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CN-121991203-A - Recombinant human III type collagen and application thereof

CN121991203ACN 121991203 ACN121991203 ACN 121991203ACN-121991203-A

Abstract

The invention discloses recombinant human III type collagen and application thereof, and belongs to the technical field of biochemical engineering. The invention obtains the target amino acid sequence through splitting according to the human III type collagen, integrates the target amino acid sequence into an expression vector to obtain a plasmid, converts the plasmid into a host, screens positive clones to obtain an expression strain, cultures the expression strain, obtains the high-yield and high-purity human III type collagen after separation and purification, and the obtained recombinant human III type collagen has a collagen triple helix region characteristic sequence (Gly-X-Y) n and has good cell migration promoting, adhesion preventing and photodamage preventing performances, thus being widely applied to the fields of medicines, biological materials, health care products, cosmetics and the like.

Inventors

  • YAN XIAOFANG
  • HUANG YUAN
  • HUANG JUNJIE
  • CHEN SHUYI
  • LI YUWU

Assignees

  • 广州诺维合成生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260209

Claims (10)

  1. 1. A recombinant human III type collagen is characterized in that the amino acid sequence is as follows: (a) SEQ ID NO. 3 or SEQ ID NO. 4, or, (B) An amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, which retains the effects of cell migration, cell adhesion and photodamage prevention of the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, or, (C) An amino acid sequence of 1 or more amino acid residues added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, which retains the cell migration, cell adhesion and photodamage preventing effects of the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
  2. 2. The recombinant human type III collagen encoding gene of claim 1.
  3. 3. The coding gene of claim 2, wherein the nucleotide sequence is shown as 2566-2844bp in SEQ ID NO. 2 or 946-1206bp in SEQ ID NO. 2.
  4. 4. A recombinant expression vector comprising the coding gene of claim 2 or 3.
  5. 5. A recombinant engineering bacterium is characterized by comprising the coding gene of claim 2 or 3 or the recombinant expression vector of claim 4.
  6. 6. The recombinant engineering bacterium according to claim 5, wherein the recombinant engineering bacterium is a host of Escherichia coli.
  7. 7. A construction method of the recombinant engineering bacterium according to claim 5 or 6 is characterized in that the recombinant expression vector according to claim 4 is transformed into escherichia coli BL21 (DE 3), and positive transformants are cultured and screened to obtain the recombinant engineering bacterium.
  8. 8. A method for rapidly preparing the recombinant human type III collagen according to claim 1 is characterized by comprising the steps of inoculating the recombinant engineering bacteria according to claim 5 or 6 into a culture medium in an inoculum size of 2-10%, adding IPTG for induction culture, collecting thalli, crushing, collecting cracking precipitate, cutting, and collecting supernatant, namely the recombinant human type III collagen according to claim 1.
  9. 9. The method according to claim 8, wherein: the induction culture conditions are 16-18 ℃, and the culture is carried out at 200-300 rpm for 20-28 hours.
  10. 10. The use of recombinant human type III collagen according to claim 1 in the fields of medicine, biomaterials, health care products, cosmetics.

Description

Recombinant human III type collagen and application thereof Technical Field The invention relates to the technical field of biochemical engineering, in particular to recombinant human III-type collagen and application thereof. Background Collagen is the highest protein content in mammals, and is the main constituent of extracellular matrix. Collagen is a wide variety, and common collagen types are generally classified into type I, type II, type III, type V, type XI and other collagen, and the molecular weight of collagen subunits is generally about 120kDa, which is far greater than that of general bioactive proteins. The III type collagen forms a supercoiled structure by three alpha chains, is homotrimer, has rich content in blood vessels, intestinal tracts and skin, and participates in basic activities of regulating and controlling cell adhesion, proliferation, migration, differentiation and the like. Collagen is widely used in cosmetics, medical appliances and other fields for repairing and regenerating tissues. At present, sources of collagen mainly include extraction from animal tissues and expression by genetic engineering techniques. The extraction of III collagen from animal tissue presents serious problems such as high difficulty, low purity, high cost, poor batch-to-batch stability, etc., and may present the risk of immunogenicity and potential pathogen infection due to its origin in animal tissue. The recombinant collagen obtained by utilizing the genetic engineering technology can relieve the pressure in the aspects of animal cultivation, environmental protection and the like, can be expressed in escherichia coli, yeast cells, animal cells and other model organisms after being modified by the source genes, can obtain a large amount of collagen products after high-density fermentation, can effectively solve the bottleneck problem of large-scale production of the collagen, and meanwhile, the produced collagen has the advantages of good water solubility, no risk of virus transmission, low immune rejection reaction, good batch-to-batch consistency and the like. The patent with publication No. CN119350505A provides a way to purify proteins by using self-aggregating peptides, which can effectively avoid the disadvantages of introducing tags, requiring protease treatment, etc. Therefore, the design and development of superior recombinant collagen and optimization are currently in need of solution. Disclosure of Invention In order to overcome the defects and shortcomings of the prior art, the invention aims to provide recombinant human type III collagen and application thereof. The invention obtains the recombinant human III type collagen with high expression and good cell activity through different sequence design and screening work, and simultaneously provides a preparation method and application thereof. The aim of the invention is achieved by the following technical scheme: the invention provides recombinant human III type collagen, which has the amino acid sequence as follows: (a) SEQ ID NO. 3 or SEQ ID NO. 4, or, (B) An amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, which retains the effects of cell migration, cell adhesion and photodamage prevention of the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, or, (C) An amino acid sequence of 1 or more amino acid residues added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4, which retains the cell migration, cell adhesion and photodamage preventing effects of the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4. Preferably, the recombinant human III type collagen is HC25, the amino acid sequence is shown as SEQ ID NO. 3 or 1023-1115aa in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as 2566-2844bp in SEQ ID NO. 2. Preferably, the recombinant human III type collagen is HC26, the amino acid sequence is shown as SEQ ID NO. 4 or 483-569aa in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as 946-1206bp in SEQ ID NO. 2. The invention also provides a coding gene of the recombinant human III type collagen. The invention also provides a recombinant expression vector containing the coding gene. The recombinant expression vector takes pET series as a vector, in particular to pET-28a (+) as a vector. The recombinant expression vector also comprises a self-aggregating peptide purifying element for subsequent separation and purification, wherein the self-aggregating peptide purifying element comprises self-assembling peptide, protein linker, pH induced C-terminal cutting intron and subtilisin propeptide. The invention also provides a recombinant engineering bacterium containing the coding gene or the recombinant expression vector. Furthermore, the recombinant engineering bacteria take escherichia coli as a host, preferably escherichia coli BL21 (DE 3) as a host. The invention