Humanized III-type collagen as well as preparation method and application thereof

Abstract

The invention discloses humanized III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. According to the method, firstly, on the basis of an original humanized III-type collagen gene, cell adhesion activity is taken as an evaluation index, a better cell adhesion sequence is screened out and spliced and integrated with the original collagen gene, and brand-new humanized III-type collagen is obtained. And constructing a recombinant expression vector of the humanized III type collagen containing the adhesion sequence by taking the probiotic EcN as a host cell, and introducing the recombinant expression vector into the EcN to obtain a strain of functional recombinant probiotic. Compared with a traditional escherichia coli engineering bacterium BL21 (DE3), the recombinant probiotics have good cell adhesion activity while efficiently expressing the humanized type III collagen, the bacterial endotoxin content of the recombinant humanized type III collagen obtained through purification meets the industrial standard, and the recombinant probiotics have good application prospects. The purification cost caused by industrial removal of endotoxin can be avoided, so that the production cost is reduced, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and biotechnology, and particularly relates to humanized type III collagen, a preparation method and application. Background Art

[0002] Collagen is the most abundant structural and functional protein in the human body, accounting for approximately 30% of the body's total protein. It is widely present in the skin, muscles, bones, and internal organs, playing a vital role in maintaining the normal physiological activities of cells, tissues, and organs and repairing damage. Collagen is typically composed of three α-chains, each consisting of repeating Gly-Xaa-Yaa segments, where Xaa is often proline (Pro) and Yaa is often hydroxyproline (Hypro) or hydroxylysine (Hylys). This structural characteristic gives it a stable triple helical structure.

[0003] Nearly 30 different types of collagen have been reported. These different types of collagen are distributed in different connective tissues and play different physiological functions. The collagen in the skin is mainly type I and type III collagen. Among them, type III collagen is very effective in promoting tissue growth, wound healing and repair, and hemostasis and coagulation, and has a wide range of applications.

[0004] The good biological activity of collagen is currently mainly achieved by modifying its active functions and using cell adhesion proteins or polypeptides to regulate the cell adhesion function. Patent application No. 202310864370.9 uses the AlphaFold2 structure prediction method to screen out the optimal amino acids that may form a triple helix structure from the full-length sequence of human type III collagen, and then splices and recombines them to obtain recombinant humanized type III collagen, which has certain cell adhesion biological activity. However, due to the limited GER or GEK ternary adhesion fragments in the collagen sequence, its cell adhesion activity is insufficient, which limits its application. Therefore, in view of the shortcomings of cell adhesion polypeptides, the design and discovery of new cell adhesion collagens are of great significance in the field of tissue repair.

[0005] In recent years, with the rapid development of molecular biology and genetic engineering technology, more and more collagens are produced by heterologous recombinant expression. As the most common prokaryotic expression system, Escherichia coli has the advantages of simple culture, short cycle, low fermentation cost, and rapid production of exogenous proteins. It is currently the most widely used expression system for recombinant collagen. However, during the expression of recombinant proteins, Escherichia coli produces a large amount of endotoxins, which triggers a pro-inflammatory immune response and limits the application of collagen in the medical field. Common methods for removing endotoxins mainly include anion exchange chromatography, affinity chromatography, molecular sieve method, etc. Although they can remove residual endotoxins in recombinant proteins to varying degrees, there are still problems such as high purification cost and low protein recovery efficiency. Therefore, in the field of collagen preparation technology, there is an urgent need to develop a method for efficiently expressing collagen with low endotoxin content and good biosafety using the Escherichia coli system, which is of practical significance. Summary of the Invention

[0006] The present invention aims to address the problems of the insufficiency of the cell adhesion polypeptide of existing humanized type III collagen, the pathogenicity, low yield and low biological activity of Escherichia coli strains producing humanized type III collagen, and provide a humanized type III collagen, a preparation method and an application.

[0007] The humanized type III collagen described in this invention is a recombinant humanized collagen protein, comprising a full-length or partial amino acid sequence fragment encoded by a specific human collagen type gene, or a combination of functional human collagen fragments, prepared by DNA recombination technology. It is further divided into type A and type B. Type A does not contain non-human collagen amino acids; type B does. This invention selects 30 amino acids from the functional region of human collagen for splicing and integration, fusing them with a linker peptide, a histidine purification tag, and an adhesion sequence, resulting in approximately 92.3% homology.

[0008] E.coli Nissle 1917, referred to as EcN, is a non-pathogenic Escherichia coli and also a probiotic. Studies have shown that Nissle 1917 does not have enterotoxins or cytotoxins associated with other pathogenic Escherichia coli, can maintain the intestinal mucosal barrier, activate the host's immune system, and prevent bacterial infection (Chen H, Lei P, Ji H, et al. Advances in Escherichia coli Nissle1917 as a customizable drug delivery system for disease treatment and diagnosis strategies. Mater Today Bio. 2023, 18: 100543.). At present, Nissle 1917 has been used as a good chassis cell for the production of various proteins. For example, CN202211716616.X discloses a method of expressing lactate oxidase using Escherichia coli Nissle 1917 to achieve efficient production of lactate oxidase preparations. CN202111328858.7 discloses a recombinant plasmid expressing Keratin 7 protein and a recombinant probiotic expressing Keratin 7 protein. The recombinant probiotic can effectively lower blood sugar while efficiently expressing Keratin 7 protein.

[0009] Therefore, to address the pathogenicity, low production, and poor cell adhesion activity of existing E. coli strains producing humanized type III collagen, the present invention proposes using the probiotic E. coli Nissle 1917 as a host cell. Based on the original collagen gene (patent application number: 202310864370.9), a cell adhesion sequence with excellent cell adhesion activity was screened. This sequence was further spliced ​​and integrated to construct a recombinant collagen expression vector, which was then introduced into E. coli Nissle 1917 to obtain a functional recombinant probiotic strain. Compared to the traditional engineered E. coli BL21 (DE3) strain, this recombinant probiotic not only efficiently expresses humanized type III collagen but also exhibits improved cell adhesion activity. Furthermore, its bacterial endotoxin content meets industry standards, avoiding the purification costs associated with industrial endotoxin removal, thereby reducing production costs and promising application prospects.

[0010] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0011] The present invention provides a humanized type III collagen, which is a recombinant humanized type III collagen. The humanized type III collagen (R8-X) comprises a collagen core functional region R8 and a cell adhesion sequence X, and has good cell adhesion activity. The collagen core functional region R8 comprises 30 amino acids from the functional region of human collagen, spliced ​​and integrated, and fused with a linker peptide and a histidine purification tag. The amino acid sequence of the collagen core functional region R8 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the collagen core functional region R8 is shown in SEQ ID NO.3.

[0012] The humanized type III collagen protein comprises a cell adhesion sequence X, wherein X is any one of the cell adhesion sequences GLPGEN, GFPGER, GPP, and GPQ. The amino acid sequence of the cell adhesion sequence X is shown in SEQ ID NOs. 6-9, and the nucleotide sequence of the gene encoding the cell adhesion sequence X is shown in SEQ ID NOs. 10-13. Preferably, the cell adhesion sequence is GLPGEN.

[0013] Preferably, the humanized type III collagen is R8-GLPGEN, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0014] The present invention also provides a method for preparing humanized type III collagen, which comprises the following steps:

[0015] 1.1 Construction of recombinant probiotics expressing humanized type III collagen:

[0016] (1) The coding gene fragment of humanized type III collagen R8-X was amplified by PCR and ligated into the pETDuet-1 plasmid to obtain the recombinant expression vector pETDuet-1-R8-X recombinant plasmid;

[0017] (2) The recombinant plasmid pETDuet-1-R8-X was transformed into E. coli Nissle 1917 by chemical transformation, and the successfully constructed recombinant strain pETDuet-1-R8-X-Nissle 1917 was screened by ampicillin antibiotic plate, which is the recombinant probiotic expressing the humanized type III collagen R8-X.

[0018] 1.2 Expression and purification of humanized type III collagen:

[0019] a) streaking the recombinant probiotic pETDuet-1-R8-X-Nissle 1917 prepared in step 1.1 onto an LB plate containing 100 μg / ml ampicillin and culturing at 37°C overnight; picking a single colony and inoculating it into 10 ml of LB medium containing 100 μg / ml ampicillin, and shaking and culturing at 37°C for 12-16 hours; inoculating it into 200 ml of TB medium containing 100 μg / ml ampicillin at a 2% inoculum, shaking at 37°C until the OD600 reaches 0.6-0.8, adding IPTG to a final concentration of 0.1 mM, and culturing at 25°C for 6-8 hours to express the humanized type III collagen, and collecting the fermentation broth;

[0020] Preferably, the recombinant probiotics are inoculated into an LB plate containing 100 μg / ml ampicillin and cultured overnight at 37°C; a single colony is picked and inoculated into 10 ml of LB medium containing 100 μg / ml ampicillin, and cultured at 37°C with shaking for 12 hours; a 2% inoculum is inoculated into 200 ml of TB medium containing 100 μg / ml ampicillin, and the culture is shaken at 37°C until the OD600 reaches 0.8. IPTG is added at a final concentration of 0.1 mM, and the culture is cultured at 25°C for 8 hours to collect the fermentation broth;

[0021] b) placing the fermentation broth obtained in step a) in a refrigerated centrifuge and centrifuging at 4°C, 8000-10000 rpm for 10-15 minutes, discarding the supernatant and collecting the bacterial precipitate; further, adding 20 mL of pre-cooled lysis buffer to the bacterial precipitate to resuspend the bacterial precipitate and transferring it to a 50 mL centrifuge tube, centrifuging at 4°C, 8000-10000 rpm for 20-30 minutes; discarding the supernatant, adding 20 mL of lysis buffer (containing PMSF at a final concentration of 1 mM) to resuspend the bacterial precipitate, and ultrasonically disrupting the precipitate in an ice bath for 30-45 minutes (350W, 5 seconds of ultrasonication, 5 seconds of rest, and a protective temperature of 25°C); centrifuging at 4°C, 8000-10000 rpm for 15-20 minutes, and collecting the supernatant solution. The supernatant solution after disruption contains recombinant collagen.

[0022] Preferably, the fermentation broth is placed in a refrigerated centrifuge and centrifuged at 4°C and 10000rpm for 10 minutes, the supernatant is discarded and the bacterial precipitate is collected; further, the bacterial precipitate is added with 20mL of pre-cooled lysis buffer to resuspend the bacteria and transfer it to a 50mL centrifuge tube, and centrifuged at 4°C and 8000rpm for 20 minutes; the supernatant is discarded, and 20mL of lysis buffer (containing PMSF with a final concentration of 1mM) is added to resuspend the bacteria, and then ultrasonically disrupted in an ice bath for 30min (350W, 5s ultrasonication, 5s interval, protection temperature is 25°C); centrifuged at 4°C and 8000rpm for 20 minutes, and the supernatant solution is collected.

[0023] c) equilibrating a nickel ion affinity column with 4 to 5 column volumes of a protein washing solution, applying the crushed supernatant solution obtained in step b) to the column at a flow rate of 4 to 6 s / drop to allow the target protein to fully bind to the nickel column, and collecting the flow-through; washing the nickel column with 4 to 5 column volumes of a pre-cooled protein washing solution at a flow rate of 4 to 6 s / drop to remove non-specifically bound proteins, and collecting the washing solution; eluting the target protein with 4 to 5 column volumes of a pre-cooled protein eluent at a flow rate of 4 to 6 s / drop, collecting the eluate, dialyzing the resulting product overnight, and concentrating it by ultrafiltration to obtain purified recombinant humanized type III collagen.

[0024] Preferably, the nickel ion affinity column is equilibrated with 5 column volumes of protein washing solution at a flow rate of 4 s / drop, and the supernatant solution after the crushing obtained in step b) is applied to the column; the nickel column is washed with 4 column volumes of pre-cooled protein washing solution at a flow rate of 4 s / drop; and the target protein is eluted with 4 column volumes of pre-cooled protein eluent at a flow rate of 6 s / drop.

[0025] In step 1.1,

[0026] Preferably, the humanized type III collagen is R8-GLPGEN;

[0027] The humanized type III collagen R8-X is expressed by regulating the expression of the Tac promoter, and the nucleotide sequence of the Tac promoter core sequence is shown in SEQ ID NO.5.

[0028] Furthermore, step (2) further comprises: adding the pETDuet-1-R8-X recombinant plasmid to the competent E. coli Nissle 1917, mixing and ice bathing for 20 to 30 minutes, heat shocking for 60 to 90 seconds, immediately ice bathing for 3 to 5 minutes, adding 700 to 800 μl of antibiotic-free LB medium, incubating at 37°C for 45 to 60 minutes, and screening positive clones on a plate containing 100 μg / ml ampicillin antibiotics, which are recombinant probiotics expressing humanized type III collagen R8-X. Preferably, the mixing and ice bathing time is 30 minutes, the heat shock time is 60 seconds, the ice bath time is 3 minutes, the volume of the antibiotic-free LB medium is 700 μl, and the incubation time is 45 minutes.

[0029] The components of the antibiotic-free LB culture medium are: 10 g / L tryptone (trypticase), 5 g / L yeast extract, and 10 g / L NaCl, and the initial pH is 7.2-7.4.

[0030] The components of the plate containing 100 μg / ml ampicillin antibiotic are: 10 g / L tryptone (trypticase), 5 g / L yeast extract, 10 g / L NaCl, and 17.5 g / L agar powder. The concentration of ampicillin antibiotic is 100 μg / ml, and the initial pH is 7.2-7.4.

[0031] In step 1.2,

[0032] Specifically, the components of the TB culture medium are: tryptone (trypticase) 11.8 g / L, yeast extract 23.6 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, 4 ml / L glycerol;

[0033] The composition of the lysis buffer is: 300mM NaCl, 50mM NaH2PO4, 10mM imidazole, pH=6.5-6.8; the parameters of the ultrasonic disruption are set as: 350W, 5s ultrasonication, 5s rest, and a protection temperature of 25°C.

[0034] The present invention also provides a recombinant probiotic expressing humanized type III collagen, characterized in that the recombinant probiotic expressing humanized type III collagen is a recombinant strain pETDuet-1-R8-X-Nissle 1917 that efficiently expresses the humanized type III collagen; wherein the probiotic used is Escherichia coli Nissle 1917;

[0035] The recombinant probiotics can efficiently express humanized type III collagen with good cell adhesion activity, and the bacterial endotoxin content of the expressed humanized type III collagen is 1.96 EU / ml, which is less than 2 EU / ml, and can effectively reduce protein production costs.

[0036] The present invention also provides a humanized type III collagen prepared by the above preparation method, wherein the humanized type III collagen has good biological activity of cell adhesion.

[0037] The present invention also provides a recombinant expression vector, which is a pETDuet-1-R8-X recombinant plasmid, and uses a Tac promoter for regulated expression.

[0038] The present invention also provides the humanized type III collagen, the preparation method of the humanized type III collagen, the recombinant probiotics, the humanized type III collagen prepared by the preparation method, or the use of the recombinant expression vector in the preparation of humanized type III collagen.

[0039] In a specific embodiment, the present invention first screens for a superior cell adhesion sequence X based on the original humanized type III collagen gene R8, splicing and integrating it with the original collagen gene to obtain a new recombinant humanized type III collagen R8-X, whose cell adhesion activity is improved. Furthermore, the present invention uses the probiotic Escherichiacoli Nissle 1917 as a host cell, and compared to the traditional engineered Escherichia coli BL21 (DE3), its collagen expression level is improved. Purification and determination of the bacterial endotoxin content of the recombinant humanized type III collagen showed that it was less than 2 EU / ml, meeting industry standards. In summary, the improved solution proposed by the present invention effectively addresses the pathogenicity, low yield, and low bioactivity problems of existing E. coli strains producing humanized type III collagen, and has significant technical benefits.

[0040] Compared with the existing technology, the beneficial effect of the present invention is that: based on the original humanized type III collagen R8 sequence, the present invention integrates the cell adhesion sequence X. Compared with the original humanized type III collagen R8, the recombinant collagen R8-X containing four cell adhesion sequences has better biological activity. The engineered bacteria of the present invention uses the probiotic E. coli Nissle 1917 as the chassis cell, constructs a recombinant expression vector and introduces it into E. coli Nissle 1917 to obtain a functional recombinant probiotic. Compared with the traditional Escherichia coli engineered bacteria BL21 (DE3), the recombinant probiotics of the present invention express humanized type III collagen with improved production, and have a wider and safer application scenario. The recombinant probiotic E. coli Nissle 1917 of the present invention does not contain enterotoxins or cytotoxins associated with other pathogenic E. coli. The bacterial endotoxin content of the recombinant humanized type III collagen obtained by purification is less than 2EU / ml, which meets industry standards. This can avoid the purification costs generated by industrial endotoxin removal, thereby reducing production costs and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1This is the structure of collagen predicted by AlphaFold2 in this invention. (AlphaFold2 website: https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb#scrollTo=kOblAo-xetgx)

[0043] Figure 2 This is an SDS-PAGE image of the recombinant collagen containing different adhesion sequences purified by the present invention, wherein M is a marker, and 1 to 5 are R8, R8-GLGEN, R8-GFPGER, R8-GPP, and R8-GPQ collagen bands, respectively.

[0044] Figure 3 This is an SDS-PAGE image of the induced expression of the recombinant probiotics used in this invention. M represents a marker; 1 represents the soluble protein after 8 hours of empty induction; 2 and 4 represent the soluble proteins after 8 hours of induction in different host bacteria, BL21(DE3) and Nissle1917, respectively; 3 and 5 represent the soluble proteins after 8 hours of induction in different host bacteria, BL21(DE3) and Nissle1917, respectively. The arrow indicates the humanized type III collagen band.

[0045] Figure 4 This is a table showing the cell adhesion activity of recombinant collagen containing different adhesion sequences purified by the present invention. DETAILED DESCRIPTION

[0046] In order to more clearly illustrate the specific embodiments of the present invention, the present invention is further described in detail with reference to the following specific examples and drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0047] Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0048] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0049] The present invention discloses a humanized type III collagen, a preparation method and an application, and belongs to the field of bioengineering technology. First, based on the original humanized type III collagen gene, the present invention uses cell adhesion activity as an evaluation index to screen out a better cell adhesion sequence and splice and integrate it with the original collagen gene to obtain a new humanized type III collagen. Then, using the probiotic EcN as a host cell, a humanized type III collagen recombinant expression vector containing the adhesion sequence is constructed, and the recombinant expression vector is introduced into EcN to obtain a functional recombinant probiotic. Compared with the traditional Escherichia coli engineered bacteria BL21 (DE3), the recombinant probiotics of the present invention have good cell adhesion activity while efficiently expressing humanized type III collagen, and the recombinant humanized type III collagen obtained by purification has a bacterial endotoxin content that meets industry standards, which can avoid the purification cost caused by industrial endotoxin removal, thereby reducing production costs and having good application prospects.

[0050] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0051] The E. coli Nissle 1917 strain used in the present invention was purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd., and the pETDuet-1 plasmid was maintained in the laboratory. The bacterial plasmid DNA extraction kit and DNA purification and recovery kit were purchased from TIANGEN; PrimeSTAR HSDNA Polymerase, T4 DNA ligase, and DNA endonucleases Nde I, Kpn I, BamH I, Bgl II, and Dpn I were all purchased from Takara.

[0052] The TB liquid culture medium of the present invention comprises 11.8 g / L tryptone (trypticase), 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, and 4 ml / L glycerol. The LB fermentation medium comprises 10 g / L tryptone (trypticase), 5 g / L yeast extract, and 10 g / L NaCl, with an initial pH of 7.2 to 7.4. The lysis buffer comprises 300 mM NaCl, 50 mM NaH2PO4, and 10 mM imidazole, with a pH of 6.5 to 6.8.

[0053] Example 1 Screening of humanized type III collagen adhesion sequences

[0054] Cell adhesion is a fundamental cellular activity, playing a key role in cell proliferation, differentiation, and migration. Based on the types of cell adhesion molecules, four different adhesion sequences were selected and named GLPGEN, GFPGER, GPP, and GPQ. The GLPGEN and GFPGER sequences are derived from the integrin α2β1 binding site, the GPP sequence is derived from a cross-linking sequence derived from type III collagen, and the GPQ sequence is derived from a fibroblast adhesion-promoting sequence in type I collagen. The amino acid sequences of these four adhesion sequences are SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively. The gene sequences were optimized based on the codon preference of Escherichia coli. The optimized nucleotide sequences are SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, respectively, as shown in the table below.

[0055]

[0056] Example 2 Construction of recombinant expression vectors of humanized type III collagen R8 and collagen R8-X containing different adhesion sequences

[0057] Humanized type III collagen R8, whose amino acid sequence is shown in SEQ ID NO. 1, is constructed by tandemly linking the type III collagen peptide (GFPGPKGNDGAPGKNGERGGPGGPGPQGPP) eight times, with the linker amino acid LD between each of the four tandem segments. The C-terminus contains six histidine residues, and the linker amino acid LE is present at the end of the gene. Its amino acid sequence is shown in SEQ ID NO. 1:

[0058] SEQ ID NO.1:

[0059] MVDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGF PGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

[0060] Humanized type III collagen R8-X containing different cell adhesion sequences (X represents a different cell adhesion sequence, and X can be any one of GLPGEN, GFPGER, GPP, and GPQ). This protein is based on the humanized type III collagen R8 sequence described above, with different adhesion sequences integrated after the terminal amino acid LE of the gene, and has six histidine residues at the C-terminus. Preferably, the amino acid sequence of the humanized type III collagen R8-GLPGEN containing the adhesion sequence GLPGEN is as follows:

[0061] SEQ ID NO.2:

[0062]

[0063]

[0064] The bold underlined portion of the above amino acid sequence is the cell adhesion sequence, which can be replaced with any of the cell adhesion amino acid sequences in Example 1. The amino acid sequences of humanized type III collagen R8-GFPGER, R8-GPP, and R8-GPQ containing different cell adhesion sequences are shown in SEQ ID NOs. 18-20, respectively:

[0065] R8-GFPGER:

[0066] MVDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGP KGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEGFPGERHHHHHH. (SEQ ID NO.18)

[0067] R8-GPP:

[0068] MVDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKG NDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEGPPGPCCGGGHHHHHH. (SEQ ID NO.19)

[0069] R8-GPQ:

[0070] MVDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGND GAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEGPQGIAGQRGVVGLPHHHHHHH. (SEQ ID NO.20)

[0071] The nucleic acids encoding the humanized type III collagen R8 and collagen R8-X containing different adhesion sequences were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for full gene synthesis based on the codon preference of Escherichia coli. Nde I and Kpn I restriction sites were added at both ends of the gene, and the collagen gene was ligated into the vector pUC19. The nucleotide sequence of R8 is shown in SEQ ID NO.3:

[0072] SEQ ID NO.3:

[0073] .

[0074] Humanized type III collagen R8-X containing different cell adhesion sequences (X represents a different cell adhesion sequence, X can be any one of GLPGEN, GFPGER, GPP, GPQ adhesion sequences, preferably, the humanized type III collagen R8-GLPGEN containing the cell adhesion sequence GLPGEN, its nucleotide sequence SEQ ID NO.4 is shown below:

[0075] SEQ ID NO.4:

[0076]

[0077] The bold underlined portion of the above amino acid sequence is the cell adhesion sequence, which can be replaced with any of the cell adhesion nucleotide sequences in Example 1. The nucleotide sequences of humanized type III collagen R8-GFPGER, R8-GPP, and R8-GPQ containing different cell adhesion sequences are shown in SEQ ID NOs. 21-23, respectively:

[0078] R8-GFPGER:

[0079] (SEQ ID NO.21)

[0080] R8-GPP:

[0081] 。(SEQ ID NO.22)

[0082] R8-GPQ:

[0083] atggtcgacggctttccgggcccgaaaggtaacgacggtgcgccgggcaaaaacggtgaacgtggtggtccgggcggtccgggcccgcaaggcccgccgggttttccgggcccaaaaggtaacgatggcgcgccgggcaaaaatggcgaacgcggcggtccgggcggtccgggcccgcaaggcccaccgggctttccgggtccgaagggcaacgacggcgcgccgggcaaaaacggcgaacgtggtggtccgggcggtccgggcccgcaaggcccgccgggttttccgggcccgaaaggcaatgacggtgcgccgggcaagaacggcgaacgtggcggtccgggcggtccgggcccgcaaggcccgccgctcgacggctttccgggcccgaaaggtaacgacggtgcgccgggcaaaaacggtgaacgtggtggtccgggcggtccgggcccgcaaggcccgccgggttttccgggcccaaaaggtaacgatggcgcgccgggcaaaaatggcgaacgcggcggtccgggcggtccgggcccgcaaggcccaccgggctttccgggtccgaagggcaacgacggcgcgccgggcaaaaacggcgaacgtggtggtccgggcggtccgggcccgcaaggcccgccgggttttccgggcccgaaaggcaatgacggtgcgccgggcaagaacggcgaacgtggcggtccgggcggtccgggcccgcaaggcccgccgctcgagggtccgcagggtatcgctggtcagcgtggtgttgttggtctgccgcaccaccaccaccaccactga。(SEQ ID NO.23)

[0084] Using the above synthesized plasmids pUC19-R8 and pUC19-R8-X containing the R8 gene and the R8-X gene as templates, PCR primers F1 and R1 were designed to amplify the target genes:

[0085] F1: CGCCATATGGTCGACGGCTTTCCGGGC(SEQ ID NO.14).

[0086] R1: CGGGGTACCTCAGGTGGTGGTGGTGGTGGTG (SEQ ID NO. 15).

[0087] The PCR reaction system is as follows:

[0088]

[0089] The PCR reaction procedure is as follows:

[0090]

[0091] The PCR product was recovered using a DNA purification and recovery kit (TIANGEN). The recovered PCR product and pETDuet-1 plasmid were double-digested with Nde I and Kpn I, respectively, and recovered. The recovered double-digested vector was further ligated with the double-digested gene using T4 ligase overnight.

[0092] Furthermore, the enzyme-linked product was added to competent Nissle 1917, mixed and incubated on ice for 30 minutes, heat-shocked at 42°C for 60 seconds, and immediately incubated on ice for 3 minutes. 700 μl of antibiotic-free LB medium was added, cultured at 37°C for 45 minutes, and spread on a plate containing 100 μg / ml ampicillin to select transformants. One single clone was picked for colony PCR verification and PCR verification was performed using universal primers pET41-F and T7t on the pETDuet-1 plasmid. The clones that were confirmed positive were sent to a sequencing company for sequencing. If they met the expected sequence, the recombinant expression vectors pETDuet-1-R8 and pETDuet-1-R8-X were successfully constructed (X represents a different cell adhesion sequence, and X can be any one of GLPGEN, GFPGER, GPP, and GPQ).

[0093] Example 3 Construction of recombinant plasmid containing Tac promoter core sequence

[0094] Using the recombinant expression vectors pETDuet-1-R8 and pETDuet-1-R8-X successfully constructed in Example 2 as templates, PCR primers F2 and R2 were designed for full plasmid PCR to replace the original promoter in the recombinant expression vectors pETDuet-1-R8 and pETDuet-1-R8-X with the Tac promoter. According to the reported Tac promoter core sequence (29 bp), the core sequence was preceded and followed by the introduction of the same tail enzyme BamH I and Bgl II restriction sites. The sequence is shown in SEQ ID NO.5, where the underlined portion is the restriction site, and the black bold font is marked as the Tac promoter core sequence.

[0095] F2:CATCGGCTCGTATAATGAGATCTGGAATTGTGAGCGGATAACAATTCCCCAT.

[0096] (SEQ ID NO.16)

[0097] R2:TCATTATACGAGCCGATGATTAATTGTCAAGGATCCATTTCGATTATGCGGC.

[0098] (SEQ ID NO.17)

[0099] Tac: The PCR reaction system is as follows:

[0100]

[0101] The PCR reaction procedure is as follows:

[0102]

[0103]

[0104] The PCR product was recovered using a DNA purification kit (TIANGEN) and the original template plasmid was digested with Dpn I enzyme. The enzyme digestion system was as follows:

[0105]

[0106] React at 37°C for 3 hours. After enzyme digestion, immediately add 10 μl of enzyme digestion to competent Nissle 1917 and mix on ice for 30 minutes. Heat shock at 42°C for 60 seconds, immediately ice bath for 3 minutes, add 700 μl of LB medium without antibiotics, expand at 37°C for 45 minutes, spread on plates containing 100 μg / ml ampicillin antibiotics to select transformants, pick one single clone for bacterial liquid PCR verification, and use pET41-F and T7t for PCR verification. The expected size is about 1300 bp. The positive clones were sent to a sequencing company for sequencing. The sequences were consistent with the expected sequences. The recombinant expression vectors pETDuet-1-R8 (PTac) and pETDuet-1-R8-X (PTac) containing the Tac promoter core sequence were successfully constructed.

[0107] Example 4 Expression and purification of recombinant humanized type III collagen R8 and R8-X in Escherichia coli

[0108] A single colony of the R8 or R8-X recombinant human type III collagen strain successfully constructed in Example 3 was inoculated into 10 ml (50 mL Erlenmeyer flask) of LB medium containing 100 μg / ml Amp (ampicillin antibiotic) and cultured overnight at 37°C. The culture was then transferred to 200 ml of TB medium containing 100 μg / ml Amp at 2% saturation and cultured at 37°C until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM and cultured at 25°C for 6-8 hours. The fermentation broth was collected. The fermentation broth was centrifuged in a refrigerated centrifuge at 4°C and 10,000 rpm for 10 minutes, and the supernatant was discarded to collect the bacterial precipitate.

[0109] The bacterial pellet was then resuspended in 20 mL of pre-chilled lysis buffer and transferred to a 50 mL centrifuge tube. The pellet was centrifuged at 8000 rpm for 20 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in 20 mL of lysis buffer (containing PMSF to a final concentration of 1 mM). The cells were then sonicated on ice for 30 minutes (350W, 5 seconds of sonication, 5 seconds of rest, with a protective temperature of 25°C). The pellet was then centrifuged at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected. This supernatant contains recombinant collagen.

[0110] The nickel ion affinity column was equilibrated with 5 column volumes of protein washing solution, and the supernatant solution obtained after the crushing was applied to the column at a flow rate of 4 s / drop to allow the target protein to fully bind to the nickel column, and the flow-through liquid was collected; the nickel column was washed with 4 column volumes of pre-cooled protein washing solution at a flow rate of 4 s / drop to remove non-specific binding proteins, and the washing liquid was collected; the target protein was eluted with 4 column volumes of pre-cooled protein eluent at a flow rate of 6 s / drop, and the eluate was collected. The obtained product was dialyzed overnight and concentrated by ultrafiltration to obtain purified recombinant humanized type III collagen. The test results are as follows: Figure 2 shown.

[0111] Example 5 Detection of cell adhesion activity of recombinant humanized type III collagen

[0112] Remove mouse embryonic fibroblast 3T3 cells from a -80°C freezer, inoculate them in cell culture medium, and culture them in a 37°C cell culture incubator. When the cells grow to 80% to 90% of the culture flask, perform cell passage. Add 2 mL of trypsin solution to the cell culture flask and digest at 37°C for 2 to 3 minutes. Observe the cell digestion under an inverted microscope. When the cells become round and close to detaching from the cell wall, pour out the trypsin solution. Add 10 mL of cell culture medium and gently pipette to detach the cells. Transfer the digested cells to a centrifuge tube, centrifuge at 1000 rpm and 22°C for 3 minutes, discard the supernatant, and add 1 mL of cell culture medium to resuspend the cells to make a cell suspension.

[0113] The purified recombinant human type III collagen prepared in step 4 of Example 1 of the present invention was configured into a 0.5 mg / ml protein solution, and 100 μL of recombinant human collagen was added to a 96-well plate as the cells to be tested, with PBS as a blank control, and incubated in a 37°C incubator for 1 hour. Remove the liquid in the wells of the 96-well plate, add 200 μL of PBS solution, and wash 2-3 times. After removing the liquid in the wells, add 100 μL of heat-inactivated 1% BSA-PBS solution (weigh 1 g of BSA (CAS: 9048-46-8) powder and dissolve it in 100 mL of PBS buffer solution. Heat inactivated at 56°C for 30 minutes), and incubate in a 37°C incubator for 1 hour. Remove the liquid in the wells, add 200 μL of PBS solution, wash 2-3 times, and remove the wells for use. Take the above cell suspension and dilute it with cell culture medium to a cell density of 1×10 6 Cells were added to a 96-well plate at a concentration of 100 μL / well and incubated at 37°C for 1 hour. After incubation for 1 hour, the 96-well plate was washed four times with 200 μL of PBS per well. The liquid in the plate was removed and set aside for further use. The assay was performed according to the instructions of the Cell Counting Kit-8 (CCK-8) and the absorbance (OD) value was measured at 450 nm.

[0114] The calculation formula for the relative adhesion rate of recombinant collagen cells is as follows:

[0115] Relative cell adhesion rate (%) = (OD of cells to be tested - OD of blank) / OD of blank × 100.

[0116] The test results are shown in Table 4. The cell adhesion activity of purified recombinant collagen R8 was 100%. Recombinant collagen R8-X containing different adhesion sequences all exhibited cell adhesion activity, and all showed higher cell adhesion activity than the original collagen R8. Among them, recombinant collagen R8-GLPGEN, containing the cell adhesion sequence GLPGEN, exhibited the highest cell adhesion activity.

[0117] Example 6 Expression of recombinant humanized type III collagen R8-GLPGEN in Escherichia coli BL21 (DE3) and Nissle 1917

[0118] The recombinant expression vector pETDuet-1-R8-GLPGEN (PTac), containing the Tac promoter core sequence, constructed in Example 3, was transformed into BL21(DE3) and Nissle1917, respectively, to obtain recombinant strains pETDuet-1-R8-GLPGEN-BL21(DE3) and pETDuet-1-R8-GLPGEN-Nissle1917. A single colony was inoculated into 10 ml (50 mL Erlenmeyer flask) of LB medium containing 100 μg / ml Amp and cultured overnight at 37°C. The strain was then transferred to 200 ml of TB medium containing 100 μg / ml Amp at 2% RT and cultured at 37°C to an OD600 of 0.6-0.8. IPTG was added to a final concentration of 0.1 mM and cultured at 25°C for 6-8 hours. The fermentation broth was then collected and centrifuged at 4°C, 10,000 rpm, and the supernatant was discarded to collect the bacterial pellet.

[0119] Further, the bacterial pellet was added to 20 mL of pre-cooled lysis buffer to resuspend the bacterial cells and transferred to a 50 mL centrifuge tube, centrifuged at 4°C, 12000 rpm for 20 minutes; the supernatant was discarded, and 20 mL of lysis buffer (containing PMSF at a final concentration of 1 mM) was added to resuspend the bacterial cells, and then ultrasonically disrupted in an ice bath for 30 minutes (350W, 5s ultrasonic, 5s intermittent, protection temperature of 25°C); centrifuged at 4°C, 8000 rpm for 20 minutes, and the supernatant was collected. The supernatant after disruption contains recombinant collagen. The test results are as follows. Figure 3 As shown, the SDS-PAGE electrophoresis diagram shows that compared with the traditional Escherichia coli engineered bacteria BL21 (DE3), the recombinant probiotics of the present invention expresses a higher yield of humanized type III collagen.

[0120] Example 7 Bacterial Endotoxin Detection of Recombinant Humanized Type III Collagen

[0121] The LPS content of recombinant collagen was determined using a quantitative chromogenic substrate method. The kit was purchased from Xiamen Limulus Amebocyte Lysate Biotechnology Co., Ltd. The lyophilized endotoxin standard solution in the kit was prepared and diluted to final concentrations of 0.1, 0.25, 0.5, and 1.0 EU / ml. Endotoxin-free water, Limulus Amebocyte Lysate standard solution, and purified collagen were added to 100 μl of Limulus Amebocyte Lysate, mixed, and incubated at 37°C for 9 minutes. At the end of the incubation period, 100 μl of chromogenic substrate solution was added, mixed, and incubated at 37°C for 6 minutes. At the end of the incubation period, 500 μl of azo reagent 1, 500 μl of azo reagent 2, and 500 μl of azo reagent 3 were added, mixed, and allowed to stand for 5 minutes. The absorbance was read at 545 nm. The endotoxin content of collagen R8-GLPGEN was determined to be 1.96 EU / ml, less than 2 EU / ml, meeting the medical device industry standard for recombinant collagen.

[0122] In summary, to address the pathogenicity, low production, and low cell adhesion activity of existing humanized type III collagen-producing E. coli strains, the present invention proposes using the probiotic E. coli Nissle 1917 as a host cell. Based on the existing collagen gene, a cell adhesion sequence with excellent cell adhesion activity was screened and further spliced ​​and integrated to construct a recombinant collagen expression vector. This vector was then introduced into E. coli Nissle 1917 to produce a functional recombinant probiotic strain. Compared to the traditional engineered E. coli BL21 (DE3) strain, this recombinant probiotic not only efficiently expresses humanized type III collagen but also exhibits improved cell adhesion activity. Furthermore, the bacterial endotoxin content of the purified recombinant humanized type III collagen meets industry standards, eliminating the purification costs associated with industrial endotoxin removal, thereby reducing production costs and promising application prospects.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0124] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention, but not excluding other aspects.

[0125] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0126] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A humanized type III collagen, characterized in that: The humanized type III collagen is a recombinant humanized type III collagen. The humanized type III collagen contains the collagen core functional region R8 and the cell adhesion sequence X, and has good cell adhesion activity.

2. The humanized type III collagen according to claim 1, wherein The collagen core functional region R8 comprises 30 amino acids in the human collagen functional region that are spliced ​​and integrated, and fused with a connecting peptide and a histidine purification tag. The amino acid sequence of the collagen core functional region R8 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the collagen core functional region R8 is shown in SEQ ID NO.

3.

3. The humanized type III collagen according to claim 1, wherein The cell adhesion sequence X is any one of the cell adhesion sequences GLPGEN, GFPGER, GPP, and GPQ; the amino acid sequence of the cell adhesion sequence X is shown in SEQ ID NOs. 6-9, and the nucleotide sequence of the gene encoding the cell adhesion sequence X is shown in SEQ ID NOs. 10-13.

4. A method for preparing humanized type III collagen according to claim 1, characterized in that: The preparation method comprises the following steps: 1.1 Construction of recombinant probiotics expressing humanized type III collagen: 1) The coding gene fragment of humanized type III collagen R8-X was amplified by PCR and ligated into the pETDuet-1 plasmid by enzyme digestion and ligation to obtain the recombinant expression vector pETDuet-1-R8-X recombinant plasmid; 2) chemically transforming the pETDuet-1-R8-X recombinant plasmid into E. coli Nissle 1917, and screening the successfully constructed recombinant strain pETDuet-1-R8-X-Nissle 1917 on an ampicillin plate, which is the recombinant probiotic expressing the humanized type III collagen; Wherein, the humanized type III collagen is expressed by regulating the expression of the Tac promoter, and the nucleotide sequence of the Tac promoter core sequence is shown in SEQ ID NO.5; 1.2 Expression and purification of humanized type III collagen: a) Streaking the recombinant probiotic pETDuet-1-R8-X-Nissle 1917 prepared in step 1.1 onto an LB plate containing 100 μg / ml ampicillin and culturing overnight at 37°C; picking a single colony and inoculating it into 10 ml of LB medium containing 100 μg / ml ampicillin, and shaking and culturing at 37°C for 12-16 hours; inoculating it into 200 ml of TB medium containing 100 μg / ml ampicillin at a 2% inoculum, shaking at 37°C to an OD600 of 0.6-0.8, adding IPTG to a final concentration of 0.1 mM, and culturing at 25°C for 6-8 hours to collect the fermentation broth; b) placing the fermentation broth obtained in step a) in a refrigerated centrifuge and centrifuging at 8,000-10,000 rpm for 10-15 minutes at 4°C, discarding the supernatant and collecting the bacterial precipitate; further, adding 20 mL of pre-cooled lysis buffer to resuspend the bacterial precipitate and transferring it to a 50 mL centrifuge tube, centrifuging at 8,000-10,000 rpm at 4°C for 20-30 minutes; discarding the supernatant, and resuspending the bacterial precipitate in 20 mL of lysis buffer containing a final concentration of 1 mM PMSF, followed by ultrasonic disruption in an ice bath for 30-45 minutes; centrifuging at 8,000-10,000 rpm at 4°C for 15-20 minutes, and collecting the supernatant; the disrupted supernatant solution contains recombinant collagen; c) equilibrating a nickel ion affinity column with 4 to 5 column volumes of a protein washing solution, applying the crushed supernatant solution obtained in step b) to the column at a flow rate of 4 to 6 s / drop to allow the target protein to fully bind to the nickel column, and collecting the flow-through; washing the nickel column with 4 to 5 column volumes of a pre-cooled protein washing solution at a flow rate of 4 to 6 s / drop to remove non-specifically bound proteins, and collecting the washing solution; eluting the target protein with 4 to 5 column volumes of a pre-cooled protein eluent at a flow rate of 4 to 6 s / drop, collecting the eluate, dialyzing the resulting product overnight, and concentrating it by ultrafiltration to obtain purified recombinant humanized type III collagen.

5. The preparation method according to claim 4, wherein The step 2) further comprises: adding the pETDuet-1-R8-X recombinant plasmid to the competent E. coli Nissle 1917, mixing and ice bathing for 20 to 30 minutes, heat shocking for 60 to 90 seconds, immediately ice bathing for 3 to 5 minutes, adding 700 to 800 μl of LB medium without antibiotics, incubating at 37° C. for 45 to 60 minutes, and screening positive clones on a plate containing 100 μg / ml ampicillin antibiotics, which are recombinant probiotics expressing humanized type III collagen R8-X; wherein, The composition of the antibiotic-free LB medium is: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, initial pH 7.2-7.4; and / or, The components of the plate containing 100 μg / ml ampicillin antibiotic are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 17.5 g / L agar powder, the concentration of ampicillin antibiotic is 100 μg / ml, and the initial pH is 7.2-7.

4.

6. The preparation method according to claim 4, wherein In step 1.2, the components of the TB culture medium are: tryptone 11.8 g / L, yeast extract 23.6 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, 4 ml / L glycerol; and / or, The composition of the lysis buffer is: 300mM NaCl, 50mM NaH2PO4, 10mM imidazole, pH=6.5-6.8; and / or, The ultrasonication parameters were set as follows: 350W, 5s ultrasonication, 5s rest, and a protection temperature of 25°C.

7. A humanized type III collagen prepared by the method according to any one of claims 4 to 6, characterized in that: The humanized type III collagen has good biological activity of cell adhesion.

8. A recombinant probiotic expressing humanized type III collagen, characterized in that: The recombinant probiotic expressing humanized type III collagen is the recombinant strain pETDuet-1-R8-X-Nissle1917 that efficiently expresses the humanized type III collagen; wherein the probiotic used is Escherichia coli Nissle 1917; and / or, The recombinant probiotics can efficiently express the humanized type III collagen with good cell adhesion activity, and the bacterial endotoxin content of the expressed humanized type III collagen is 1.96 EU / ml, which is less than 2 EU / ml, and can effectively reduce the cost of protein production.

9. A recombinant expression vector, characterized in that: The recombinant expression vector is a pETDuet-1-R8-X recombinant plasmid, which is characterized by using a Tac promoter for regulated expression.

10. An application, characterized in that: Use of the humanized type III collagen according to any one of claims 1 to 3, the preparation method according to any one of claims 4 to 6, the humanized type III collagen according to claim 7, the recombinant probiotic according to claim 8, or the recombinant expression vector according to claim 9 in the preparation of humanized type III collagen.

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