CN114085284A

CN114085284A - Recombinant III-type humanized collagen, nucleic acid, vector and implant

Info

Publication number: CN114085284A
Application number: CN202111391978.1A
Authority: CN
Inventors: 侯增淼; 魏文培; 周浩; 郝东; 赵硕文
Original assignee: Xi'an Denos Medical Technology Co ltd
Current assignee: Xi'an Denos Medical Technology Co ltd
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-02-25
Anticipated expiration: 2041-11-19
Also published as: CN114085284B

Abstract

The invention discloses a recombinant III-type humanized collagen, nucleic acid, a carrier and an implant, relating to the technical field of biological genes; the invention selects the sequence peptide sections 549-560aa, 597-613aa, 881-902aa and 648-662aa with good stability obtained by long-term screening in the natural human III collagen alpha 1 chain, splices the four sections of peptide chains in sequence to form monomers, and then carries out tandem expression on the monomers. Through fermentation and purification, the obtained recombinant III-type humanized collagen has high stability, effectively solves the problem of degradation of the recombinant III-type humanized collagen in the clinical use process, and can be prepared into a liquid preparation for storage and use.

Description

Recombinant III-type humanized collagen, nucleic acid, vector and implant

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant III-type humanized collagen, nucleic acid, a vector and an implant.

Background

Collagen (collagen) is a protein with the largest content in vivo, is a main component of extracellular matrix (ECM), has important effects on maintaining normal physiological functions of cells, tissues and organs and repairing damage, and is widely applied to the industries of medicines, health care products and cosmetics. In the molecular structure, each collagen peptide chain is mainly composed of peptide chains with a Gly-X-Y structure (X, Y is any amino acid residue except Gly), and the peptide chain structure is necessary for forming a collagen fiber higher-order structure, so that the excellent biocompatibility and the low immunogenicity of the collagen are determined.

The main source of collagen is extracted from animal tissues, and along with the development of biotechnology, recombinant collagen obtained by microbial fermentation using genetic recombination technology has been widely used. The national drug administration of 3/15/2021 issues a "guidance principle for naming recombinant collagen biomaterials", wherein in the principle, recombinant collagens are named in a classified manner, and the current recombinant collagens are divided into three types, namely recombinant human collagens, recombinant humanized collagens and recombinant collagens, wherein the recombinant human collagens refer to full-length amino acid sequences coded by specific types of genes of the human collagens and have a triple-helix structure, but the recombinant collagens have technical problems and are not applied in the market temporarily; the second kind is recombinant humanized collagen, full-length or partial amino acid sequence fragment of specific type gene code of human collagen prepared by DNA recombination technology, or combination containing human collagen functional fragment; the third kind is recombinant collagen-like protein, amino acid sequence or its segment produced by DNA recombination technology and encoded by specific gene after design and modification, or the combination of these functional amino acid sequence segments. The gene coding sequence or amino acid sequence has low homology with the gene coding sequence or amino acid sequence of human collagen. At present, the products applied and formed in the market mainly comprise recombinant humanized collagen and recombinant collagen-like protein, but the recombinant humanized collagen and the recombinant collagen-like protein have the advantages and the disadvantages respectively, the amino acid sequence of the recombinant humanized collagen is the same as that of a natural human sequence, the biocompatibility is good, the safety is higher, but the problems of poor stability and easy degradation exist in the actual production and the actual application, and in order to improve the stability of a final product, the implanted recombinant human collagen product preparation on the market at present is stored in the form of being prepared into freeze-dried powder; the recombinant collagen-like protein optimizes and modifies partial amino acids in the sequence in order to change the stability, and although the stability is greatly improved, the improved sequence has low homology with the natural human sequence, so that the compatibility and safety are reduced, and the application of the recombinant collagen-like protein is influenced.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides a recombinant III-type humanized collagen, a nucleic acid, a carrier and an implant, so as to overcome the defects that the recombinant humanized collagen in the prior art is poor in stability and easy to degrade in the storage and use processes of a liquid formulation, and the application of the recombinant humanized collagen in clinic is limited.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, a recombinant type III humanized collagen is provided, wherein the recombinant type III humanized collagen is formed by connecting monomers in series repeatedly, and the amino acid sequence of the monomers is shown as SEQ.ID.NO. 5; the number of the monomers connected in series is 2-15.

Preferably, the monomer is formed by splicing the following amino acid peptide fragments in sequence:

the amino acid sequence of the 549-560aa peptide segment in the III type human collagen alpha 1 chain is shown in SEQ.ID.NO. 1;

597-613aa peptide segment in the III type human collagen alpha 1 chain, the amino acid sequence is shown in SEQ.ID.NO. 2;

the amino acid sequence of the 881-902aa peptide segment in the III type human collagen alpha 1 chain is shown as SEQ ID NO. 3;

the amino acid sequence of 648-662aa peptide segment in the III type human collagen alpha 1 chain is shown in SEQ ID.

Preferably, the molecular weight of the recombinant III type humanized collagen is 10 KD-100 KD.

The preparation process of the recombinant III-type humanized collagen comprises the following steps:

(1) structure design and gene acquisition of recombinant type III humanized collagen

Four peptide chains of 549-560aa, 597-613aa, 881-902aa and 648-662aa in the type III collagen alpha 1 chain are spliced in sequence to form a monomer, and the monomer sequence is shown in SEQ ID No.5 and comprises 66 amino acids.

The corresponding nucleotide sequence is designed according to the codon preference of pichia pastoris in the monomer amino acid sequence, and meanwhile, for subsequent enzyme digestion connection, Xho I restriction endonuclease digestion sites CTCGAG, KEX2 digestion sites AAAAGA and Xba I restriction endonuclease digestion sites GCTAGC are added to the 5 'end of the DNA sequence, and Nhe I restriction endonuclease digestion sites TCTAGA and EcoR I restriction endonuclease digestion sites GAATTC are added to the 3' end of the DNA sequence; the designed nucleotide sequence of SEQ.ID.NO.6 was subjected to whole gene synthesis (Beijing engine family organism). Then, according to the characteristic that Xba I and Nhe I in the designed sequence are isocaudarner, repeatedly carrying out double enzyme digestion and connection on Xba I + EcoR I and Nhe I + EcoR I in a plasmid (pUC57-COL3) synthesized by a whole gene to obtain plasmids (2-15) with different repeated monomer tandem sequences, wherein the plasmids are named as pUC57-COL3-2, pUC57-COL3-3 and pUC57-COL3-4.

(2) Construction of expression vectors

Carrying out Xho I and EcoR I double enzyme digestion on the obtained multiple repeat plasmid and a pichia pastoris expression vector pPIC9K at the same time, and then connecting and transforming to obtain multiple tandem expression plasmids: pPIC9K-COL3-2, pPIC9K-COL3-3, pPIC9K-COL3-4.

(3) Construction of genetically engineered bacteria

The expression vectors obtained above, such as pPIC9K-COL3-2, pPIC9K-COL3-3, pPIC9K-COL3-4.

(4) Fermentation and purification of recombinant type III humanized collagen

Taking an inorganic salt BSM culture medium as a bottom material, fermenting by using a 10L fermentation tank, regulating pH to 5.0 by ammonia water, controlling the temperature to be 29.0 ℃, controlling dissolved oxygen to be about 30%, starting methanol induction when the wet weight of the thalli reaches 180-200 mg/mL, inducing for 40-50 hours, putting the thalli into the tank, and centrifuging to collect fermentation supernatant. And (3) removing impurities, filtering and concentrating the fermentation supernatant by using a hollow fiber ultrafiltration system, and then performing ion exchange chromatography by using MMC Bestarose6FF to obtain the recombinant III-type humanized collagen with the purity of more than 95%.

In a second aspect, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the recombinant humanized type III collagen of the first aspect or a complement thereof.

Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in seq.id No. 6.

In a third aspect, there is provided a vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, there is provided an implant comprising the recombinant type III humanized collagen of the first aspect.

Preferably, the implant is made of the following materials by mass percent: recombinant type III humanized collagen: 0.1-2%; sodium hyaluronate: 1 to 10 percent; water for injection: and (4) the balance.

Preferably, the implant is made of the following materials by mass percent: recombinant type III humanized collagen: 1 percent; sodium hyaluronate: 5 percent; water for injection: and (4) the balance.

In a fifth aspect, there is provided a recombinant humanized type III collagen according to the first aspect, a nucleic acid molecule according to the second aspect, a vector according to the third aspect, an implant according to the fourth aspect for use in tissue engineering and medical and cosmetic products.

Compared with the prior art, the invention has the following beneficial effects:

the invention selects the sequence peptide sections 549-560aa, 597-613aa, 881-902aa and 648-662aa which have good stability and are obtained by long-term screening of the natural human III type collagen alpha 1 chain, sequentially splices the four sections of peptide chains to form monomers, and then carries out tandem expression on the monomers. Through fermentation and purification, the obtained recombinant III-type humanized collagen has high stability, effectively solves the problem of degradation of the recombinant III-type humanized collagen in the clinical use process, and can be prepared into a liquid preparation for storage and use. Meanwhile, the invention can also produce recombinant III-type humanized collagen with different molecular weights by serial expression so as to meet the requirements of downstream application.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1 is a schematic diagram showing the shake-flask expression result of monomeric ten-repeat recombinant type III humanized collagen;

FIG. 2 is a schematic diagram showing the sampling electrophoresis results of the ten-fold single recombinant type III humanized collagen 10L fermentation at different time periods;

FIG. 3 is a schematic diagram showing the electrophoresis result of purified recombinant type III humanized collagen;

FIG. 4 is a diagram showing electrophoresis results of accelerated stability and room temperature stability of monomeric ten-fold recombinant type III humanized collagen.

Detailed Description

For a fuller understanding of the technical content of the present invention, reference should be made to the following detailed description taken together with the accompanying drawings; it is to be understood that the embodiments described below are only a few embodiments of the present invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The features, benefits and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.

All percentages, fractions and ratios are calculated on the total mass of the composition of the invention, unless otherwise indicated. The term "mass content" herein may be represented by the symbol "%".

In the present invention, the ratio of each component in the composition and the cosmetic is the same.

The use of "including," "comprising," "containing," "having," or other variations thereof herein, is meant to encompass the non-exclusive inclusion, as such terms are not to be construed. The term "comprising" means that other steps and ingredients can be added that do not affect the end result. The term "comprising" also includes the terms "consisting of and" consisting essentially of. The compositions and methods/processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.

Example 1

Structural design of recombinant type III humanized collagen

The invention sequentially splices four peptide chains of 560aa, 597-613aa, 881-902aa and 648-662aa according to a natural human type III collagen alpha 1 chain sequence (reference sequence Genebank accession number: NM-000090.3) 549-containing peptides to form a monomer, and the amino acid sequence of the monomer is shown in SEQ. ID. NO. 5.

The invention carries out repeated tandem expression on monomers consisting of the tetrapeptide segments.

(II) preparation of recombinant type III humanized collagen

2.1 obtaining of the Gene

The corresponding nucleotide sequence is designed according to the codon preference of pichia pastoris in the monomer amino acid sequence, and meanwhile, for subsequent molecular operation, Xho I restriction endonuclease cleavage site CTCGAG, KEX2 cleavage site AAAAGA and Xba I restriction endonuclease cleavage site GCTAGC are added at the 5 'end of the DNA sequence, Nhe I restriction endonuclease cleavage site TCTAGA and EcoR I restriction endonuclease cleavage site GAATTC are added at the 3' end, the sequence is shown in SEQ ID.NO.6, and the designed nucleotide sequence is subjected to whole gene synthesis (Beijing optimae). Then, according to the characteristic that Xba I and Nhe I in the designed sequence are isocaudarner, Xba I + EcoR I and Nhe I + EcoR I are subjected to double enzyme digestion and connection in a plasmid (pUC57-COL3) synthesized by a whole gene, so that plasmids (2-15) with different repetitive monomer tandem sequences can be obtained, and the plasmids can be named as pUC57-COL3-2, pUC57-COL3-3, pUC57-COL3-4.

2.2 construction of expression vectors

Carrying out Xho I and EcoR I double enzyme digestion on the obtained multiple plasmid pUC57-COL3 (2-15) and a pichia pastoris expression vector pPIC9K at the same time, then connecting T4 DNA ligase, transforming escherichia coli DH5 alpha, screening ampicillin, and obtaining multiple tandem expression plasmids through sequencing identification: pPIC9K-COL3-2, pPIC9K-COL3-3, pPIC9K-COL3-4.

2.3 construction of genetically engineered bacteria

The obtained multiple tandem expression plasmid is subjected to medium extraction, enzyme digestion linearization is carried out by using a restriction enzyme Sal I, a pichia host strain GS115(Invitrogen) is transformed by an electroporator, a high-copy transformant is screened by G418 (4mg/mL, YPD culture medium), and the expression condition is determined by shake flask fermentation (29 ℃, 200rpm, BMMY culture medium and methanol feeding 1% at intervals of 24 hours). And detecting the expression condition of the target protein by SDS-PAGE electrophoresis of the supernatant obtained by the shake flask fermentation.

(III) fermentation and purification production of recombinant III-type humanized collagen

Performing fermentation tank fermentation on the multiple recombinant III type humanized collagen genetic engineering bacteria, using an inorganic salt BSM culture medium as a base material, performing fermentation by using a 10L fermentation tank (the pH value is controlled by ammonia water at 29.0 ℃, the dissolved oxygen is controlled at about 30%), starting methanol induction when the wet weight of the bacteria reaches 180-200 mg/mL, performing induction for 40-50 hours, putting the bacteria into the tank, centrifuging to collect fermentation supernatant, performing microfiltration on the fermentation supernatant by using a hollow fiber microfiltration system with the aperture of 0.22 mu m, collecting filtrate, performing chromatographic purification by using MMC Bestarose6FF, performing column equilibrium by using 20mM pH5.0 citric acid buffer solution, performing column equilibrium on a sample with the pH value of 5.0, performing sample introduction with the conductance of about 4.0ms/cm, and eluting by using 20mM pH5.5 buffer solution to obtain the recombinant III type humanized collagen with the purity of more than 95%.

Example 2:

preparation of monomer ten-repeat recombination III type humanized collagen

Construction of genetically engineered bacteria

After mass extraction of the multiple tandem expression plasmid obtained in example one, preferably the ten-fold tandem pPIC9K-COL3-10 plasmid, linearization was performed by restriction enzyme Sal I digestion, then the Pichia host strain GS115(Invitrogen) was transformed by electroporator, high copy transformants were screened (4mg/mL, YPD medium) by G418, and expression was confirmed by shake flask fermentation (29 ℃, 200rpm, BMMY medium, 1% methanol feed at 24 h intervals). The supernatant obtained by the shake flask fermentation is detected by SDS-PAGE electrophoresis, and as can be seen from figure 1, an obvious electrophoresis band is formed at the position of about 60KD, so that the monomer ten-repeat recombination type III humanized collagen gene engineering bacteria can be obtained.

(II) fermentation and purification

Fermenting the monomer ten-repeat recombination III type humanized collagen gene engineering bacteria in a fermentation tank, taking inorganic salt BSM culture medium as a substrate, fermenting in a 10L fermentation tank (pH 5.0 regulated by ammonia water, temperature 29.0 ℃, dissolved oxygen controlled at about 30%), inducing with methanol for 40-50 hours when the wet weight of thallus reaches 180-200 mg/mL, placing in a tank (the detection of fermentation process is shown in figure 2), centrifuging to collect fermentation supernatant, microfiltering the fermented supernatant with a hollow fiber microfiltration system with pore diameter of 0.22 μm, collecting filtrate, then performing chromatographic purification by MMC Bestarose6FF, performing column balance by 20mM pH5.0 citric acid buffer solution, loading a sample with pH5.0 and conductance of about 4.0ms/cm, eluting by 20mM pH5.5 buffer solution, finally performing ultrafiltration desalination by using a roll-type ultrafiltration membrane with cut-off molecular weight of 10KD, freeze-drying, thus obtaining the recombinant III type humanized collagen with the purity of more than 95% (the purity electrophoretic analysis is shown in figure 3).

Example 3:

preparing the obtained recombinant III type humanized collagen freeze-dried powder into 0.1% liquid by using purified water, sealing and subpackaging after sterile filtration, carrying out an accelerated experiment under the conditions of the temperature of 40 +/-2 ℃ and the relative humidity of 75 +/-5%, carrying out a normal temperature experiment at the temperature of 25 +/-2 ℃ and the relative humidity of 60 +/-5%, sampling at different time points, and detecting the integrity of the recombinant III type humanized collagen by SDS-PAGE electrophoresis. The test results are shown in fig. 4; as can be seen from FIG. 4, the protein band was intact and hardly degraded when sampled at 25 ℃ at room temperature for 3 months and 6 months; sampling at 40 ℃ under the acceleration condition for 3 months and 6 months, wherein the protein is complete and almost not degraded after 3 months, and little degradation occurs in the protein after sampling for 6 months. From the above experimental results, the recombinant type III humanized collagen obtained by the present invention can be used as a product formulation in a liquid form without being made into a lyophilized formulation, which allows the final product to be produced with substantial savings in equipment, labor and time costs.

The data of the embodiment show that the recombinant III-type humanized collagen designed and prepared by the invention has high purity and good stability, lays a foundation for the application of the recombinant III-type humanized collagen, and simultaneously prompts the application of the recombinant III-type humanized collagen in tissue engineering and medical products.

Example 4:

in the embodiment, the recombinant III-type humanized collagen with the number of monomers connected in series of 2-15 is further prepared, and the preparation method refers to the embodiment 2; in this embodiment, as shown in example 3, the recombinant type III humanized collagen prepared by using monomers in a number of 2-15 in series has the advantages of high purity and good stability, similar to the monomeric ten-repeat recombinant type III humanized collagen of example 2.

Example 5:

this example provides an implant prepared from the recombinant type III humanized collagen obtained in example 2 or example 4 of the present invention, wherein the implant is prepared from the following materials by weight:

recombinant type III humanized collagen (lyophilized powder): 0.1-2%; sodium hyaluronate: 1 to 10 percent; water for injection: and (4) the balance.

More preferably, the implant is made of: recombinant type III humanized collagen: 1 percent; sodium hyaluronate: 5 percent; water for injection: and (4) the balance.

The preparation method of the implant of this example is as follows:

dissolving the recombinant III type humanized collagen freeze-dried powder and sodium hyaluronate in water for injection, sterile filtering with 0.22um, subpackaging in a penicillin bottle, storing at 4 ℃, and filling facial dermal tissues to achieve the aim of removing wrinkles.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, the specific implementation manners and the application ranges may be changed, and in conclusion, the content of the present specification should not be construed as limiting the invention.

Sequence listing

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Claims

1. A recombinant type III humanized collagen is characterized by being formed by connecting monomers in series repeatedly, wherein the amino acid sequence of the monomers is shown in SEQ.ID.NO. 5; the number of the monomers connected in series is 2-15.

2. The recombinant humanized type III collagen according to claim 1, wherein said monomer is formed by sequentially splicing the following amino acid peptide fragments:

3. The recombinant type III humanized collagen according to claim 1, wherein the molecular weight of the recombinant type III humanized collagen is 10 KD-100 KD.

4. A nucleic acid molecule comprising a nucleotide sequence encoding the recombinant type III humanized collagen according to any one of claims 1 to 3 or a complementary sequence thereof.

5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID No. 6.

6. A vector comprising the nucleic acid molecule of claim 4 or 5.

7. An implant comprising the recombinant type III humanized collagen according to any one of claims 1 to 3.

8. The implant of claim 7, wherein the implant is made of, in mass percent: recombinant type III humanized collagen: 0.1-2%; sodium hyaluronate: 1 to 10 percent; water for injection: and (4) the balance.

9. The implant of claim 8, wherein the implant is made from the following materials, by mass percent: recombinant type III humanized collagen: 1 percent; sodium hyaluronate: 5 percent; water for injection: and (4) the balance.

10. Use of the recombinant humanized type III collagen according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the vector according to claim 6, the implant according to any one of claims 7 to 9 in tissue engineering and medical and cosmetic products.