Recombinant hydroxylated human collagen type iii with cell adhesion properties, process for its preparation and use

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant hydroxylated human type III collagen as well as a preparation method and application thereof. The recombinant hydroxylated human type III collagen has cell adhesion, and an amino acid sequence thereof is shown as SEQ ID NO. 1. The application replaces 4 natural integrin sites with lower adhesion at 9 different sites of full-length natural type III collagen without changing the total length of the natural full-length amino acid, and retains the C-terminal peptide so as to have the potential to form a triple helix structure. The adhesion of the collagen is improved by more than 27% through the modification mode, the molecular weight is moderate, the collagen is easy to prepare, and the hydroxylation makes the expression more stable.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant hydroxylated human type III collagen with cell adhesion and a preparation method and application thereof. BACKGROUND

[0002] Collagen is one of the most important and abundant proteins in mammals, and is a structural protein found in the skin, connective tissue and bone of the human body and other tissues. The content of collagen in the human body is about 30% of the total protein. Collagen is a structural protein and the main component of the extracellular matrix. Type III collagen is twisted into a triple helix by three peptide chains curling to the right. Primary structure analysis shows that the long segment sequence of the polypeptide chain is repeated by Gly-X-Y amino acid sequence. Among them, X is usually proline, and Y is usually hydroxyproline and hydroxylysine, which are rarely seen in other proteins.

[0003] Due to the existence of four natural integrin sites with lower adhesion in the sequence of natural collagen, the adhesion is poor. SUMMARY

[0004] The purpose of the present application is to provide a recombinant hydroxylated human type III collagen with cell adhesion and a preparation method and application thereof, and to improve the adhesion of collagen.

[0005] The technical solution adopted by the present application is a recombinant human type III collagen with cell adhesion, the amino acid sequence of which is shown in SEQ ID NO. 1, and the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0006] A sequence with hydroxylation function and enhanced adhesion of the protein encoded by SEQ ID NO. 1, the amino acid sequence of which is shown in SEQ ID NO. 4, and the nucleotide sequence of which is shown in SEQ ID NO. 5.

[0007] The recombinant human type III collagen is used for preparing a cell adhesion promoter.

[0008] An expression vector comprising the nucleotide sequence shown in SEQ ID NO. 2.

[0009] An expression vector comprising the nucleotide sequence shown in SEQ ID NO. 5.

[0010] A host cell comprising the expression vector of any one of the above.

[0011] The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

[0012] The method for preparing the recombinant human type III collagen by the host cell comprises the following steps: culturing the host cell in a culture medium, purifying the recombinant human type III collagen after inducing the expression of the recombinant human type III collagen, and obtaining the recombinant human type III collagen.

[0013] The purification method is selected from any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.

[0014] Compared with the prior art, the method has the advantages that the four natural integrin sites with lower adhesion are replaced without changing the total length of the natural full-length amino acid, and the C-terminal and N-terminal full-length chain sequences of the collagen coding region are not removed, so that the collagen has the potential to form a triple helix structure.

[0015] The recombinant collagen protein has the potential to form a triple helix structure.

[0016] The recombinant collagen protein is expressed by the Pichia pastoris engineering bacteria, and the protein has no endotoxin hidden danger, and the protein does not carry a histidine marker, so that the protein can be purified by a molecular sieve, and the target protein can be directly obtained without additional removal of the histidine marker sequence.

[0017] The recombinant collagen protein prepared by the method can effectively perform immune response, inflammatory response, blood coagulation, tumor metastasis and wound healing.

[0018] The preparation method of the recombinant human hydroxylated type III collagen is suitable for industrial large-scale production, and the prepared product has no animal source infection source, so that the biological safety is higher. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A is a target gene plasmid restriction enzyme electrophoresis map, and B is a hydroxylated gene plasmid restriction enzyme electrophoresis map.

[0020] Figure 2 The colony PCR plasmid transformant contains a target gene.

[0021] Figure 3 The colony PCR plasmid transformant contains a hydroxylated gene.

[0022] Figure 4 The crystal violet staining result map is shown from left to right as BSA, type III natural collagen, type III recombinant collagen and type III recombinant hydroxylated collagen.

[0023] Figure 5 The AO / EB staining result chart from left to right is BSA, type III natural collagen, type III recombinant collagen, and type III recombinant hydroxylated collagen. DETAILED DESCRIPTION

[0024] In order for those skilled in the art to better understand the technical solutions of the present application, the present application is further described below in conjunction with specific examples and drawings.

[0025] In the description of the present application, if not specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0026] The inventive concept of the present application is as follows: by replacing 9 different sites of full-length natural type III collagen, the function of the full-length natural type III collagen is enhanced without changing the total length of the natural full-length amino acid, and 4 natural integrin sites with lower adhesion are replaced, while the C-terminal peptide is retained to have the potential to form a triple helix structure. Hydroxyproline plays an important role in maintaining the triple helix structure of collagen and its biological properties during synthesis. Mammalian cells with prolyl 4-hydroxylase can directly produce hydroxylated collagen through post-translational modification.

[0027] The purpose of the present application is to provide a recombinant hydroxylated collagen with cell adhesion, which can effectively support cell adhesion and is an important molecular basis for a series of important physiological and pathological processes such as immune response, inflammatory response, blood coagulation, tumor metastasis, and wound healing. It can be widely used in food, health care products, biological medicine and other fields.

[0028] The present application provides a type III recombinant collagen with cell adhesion, and the amino acid sequence is shown in SEQ ID NO. 1.

[0029] SEQ ID NO. 1:

[0030]

[0031] The present application provides a type III recombinant collagen with cell adhesion, which is codon-optimized for host cell expression, and has a signal peptide cleavage site and EcoR I and Not I enzyme cleavage sites added at both ends during design to facilitate later gene manipulation. The nucleotide sequence of the above-optimized collagen is shown in SEQ ID NO. 2.

[0032] SEQ ID NO. 2:

[0033]

[0034] The present application provides an expression vector comprising the above nucleic acid molecule. The expression vector can contain regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host into which the vector is to be introduced, e.g., bacterial, fungal, plant, or animal, as appropriate and taking into consideration whether the vector is DNA- or RNA-based. In a specific embodiment, the expression vector is pPIC9k, the nucleotide sequence of which is shown in SEQ ID NO. 3.

[0035] SEQ ID NO. 3:

[0036]

[0037] The present application provides a sequence with hydroxylation function and enhanced collagen adhesion, and the amino acid sequence is shown as SEQ ID NO. 4.

[0038] SEQ ID NO. 4:

[0039] KYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYHHYDGDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGTSIFFWVADPVTRKLYKETLHAGLPVKSGEKIIANQWIRAVK.

[0040] The present application provides a sequence with hydroxylation function and enhanced collagen adhesion, and the amino acid sequence is shown as SEQ ID NO. 4.

[0041] SEQ ID NO. 4:

[0042] ATGACCAACAAGTTCATCTCTTACAACAAGATGGAAACTCGTGAATACTTGCTGACCATTCTGTTCGTTATCGCTTGTTTCATGGTCTTGAACCTGGAAAGAAGAGAAGGTTTCGAGACTTCTGATAGACCAGGTGTTTGTGACGGTAAGTACTACGAGAAGATCGATGGATTTCTGTCTGACATCGAATGTGATGTGCTGATTAACGCTGCTATCAAGAAGGGTCTGATCAAGTCCGAAGTTGGTGGTGCTACTGAGAACGATCCAATCAAACTTGATCCAAAGTCTCGTAACTCCGAACAAACCTGGTTCATGCCAGGTGAACATGAGGTTATCGACAAGATCCAGAAGAAAACACGAGAGTTTCTGAACTCCAAGAAGCATTGCATCGACAAGTACAACTTCGAAGATGTTCAAGTTGCTAGGTACAAACCAGGTCAGTACTACTACCATCACTACGATGGTGATGATTGCGATGATGCTTGTCCAAAGGATCAGAGACTGGCTACTCTGATGGTTTACCTGAAGGCACCTGAAGAAGGTGGTGGAGGTGAAACCGATTTCCCTACTCTTAAGACCAAGATCAAGCCAAAGAAGGGTACTTCTATCTTCTTCTGGGTTGCTGATCCAGTCACCAGAAAGTTGTACAAAGAGACTTTGCATGCTGGTTTGCCAGTCAAGTCTGGTGAGAAGATTATCGCCAATCAGTGGATCAGAGCTGTTAAG.

[0043] The present application provides an expression vector comprising the above nucleic acid molecule. The expression vector can contain regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host, e.g. bacteria, fungi, plants or animals, into which the vector is to be introduced, and taking into account whether the vector is DNA-based or RNA-based, as appropriate. In a particular embodiment, the expression vector is pPICZA, the nucleotide sequence of which is shown in SEQ ID NO. 6.

[0044] SEQ ID NO. 6:

[0045]

[0046] The present application provides a host cell comprising the above-mentioned nucleic acid molecule.

[0047] The host cell refers to a cell into which a foreign nucleic acid has been introduced, including the progeny of such a cell. The host cell includes transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny can not be completely identical to the parent cell in nucleic acid content, but can contain mutations.

[0048] In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0049] In a specific embodiment, the host cell is Pichia pastoris GS115.

[0050] The present application provides a method for preparing any one of the aforementioned recombinant collagens, comprising the following steps:

[0051] The host cell is expressed using the above-mentioned host cell, and then separated and purified; the expression of the host cell refers to the cultivation of the host cell, and the culture medium and culture conditions are well known to those skilled in the art.

[0052] In a specific embodiment, the host cell is Pichia pastoris, and after obtaining the Pichia pastoris genetically engineered bacteria, the specific culture conditions are as follows: inoculate the Pichia pastoris genetically engineered bacteria into YPD culture medium, cultivate at 30°C and 220 rpm for 22h-24h until the OD600 is 18-20 as the upper tank seed liquid, inoculate the seed liquid into an NBS 415 fermenter with an initial volume of 5L at a volume of 10%, the culture temperature is 28°C-30°C, the pH is 5.0-6.0, the dissolved oxygen is controlled at 20%-30%, and when the glycerol is consumed, start the glycerol feeding culture, and when the wet weight of the bacteria reaches more than 180g / L, start the induction culture.

[0053] The present application does not make any limitation on the expression method, which can be determined as needed, for example, expression as induced expression, and for induced expression, the inducer is methanol.

[0054] In a specific embodiment, methanol is added for induction culture, the induction stage temperature is 28°C, the pH is 5.0, and the induction is performed for 48h before the tank is removed.

[0055] The present application does not make any limitation on the method for separation and purification, which can be determined as needed, for example, salt precipitation method, ultrafiltration method, affinity chromatography method, and gel filtration chromatography method can be used.

[0056] The application also provides the use of the recombinant hydroxylated collagen protein, the recombinant collagen protein encoded by the nucleic acid molecule, the recombinant hydroxylated collagen protein expressed by the expression vector, or the recombinant hydroxylated collagen protein produced by the host cell in immune response, inflammation, blood coagulation, tumor metastasis and wound healing.

[0057] Example 1: Expression of recombinant hydroxylated human type III collagen

[0058] The recombinant collagen protein gene of the application, i.e. the target gene, has a nucleotide sequence as shown in SEQ ID NO. 2. EcoR I and Not I recognition sites and signal peptide recognition sites are added at the 5' end and 3' end respectively during synthesis, and the linearized by restriction enzyme Sac I, and then cloned into the expression vector pPIC9K to obtain the pPIC9K-RCOL(III) cloning plasmid. The pPIC9K-RCOL(III) cloning plasmid is linearized by electroporation and then transformed into the GS115-pPIC9K-RCOL(III) to obtain the GS115-pPIC9K-RCOL(III)-pPICZA-PH4, which is screened by bleomycin. The linearization result of the pPICZA-PH4 plasmid is shown in FIG. 2B. Figure 1

[0059] The hydroxylated gene of the application is synthesized, and has a nucleotide sequence as shown in SEQ ID NO. 5. EcoR I and Kpn I recognition sites and signal peptide recognition sites are added at the 5' end and 3' end respectively during synthesis, and the linearized by restriction enzyme Sac I, and then cloned into the expression vector pPICZA to obtain the vector pPICZA-PH4. The above-mentioned Pichia pastoris GS115-pPIC9K-RCOL(III) is used as the expression host strain, and the obtained pPICZA-PH4 cloning plasmid is linearized by electroporation and then transformed into the recombinant strain GS115-pPIC9K-RCOL(III) to obtain the GS115-pPIC9K-RCOL(III)-pPICZA-PH4, which is screened by bleomycin. The linearization result of the pPICZA-PH4 plasmid is shown in FIG. 2B. Figure 1

[0060] ​​Screening of positive transformants: In the G418 gradient method to select high copy positive clones, some transformants were selected with a marker pen, a small amount of bacteria of the selected transformants were picked and smeared on the bottom of a PCR tube, then 50 μL of enzyme-free water was added and mixed by blowing; first heated in a microwave oven for 10 min, then immediately frozen in a -80°C refrigerator for 10 min, repeated 5 times; centrifuged at 12000 rpm for 1 min, and 3 μL of supernatant was taken as a template for PCR reaction; GS115-pPIC9K-RCOL(III)-pPICZA-PH4 was screened with zeo + The method is the same as above. The agarose gel electrophoresis diagram of GS115-pPIC9K-RCOL(III) and GS115-pPIC9K-RCOL(III)-pPICZA-PH4 is shown in Figure 2 and Figure 3 .

[0061] The above obtained Pichia pastoris genetically engineered bacteria were inoculated in YPD culture medium and cultured to OD600 of 19.88, then inoculated into an initial volume of 5 L NBS 415 fermenter at a volume of 10% of the inoculation amount, the culture temperature was 30°C, the pH was 5.5, and the dissolved oxygen was controlled at 20%; after the glycerol was consumed, glycerol feeding culture was started, and when the wet weight of the bacteria reached more than 190 g / L, methanol was added, and the induction culture was carried out at a methanol flow rate of 80 mL / h, the induction stage temperature was 28°C, and the pH was 5.0; after 48 h of induction, the tank was discharged, and the supernatant was collected by centrifugation.

[0062] Example 2: Purification of recombinant hydroxylated human type III collagen

[0063] 1. The centrifugally collected supernatant was ultrafiltrated to 50% of the initial volume, then 5 times the volume of pure water was added, and the supernatant was concentrated to 5% of the initial volume by ultrafiltration.

[0064] 2. The concentrated supernatant was added to saturated ammonium sulfate, the amount of which was 60% of the volume of the concentrated supernatant, stirred at room temperature for 30 min, and then the precipitate was collected by centrifugation at 9000 rpm for 10 min; the obtained precipitate was dissolved in 500 mL of 0.05 M PBS with pH of 7.0 and filtered through a 0.22 μm filter membrane.

[0065] 3. Prepare the equilibrium buffer according to the isoelectric point of the protein: 20 mmol / L sodium phosphate buffer, pH 6.0, noted as A; and eluent prepared by mixing 20 mmol / L sodium phosphate and 1.0 mol / L NaCl, pH 6.0, noted as B. Prepare the sample solution by diluting the PBS protein solution obtained in the previous step with A at a ratio of 10:1, and then filter and load the sample solution into a 25 mL CM-Sepharose cation exchange chromatography column, which has been equilibrated with the equilibrium buffer. After loading the sample, first wash the column with A for 2 column volumes, and then perform gradient elution with 70% A and 30% B at a flow rate of 2 mL / min. Collect the eluted fractions and test them by SDS-PAGE.

[0066] 4. According to the molecular weight distribution of the protein obtained after ion exchange chromatography, select a Sephadex 200 gel column to further purify the target protein. AKTA operation: first wash to the baseline with the equilibrium buffer, i.e., 0.01 mol / L PBS and 0.05 mol / L NaCl, and then load the recombinant GS115-pPIC9K-RCOL(III)-pPICZA-PH4 protein fraction obtained after ion exchange column elution in the previous step into a gel filtration chromatography column packed with Superdex 200, and elute with the eluent at a flow rate of 10 mL / min and a UV detection wavelength of 215 nm. Finally, collect the target protein fraction after detecting by SDS-PAGE electrophoresis.

[0067] 5. Ultrafiltration desalting; G25 desalting column desalting, i.e., use 25 mL of G25 packing, and the operation process is similar to the gel filtration chromatography step. Load 6.5 mL each time, and collect about 8 mL. Desalting can be completed after 10 min of loading.

[0068] 6. Concentrate to 30% of the initial volume by ultrafiltration, and then pre-freeze in a -20°C refrigerator for 4 h, and then transfer to a vacuum freeze dryer for freeze-drying. After 48 h, collect the freeze-dried protein, and store the freeze-dried protein sample in a -80°C refrigerator for later use.

[0069] Example 3: Application of recombinant hydroxylated human type III collagen in preparing a cell adhesion promoter

[0070] 1. Cell adhesion experiment

[0071] Two 48-well plates were coated with 300 μL of 0.2 mg / mL bovine serum albumin (BSA), native human type III collagen, recombinant human type III collagen and recombinant hydroxylated human type III collagen solutions, respectively, at 4°C overnight, blocked with 1% heat denatured BSA for 1 h at room temperature, and washed twice with PBS. L929 cells in logarithmic growth phase were diluted and counted, and finally a cell suspension with a cell density of 2.0 x 105cells / mL was inoculated in the 48-well plates coated with different proteins, and the two plates were incubated in a CO2incubator for 4 h and 24 h, respectively. 5 cell / mL of cell suspension with a cell density of 2.0 x 105cells / mL was inoculated in the 48-well plates coated with different proteins, and the two plates were incubated in a CO2incubator for 4 h and 24 h, respectively.

[0072] After 4 h of incubation, the culture supernatant was discarded, the L929 cells were washed three times with PBS, 1% SDS solution was added to the plates, and the OD value was measured at 590 nm. The control group was the total number of cells without PBS washing. The cell adhesion force was calculated using the formula:

[0073] Cell adhesion rate = [(remaining cell OD590) / (Control cell OD590)]

[0074] After 24 h of incubation, the L929 cells were washed three times with PBS, the remaining cells were fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet for 20 min, after which the cell morphology was observed under an optical microscope and analyzed.

[0075] The results of the relative adhesion rate of the cells were calculated according to the absorbance values, with BSA set at 100%, the cell adhesion map is shown in Figure 4 , and the cell adhesion rate statistics are shown in Table 1

[0076] 2. Cell proliferation-promoting experiment

[0077] L929 cells in logarithmic growth phase were inoculated in 96-well tissue culture plates and incubated in a 37°C, 5% CO2incubator for 1 d. The old culture medium was discarded, and BSA, native human type III collagen, recombinant human type III collagen and recombinant hydroxylated human type III collagen were sequentially dissolved in DMEM medium at a concentration of 0.2 mg / ml, filtered and sterilized and added to the above 96-well plates. After 24 h of incubation, all the incubated L929 cells were stained with AO / EB, and their growth after incubation in collagen medium was observed under a fluorescence microscope, and the AO / EB staining map is shown in Figure 5 .

[0078] BSA native type III collagen recombinant type III collagen recombinant hydroxylated type III collagen adhesion rate 100％ 109％ 126％ 136％

[0079] The results show that the type III recombinant hydroxylated collagen can obviously promote L929 cell adhesion and is higher than natural collagen and BSA, has good cell compatibility and shows better promotion, which is consistent with the light microscope observation result.

[0080] The technical features of the above-described embodiments can be combined arbitrarily, and to make the description simple, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, it should be considered that it is within the scope of the present disclosure.

[0081] The above-described embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application.

Claims

1. A recombinant human collagen type III having cell adhesiveness, characterized by comprising a sequence represented by SEQ ID NO:

1. The amino acid sequence is shown as SEQ ID NO.

1.

2. A gene encoding the recombinant human collagen type III according to claim 1, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

3. The recombinant human collagen type III according to claim 1, wherein, The recombinant human collagen type III is used for preparing a cell adhesion promoter.

4. An expression vector, characterized by, The nucleotide sequence is shown as SEQ ID NO.

2.

5. A host cell, characterized in that, The expression vector is shown as SEQ ID NO.

4.

6. The host cell of claim 5, wherein, The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

7. A method for producing recombinant human collagen type III using the host cell of claim 5, characterized by, The host cell is cultured in a culture medium, and after the expression of the recombinant human collagen type III is induced, the recombinant human collagen type III is obtained through purification.

8. The method of claim 7, wherein, The purification method is any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.

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