medium was aspirated, and 100 μL of DMEM medium containing 10% CCK8 was added to each group. The plates were incubated for 2 h, and the absorbance (OD) at 450 nm was measured using a microplate reader.

[0160] The results are as follows Figure 7 As shown, compared with wild-type cocoon extract and control group, FH-mp cocoon extract significantly enhanced cell proliferation activity.

[0161] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant human type III collagen fragment, characterized in that, The amino acid sequence of the recombinant human type III collagen fragment is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant human type III collagen fragment, characterized in that, After optimization based on the codon preference of silkworms, the nucleotide sequence of the gene encoding the recombinant human type III collagen fragment is shown in SEQ ID NO.3, and is used to encode the recombinant human type III collagen fragment of claim 1.

3. A full-length recombinant human type III collagen containing the recombinant human type III collagen fragment of claim 1, characterized in that, The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.

2.

4. A gene encoding a full-length recombinant human type III collagen, characterized in that, After optimization based on the codon preference of silkworms, the nucleotide sequence of the gene encoding the full-length recombinant human type III collagen is shown in SEQ ID NO.4, which is used to encode the full-length recombinant human type III collagen as described in claim 3.

5. A transgenic plasmid, characterized in that, The gene encoding the recombinant human type III collagen fragment as described in claim 2 or the gene encoding the full-length recombinant human type III collagen as described in claim 4.

6. A method for constructing recombinant human type III collagen from silkworms, characterized in that, The transgenic plasmid described in claim 5 was introduced into silkworm cells.

7. The construction method according to claim 6, characterized in that, Includes the following steps: Step 1, constructing transgenic plasmids Using the transgenic vector pXL-BacII-IE1-DsRed2-FibH-P, which is the promoter of the silkworm fibroin heavy chain gene, as the original plasmid, the gene encoding the recombinant human type III collagen fragment described in claim 2 and the gene encoding enhanced green fluorescent protein EGFP or the full-length recombinant human type III collagen described in claim 4 were ligated into the original plasmid through homologous recombination to obtain the transgenic plasmid. Step 2, genetic transformation of silkworms The transgenic plasmid constructed in step 1 is mixed with the helper plasmid and injected into silkworm embryos. After induction, hatching, rearing, and moth emergence, G0 generation silkworm moths are obtained. Male and female moths of the G0 generation mate, and the extra moths mate with wild-type silkworm moths. Then, the female moths are placed on oviposition paper to lay eggs, and the eggs laid are the G1 generation. The G1 offspring are selected by fluorescent selection to obtain transgenic positive silkworm individuals, that is, silkworms that produce recombinant human type III collagen. The helper plasmid is a recombinant plasmid expressing transposase. The mixing mass ratio of the transgenic plasmid to the helper plasmid is 0.5-10:

1.

8. The construction method according to claim 7, characterized in that, The original transgenic vector mentioned in step 1 is pXL-BacII-IE1-DsRed2-FibH-P; the linker elements of the transgenic vector from the 5' end to the 3' end are, in sequence, the silk fibroin heavy chain promoter FibH-P, the silk fibroin heavy chain N-terminal domain FibH-NTD, and the silk fibroin heavy chain C-terminal domain FibH-CTD.

9. The construction method according to claim 7, characterized in that, In step 1, the amplification primers used to amplify the recombinant human type III collagen fragment were pp_F and pp_R, and their nucleotide sequences are shown in SEQ ID NO. 6-7; the amplification primers used to amplify the full-length recombinant human type III collagen were mp_F and mp_R, and their nucleotide sequences are shown in SEQ ID NO. 8-9; the amplification primers used to amplify enhanced green fluorescent protein (EGFP) were EGFP_F and EGFP_R, and their nucleotide sequences are shown in SEQ ID NO. 10-11; the nucleotide sequence of enhanced green fluorescent protein (EGFP) is shown in SEQ ID NO.

5.

10. A method for producing recombinant human type III collagen using silkworms, characterized in that, The method involves collecting the posterior silk glands or cocoons of silkworms constructed using the method described in any one of claims 6-9, and extracting recombinant human type III collagen.

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