CN117964741A - Recombinant human VI type collagen with antibacterial and anti-inflammatory activities and external use ophthalmic medicine - Google Patents
Recombinant human VI type collagen with antibacterial and anti-inflammatory activities and external use ophthalmic medicine Download PDFInfo
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Abstract
The application discloses recombinant human type VI collagen with antibacterial and anti-inflammatory activities and an ophthalmic external medicine. The amino acid sequence of the recombinant human VI type collagen is (GYQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPG LPGCKGSPG) n, and n is a natural number greater than 1. The recombinant human type VI collagen has obvious inhibition effect on common pathogenic bacteria of a medical system, and particularly has the inhibition effect on staphylococcus aureus which is a causative factor of hordeolum and even exceeds the first-line clinical erythromycin for treating hordeolum. Moreover, the polypeptide has cell adhesion promoting and cell proliferation promoting effects, and has potential to be developed into an ophthalmic external medicament (such as eye drops) capable of treating bacterial infection and promoting wound healing, and is particularly suitable for patients with ocular trauma and suffering from pathogenic bacterial infection.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and in particular relates to human type VI collagen which is produced by using genetic engineering technology, fermentation technology and purification technology and has antibacterial, anti-inflammatory, cell adhesion promoting and proliferation effects.
Background
Hordeolum is also known as needle eye and hordeolum. Is a common disease and frequently occurring disease of people in ophthalmology. The mild early stage can relieve pain and detumescence by hot towel hot compress, and when furuncle is large, the furuncle needs to be treated by oral administration of antibiotics, external dripping of anti-inflammatory eyedrops and the like. If the furuncle is found to be serious and even suppurative symptoms appear, the furuncle needs to be excised and pus discharged by surgical means. Hordeolum was found to be an ophthalmic disease caused by staphylococcus aureus infection.
Collagen has low immunogenicity, has effects of promoting tissue repair and hemostasis, and has been widely used in foods, cosmetics, biomedical materials, medical devices and medicines. Is a well-known protein with better compatibility with human body.
Type VI collagen is a ubiquitous extracellular matrix component that is present in all connective tissues and often binds to the basement membrane. Type VI collagen forms a complex and extensive network of beaded microfibrils in most connective tissue.
There are few reports on the bacteriostatic and anti-inflammatory effects of type VI collagen. Based on the current results, it is generally believed that polypeptides from the N-or C-terminal region of type VI collagen may have antimicrobial activity against gram-positive and gram-negative bacteria.
Disclosure of Invention
The inventor unexpectedly found in the study that a polypeptide from a collagen domain (not an N-terminal or C-terminal domain) of the type VI collagen has obvious inhibition effect on common pathogenic bacteria of a medical system, such as staphylococcus aureus, pseudomonas aeruginosa and escherichia coli, and especially the inhibition effect on staphylococcus aureus which is a causative factor of hordeolum even exceeds that of erythromycin which is a first-line clinical medication for treating hordeolum. Moreover, the polypeptide has cell adhesion promoting and cell proliferation promoting effects, and has potential to be developed into an ophthalmic external medicament (such as eye drops) capable of treating bacterial infection and promoting wound healing, and is particularly suitable for patients with ocular trauma and suffering from pathogenic bacterial infection.
Namely, the present invention includes:
1. A recombinant human type VI collagen with antibacterial effect has an amino acid sequence of (GYQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPG) n, wherein n is a natural number greater than 1. The natural number n may be less than 20, preferably less than 10, more preferably less than 6, for example may be 2,3, 4, 5.
2. The recombinant human type VI collagen according to item 1, which has an amino acid sequence as shown in SEQ ID NO: 1.
SEQ ID NO：1
1GYQGMKGEKG SRGEKGSRGP KGYKGEKGKR GIDGVDGVKG EMGYPGLPGC KGSPG
56GYQGMKGEKG SRGEKGSRGP KGYKGEKGKR GIDGVDGVKG EMGYPGLPGC KGSPG
3. Nucleic acid encoding the recombinant human type VI collagen of item 1.
4. Nucleic acid encoding recombinant human type VI collagen according to item 2, preferably having a nucleotide sequence as set forth in SEQ ID NO: 2.
SEQ ID NO：2
1GGTTACCAGG GCATGAAAGG TGAAAAAGGC TCCCGCGGCG AAAAAGGCTC TCGTGGCCCG AAGGGCTACA
71AAGGTGAAAA AGGCAAACGT GGCATCGATG GTGTTGACGG CGTGAAAGGC GAAATGGGTT ACCCGGGTCT
141GCCTGGTTGC AAAGGCAGCC CTGGTGGCTA TCAGGGTATG AAAGGTGAGA AGGGTTCTCG TGGTGAAAAA
211GGTTCCCGTG GCCCGAAGGG TTACAAGGGC GAAAAGGGTA AACGCGGTAT CGATGGCGTT GACGGCGTTA
281AAGGTGAAAT GGGCTACCCAGGTCTGCCGG GTTGTAAAGG CTCTCCGGGC
5. An expression vector comprising the nucleic acid of item 3. Preferably, the expression vector is pET28a.
6. A host cell into which the expression vector according to claim 5 has been introduced. The host cell may be E.coli, saccharomyces cerevisiae, pichia pastoris or Bacillus subtilis, preferably E.coli (e.g.BL 21 (DE 3) may be used).
7. A method of producing the recombinant human type VI collagen of claim 1 comprising: culturing the host cell according to item 6 and collecting the cell culture.
8. The method of item 7, further comprising the step of subjecting the cell culture to separation and purification using one or a combination of several of isoelectric precipitation, salting out, alcohol precipitation, molecular sieves, ultrafiltration, ion exchange chromatography, affinity chromatography, and hydrophobic chromatography.
9. An ophthalmic topical comprising the recombinant human type VI collagen of claim 1 and a pharmaceutically acceptable carrier.
10. The ophthalmic external preparation according to item 9, which is an eye ointment or an eye drop.
11. The use of recombinant human type VI collagen according to item 1 in the preparation of an ophthalmic topical. The ophthalmic topical agent may be, for example, an eye ointment or an eye drop.
12. The use according to claim 11, wherein the ophthalmic topical medicament is for the treatment of bacterial-induced ocular infections, in particular ocular infections caused by staphylococcus aureus, pseudomonas aeruginosa and/or escherichia coli.
According to the invention, the recombinant human VI type collagen is fully human, and has good biological safety; the production process is simple, the yield is high, the preparation can be rapidly carried out in large quantity through an escherichia coli expression system, and the production performance is good; it has good antibacterial activity, good cell adhesion promoting activity and cell proliferation promoting activity, and has potential to be developed into an ophthalmic external medicine (such as eye drops).
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of recombinant human type VI collagen-1 prepared in the examples.
FIG. 2 is an SDS-PAGE electrophoresis of recombinant human type VI collagen-2 prepared in the examples.
FIG. 3 is an SDS-PAGE electrophoresis of recombinant human type VI collagen-3 prepared in the examples.
Detailed Description
The application will be further illustrated with reference to the following examples, which are to be understood as merely further illustrating and explaining the application and are not to be construed as limiting the application.
Example 1 construction of a strain expressing recombinant human type VI collagen with bacteriostatic action.
The delegated Huada gene company synthesized SEQ ID NO:2, which encodes the gene set forth in SEQ ID NO:1 (amino acid sequence of 296-350 of alpha 1 chain of natural human VI type collagen is repeated 2 times), the gene is inoculated into a pET28a carrier, then BL21 (DE 3) competent cells purchased from the Optimago are introduced, and the operation process is referred to the specification attached to the competent cells. Coating a kanamycin LB solid plate containing 50 mug/ml, standing at 37 ℃ for culturing for 24 hours, picking 3-8 larger single colonies, respectively inoculating into LB liquid culture medium containing 50 mug/ml kanamycin, shaking at 220rpm for 18-20 hours at 37 ℃, respectively transferring into LB liquid culture medium, shaking at 220rpm for 6-8 hours at 37 ℃, adding 0.5mmol/l IPTG, cooling to 28 ℃ and continuously culturing for 8 hours. Respectively collecting bacterial sludge before and after IPTG induction, performing SDS-PAGE electrophoresis after ultrasonic crushing, and carrying out mass spectrum identification on newly appeared band gel near the theoretical molecular weight by cutting and sending to Beijing Baitai Pike, wherein mass spectrum results of several strains of bacteria are the same as SEQ ID NO:1 are consistent. Through shake flask verification, a recombinant escherichia coli with large yield and stable expression is further screened from several strains, and the genetic engineering strain is successfully constructed.
By adopting the same method, the recombinant human VI type collagen gene engineering strain is successfully constructed by repeating the amino acid sequence of 296-350 sites of the natural human VI type collagen alpha 1 chain for 1 time or 3 times.
EXAMPLE 2 expression of recombinant human type VI collagen with antibacterial and anti-inflammatory effects
First step, seed activation: each of the genetically engineered strains constructed in example 1 was inoculated into LB solid medium by streaking. Culturing at 37 ℃ for 36h.
And step two, preparing primary seed liquid: the single colony activated in the first step is inoculated in LB liquid medium as primary seed liquid.
And step three, preparing secondary seed liquid: inoculating the first-stage seed liquid produced in the second step into LB liquid culture medium according to a certain inoculation proportion, and performing shake culture to obtain the second-stage seed liquid.
Fourth step, collagen fermentation production: inoculating the secondary seed liquid into a fermentation culture medium according to a certain inoculation proportion, and placing the fermentation culture medium into a 20L reactor. As the bacterial grows after inoculation, the bacterial concentration increases, the system can produce acid substances, and the pH is controlled by 30% ammonia water. And when the bacterial concentration is increased to 0 D600=40-50, inducing, supplementing nutrient substances according to the requirement, and controlling the growth parameters to enable microorganisms to rapidly reproduce and grow. And judging the end of the culture according to the process requirement.
Example 3 separation and purification of recombinant human type VI collagen with antibacterial and anti-inflammatory effects
And (3) collecting bacterial sludge from the escherichia coli which is fermented and produces recombinant human type VI collagen. And release the target protein in the microbial cells through the operation means such as ultrasonic disruption or homogenization disruption. After disruption, cell debris and unbroken cells are removed by centrifugation, filtration, and the like. Salting out the fermentation broth with the fragments removed, isoelectric point precipitation, alcohol precipitation, acid precipitation and other operations to remove the impurity protein, wherein the target protein is located in the precipitate. Re-dissolving target protein precipitate with double distilled water or buffer solution, subjecting the re-dissolved protein solution to ion exchange column, hydrophobic chromatography column, affinity chromatography column or size exclusion chromatography column, and subjecting to concentration or non-concentration as required.
The recombinant human type VI collagen obtained by repeating the amino acid sequence (GYQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLP GCKGSPG) obtained by purification for 1 time, 2 times or 3 times is respectively named as recombinant human type VI collagen-1, recombinant human type VI collagen-2 and recombinant human type VI collagen-3. SDS-PAGE electrophoresis of the recombinant human type VI collagens-1, 2 and 3 is shown in figures 1-3.
Example 3 antibacterial experiments on recombinant human type VI collagen with anti-inflammatory antibacterial Activity
The experiment adopts three common pathogenic bacteria in hospitals as indicator bacteria, namely staphylococcus aureus, pseudomonas aeruginosa and escherichia coli as indicator bacteria, and adopts an oxford cup transparent circle method to judge the antibacterial capability. Setting 0.1mg/l;0.4mg/l;0.8mg/l;1.2mg/l;1.6mg/l and 2mg/l of collagen are taken as experimental groups, water for injection is taken as a solvent, and protein concentration is obtained by diluting protein mother liquor detected by a biuret method; erythromycin was diluted with water for injection using 10mg/l erythromycin as a positive control. The antibacterial result is as follows, and the diameter unit of the antibacterial ring is mm. The experimental results are shown in tables 1 and 2. It should be noted that the recombinant human type VI collagen-1 has no inhibitory effect on various indicator bacteria under the conditions of each test concentration in the experiment.
TABLE 1
TABLE 2
From the above statistical data, it can be seen that the recombinant human type VI collagen-2 and 3 has obvious inhibition effects on staphylococcus aureus, pseudomonas aeruginosa and escherichia coli, wherein the inhibition effect on staphylococcus aureus is optimal, and the inhibition effect even exceeds the common erythromycin for treating hordeolum. However, it is unexpected that the recombinant human type VI collagen-1 has no obvious inhibition effect on staphylococcus aureus, pseudomonas aeruginosa and escherichia coli. The inventors speculate that the inhibitory effect of this type of recombinant human type VI collagen may depend on the formation of specific secondary and tertiary structures, which are not yet formed by the short amino acid sequence of the recombinant human type VI collagen-1.
EXAMPLE 4 Effect of recombinant human type VI collagen-2 on the promotion of cell proliferation
Cell proliferation experiments were performed using recombinant human type VI collagen-2 as a sample for HSF (human skin fibroblasts) cells. The specific operation is as follows: human HSF cells (purchased from ATCC cell bank) were inoculated into sterile 96-well plates at 200. Mu.L per well of medium, and the cells were cultured using DMEM/F12 complete medium with 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6mg/mL of recombinant human type VI collagen-2 added to each well, respectively. Three duplicate wells are arranged at each group concentration, the blank control group is added with an equal volume of culture solution, and the culture solution is placed in a 37 ℃ incubator for culturing for 48 hours. Removing supernatant, adding 100 μl culture solution and 50 μl MTT solution into each well, shaking, incubating in an incubator for 4h, sucking out the mixture, adding 150 μl DMSO to dissolve purple formazan crystal, shaking for 10min, placing in an ELISA reader, measuring absorbance value of each well, and determining HSF (human skin fibroblast) cell proliferation promoting effect of recombinant type VI collagen, positive control (collagen purchased from Abcam company) and blank control data as follows
Proliferation rate = (experimental OD-blank OD) ×100%/blank.
| Parallel 1 | Parallel 2 | Parallel 3 | |
| Blank control | 0.325 | 0.319 | 0.320 |
| 0.2mg/l | 0.411 | 0.413 | 0.416 |
| 0.4mg/l | 0.489 | 0.512 | 0.509 |
| 0.8mg/l | 0.567 | 0.545 | 0.548 |
| 1.6mg/l | 0.611 | 0.617 | 0.605 |
| Positive control 1mg/l | 0.458 | 0.446 | 0.457 |
From the above statistics, it was found that the recombinant human type VI collagen-2 has an obvious effect of promoting cell proliferation, the proliferation rate is approximately 80%, and the positive control group is only about 40%.
EXAMPLE 5 Effect of recombinant human type VI collagen-2 on cell adhesion
The experiment was performed using recombinant human type VI collagen-2 as a sample, specifically, the cell adhesion promotion experiment was performed on HSF (human skin fibroblast) cells. The method comprises the following specific steps: human HSF cells were inoculated into a sterile 96-well plate, 200. Mu.L of medium per well, PBS alone was used as a blank control group, the groups were washed 3 times with PBS after standing at 37℃for 2 hours, non-adherent cells were washed off, 100. Mu.L of medium and 50. Mu. LMTT of solution were added per well, shaken well, incubated in an incubator for 4 hours, the mixture was aspirated, 150. Mu.L of DMSO was added to dissolve purple crystal formazan, shaken for 10 minutes, placed in an microplate reader, the wavelength was measured at 570nm, and the absorbance value of each well was measured. The adhesion to human skin fibroblasts with commercially available collagen (from Abcam corporation) was used as a positive control. The experimental group was designed with several concentrations of recombinant type VI collagen of 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6 mg/mL. The adhesion promotion calculation method comprises the following steps:
Cell adhesion rate= (experimental OD-blank OD) ×100%/blank.
The absorbance data statistics for the experimental and control groups are as follows:
| Parallel 1 | Parallel 2 | Parallel 3 | |
| Blank control | 0.258 | 0.248 | 0.251 |
| 0.2mg/l | 0.309 | 0.312 | 0.308 |
| 0.4mg/l | 0.367 | 0.356 | 0.344 |
| 0.8mg/l | 0.415 | 0.417 | 0.406 |
| 1.6mg/l | 0.467 | 0.462 | 0.459 |
| Positive control 1mg/l | 0.389 | 0.376 | 0.377 |
The adhesion rate of the recombinant human type VI collagen-2 is more than or equal to 60%, and the positive control group is about 50%, so that the recombinant human type VI collagen-2 has obvious adhesion promoting effect.
In conclusion, the recombinant human type VI collagen has antibacterial effect, cell proliferation promoting effect and cell adhesion promoting effect, so that the recombinant human type VI collagen can be used in the field of biological medicine and has positive curative effects on common diseases such as hordeolum and the like caused by staphylococcus aureus infection and other bacterial diseases and wound diseases caused by wounds.
Claims (10)
1. A recombinant human type VI collagen with antibacterial effect has an amino acid sequence of (GYQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGC KGSPG) n, wherein n is a natural number greater than 1, and the natural number n can be less than 20, preferably less than 10, more preferably less than 6, such as 2,3, 4, and 5.
2. The recombinant human type VI collagen according to claim 1, having an amino acid sequence as set forth in SEQ ID NO: 1.
3. A nucleic acid encoding the recombinant human type VI collagen of claim 1.
4. A nucleic acid encoding the recombinant human type VI collagen of claim 2. The nucleotide sequence of the nucleic acid can be shown as SEQ ID NO: 2.
5. An expression vector comprising the nucleic acid of claim 3. Preferably, the expression vector is pET28a.
6. A host cell into which the expression vector according to claim 5 has been introduced. The host cell may be E.coli, saccharomyces cerevisiae, pichia pastoris or Bacillus subtilis, preferably E.coli (e.g.BL 21 (DE 3) may be used).
7. A method of producing the recombinant human type VI collagen of claim 1 comprising: a step of culturing the host cell of claim 6 and collecting the cell culture.
8. The method of claim 7, further comprising the step of subjecting the cell culture to separation and purification using one or a combination of several of isoelectric precipitation, salting out, alcohol precipitation, molecular sieves, ultrafiltration, ion exchange chromatography, affinity chromatography, and hydrophobic chromatography.
9. An ophthalmic topical comprising the recombinant human type VI collagen of claim 1 and a pharmaceutically acceptable carrier. Preferably, the ophthalmic external use agent may be, for example, an eye ointment or an eye drop.
10. Use of recombinant human type VI collagen according to claim 1 in the manufacture of an ophthalmic topical. Preferably, the ophthalmic external use agent may be, for example, an eye ointment or an eye drop. Preferably, the ophthalmic topical formulation is used for the treatment of bacterial ocular infections, in particular ocular infections caused by staphylococcus aureus, pseudomonas aeruginosa and/or escherichia coli.
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