CN-121736084-B - Stable recombinant type I collagen and application thereof

Abstract

The invention provides stable recombinant type I collagen and application thereof, wherein the recombinant type I collagen is designed based on the amino acid sequence of human type I collagen and comprises 19 basic units which are repeatedly connected in series, the amino acid sequence of the basic units is shown as SEQ ID NO.1, and the amino acid sequence of the recombinant type I collagen is shown as SEQ ID NO. 6. The invention also establishes a high-efficiency recombinant expression system and a label-free protein purification process, avoids potential influence of exogenous label residues on the natural conformation and functional integrity of the protein, ensures that the recombinant type I collagen is more similar to the collagen of a human body in structure and biological activity, has high purity, excellent water solubility and stability, and improves the reliability of clinical application. Lays a solid foundation for realizing stable and low-cost large-scale industrial production of the type I collagen, and is beneficial to meeting the wide market demand for high-quality collagen.

Inventors

• AN HAIQIAN
• ZHU LUYU

Assignees

• 江苏金普诺安生物科技股份有限公司

Dates

Publication Date

20260508

Application Date

20260225

Claims (8)

1. 1. The recombinant type I collagen is characterized in that the amino acid sequence of the recombinant type I collagen is shown as SEQ ID NO. 6.
2. 2. A nucleic acid molecule encoding the recombinant type I collagen of claim 1.
3. 3. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a vector, a cell or a microorganism.
4. 4. Use of the recombinant type I collagen of claim 1, the nucleic acid molecule of claim 2 or the biomaterial of claim 3 for the preparation of a medicament, a medical biomaterial or a medical device.
5. 5. A product comprising the recombinant type I collagen of claim 1, wherein the product is a pharmaceutical product, a medical biomaterial, or a medical device.
6. 6. A process for producing a recombinant type I collagen, comprising culturing the microorganism described in claim 3 and recovering the recombinant type I collagen from the culture product.
7. 7. The method of claim 6, wherein the microorganism comprises yeast.
8. 8. The method according to claim 6 or 7, wherein the recombinant type I collagen obtained by the recovery is purified by any one or more of affinity chromatography, ion exchange, hydrophobic chromatography and salting out.

Description

Stable recombinant type I collagen and application thereof Technical Field The invention relates to the technical field of bioengineering, in particular to stable recombinant type I collagen and application thereof. Background The type I collagen is a key functional component of a human extracellular matrix, and has irreplaceable application value in the medical field by virtue of excellent biocompatibility, cell adhesion activity and tissue repair capability. However, the existing technology for obtaining and preparing the type I collagen has a remarkable bottleneck, and severely restricts the industrial application of the type I collagen in high quality and large scale. Animal tissue extraction is the main way to obtain type I collagen, but the operation flow of the process is complex, and the obtained product has animal origin, possibly causes immune rejection reaction and is accompanied with virus pollution risk. In addition, the quality difference among batches is large, the controllability is poor, the large-scale production is limited, and the high standard requirements of modern medical treatment on the consistency and the safety of materials are difficult to meet. The rise of genetic engineering technology provides a new direction for the production of type I collagen. However, currently, the type I collagen prepared by recombinant expression techniques generally faces the challenge of insufficient stability, and is easily denatured, degraded or lost in function during storage and application. Therefore, a new generation of preparation process capable of integrating human type I collagen sequences to safely, efficiently, highly-purity and stably produce type I collagen in a large scale is needed to be developed in the field, so as to fill the gap of urgent demands of industries such as biology, medical treatment and the like for high-quality collagen raw materials. Disclosure of Invention In order to solve the problems in the prior art, the invention provides stable recombinant type I collagen and application thereof. In a first aspect, the present invention provides a recombinant type I collagen comprising an amino acid sequence as shown in SEQ ID NO. 6. According to the recombinant type I collagen provided by the invention, preferably, the recombinant type I collagen does not include a tag. More preferably, the tag is selected from at least one of a His tag, a GST tag, an MBP tag, a Strep tag, and a FLAG tag. Further preferably, the tag is a His tag. More preferably, the amino acid sequence of the recombinant type I collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity and has the same function as the amino acid sequence shown in SEQ ID No. 6. In some embodiments, the amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO. 6. In a second aspect, the invention provides a nucleic acid molecule encoding a recombinant type I collagen comprising an amino acid sequence as shown in SEQ ID NO. 6. According to one of the nucleic acid molecules provided herein, preferably, the recombinant type I collagen does not include a tag. More preferably, the tag is selected from at least one of a His tag, a GST tag, an MBP tag, a Strep tag, and a FLAG tag. Further preferably, the tag is a His tag. More preferably, the amino acid sequence of the recombinant type I collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity and has the same function as the amino acid sequence shown in SEQ ID No. 6. In some embodiments, the amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO. 6. According to one of the nucleic acid molecules provided by the invention, preferably, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO. 7. More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as set forth in SEQ ID No.7 and encodes a protein having the same function. In some embodiments, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO. 7. In a third aspect, the invention provides a biological material comprising the nucleic acid molecule, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a vector, a cell or a microorganism. According to the biomaterial provided by the invention, preferably, the vector takes the yeast expression vector as a framework. More preferably, the vector has a ppiczαa vector or pPIC9k vector as a backbone. Further preferably, the vector has the ppiczαa vector as a backbone. Further preferably, the nucleic acid molecule is located between the EcoR I site and the Not I site of the backbone. According to one of the biological materials provided by the present invention, preferably the microorganism comprises a yeast. More preferably, the yeast is pichia pastoris. Further preferably, the pichia is pichia X33 strain, pichia GS115 strain, pichi