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Transformed silkworm producing human collagen

A technology of human collagen and collagen, applied in the fields of biochemical equipment and methods, oxidoreductases, animal/human peptides, etc.

Inactive Publication Date: 2004-07-28
JAPAN SCI & TECH CORP +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As mentioned above, methods using insect cells, mammalian cells, and yeast have been conceived as methods for producing recombinant human collagen. Such a high output is more difficult

Method used

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  • Transformed silkworm producing human collagen
  • Transformed silkworm producing human collagen
  • Transformed silkworm producing human collagen

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Production of transformed silkworm by gene targeting method using AcNPV vector

[0052] The cDNA encoding human type III procollagen is based on the cDNA cloned by the inventor of the present application (Japanese Patent Laid-Open No. 8-23979: GeneBank Accession No. X14420), while the silk fibroin L chain gene and its downstream silkworm genomic DNA The sequence was isolated according to the following method based on the known base sequence of the silk fibroin L chain gene (Gene 100: 151-158, 1992: GeneBank database accession number M76430).

[0053] In the following description, the base numbering of the cDNA of human type III procollagen and the silk fibroin L chain gene is based on the base numbering described in the aforementioned GeneBank database.

[0054] (1) Isolation of silk fibroin L chain gene and its downstream silkworm genomic DNA sequence

[0055] The PCR method was used to amplify the gene fragments in intron 6 of the fibroin L chain. The PCR primers ...

Embodiment 2

[0077] Production of transformed silkworm by injecting piggyBac vector into eggs

[0078] (1) Preparation of piggyBac plasmid vector

[0079] The human type III procollagen cDNA (base number 92-4550: GeneBank database accession number X14420) was digested with XhoI, the base sequence region with base number 1075-3545 was removed, and the cut end was connected by self-ligation. Make small type III collagen cDNA. The small type III collagen cDNA is composed of an amino propeptide, a part of the triple helix region (approximately 1 / 5 of the triple helix region of the full-length collagen), and a base sequence encoding a carboxy propeptide. Based on this, the following 3 Kind of carrier.

[0080] i) pMOSRA-4B

[0081] The inserted DNA fragment contained in the vector is composed of a silk jelly (protein) promoter, a small collagen cDNA, and a silk fibroin L chain plus a polyA signal peptide. The silkworm sericin (protein) promoter (base number 1299-1622: GeneBank database accession...

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Abstract

A transformed silkworm which has a polynucleotide encoding human collagen in its genomic DNA and produces recombinant human collagen as a part of proteins in its cocoon or silk gland; a process for producing human collagen by using this transformed silkworm; and a recombinant vector to be used in the constitution of the transformed silkworm. Since human collagen is collected from the cocoon discharged by this transformed silkworm or its silk gland, highly pure human collagen can be conveniently obtained in a large amount. Moreover, the recombinant human collagen produced by the transformed silkworm is a highly safe collagen which is free from any fear of the contamination with pathogens such as viruses or prions and shows no antigenicity on humans. Thus, it is usable in various industrial fields including medicines, foods and cosmetics.

Description

Technical field [0001] The present invention relates to a transformed silkworm that produces recombinant human collagen. In more detail, the invention of this application relates to a transformed silkworm that produces recombinant human collagen, a recombinant vector for producing the transformed silkworm, and a method for producing recombinant human collagen. Background technique [0002] Collagen is a representative protein that constitutes the extracellular matrix. In addition to the so-called mechanical function of maintaining the biological structure that becomes the foothold of the cell, collagen also has various physiological functions such as regulating cell proliferation, differentiation, and migration. Therefore, collagen is used as a biological material for repairing biological damage (J. Surg. Res. 10: 485-491, 1970) and as a carrier for slow release of a certain agent (J. Controlled Release 33: 307-315). , 1995) is widely used in the medical field. However, most of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/033A01K67/04C07K14/435C07K14/47C07K19/00C12N5/10C12N9/02C12N15/09C12N15/85
CPCA01K2267/01A01K2217/05A01K67/0333A01K67/0339C12N15/8509
Inventor 吉里胜利富田正浩佐藤勉森肇田村俊树安达敬泰宗纲洋人
Owner JAPAN SCI & TECH CORP
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