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Method for producing human collagen type II

A technology of human collagen and fluorescent protein, applied in the field of production of recombinant human collagen type II, which can solve the problems of inability to carry out large-scale production, low expression level, and hidden dangers of viruses

Active Publication Date: 2015-01-28
SOUTH CHINA AGRI UNIV +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these materials are currently derived from animals, and animal-derived collagens can produce immune responses and have potential viral hazards. In addition, the lack of large amounts of tissue and the challenges of large-scale purification of collagen make it difficult to use these materials. Recombinant expression technology can solve the above problems
Although recombinant collagen has been successfully expressed in mammalian cells, insects, yeast, Escherichia coli, tobacco, mice and silkworms, the collagen gene can only be expressed in mammalian cells when the prolyl 4-hydroxylase gene is not expressed. Hydroxylated collagen is expressed, but its expression level is too low for large-scale production

Method used

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  • Method for producing human collagen type II
  • Method for producing human collagen type II
  • Method for producing human collagen type II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: the acquisition of recombinant plasmid

[0080] According to the polymorphism of EX-5512-B31 (synthesized by GeneCopoeia, Accession No.: NM_033150, its ORF sequence is shown in SEQ ID No.4, which contains the full-length sequence of human collagen type II (SEQ ID No.1)) Cloning site, select restriction site Xba I and Avr II to carry out double enzyme digestion on EX-5512-B31 to obtain the desired target gene, that is, human collagen type II α1 chain full-length gene (sequence such as SEQ ID No .1). And the genes IRES (sequence shown in SEQ ID No.5) and mCherry (sequence shown in SEQ ID No.6) were synthesized. Then, the human collagen type II α1 chain full-length gene (sequence shown in SEQ ID No.1) was connected to the transfer vector pFBDM (purchased from Shenzhen Baozhu Biotechnology Co., Ltd., Cat. No. zt323), construct pFBDM-col II, link the synthetic IRES and mCherry into pFBDM-col II, and construct the recombinant plasmid pFBDM-IM-col II.

[0081]...

Embodiment 2

[0082] Example 2: Recombinant Human Collagen Type II Virus Infects BmN to Construct Expression Vector Virus

[0083] The recombinant plasmids pFBDM-IM-col II and pUCDM-P4Hα-P4Hβ constructed in Example 1 were introduced into the asd auxotrophic host Escherichia coli SW106 by transposition (this laboratory preserved and transformed, and the transformation methods refer to Sun, Jingchen et al, Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid, Biotechnol. Appl. Biochem. (2010) 57, 117-125 (Printed in Great Britain. 20BA) doi 04: The recombinant genetically engineered bacteria SW106-pFBDM-col II-IM-pUCDM-P4Hα-P4Hβ producing human collagen type II.

[0084] In LB-Kan-Tet-Spe-Gm-Cm-DAP solid medium (medium composition: ordinary LB medium, Kan-Tet-Spe-Gm-Cm-DAP is antibiotics and nutritional ingredients, which contains 50ug / ml Kanamycin (Kan), 10ug / ml...

Embodiment 3

[0086] Example 3: Expression of Collagen Type II by Virus Injection Bombyx mori

[0087]The recombinant Escherichia coli successfully transformed in Example 2 is directly infected with silkworm cell BmN (ATCC CRL-8910) in Grace medium (purchased from Gibco), and can produce baculovirus BmMNPV-hcol II, and the virus supernatant is 12000rpm Centrifuge for 1 minute to remove residual BmN cells and take the supernatant. Inject the virus supernatant into the penultimate segment of the 5th instar larvae of the silkworm with a micro-injection syringe, inject 10ul per silkworm, 28°C, humidity 60% to 70%, after feeding for 4 days, select the epidermis under the observation of a stereofluorescence microscope to show red fluorescence (caused by mCherry) of the silkworm.

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Abstract

The invention discloses a method for producing human collagen type II, and belongs to the technical field of gene engineering. The method is characterized in that a baculovirus multi-gene expression system is adopted, insect cells are infected by the bacteria converting the baculovirus multi-gene expression system to produce baculovirus with multiple genes expressed, the virus liquid is infected in the insect cells or injected into silkworm larva bodies to produce recombinant human collagen type II full-length protein, the protein structure is that 3 peptide chains with (G-X-Y) n repetitive sequences form a three-strand helical structure, to both keep the conformation of the natural collagen type II and have the activity of the natural collagen type II. By adopting the method disclosed by the invention, multiple genes can be expressed to facilitate the production of then collagen type II full-length protein; the method disclosed by the invention is simple in process method, simple and convenient to operate and short in process route.

Description

technical field [0001] The invention relates to a method for producing recombinant human collagen type II, which belongs to the technical field of genetic engineering. Background technique [0002] Collagen, also known as collagen, is the most abundant protein in mammals, accounting for about 25%-30% of the total protein in the body. It is widely found in animal skin, bone, cartilage, teeth, tendons, ligaments and blood vessels. It is an important structural protein of connective tissue and has the functions of supporting organs and protecting the body. [0003] Current research reports show that since Miller and Matukas discovered type II collagen in 1969, more than 28 different types of collagen have been found in vertebrates, which consist of at least 46 distinct polypeptide chains. These different types of collagen all contain at least one triple helical domain, some as many as 96% (type I), and as little as 10% (type XII). The basic composition of collagen is similar,...

Claims

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Application Information

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IPC IPC(8): C12N15/866C07K14/78C07K1/22
Inventor 孙京臣黄亚东梁智升齐琦黄秋生项琪贝煜
Owner SOUTH CHINA AGRI UNIV
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