JP2001161214A - Transformed silkworm - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transformed silkworm producing a recombinant human collagen as a part of protein contained in cocoon and obtain a recombinant human collagen produced by the silkworm. SOLUTION: The transformed silkworm contains a hybrid gene composed of a human collagen gene and a silkworm fibroin L chain gene and produces a recombinant hybrid protein containing the recombinant human collagen as a part of protein in a cocoon or a sericterium. The invention also provides a recombinant human collagen produced by the silkworm.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The invention of this application relates to a recombinant human
The present invention relates to a transgenic silkworm that produces collagen. More specifically, the invention of this application relates to recombinant human
The present invention relates to a transformed silkworm that produces collagen, a recombinant vector for producing the transformed silkworm, a recombinant human collagen produced by the transformed silkworm, and a method for producing a recombinant human collagen.

[0002]

2. Description of the Related Art Collagen is a typical protein constituting the extracellular matrix, and serves as a scaffold for cells to maintain the structure of living organisms, as well as for proliferation, differentiation and migration of cells. It has various physiological functions to control. Therefore, collagen is used as a biomaterial for repairing damage to living bodies (J. Surg. Res. 10: 485-4
91, 1970) and as a carrier for the sustained release of certain drugs (J. Controlled Release 33: 307-315, 199).
5), widely used in medical field. However, most of the collagen currently used is derived from animal tissues such as cows and pigs, and it is known that allergic reactions occur in about 3% of patients when these collagens are transplanted into humans. (J. Immunol. 136: 877-882, 1986; Bio
materials 11: 176-180, 1990). In addition, the danger of contamination with pathogens such as viruses and brions in animal tissue-derived collagen has become a serious problem in recent years. Therefore, a system for producing recombinant human collagen which has no antigenicity and has no risk of pathogen contamination is desired. Therefore,
The inventors of the present application produced recombinant human collagen having a triple helix structure equivalent to that in a human body by infecting insect cells with a recombinant virus into which cDNA encoding human collagen was inserted. And a patent application has been filed (JP-A-8-23979). Also, a method of producing human collagen using mammalian cells or yeast has been devised (Japanese Patent Application Laid-Open No. 7-50 / 1990).
1939).

[0003]

As described above, as a method for producing recombinant human collagen, methods using insect cells, mammalian cells, and yeast have been devised, but insect cells and mammalian cells are used. It is difficult for the method to increase the production so high that it can be used in the medical field. Further, in the method using yeast, purification of recombinant human collagen is not always easy because a recombinant product is produced in the cells.

[0004] The invention of this application has been made in view of the above circumstances, and solves the problems of the prior art, and provides a method for producing recombinant human collagen which has both high productivity and ease of purification. The challenge is to provide genetic engineering materials for that purpose.

[0005]

This application provides the following inventions (1) to (6) to solve the above-mentioned problems. (1) The fusion gene of the human collagen gene and the silkworm fibroin L chain gene is incorporated,
A transformed silkworm that produces a recombinant fusion protein containing collagen as a part of the protein in the cocoon or silk gland. (2) A recombinant vector containing a fusion gene of a human collagen gene and a silkworm fibroin L chain gene, and capable of incorporating this fusion gene into silkworm genomic DNA. (3) The recombinant vector according to the invention (2), which is an Autographa californica nucleopolykaryotic virus containing a fusion gene. (4) A recombinant fusion protein of a fibroin L chain produced by the transformed silkworm of the invention (1) and human collagen. (5) Recombinant human collagen obtained by removing the fibroin L chain from the recombinant fusion protein of the invention (4). (6) From the cocoon or silk gland of the transformed silkworm of the invention (1),
A recombinant fusion protein containing recombinant human collagen is isolated, and fibroin L is isolated from the recombinant fusion protein.
A method for producing recombinant human collagen, comprising removing chains.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been known that there are nineteen different types of collagen from type I to type XIX to date. Each of these collagens is a trimer molecule formed from three subunits (α chains), and is characterized by having a triple helical structure in the molecule. Some collagen is first synthesized as a precursor, procollagen, and then converted to a mature collagen molecule. For example, fibrous collagen such as type I, II, and III collagen is synthesized as a procollagen having a non-triple helix aminopropeptide and a carboxyl propeptide at the amino terminal side and the carboxyl terminal side of the triple helix region which is the main body thereof. And after both propeptides have been cleaved by specific proteases,
It becomes mature collagen.

[0007] The human collagen to which the invention of this application is directed is any collagen including type I to type XIX collagen. Moreover, in addition to mature collagen, the precursor includes procollagen and propeptide which are partially cut.

[0008] 97% of cocoons produced by silkworms are composed of proteins called fibroin and sericin, and fibroin is the most important protein, accounting for 70-80% of silk proteins. Fibroin is a complex composed of an H chain having a molecular weight of about 350 kDa and an L chain having a molecular weight of 25 kDa, and is synthesized in large amounts in the posterior silk gland at the age of 5 years. In the posterior silk gland, DNA replication without cell division was repeated as the larva grew, and on day 5 of age 5 the amount of nuclear DNA in the posterior silk gland reached 200,000 times the diploid genome size and was synthesized there. The amount of synthesized fibroin is 1 cocoon length per silkworm, 1,
It is huge, up to 300m. Therefore, the inventors of the present application focused on the superior fibroin synthesis ability of silkworms and the uniformity of proteins in cocoons, and by using a biosynthesis system for fibroin, achieved high productivity and an easy purification method. A combined human collagen production method was invented.

As a transient foreign gene expression system in Bombyx mori, a method has been established in which Bombybean nucleopolynucleovirus (BmNPV) is used as a vector (Japanese Patent Publication 7-97).
No. 995). However, in this method, since silkworms are killed by virus infection, foreign genes cannot be transmitted to subsequent generations. On the other hand, Mori et al. Autographaca
By infecting silkworms with lifornica nucleopolynuclear virus (AcNPV), we have developed a system that introduces genes without lethaling the silkworms. In addition, when a female is infected, foreign genes are transmitted to the next generation through germ cells. (JP-A-6-277051). Furthermore, after constructing an AcNPV that incorporates a part of the gene sequence in the silkworm genome, Yamao et al. Succeeded in producing a transgenic silkworm into which a foreign gene was inserted by infecting this and then transfecting the gene (Gene
s Dev. 13: 511-516, 1996).

In the invention of this application, AcNPV is used as a vector according to the method of Yamao et al. The collagen cDNA to be incorporated into the vector is incorporated as a fusion gene with the fibroin light chain gene. After being synthesized in the posterior silk gland cells, the fibroin L chain forms a complex with the fibroin H chain through disulfide bonds and is secreted as a main component of the cocoon filament. Therefore, the fusion protein synthesized from the fusion gene of the fibroin L chain gene and the collagen cDNA also forms a complex with the H chain and is efficiently secreted from the silk gland as a cocoon thread component. The collagen cDNA may be any type I to type XIX collagen cDNA. The nucleotide sequences of these cDNAs are described in the literature (eg, Essays Biochem. 27: 49-67, 1992; An
nu. Rev. Biochem. 64: 403-434, 1995), for example, a method for screening a human cDNA library using oligonucleotides prepared based on these cDNA sequences as probes. Alternatively, using the oligonucleotides corresponding to both ends of the cDNA sequence as primers and PCR using human genes as templates, each c
DNA can be obtained.

[0011] Gene ligation can be performed, for example, by incorporating cDNA
If encodes fibrous collagen,
A fusion gene is prepared by ligating the 5 'end of the cDNA encoding fibrous procollagen to the 3' end of the fibroin L chain gene so that the amino acid frame is continuous. From this fusion gene, a fusion protein in which a fibroin L chain is linked to the amino terminal side of procollagen is synthesized. The reason that the fibroin L chain is linked to the amino terminal side of fibrous procollagen rather than the carboxyl terminal side is that the carboxyl propeptide present at the carboxyl terminal of procollagen plays an important role in the association with procollagen trimer. This is because if a fibroin L chain is linked to the end of this region, the trimerization ability of procollagen may be lost. The ligation of the fibroin L chain gene and the procollagen cDNA is performed, for example, by introducing the same restriction enzyme site immediately before the stop codon in the exon 7 of the fibroin L chain gene and in the nucleotide sequence encoding the aminopropeptide region of the procollagen cDNA. These are cleaved with restriction enzymes and then ligated. The fusion gene prepared by the above method is inserted into the fibroin L chain locus in the silkworm genome by gene targeting. For this purpose, it is necessary to insert the genomic DNA sequences upstream and downstream from the exon 7 of the fibroin L chain, which is the portion into which the collagen cDNA is inserted, into the vector AcPNV at about 0.5 kb to 6.0 kb, respectively. For example, the fibroin light chain gene upstream of exon 7 is inserted as a fusion gene with collagen cDNA,
The silkworm genomic DNA sequence downstream from exon 7 should be incorporated downstream of the collagen cDNA.

Recombinant AcNP incorporating the above DNA sequence
The V vector was prepared according to the method of Yamao et al. (GenesDev. 13: 511-516, 1
According 999) was inoculated into 5 old female larvae from F ₁ and F ₂ generations derived from that silkworm, using methods such as PCR or Southern blot, collagen cDNA is incorporated into fibroin L chain locus of the genome The transformed silkworms are selected. To ensure selection, a marker gene such as GFP (jellyfish-derived fluorescent protein) can be incorporated into the vector. In this case, a promoter sequence such as the Drosophila HSP70 promoter is incorporated upstream of the marker gene, and the marker gene is expressed by its action. Places where the promoter and marker genes are incorporated into the vector include, for example, procollagen cDNA and fibroin L
Suitable is between silkworm genomic DNA sequences downstream of exon 7 of the strand.

[0013] Collagen cDNA is incorporated in the fibroin L chain gene of the selected transgenic silkworm, and collagen is produced as a fusion protein with the fibroin L chain. Since the fusion gene is transcribed by the endogenous fibroin L chain promoter, the fusion protein is synthesized only in the posterior silk gland, and its synthesis is maximized at the fifth infancy when silk is formed. The fusion protein of fibroin L chain and collagen synthesized in posterior silk gland cells
After being linked to the fibroin H chain by a disulfide bond, it is secreted efficiently out of the cell and exhaled from the mouth of the silkworm as a part of a cocoon thread. About 70% of cocoon protein
Up to 80% is fibroin, and the bond between the fusion protein and the fibroin H chain can be cleaved by reducing it, so that the fusion protein can be easily purified from the cocoon. When the collagen contained in the fusion protein is fibrous collagen and only the triple helix region (atelocollagen) is purified, the cocoon protein containing the fusion protein is treated with a protease such as pepsin. By this operation, it is possible to isolate only atelocollagen that is not digested by the protease.

[0014]

EXAMPLES The invention of this application will be more specifically described below with reference to examples relating to a method for producing human type III collagen, but the invention of this application is not limited to these examples. A. Preparation of targeting vector cDNA encoding human type III procollagen
CDNA cloned by the inventors of this application (Japanese Patent Laid-Open No. 8-23979: GeneBank database registration number X1442)
0) In addition, the silkworm fibroin L chain gene and the downstream silkworm genomic DNA sequence
Nucleotide sequence of fibroin L chain gene (Gene 100: 151-15
8, 1992: GeneBank database registration number M76430).

In the following description, human type III procollagen cDNA and silkworm fibroin L
The base numbers of the strand genes are in accordance with the base numbers described in the GeneBank database. A-1. Isolation of Bombyx mori fibroin L chain gene and genomic DNA sequence downstream thereof A gene fragment in fibroin L chain intron 6 was amplified by PCR. Oligonucleotides (SEQ ID NO: 1 and SEQ ID NO: 2) synthesized based on the sequence of the database were used as PCR primers, and genomic DNA of silkworm tokai × asahi strain was used as a template. Next, using the obtained DNA fragment in a prog, λEMBL
The silkworm kinshu × showa strain genomic library constructed in 3 was screened according to a standard method. As a result, a silkworm genomic DNA fragment (pRI / 10k) containing a region of about 15 kb downstream from base number 9600 of the fibroin L chain gene was obtained. A-2. Preparation of Restriction Enzyme Recognition Sequence by Site-Directed Mutagenesis In order to connect the type III procollagen cDNA to exon 7 of the fibroin L chain gene, a new restriction enzyme site was provided in the fibroin gene by site-directed mutagenesis. Mutagenesis is from Clontech's Mutagenests Kit
It was used. The primer (SEQ ID NO: 3) is a restriction enzyme immediately before the stop codon of exon 7 of the fibroin L chain gene.
It was designed to provide an Xho I site (positions 8-12 of SEQ ID NO: 3). The template plasmid contains EcoRI-SphI from pRI / 10k.
A fragment (pRISphI / 2.9k) obtained by subcloning a fragment (base numbers 12000-14800) into pUC18 was used (Mut pRISph).
I-XhoI). Similarly, an XbaI site was newly provided by mutagenesis in order to insert a fragment of the downstream region of the fibroin L chain gene (base numbers 14200-18900). The primer (SEQ ID NO: 4) was designed to provide an XbaI site (positions 12-17 of SEQ ID NO: 4) immediately before the stop codon of fibroin L chain gene exon 7. The same pRISphI / 2.9k was used as the template plasmid (Mut pRISphI-
XhoI).

The site of the restriction enzyme XhoI was also introduced into the nucleotide sequence encoding the aminopropeptide of the type III procollagen cDNA using the same site-directed mutagenesis method as described above. The nucleotide sequence of the primer (SEQ ID NO: 5) corresponds to nucleotide numbers 292 to 324 of type III procollagen cDNA, and a restriction enzyme XhoI site (positions 17-22 of SEQ ID NO: 5) has been inserted. A-3. Construction of transfer vector Clontech baculovirus transfer vector pBacPAK9 was digested with restriction enzymes SmaI and EcoRI, and pCa
A DNA fragment (EcoRV-E) obtained by ligating the Drosophila HSP70 promoter excised from SepR-hsp (GeneBank accession number U59056) and EGFP cDNA (Clontech)
(coRI), and transformed into E. coli DH5α (pBacPAKhsEGFP). Ligation was performed using TaKaRa Ligation Kit Ver. 1, and transformation was performed according to a standard method.

The previously completed mutant plasmid Mut
A fragment of the insert sequence from pRISphI-XhoI (Eco
RV-XhoI), type III procollagen cDNA
XhoI site (constructed by mutagenesis). Subsequently, the SV40 polyA additional signal sequence amplified from pCEP4 (Invitrogen) by PCR was added to collagen cDN.
It was inserted downstream of A (BglI site). Then, the completed fibroin / collagen / polyA additional signal sequence fragment (EcoRV-BglI) was ligated to EcoRV-BglI of pBacPAKhsEGFP.
Connected to the site. Next, a part of intron 2 to a part of exon 7 of the fibroin L chain gene (base number about 10,000-1
3100) was inserted into the EcoRV site (pBacPAKhsEGFP-Fib).
1).

Subsequently, the mutant plasmid Mut pRISph
Fragment of the insert sequence EcoRV-SphI from I-XbaI
1.7 kbp (base number about 13100-14800) was cut out and pRI / 1
Inserted at the 0k SmaI-SphI site (by partial digestion).
An XbaI digested fragment (part of fibroin gene exon 7 and its downstream region / base number of about 14200-18900) cut out from the completed Mut RI / 10k was pBacPAKhsEGFP-Fib.
1 (pMOSRA-1: FIG. 1). A-4. Preparation of Recombinant Virus Recombinant virus was prepared by co-transfection of the prepared targeting vector pMOSRA-1 and baculovirus DNA into cultured Spodoptera litura cells Sf9. After adding 4 μl of lipofectin (Gibco) and 4 μl of water to 7 μl of pMOSRA-1 0.4 μg / μl and 1 μl of linear baculovirus DNA (Pharmingen) 0.1 μg / μl, and mixing well, the serum-free medium SF900-II (Gibco) was added. Replaced Sf9
The cells were dropped on 1 × 10 ⁶ cells and cultured. 10% after 24 hours
After adding 1 ml of serum-containing Grace medium and culturing for further 3 days, the culture supernatant was recovered. A-5. Screening and purification of recombinant virus 100 μl of the above virus solution was added to 1 × 10 ⁶ Sf9 cells,
Infect at 1 hour at 28 ° C. The supernatant was removed, and 1 ml of Grace medium containing 1% low boiling point agarose solution (SEAPLAQUE, FMC) was added to solidify. After 3 days of culture, the resulting plaques
Irradiate with 0 nm excitation light and plaques that emit green fluorescence, i.e., plaques of cells expressing EGFP,
Pieces and agar were cut out. Next, 1 × 10 ⁶ Sf9 cells were infected again with the recombinant virus recovered from these plaques, DNA in the cells was extracted, and dot plotting was performed. Two kinds of probes, a probe recognizing the fibroin L chain gene and a probe recognizing the procollagen cDNA, were used. As a result, virus clones derived from four plaques were positive. Next, 1 × 10 ⁶ Sf9 cells were infected with these positive viruses, and the culture supernatant was collected 3 days later. The virus in the culture supernatant was similarly reinfected into Sf9 cells to obtain a high titer virus stock. B. Preparation of transgenic silkworm B-1. Infection of the 5th-instar larvae of the silkworm, Bombyx mori, with 50 μl of the virus solution (5
× 10 ⁶ pfu) was injected subcutaneously. 20-hydroxyec dissolved in methanol 3-4 days after inoculation of larvae
10 μl of dysone (2 mg / ml) was administered. After eclosion, they were crossed with normal silkworm males and laid eggs. B-2. F ₁ silkworm F ₁ eggs gave birth Mesukaiko screening one animal of the (100 to 300 grains) and one group was sampled approximately 50 grain of eggs from each group continued to breeding for the remaining eggs. DNA was extracted from the sampled eggs by a conventional method, and the presence or absence of a foreign gene was determined by a PCR method. PCR was performed to detect the SV40 polyA additional signal using primers (SEQ ID NOs: 6 and 7) or to detect the HSP70 promoter using primers (SEQ ID NOs: 8 and 9).

[0019] The total 1800 group eggs a result of the screening of a 15 group is positive, were hatched the rest of the eggs of these groups, and the hatched F ₁ larvae were reared up to 5 age. On the third day of the fifth instar, about 100 μl of body fluid was collected from each larva, the body fluid cells were separated from these body fluids by centrifugation, DNA was extracted, and screening was performed by PCR. As a result of screening a total of 3,400 5th instar larvae, 8 were positive. These silkworms continued to be bred, and the emerged silkworms were crossed with normal silkworms to lay eggs. B-3. F ₂ silkworm screening F ₂ silkworms screening was performed by green fluorescence.
Exciting light having a wavelength of 360 nm was applied to the first instar larvae to select individuals that emitted green fluorescence of EGFP, that is, individuals that expressed EGFP inserted as a marker gene. 8 groups of F
As a result of screening _two eggs, two positive silkworms could be obtained. B-4. Were housed detected positive F ₂ silkworms of recombinant human collagen to phase the spinning, the proteins in the cocoon SDS- sample buffer (0.125 M Tris-HCl buffer,
pH 6.8 / 4% SDS / 10% 2-mercaptoethanol / 20
% Glycerol), mixed and heat-treated at 100 ° C. for 5 minutes to extract. This sample was subjected to SDS polyacrylamide gel electrophoresis (Nature 227: 680-685, 1970), and the method of Matudaira et al. (J. Biol. Chem. 261: 10035-10) was used.
038, 1987), the electrophoresed protein was transferred to a nitrocellulose membrane BA85 (S & S). next,
The nitrocellulose membrane onto which the protein was transferred was blocked with a blocking solution (3% BSA / 50 mM Tris-HCl buffer pH 7.5 / 1
After treatment with 50 mM NaCl) at 4 ° C. for 16 hours, the mixture was reacted with an anti-human / bovine type III collagen antibody diluted 200-fold with a blocking solution for 1 hour at room temperature. Proteins to which these antibodies react were detected with Vectastain ABC kit (Vector Laboratories). As a result, human type III collagen could be detected from the cocoon. B-5. The Purification of recombinant F ₂ silkworm recombinant collagen mated with normal silkworms to obtain F ₃ silkworm. The silkworms were bred to the fifth instar, body fluids were collected from all the silkworms, and the presence or absence of the recombinant gene was determined by PCR. As a result, the human collagen gene was detected in about 50% of the silkworms. Since these recombinant F ₃ silkworms spun normally and formed cocoons, extraction and purification of recombinant collagen from cocoons was attempted. Ten cocoons were treated with 50% Tris-HCl buffer containing 5% 2-mercaptoethanol pH
After crushing in 7.5, protein extraction was performed at 4 ° C. for 24 hours. Next, the extract was dialyzed against 0.5 M acetic acid, and then pepsin was added to digest the non-collagen protein and the fibroin L chain portion of the recombinant fusion protein. Further, collagen was purified according to a standard method. As a result, with the above simple operation, high-purity human type III collagen
g (10 mg / cocoon) could be obtained.

[0020]

As described above in detail, according to the invention of this application, a transgenic silkworm that produces recombinant human collagen as a part of the protein contained in the cocoon, and a recombinant human collagen produced by the silkworm, Provided. Since the recombinant human collagen is recovered from the cocoon of the transgenic silkworm, the collagen can be easily extracted and high-purity collagen can be easily obtained. In addition, the recombinant human collagen produced by the transgenic silkworm has no danger of contamination with pathogens such as viruses and prions, and is safe human collagen without antigenicity to humans. Can be used in various industrial fields.

[0021]

[Sequence List] <110> Japan Science and Technology Agency <110> Hiroshima Prefectural Industrial Technology Promotion Organization <110> Terumo Corporation <110> Koken Co., Ltd. <120> Transgenic silkworm <130> NP99491 <160> 9 <210> 1 <211> 22 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 1 tcgtaactgc ctacacgttt gc 22 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 2 agacgtgaac ctggctggct 20 <210> 3 <211> 15 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 3 tttagactcg agcct 15 <210 > 4 <211> 25 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 4 tacagttctt atctagacgt gaacc 25 <210> 5 <211> 33 <212> DNA <213> Artificial sequence <220 > <213> Synthesized oligonucleotide <400> 5 gacataatat gtgacgctcg agaattagac tgc 33 <210> 6 <211> 22 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 6 ggatccagac atgataagat ac 22 <210 > 7 <211> 21 <21 2> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 7 gatcataatc agccatacca c 21 <210> 8 <211> 22 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide < 400> 8 gatcccccta gaatcccaaa ac 22 <210> 9 <211> 22 <212> DNA <213> Artificial sequence <220> <213> Synthesized oligonucleotide <400> 9 cttagcgacg tgttcacttt gc 22

[Brief description of the drawings]

FIG. 1 is a restriction map of a transfer vector pMOSRA-1.

 ──────────────────────────────────────────────────続 き Continuation of the front page (71) Applicant 591071104 Koken Co., Ltd. 3- 14-3 Mejiro, Toshima-ku, Tokyo (72) Inventor Katsutoshi Yoshizato Inventor Masahiro Tomita 5-1-19 Chuo Saijo Chuo, Higashihiroshima City, Hiroshima Prefecture (72) Inventor Tsutomu 7-21-36 Chuo Saijo Chuo, Higashihiroshima City Hiroshima Pref. 4215-1

Claims (6)

[Claims] 1. A trait in which a fusion gene of a human collagen gene and a silkworm fibroin L chain gene is incorporated and produces a recombinant fusion protein containing recombinant human collagen as a part of a protein in a cocoon or silk gland. Converted silkworm. 2. A recombinant vector comprising a fusion gene of a human collagen gene and a silkworm fibroin L chain gene, wherein this fusion gene can be incorporated into silkworm genomic DNA. 3. An Autographa califor containing a fusion gene.
3. The recombinant vector of claim 2, which is a nica nucleopolykaryotic virus. 4. A recombinant fusion protein of a fibroin L chain produced by the transgenic silkworm of claim 1 and human collagen. 5. A recombinant human collagen obtained by removing the fibroin L chain from the recombinant fusion protein according to claim 4. 6. A recombinant fusion protein containing recombinant human collagen is isolated from the cocoon or silk gland of the transformed silkworm according to claim 1, and the fibroin L chain is removed from the recombinant fusion protein. For producing recombinant human collagen.