Recombinant hydroxylated human III-type collagen with cell adhesion as well as preparation method and application of recombinant hydroxylated human III-type collagen

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant hydroxylated human III-type collagen as well as a preparation method and application thereof. The invention relates to a recombinant hydroxylated human III-type collagen with cell adhesion. The amino acid sequence of the recombinant hydroxylated human III-type collagen is shown as SEQ ID NO. 1. Nine different sites of the full-length natural III-type collagen are replaced, four natural integrin sites with low adhesion are replaced under the condition that the total length of natural full-length amino acid is not changed, and meanwhile, terminal peptide is reserved, so that the collagen has the potential of forming a triple helix structure. According to the modification mode, the adhesion of the collagen is improved by 27% or above, the molecular weight of the collagen is moderate, the collagen is easy to prepare, and hydroxylation enables the collagen to be expressed more stably.

Description

Technical Field

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

[0002] Collagen is one of the most important and abundant proteins in mammals. It is a structural protein found in human skin, connective tissue, bones 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 composed of three peptide chains curled to the right and twisted into a triple helix. Primary structure analysis shows that the long segment sequence of its polypeptide chain is composed of repeated Gly-XY amino acid sequences. Among them, X is usually proline, and Y is usually hydroxyproline and hydroxylysine. The latter two amino acids are rarely found in other proteins.

[0003] The adhesion is poor due to the presence of four natural integrin sites with low adhesion on the natural collagen sequence. Summary of the invention

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

[0005] The technical solution adopted by the present invention 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 having a hydroxylation function and enhancing the adhesion of the protein encoded by SEQ ID NO.1, wherein the amino acid sequence is shown in SEQ ID NO.4 and the nucleotide sequence 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 comprises the nucleotide sequence shown as SEQ ID NO.2.

[0009] An expression vector comprises the nucleotide sequence shown as SEQ ID NO.5.

[0010] A host cell comprising any of the expression vectors described above.

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

[0012] A method for preparing recombinant human type III collagen using the host cell comprises culturing the host cell in a culture medium, inducing the expression of the recombinant human type III collagen, and then purifying the recombinant human type III collagen to obtain 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 present invention has the following beneficial effects: by replacing 9 different sites of full-length natural type III collagen, the present invention replaces 4 natural integrin sites with low adhesion without changing the total length of the natural full-length amino acids, while retaining the terminal peptide to enable it to have the potential to form a triple helix structure. This modification method improves the adhesion of collagen by more than 27%, and its molecular weight is moderate, easy to prepare, and hydroxylation makes its expression more stable

[0015] The recombinant collagen of the present invention does not remove the full-length chain sequence of the C-terminus and the N-terminus of the collagen coding region, and has the ability to form a triple helix structure in combination with hydroxylation.

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

[0017] The recombinant collagen prepared by the method of the present invention can effectively carry out immune response, inflammatory response, blood coagulation, tumor metastasis and wound healing.

[0018] The method for preparing recombinant human hydroxylated type III collagen of the present invention is suitable for industrial large-scale production, and the prepared product has no infection source of animal origin, and thus has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are restriction enzyme electrophoresis diagrams, A: restriction enzyme electrophoresis diagram of target gene plasmid; B: restriction enzyme electrophoresis diagram of hydroxylation gene plasmid.

[0020] Figure 2 It is the plasmid transformant containing the target gene by colony PCR.

[0021] Figure 3 This is a plasmid transformant containing the hydroxylation gene by colony PCR.

[0022] Figure 4 This is the result of crystal violet staining, from left to right: BSA, type III natural collagen, type III recombinant collagen, and type III recombinant hydroxylated collagen.

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

[0024] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0025] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0026] The inventive concept of the present invention is as follows: by replacing 9 different sites of full-length natural type III collagen, its function is enhanced without changing the total length of the natural full-length amino acids, and 4 natural integrin sites with low adhesion are replaced, while retaining the terminal peptide so that it has 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 the synthesis process. Mammalian cells with prolyl 4-hydroxylase can directly produce hydroxylated collagen through post-translational modification.

[0027] The purpose of the present invention 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, coagulation, tumor metastasis and wound healing. It can be widely used in the fields of food, health products, biomedicine, etc.

[0028] The present invention provides a type III recombinant collagen with cell adhesion, the amino acid sequence of which is shown as SEQ ID NO.1.

[0029] SEQ ID NO.1:

[0030]

[0031] The present invention provides a type III recombinant collagen with cell adhesion, wherein codon optimization is performed for host cell expression, and signal peptide cleavage sites and EcoR I and Not I restriction sites are added to both ends during the design process to facilitate later gene manipulation. After the above optimization, the nucleotide sequence is shown in SEQ ID NO.2.

[0032] SEQ ID NO.2:

[0033]

[0034] The present invention provides an expression vector comprising the above nucleic acid molecule. The expression vector may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host to be introduced into the vector, for example, bacteria, fungi, plants or animals, as appropriate and taking into account whether the vector is based on DNA or RNA. In a specific embodiment, the expression vector is pPIC9k, and its nucleotide sequence is shown in SEQ ID NO.3.

[0035] SEQ ID NO.3:

[0036]

[0037] The present invention provides a sequence having a hydroxylation function and enhancing the adhesion of collagen, and the amino acid sequence thereof is shown in SEQ ID NO.4.

[0038] SEQ ID NO.4:

[0039] KYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYHHYDGDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGTSIFFWVADPVTRKLYKETLHAGLPVKSGEKIIANQWIRAVK.

[0040] The present invention provides a sequence with hydroxylation function and enhanced collagen adhesion, codon optimization is performed for host cell expression, and signal peptide cleavage sites and EcoR I and Kpn I restriction sites are added at both ends during the design process to facilitate later gene manipulation. After the above optimization, its nucleotide sequence is shown as SEQ ID NO.5.

[0041] SEQ ID NO.5:

[0042] .

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

[0044] SEQ ID NO.6:

[0045]

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

[0047] The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to parent cells in nucleic acid content, but may 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 invention provides a method for preparing any of the aforementioned recombinant collagen, comprising the following steps:

[0051] The above host cells are used for expression and then isolated and purified to obtain the protein; the host cells for expression refer to culturing the host cells, 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. After obtaining the genetically engineered Pichia pastoris, the specific culture conditions are as follows: the genetically engineered Pichia pastoris is inoculated into a YPD medium and cultured at 30°C and 220rpm for 22h to 24h until the OD600 is 18 to 20 as the seed solution for the upper tank. After the seed solution is expanded, it is inoculated into an NBS 415 fermenter with an initial volume of 5L at a volumetric inoculation amount of 10%. The culture temperature is 28°C to 30°C, the pH is 5.0 to 6.0, and the dissolved oxygen is controlled at 20% to 30%. When the glycerol is exhausted, glycerol fed culture is started, and when the wet weight of the bacteria reaches 180g / L or more, induction culture is started.

[0053] The present invention does not impose any limitation on the expression mode, which can be confirmed as needed, for example, the expression is induced expression, and the inducer for induced expression is methanol.

[0054] In a specific embodiment, methanol is added for induction culture, the temperature during the induction stage is 28° C., the pH is 5.0, and the culture is released after 48 hours of induction.

[0055] The present application does not impose any limitation on the separation and purification method, which can be determined according to the method, for example, salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method can be used.

[0056] The present invention also provides the above-mentioned recombinant hydroxylated collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant hydroxylated collagen expressed by the above-mentioned expression vector, or the recombinant hydroxylated collagen produced by the above-mentioned host cell, which has applications in immune response, inflammatory response, coagulation, tumor metastasis and wound healing.

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

[0058] The recombinant collagen gene of the present invention, i.e., the target gene, is chemically synthesized, and the nucleotide sequence is shown in SEQ ID NO.2. During the synthesis, EcoR I and Not I recognition sites and signal peptide recognition sites are added to the 5' and 3' ends respectively, and then linearized with restriction endonuclease Sac I and cloned into the expression vector pPIC9K to obtain the pPIC9K-RCOL (III) cloning plasmid. Pichia pastoris GS115 is used as the expression host bacteria, and the obtained pPIC9K-RCOL (III) cloning plasmid is linearized by electroporation and then transformed into GS115 to obtain GS115-pPIC9K-RCOL (III). High-copy positive clones are selected by the G418 gradient method, and the Pichia pastoris genetically engineered bacteria are obtained by culturing at 30°C for 72 hours. The linearization results of the pPIC9K-RCOL (III) cloning plasmid are shown as follows: Figure 1 As shown in A.

[0059] The hydroxylation gene of the present invention was chemically synthesized, and the nucleotide sequence was shown in SEQ ID NO.5. During the synthesis, EcoR I and Kpn I recognition sites and signal peptide recognition sites were added to the 5' and 3' ends respectively, and the residues were linearized with restriction endonuclease Sac I and cloned into the expression vector pPICZA to obtain the vector pPICZA-PH4. The above-mentioned Pichia pastoris GS115-pPIC9K-RCOL (III) was used as the expression host bacteria, and the obtained pPICZA-PH4 clone plasmid was linearized by electroporation and transformed into the recombinant strain GS115-pPIC9K-RCOL (III) to obtain GS115-pPIC9K-RCOL (III)-pPICZA-PH4, which was screened with bleomycin. The linearization results of pPICZA-PH4 plasmid are shown in Figure 5. Figure 1 As shown in B.

[0060] Screening of positive transformants: In the selection of high-copy positive clones by the G418 gradient method, select some transformants with a marker pen, pick a small amount of selected transformants, smear them on the bottom of the PCR tube, then add 50 μL of enzyme-free water and mix them by pipetting; first heat at medium-high temperature in a microwave oven for 10 minutes, then immediately put it in a -80℃ refrigerator and freeze it for 10 minutes, repeat 5 times; centrifuge at 12000rpm for 1 minute, take 3 μL of supernatant as a template for PCR reaction; GS115-pPIC9K-RCOL(III)-pPICZA-PH4 was used as zeo + The screening method is the same as above. The agarose gel electrophoresis images of GS115-pPIC9K-RCOL(III) and GS115-pPIC9K-RCOL(III)-pPICZA-PH4 are shown in Figure 2 and Figure 3 shown.

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

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

[0063] 1. When the supernatant collected by centrifugation is ultrafiltered to 50% of the initial volume, 5 times the volume of pure water is added and then ultrafiltered to concentrate to 5% of the initial volume.

[0064] 2. Add saturated ammonium sulfate to the concentrated supernatant in an amount of 60% of the volume of the concentrated supernatant, stir at room temperature for 30 minutes, centrifuge at 9000 rpm for 10 minutes, collect the precipitate, dissolve the precipitate in 500 mL of 0.05 M PBS, pH 7.0, and filter through a 0.22 μm filter membrane.

[0065] 3. Prepare the equilibrium buffer according to the isoelectric point of the protein: 20mmol / L sodium phosphate buffer at pH 6.0, recorded as A solution; prepare the eluent at pH 6.0 by mixing 20mmol / L sodium phosphate and 1.0mol / L NaCl, recorded as B solution. Dilute the PBS protein solution obtained in the previous step with A solution at a ratio of 10:1 to prepare the loading solution, and then load it on a 25mL CM-Sepharose cation exchange chromatography column after filtration. Equilibrate the column with equilibrium buffer before loading. After loading, rinse 2 column volumes with A solution, then use 70% A solution and 30% B solution for gradient elution at a flow rate of 2mL / min. Collect the eluted components and detect them by SDS-PAGE.

[0066] 4. According to the distribution range of protein molecular weight obtained after ion exchange chromatography, select Sephadex 200 gel column to further purify the target protein. AKTA operation process: first use equilibrium buffer, i.e. 0.01mol / L PBS, 0.05mol / L NaCl, to rinse until the baseline is stable, and then load the recombinant GS115-pPIC9K-RCOL(III)-pPICZA-PH4 protein component eluted from the ion exchange column obtained in the previous step onto a gel filtration chromatography column filled with Superdex 200, eluted with eluent, the flow rate is set to 10mL / min, and the ultraviolet detection wavelength is 215nm. Finally, collect the outflowing target protein component after SDS-PAGE electrophoresis detection.

[0067] 5. Ultrafiltration desalting: G25 desalting column desalting, that is, using 25mL of G25 filler, the operation process is similar to the gel filtration chromatography step, each time 6.5mL is loaded, and about 8mL is collected. Desalting can be completed 10 minutes after loading.

[0068] 6. Concentrate to 30% of the initial volume by ultrafiltration, then pre-freeze in a -20°C refrigerator for 4 hours, then transfer to a vacuum freeze dryer for freeze-drying, collect the freeze-dried protein after 48 hours, 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 the preparation of cell adhesion promoter

[0070] 1. Cell Adhesion Assay

[0071] Two 48-well plates were coated with 300 μL of 0.2 mg / mL bovine serum albumin BSA, natural human type III collagen, recombinant human type III collagen, and recombinant hydroxylated human type III collagen solution, respectively, at 4°C overnight, blocked with 1% heat-denatured BSA at room temperature for 1 h, and then washed twice with PBS. L929 cells in the logarithmic growth phase were diluted and counted, and finally the cell density was 2.0 × 10 5 The cell suspension of 100 cell / mL was inoculated on a 48-well culture plate coated with different proteins, and the two well plates were cultured in a CO2 biochemical incubator for 4 h and 24 h, respectively.

[0072] After incubation for 4 hours, the culture supernatant was discarded, the L929 cells were washed three times with PBS, 1% SDS solution was added to the well plate, and its OD value was measured at 590nm. The control group was the total number of cells not washed with PBS. The cell adhesion force was calculated using the formula:

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

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

[0075] The relative adhesion rate of cells was calculated based on the absorbance value, and BSA was set as 100%. The cell adhesion graph is shown in Figure 4 The cell adhesion rate statistics are shown in Table 1

[0076] 2. Cell proliferation assay

[0077] L929 cells in the logarithmic growth phase were seeded on a 96-well tissue culture plate and cultured for 1 day at 37°C in a biochemical incubator with a volume fraction of 5% CO2. The old culture medium was discarded, and BSA, natural human type III collagen, recombinant human type III collagen, and recombinant hydroxylated human type III collagen were dissolved in DMEM culture medium at a concentration of 0.2 mg / ml, filtered and sterilized, and added to the above 96-well plate. After culturing for 24 hours, all incubated L929 cells were stained with AO / EB, and their growth after incubation in collagen culture medium was observed under a fluorescence microscope. The AO / EB staining picture is shown in Figure 5 .

[0078] BSA Natural 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 of the present invention can significantly promote the adhesion of L929 cells to the wall, and its adhesion promoting effect is higher than that of natural collagen and significantly higher than that of BSA. It has good cell compatibility and shows a good promoting effect, which is consistent with the results of light microscopy observation.

[0080] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0081] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A recombinant human type III collagen with cell adhesion, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

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

2.

3. A sequence having a hydroxylation function and enhancing the adhesion of the recombinant human type III collagen as claimed in claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.

5.

4. The use of recombinant human type III collagen according to claim 1, characterized in that: The recombinant human type III collagen is used for preparing a cell adhesion promoter.

5. An expression vector, characterized in that: Comprising the nucleotide sequence as claimed in claim 2.

6. An expression vector, characterized in that: Comprising the nucleotide sequence as claimed in claim 3.

7. A host cell, characterized in that Comprising the expression vector according to any one of claim 5 or claim 6.

8. The host cell according to claim 7, characterized in that The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

9. A method for preparing recombinant human type III collagen using the host cell according to claim 7, characterized in that: The host cells are cultured in a culture medium, and the expression of recombinant human type III collagen is induced and then purified to obtain the recombinant human type III collagen.

10. The method according to claim 9, characterized in that The purification method is selected from any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.

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