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

Abstract

The invention discloses a preparation method of recombinant III-type collagen, which comprises the following steps: adding cation exchange chromatography filler into recombinant III-type collagen fermentation liquor, centrifuging, collecting filtrate, purifying through a cation exchange chromatography column, and finally concentrating to obtain the III-type recombinant collagen. The invention also provides the recombinant III-type collagen obtained by the preparation method and application thereof. The recombinant III-type collagen, the preparation method and the application thereof solve the problem that the protein stability is influenced in the purification process of the existing III-type collagen production method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical materials, and specifically relates to a method for preparing recombinant type III collagen, and also relates to the recombinant type III collagen obtained by the preparation method and its application. Background Art

[0002] Collagen is the most abundant protein in mammals. It is a structural protein and is widely found in connective tissue. At least 28 types of collagen have been discovered, of which types I, II, and III are the most common and abundant. Type III collagen is primarily found in infant skin, blood vessel walls, and internal organs. Its fine fibrils impart elasticity and flexibility to tissues, making it a highly sought-after material in the medical aesthetics field, biomedical materials, and drug delivery systems, as well as in traditional cosmetics.

[0003] The traditional method of producing collagen is to use acid and alkali methods to treat animal tissue. Collagen from this source is pure and has a series of safety risks, and it is difficult to isolate high-purity type III collagen. To address these problems, some research institutions and biotechnology companies at home and abroad have successively invested in the research and development of recombinant collagen. Chinese patent CN108070032B (application date: 2018.01.23) discloses a method for purifying recombinant human collagen, and Chinese patent CN119775386A (application date: 2024.11.01) discloses a method for isolating and purifying recombinant human collagen. These two methods use Pichia pastoris as the genetically engineered bacteria for recombinant collagen to obtain recombinant human collagen. The collagen produced by recombinant technology is not only high in purity, good in water solubility, and high in biological activity, but also has better performance than traditional collagen.

[0004] While all of the above methods have been commercialized, the actual production of recombinant collagen remains primarily challenging due to production costs and quality control challenges. Escherichia coli requires cell collection and cell disruption, followed by protein refolding and endotoxin control. The isolation and purification of recombinant human collagen from Pichia pastoris involves cumbersome pigment removal and purification steps. The initial stages of the process require complex solid-liquid separation and ultrafiltration desalination, which can negatively impact protein stability. Summary of the Invention

[0005] The first purpose of the present invention is to provide a method for preparing recombinant type III collagen, which solves the problem that the purification process of existing type III collagen production methods affects protein stability.

[0006] The second object of the present invention is to provide recombinant type III collagen obtained by the above method.

[0007] The third object of the present invention is to provide the use of the recombinant type III collagen obtained by the above method in the field of medicine or cosmetics.

[0008] The first technical solution adopted by the present invention is: a method for preparing recombinant type III collagen, which includes adding a cation exchange chromatography filler to the recombinant type III collagen fermentation broth, collecting the filtrate after centrifugation, purifying it through a cation exchange chromatography column, and finally concentrating it to obtain the recombinant type III collagen.

[0009] The first technical solution adopted by the present invention is also characterized in that: Furthermore, the method for preparing recombinant type III collagen is specifically implemented according to the following steps: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted, the pH value and conductivity were adjusted, a cation exchange chromatography filler was added to the fermentation broth, and the mixture was stirred slowly to allow the target protein to fully bind to the chromatography filler; The mixture was washed with buffer A and centrifuged until the filtrate was basically clear. The solid filler was collected and eluent B was added. After stirring and mixing thoroughly, the mixture was centrifuged and the filtrate was collected to obtain the desired captured recombinant type III collagen. Step 2, cation exchange chromatography: First, balance the cation exchange chromatography column with equilibrium solution C, then add citric acid to the filtrate collected in step 1 to adjust the pH and then pass it through the cation exchange chromatography column for adsorption. Finally, balance solution C and buffer D are mixed in proportion and the target protein is eluted. The eluate is recombinant type III collagen.

[0010] Step 3: Ultrafiltration and concentration: The sample collected in step 2 was concentrated using an ultrafiltration membrane and the liquid was replaced; type III recombinant collagen with a purity greater than 98% was obtained.

[0011] Furthermore, in step 1, the pH is adjusted to 4.0-5.5, and the conductivity is adjusted to no more than 5.0 ms / cm; and the stirring time is 20-40 min.

[0012] Furthermore, in step 1, buffer A is a citric acid solution, and eluent B is a phosphate buffer solution.

[0013] Furthermore, the cation exchange chromatography filler in step 1 is specifically an agarose filler with a particle size of not less than 60 μm and a pore size of not less than 30 nm, and the amount of the cation exchange chromatography filler added to the fermentation broth is: 100 g per 3-6 L of fermentation broth.

[0014] Furthermore, the filler of the cation exchange chromatography column in step 2 is SP sepharose Fast Flow; the equilibration solution C in step 2 is citric acid plus a low concentration NaCl solution; the eluent D is citric acid plus a high concentration NaCl solution; the elution method for protein elution in step 2 is constant flow gradient elution, the pH value is adjusted to 4.0-5.5, and a mixture of equilibration solution C and buffer D is used to elute the target protein, and the ratio of the concentration of equilibration solution C to the concentration of buffer D is 4:1-9:1.

[0015] Furthermore, the ultrafiltration membrane in step 3 is specifically a 10kd hollow fiber membrane.

[0016] The second technical solution adopted by the present invention is: the nucleotide sequence of the above-mentioned recombinant type III collagen is shown as SEQ ID NO.1.

[0017] The second technical solution adopted by the present invention is also characterized in that the amino acid sequence of the recombinant type III collagen is shown as SEQ ID NO.2.

[0018] The third technical solution adopted by the present invention is: application of the recombinant type III collagen obtained by the above-mentioned recombinant type III collagen preparation method in the field of medicine or cosmetics.

[0019] The beneficial effects of the present invention are: This method uses fermentation broth to directly absorb and capture the target protein, replacing the traditional crude and pure capture method of solid-liquid separation, sterile filtration, and ultrafiltration concentration. This method saves process steps, reduces time and cost, and reduces equipment requirements. It is simple to operate. Combined with a one-step ion exchange chromatography method, it achieves a purity of over 98% for recombinant type III collagen and a yield of over 80%. This method is an ideal large-scale method for producing recombinant type III collagen. It can also be used for the separation and purification of other recombinant proteins.

[0020] The present invention selects two segments of fully human type III collagen sequence for splicing, thereby ensuring the function of type III collagen and being applicable to the preparation and production of cosmetics and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is the SDS-PAGE result of the purification process of the method of the present invention.

[0022] Figure 2 This is the purity analysis result of type III collagen after purification by the method of the present invention. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and examples.

[0024] The present invention provides a method for preparing recombinant type III collagen and its application, which mainly comprises the following steps: (1) Target protein capture Dilute the fermentation broth of recombinant type III collagen expressed in Pichia pastoris with purified water, adjust the pH to 4.0-5.5, and adjust the conductivity to no greater than 5.0 ms / cm. Add an appropriate amount of cation exchange chromatography medium to the fermentation broth and gently stir for 30 minutes to allow the target protein to fully bind to the chromatography medium.

[0025] The mixture is placed in a sedimentation centrifuge, the centrifuge is pre-laid with filter cloth, the centrifugation is started, and no less than 5CV volume of equilibration buffer A is introduced into the centrifuge feed port for cleaning until the centrifugal filtrate is basically clear. The filler retained by the filter cloth is collected and placed in a stainless steel container, 3CV of eluent B is added and mixed thoroughly for 5 minutes, the mixture is placed in a centrifuge for centrifugation, and the filtrate is collected, which is the recombinant type III collagen to be captured.

[0026] The mesh size of the filter cloth used for centrifugation in step (1) should be between 100 and 550 meshes.

[0027] In step (1), the cation exchange chromatography filler has a particle size of not less than 90 μm and a pore size of not less than 100 nm, which is an ultra-large pore agarose filler.

[0028] In step (1), buffer A should be a 10-50 mM citric acid solution with a pH of 4.0-5.5; and eluent B should be a 5-20 mM phosphate buffer solution with a pH of 6.0-8.5.

[0029] (2) Cation exchange chromatography The filtrate collected in step (1) is added with 10 mM to 50 mM citric acid, the pH is adjusted to 4.5 to 6.5, and then purified by a cation exchange chromatography column.

[0030] First, the cation exchange chromatography column is equilibrated with equilibration solution C. Then, the filtrate collected in step 1 is added with citric acid to adjust the pH and then passed through the cation exchange chromatography column for adsorption. Finally, the target protein is eluted with a mixture of equilibration solution C and buffer D in a certain proportion. The eluate is the recombinant type III collagen.

[0031] The filler of the cation exchange chromatography column in step (2) is SP sepharose High Performance.

[0032] In step (2), the equilibration solution C is a 4.0-6.0 μg 10-50 mM citric acid buffer solution plus a 20 mM NaCl buffer solution. The eluent D is a 4.0-6.0 μg 10-50 mM citric acid buffer solution plus a 0.5-1.5 M NaCl buffer solution.

[0033] In step (2), the protein solution collected in step (1) is added with 10-50 mM citric acid to adjust the conductivity to 2.5-5.8 mS / cm and the pH to 4.0-6.0, and then separated and purified by a strong cation exchange chromatography column.

[0034] In step (2), the elution method for eluting the target protein is constant flow gradient elution.

[0035] In step (2), the ratio of the concentration of the target protein elution gradient equilibrium solution C to the concentration of the elution solution D is 4:1 to 9:1.

[0036] (3) Ultrafiltration concentration The sample collected in step (2) was concentrated and replaced with a 10 kd hollow fiber membrane; type III recombinant collagen with a purity greater than 98% was obtained, and its SDS-PAGE results were as follows: Figure 1 As shown, the purity analysis test results are as follows Figure 2 shown.

[0037] The present invention also provides recombinant type III collagen obtained by the above preparation method, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0038] The amino acid sequence of the recombinant type III collagen of the present invention is shown in SEQ ID NO. 2, which contains 374 amino acids in total and has a theoretical molecular weight of 34 kd.

[0039] The present invention also provides the use of the recombinant type III collagen obtained by the above preparation method in the field of medicine or cosmetics.

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Example 1 1. Preparation of Recombinant Type III Collagen in Pichia pastoris (1) Preparation of expression strain Codon optimization of the yeast expression system was performed according to the amino acid sequence shown in SEQ ID No: 2 to obtain a type III target gene sequence as shown in SEQ ID No: 1. The obtained target gene was synthesized, and the synthesized gene was ligated into the PPIc9K plasmid. After linearization, the gene was transformed into a Pichia pastoris competent cell, and transformants were screened to obtain a yeast expression strain.

[0042] (2) Protein induced expression A single colony of the yeast expression strain was picked and added to 200 ml of YPD liquid culture, and activated at 30°C and 200 rpm; the recombinant human collagen engineering bacteria seed liquid was inoculated into 1 L of BMGY liquid culture medium at a volume ratio of 1:10, and cultured at 30°C and 200 r / min for 48 hours; finally, the recombinant human collagen engineering bacteria activation liquid was inoculated into 50 L of BMGY liquid culture medium at a volume ratio of 1:25. During this process, methanol was continuously added to induce fermentation, and the dissolved oxygen was controlled to be 30%, the pH was 5.0, and the temperature was 30°C to induce fermentation for 68 hours.

[0043] 2. Isolation of Recombinant Type III Collagen Buffer A: pH 4.4 20 mM citric acid solution.

[0044] Eluent B: pH 7.5 10mM phosphate buffer solution.

[0045] Balanced solution C: pH 4.4 10nM citric acid + 20mM NaCl buffer solution.

[0046] Eluent D: pH 4.4 10 mM citric acid + 1.0 M NaCl buffer solution.

[0047] (1) Target protein capture Dilute 30 L of recombinant type III collagen fermentation broth expressed in Pichia pastoris with purified water until the conductivity reaches 4.0 ms / cm and the pH is adjusted to 4.4. Add 500 g of cation exchange chromatography medium with a pore size greater than 100 nm and a particle size greater than 120 μm to the fermentation broth and gently stir for 30 minutes to allow the target protein to fully bind to the chromatography medium.

[0048] The mixture was placed in a sedimentation centrifuge, which was pre-laid with a 150-mesh filter cloth. The centrifuge was started, and 7CV of gel volume equilibration buffer A was introduced into the centrifuge feed port for cleaning until the centrifugal filtrate was basically clear. The filler retained by the filter cloth was collected and placed in a stainless steel container. 3CV of eluent B was added and mixed thoroughly for 5 minutes. The mixture was centrifuged in a centrifuge, and the filtrate was collected, which was the captured recombinant type III collagen.

[0049] (2) Cation exchange chromatography The filtrate collected from the target protein capture in step (1) was added with 50 nM citric acid and the pH was adjusted to 4.4 and the conductivity to 4.5 ms / cm.

[0050] Equilibrate a high-resolution, strong cationic composite chromatography column packed with SP sepharose High Performance chromatographic medium for 5 column volumes using equilibration buffer C at a flow rate of 200 mL / min and a residence time of 5 minutes. Load the protein filtrate, after adjusting the pH and conductivity, onto the column. Equilibrate the column with buffer A for 5 column volumes to essentially reset the chromatographic baseline. Elute the protein for 5 column volumes using a equilibration buffer C:elution buffer D ratio of 17:3. Collect the eluate, which is the target protein.

[0051] (3) Ultrafiltration concentration The target protein sample collected by strong cation exchange chromatography was transferred to a 10kd hollow fiber membrane device for ultrafiltration concentration and desalted with a pure chemical until the conductivity reached 0.8ms / cm. The resulting recombinant type III collagen sample was tested by gel chromatography and had a purity of 99.05%. The total yield of the purification process was 82%, and its endotoxin content was detected to be 0.38EU / ml.

[0052] 3. Efficacy Determination of Recombinant Type III Collagen (1) Promoting cell migration After thawing, stored HSF cells were cultured in complete DMEM at 37°C, 5% CO2, and 95% relative humidity. HSF cells in the logarithmic growth phase were seeded into 6-well plates at a density of 6 × 105 cells / well and incubated overnight in an incubator before being wound. After wounding, the cells were washed three times with PBS and administered with 2 mL of 0.5 mg / mL recombinant type III collagen solution per well. The control group (SC) received 2 mL of serum-free medium. Culture was continued for 24 hours, and the wounds were photographed. The SC migration rate was normalized, and the relative migration rate of recombinant type III collagen was calculated as follows: Table 1 Relative mobility of recombinant type III collagen

[0053] From the results in Table 1 above, it can be seen that recombinant type III collagen has a significant effect in promoting cell migration.

[0054] (2) Promote the expression of other types of collagen genes Using real-time fluorescence quantitative PCR, logarithmically growing HSF cells were seeded into 6-well plates at a density of 6×105 cells / well and incubated in an incubator for 24 hours. 2 mL of 0.5 mg / mL recombinant type III collagen was then added to each well. The plates were then incubated for an additional 24 hours. After incubation, the plates were washed three times with PBS, and 1 mL of RNAiso Plus was added to each well. After pipetting and lysis, the cells were lysed. The cell lysate was collected for RNA extraction, reverse transcription, and quantitative fluorescence PCR analysis of gene expression, and the results were calculated.

[0055] Table 2 Recombinant type III collagen promotes the expression of other collagen genes

[0056] As shown in Table 2 above, recombinant type III collagen can increase the gene expression levels of collagen IV and collagen VII by 131.2% and 164.6%, respectively. This significantly increases the gene expression of these two collagens.

[0057] Example 2 Buffer A: pH 4.6 20 mM citric acid solution.

[0058] Eluent B: pH 7.0 10mM phosphate buffer solution.

[0059] Balanced solution C: pH 4.6 20mM acetic acid-sodium acetate solution.

[0060] Eluent D: pH 4.6 20 mM acetic acid-sodium acetate + 1.0 M NaCl buffer solution.

[0061] (1) Target protein capture Dilute 30 L of recombinant type III collagen fermentation broth expressed in Pichia pastoris with purified water until the conductivity reaches 4.0 ms / cm and the pH is adjusted to 4.6. Add 500 g of cation exchange chromatography medium with a pore size greater than 100 nm and a particle size greater than 120 μm to the fermentation broth and gently stir for 30 minutes to allow the target protein to fully bind to the chromatography medium.

[0062] The mixture was placed in a sedimentation centrifuge, which was pre-laid with a 150-mesh filter cloth. The centrifuge was started, and 7CV of gel volume equilibration buffer A was introduced into the centrifuge feed port for cleaning until the centrifugal filtrate was basically clear. The filler retained by the filter cloth was collected and placed in a stainless steel container. 3CV of eluent B was added and mixed thoroughly for 5 minutes. The mixture was centrifuged in a centrifuge, and the filtrate was collected, which was the captured recombinant type III collagen.

[0063] (2) Cation exchange chromatography The filtrate collected from the target protein capture in step (1) was added with 1 M acetic acid and the pH was adjusted to 4.6 with NaOH, and the conductivity was 2.8 ms / cm.

[0064] Equilibrate a high-resolution, strong cationic composite chromatography column packed with SP sepharose High Performance chromatographic medium for 5 column volumes using equilibration buffer C at a flow rate of 200 mL / min and a residence time of 5 minutes. Load the protein filtrate, after adjusting the pH and conductivity, onto the column. Equilibrate with buffer A for 5 column volumes to essentially reset the chromatographic baseline. Elute the protein for 5 column volumes using a 17:3 ratio of equilibration buffer C to eluent D. Collect the eluate, which is the target protein.

[0065] (3) Ultrafiltration concentration The target protein sample collected by strong cation exchange chromatography was concentrated by ultrafiltration using a 10 kD hollow fiber membrane device and desalted using a purified solution until the conductivity reached 0.8 mS / cm. The resulting recombinant type III collagen sample was tested by gel permeation chromatography and showed a purity of 98.65%. The overall yield of the purification process was 74%. The endotoxin content was 0.42 EU / ml.

[0066] Example 3 The method for preparing recombinant type III collagen comprises adding a cation exchange chromatography filler to a fermentation broth of the recombinant type III collagen, collecting the filtrate after centrifugation, purifying it through a cation exchange chromatography column, and finally concentrating it to obtain the recombinant type III collagen.

[0067] Please follow the steps below to implement: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted, the pH value and conductivity were adjusted, a cation exchange chromatography filler was added to the fermentation broth, and the mixture was stirred slowly to allow the target protein to fully bind to the chromatography filler; The mixture is placed in a decanter centrifuge with filter cloth laid in advance. The centrifuge is started and no less than 5CV of gel volume of equilibration buffer A is passed through the centrifuge feed port for cleaning until the centrifugal filtrate is basically clear. The filler retained by the filter cloth is collected and placed in a stainless steel container. 3CV of eluent B is added and thoroughly mixed for 5 minutes. The mixture is then centrifuged in a centrifuge and the filtrate is collected, which is the recombinant type III collagen to be captured. Step 2, cation exchange chromatography: The filtrate collected in step 1 was added with citric acid, the pH was adjusted and then purified by a cation exchange chromatography column; Step 3: Ultrafiltration and concentration: The sample collected in step 2 was concentrated using an ultrafiltration membrane and the liquid was replaced; type III recombinant collagen with a purity greater than 98% was obtained.

[0068] Example 4 The method for preparing recombinant type III collagen comprises adding a cation exchange chromatography filler to a fermentation broth of the recombinant type III collagen, collecting the filtrate after centrifugation, purifying it through a cation exchange chromatography column, and finally concentrating it to obtain the recombinant type III collagen.

[0069] Please follow the steps below to implement: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted and adjusted to a pH of 5.5 and a conductivity of 3.0 ms / cm. A cation exchange chromatography filler was added to the fermentation broth at a rate of 5 L / 100 g. The mixture was stirred slowly for 40 minutes to allow the target protein to fully bind to the chromatography filler. The mixture is placed in a decanter centrifuge, which is pre-laid with filter cloth. The centrifuge is started, and at least 5 CV of equilibration buffer A is introduced into the centrifuge feed port for cleaning until the centrifugal filtrate is substantially clear. The filler retained by the filter cloth is collected and placed in a stainless steel container. 3 CV of eluent B is added and thoroughly mixed for 5 minutes. The mixture is then centrifuged in a centrifuge, and the filtrate is collected, which is the recombinant type III collagen to be captured. Buffer A is a citric acid solution, and eluent B is a phosphate buffer solution. Step 2, cation exchange chromatography: The filtrate collected in step 1 was added with citric acid, the pH was adjusted and then purified by a cation exchange chromatography column; First, the cation exchange chromatography column was equilibrated with equilibration solution C. Then, the filtrate collected in step 1 was added with citric acid to adjust the pH and then passed through the cation exchange chromatography column for adsorption. Finally, the target protein was eluted with a mixture of equilibration solution C and buffer D in a ratio of 4:1. The eluate was the recombinant type III collagen.

[0070] The cation exchange chromatography column was filled with SP sepharose Fast Flow; the equilibration solution C was citric acid plus a low-concentration NaCl buffer solution; the eluent D was citric acid plus a high-concentration NaCl buffer solution; the protein was eluted using a constant-current gradient elution method; Step 3: Ultrafiltration and concentration: The sample collected in step 2 is concentrated and replaced with an ultrafiltration membrane to obtain type III recombinant collagen with a purity greater than 98%; the ultrafiltration membrane is specifically a 10kd hollow fiber membrane.

[0071] Example 5 The method for preparing recombinant type III collagen comprises adding a cation exchange chromatography filler to a fermentation broth of the recombinant type III collagen, collecting the filtrate after centrifugation, purifying it through a cation exchange chromatography column, and finally concentrating it to obtain the recombinant type III collagen.

[0072] Please follow the steps below to implement: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted and adjusted to a pH of 4.0 and a conductivity of 2.0 ms / cm. A cation exchange chromatography filler was added to the fermentation broth at a rate of 3 L / 100 g. The mixture was stirred slowly for 20 minutes to allow the target protein to fully bind to the chromatography filler. The mixture is placed in a decanter centrifuge, which is pre-laid with filter cloth. The centrifuge is started, and at least 5 CV of equilibration buffer A is introduced into the centrifuge feed port for cleaning until the centrifugal filtrate is substantially clear. The filler retained by the filter cloth is collected and placed in a stainless steel container. 3 CV of eluent B is added and thoroughly mixed for 5 minutes. The mixture is then centrifuged in a centrifuge, and the filtrate is collected, which is the recombinant type III collagen to be captured. Buffer A is a citric acid solution, and eluent B is a phosphate buffer solution. Step 2, cation exchange chromatography: The filtrate collected in step 1 was added with citric acid, the pH was adjusted and then purified by a cation exchange chromatography column; First, the cation exchange chromatography column was equilibrated with equilibration solution C. Then, the filtrate collected in step 1 was added with citric acid to adjust the pH and then passed through the cation exchange chromatography column for adsorption. Finally, the target protein was eluted with a mixture of equilibration solution C and buffer D in a ratio of 9:1. The eluate was the recombinant type III collagen.

[0073] The cation exchange chromatography column was filled with SP sepharose Fast Flow; the equilibration solution C was citric acid plus a low-concentration NaCl buffer solution; the eluent D was citric acid plus a high-concentration NaCl buffer solution; the protein was eluted using a constant-current gradient elution method; Step 3: Ultrafiltration and concentration: The sample collected in step 2 is concentrated and replaced with an ultrafiltration membrane to obtain type III recombinant collagen with a purity greater than 98%; the ultrafiltration membrane is specifically a 10kd hollow fiber membrane.

[0074] Example 6 The method for preparing recombinant type III collagen comprises adding a cation exchange chromatography filler to a fermentation broth of the recombinant type III collagen, collecting the filtrate after centrifugation, purifying it through a cation exchange chromatography column, and finally concentrating it to obtain the recombinant type III collagen.

[0075] Please follow the steps below to implement: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted and adjusted to a pH of 5.5 and a conductivity of 5.0 ms / cm. A cation exchange chromatography filler was added to the fermentation broth at a rate of 6 L / 100 g. The mixture was stirred slowly for 40 minutes to allow the target protein to fully bind to the chromatography filler. The mixture is placed in a decanter centrifuge, which is pre-laid with filter cloth. The centrifuge is started, and at least 5 CV of equilibration buffer A is introduced into the centrifuge feed port for cleaning until the centrifugal filtrate is substantially clear. The filler retained by the filter cloth is collected and placed in a stainless steel container. 3 CV of eluent B is added and thoroughly mixed for 5 minutes. The mixture is then centrifuged in a centrifuge, and the filtrate is collected, which is the recombinant type III collagen to be captured. Buffer A is a citric acid solution, and eluent B is a phosphate buffer solution. Step 2, cation exchange chromatography: The filtrate collected in step 1 was added with citric acid, the pH was adjusted and then purified by a cation exchange chromatography column; First, the cation exchange chromatography column was equilibrated with equilibrium solution C. Then, the filtrate collected in step 1 was added with citric acid to adjust the pH and then passed through the cation exchange chromatography column for adsorption. Finally, the target protein was eluted with a mixture of equilibrium solution C and buffer D in a ratio of 17:3. The eluate was the recombinant type III collagen.

[0076] The cation exchange chromatography column was filled with SP sepharose Fast Flow; the equilibration solution C was citric acid plus a low-concentration NaCl buffer solution; the eluent D was citric acid plus a high-concentration NaCl buffer solution; the protein was eluted using a constant-current gradient elution method; Step 3: Ultrafiltration and concentration: The sample collected in step 2 is concentrated and replaced with an ultrafiltration membrane to obtain type III recombinant collagen with a purity greater than 98%; the ultrafiltration membrane is specifically a 10kd hollow fiber membrane.

[0077] Comparative Example 1 This comparative example provides a method for preparing and applying recombinant type III collagen. The specific steps and parameters are the same as those in Example 1, except that in step (1), the filter cloth used in the process of capturing the target protein is 400 mesh, and the ion exchange chromatography filler used is a SP chromatography filler with a conventional pore size; buffer A: 20 mM P5.0 citric acid buffer solution; eluent B: 20 ​​mM PB buffer pH 6.5; and the process conditions in step (2) remain unchanged.

[0078] The results showed that during step 1, the amount of equilibration solution increased by about 3 times. During the actual cleaning process, the chromatography filler had obvious yellow matter that was not cleaned, and the collected eluate was slightly turbid.

[0079] Comparative Example 2 This comparative example provides a method for preparing and applying recombinant type III collagen. The specific steps and parameters are the same as those in Example 1, except that the ion exchange chromatography filler used in step (2) is a weak cation exchange filler (CMff); the balance solution C is a pH 4.6 20mM acetic acid-sodium acetate elution solution. The eluent D is a pH 4.6 20mM acetic acid-sodium acetate + 1.0M NaCl buffer solution; during the elution process, 3CV is eluted at a balance solution C: eluent D ratio of 19:1, and the impurities are eluted. Then, 3CV is eluted at a balance solution C: eluent D ratio of 9:1, and the eluent is collected to obtain the target protein and detected.

[0080] Comparative Example 3 This comparative example provides a method for preparing and applying recombinant type III collagen. The specific steps and parameters are the same as those in Example 2, except that the ion exchange chromatography filler used in step (2) is a strong cation exchange filler (SPff); the balance solution C is a 10 nM citric acid + 20 mM NaCl buffer solution. The eluent D is a pH 4.4 10 mM citric acid + 1.0 M NaCl buffer solution; during the elution process, the balance solution C: eluent D ratio is 19:1, and the elution is performed for 2 CV. The impurities are then eluted for 3 CV using a balance solution C: eluent D ratio of 17:3. The eluent is collected as the target protein and detected.

[0081] The recombinant human collagen solution obtained after the final purification of Example 1-2 and Comparative Example 1-3 was tested using a recombinant human collagen-related detection method to obtain the purity, recovery rate and endotoxin content of the recombinant human collagen. The test results are shown in Table 3 below. Table 3 Purity, recovery and endotoxin content test results of Examples 1, 2 and Comparative Example

[0082] Sequence Listing <110> Xi'an Peihua College <120> Preparation method and application of recombinant type III collagen <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1122 <212> DNA <213> Recombinant type III collagen <400> 1 ggtgaccctg gtattccagg acaaccaggg tcccctggtt ctcctggccc ccctggatc60 tgtgaatcat gccctactgg tcctcagaac tattctcccc agtatgattc atatgatgtc1 aagtctggag tagcagtagg aggactcgca ggctatcctg gaccagctgg ccccccaggc1 cctcccggtc cccctggtac atctggtcat cctggttccc ctggatctcc aggataccaa2 ggaccccctg gtgaacctgg gcaagctggt ccttcaggcc ctccaggacc tcctggtgct300 ataggtccat ctggtcctgc tggaaagat ggagaatcag gtagacccgg acgacctgga360 gagcgaggat tgcctggacc tccaggtatc aaaggtccag ctgggatacc tggattccct420 ggtatgaaag gacacagagg cttcgatgga cgaatggag aaaagggtga aacaggtgct4 cctggattaa agggtgaaaa tggtcttcca ggcgaaaatg gagctcctgg acccatgggt5 ccaagagggg ctcctggtga gcgaggacgg ccaggacttc ctggggctgc aggtgctcgg600 ggtaatgacg gtgctcgagg cagtgatggt caaccaggcc ctcctggtcc tcctggaact660 gccggattcc ctggatcccc tggtgctaag ggtgaagttg gacctgcagg tactcctggt720 ctgcaaggaa tgcctggaga aagaggaggt cttggaagtc ctggtccaaa gggtgacaag780 ctgcaaggaa tgcctggaga aagaggaggt cttggaagtc ctggtccaaa gggtgacaag780 ggtgaaccag gcggtccagg tgctgatggt gtcccaggga aagatggccc aaggggtcct840 ggtgaaccag gcggtccagg tgctgatggt gtcccaggga aagatggccc aaggggtcct840 actggtccta ttggtcctcc tggcccagct ggccagcctg gagataaggg tgaaggtggt900 actggtccta ttggtcctcc tggcccagct ggccagcctg gagataaggg tgaaggtggt900 gcccccggac ttccaggtat agctggacct cgtggtagcc ctggtgagag aggtgaaact960 gcccccggac ttccaggtat agctggacct cgtggtagcc ctggtgagag aggtgaaact960 ggccctccag gacctgctgg tttccctggt gctcctggac agaatggtga acctggtggt1020 ggccctccag gacctgctgg tttccctggt gctcctggac agaatggtga acctggtggt1020 aaaggagaaa gaggggctcc gggtgagaaa ggtgaaggag gccctcctgg agttgcagga1080 aaaggagaaa gaggggctcc gggtgagaaa ggtgaaggag gccctcctgg agttgcagga1080 ccccctggag gttctggacc tgctggtcct cctggtcccc aa1122 ccccctggag gttctggacc tgctggtcct cctggtcccc aa1122 <210>2<210>2 <211>374<211>374 <212>PRT<212>PRT <213>重组Ⅲ型胶原蛋白<213>Recombinant type III collagen <400>2 <400>2 Gly Asp Pro Gly Ile Pro Gly Gln Pro Gly Ser Pro Gly Ser Pro Gly Gly Asp Pro Gly Ile Pro Gly Gln Pro Gly Ser Pro Gly Ser Pro Gly 1 5 10 15 1 5 10 15 Pro Pro Gly Ile Cys Glu Ser Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro Pro Gly Ile Cys Glu Ser Cys Pro Thr Gly Pro Gln Asn Tyr Ser 20 25 30 20 25 30 Pro Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly Val Ala Val Gly Gly Pro Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly Val Ala Val Gly Gly 35 40 45 35 40 45 Leu Ala Gly Tyr Pro Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro 50 55 60 Pro Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln 65 70 75 80 Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly 85 90 95 Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu 100 105 110 Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro 115 120 125 Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly 130 135 140 His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala 145 150 155 160 Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro 165 170 175 Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly 180 185 190 Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Ser 195 200 205 Asp Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly Thr Ala Gly Phe Pro 210 215 220 Gly Ser Pro Gly Ala Lys Gly Glu Val Gly Pro Ala Gly Thr Pro Gly 225 230 235 240 Leu Gln Gly Met Pro Gly Glu Arg Gly Gly Leu Gly Ser Pro Gly Pro 245 250 255 Lys Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Ala Asp Gly Val Pro 260 265 270 Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly Pro Pro Gly 275 280 285 Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gly Ala Pro Gly Leu 290 295 300 Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Thr 305 310 315 320 Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly Gln Asn Gly 325 330 335 Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gly Glu Lys Gly Glu 340 345 350 Gly Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Gly Ser Gly Pro Ala 355 360 365 Gly Pro Pro Gly Pro Gln 370

Claims

1. A method for preparing recombinant type III collagen, characterized in that: A cation exchange chromatography filler is added to the recombinant type III collagen fermentation broth, the filtrate is collected after centrifugation, purified by a cation exchange chromatography column, and finally concentrated to obtain the type III recombinant collagen.

2. The method for preparing recombinant type III collagen according to claim 1, characterized in that: Please follow the steps below to implement: Step 1: Capture of target protein: The fermentation broth of recombinant type III collagen expressed by the recombinant strain was diluted, the pH value and conductivity were adjusted, a cation exchange chromatography filler was added to the fermentation broth, and the mixture was stirred slowly to allow the target protein to fully bind to the chromatography filler; The mixture was washed with buffer A and centrifuged until the filtrate was basically clear. The solid filler was collected and eluent B was added. After stirring and mixing thoroughly, the mixture was centrifuged and the filtrate was collected to obtain the desired captured recombinant type III collagen. Step 2, cation exchange chromatography: First, the cation exchange chromatography column is equilibrated with equilibration solution C. Then, the filtrate collected in step 1 is added with citric acid to adjust the pH and then passed through the cation exchange chromatography column for adsorption. Finally, the target protein is eluted with a mixture of equilibration solution C and buffer D in a certain proportion. The eluate is the recombinant type III collagen. Step 3: Ultrafiltration and concentration: The sample collected in step 2 was concentrated using an ultrafiltration membrane and the liquid was replaced; type III recombinant collagen with a purity greater than 98% was obtained.

3. The method for preparing recombinant type III collagen according to claim 2, characterized in that: In step 1, the pH is adjusted to 4.0-5.5, and the conductivity is adjusted to no more than 5.0 ms / cm; and the stirring time is 20-40 min.

4. The method for preparing recombinant type III collagen according to claim 2, wherein: In step 1, buffer A is a citric acid solution, and eluent B is a phosphate buffer solution.

5. The method for preparing recombinant type III collagen according to claim 2, characterized in that: The cation exchange chromatography filler in step 1 is specifically an agarose filler with a particle size of not less than 60 μm and a pore size of not less than 30 nm. The amount of the cation exchange chromatography filler added to the fermentation broth is: 100 g per 3-6 L of fermentation broth.

6. The method for preparing recombinant type III collagen according to claim 2, wherein: The filler of the cation exchange chromatography column in step 2 is SP sepharose Fast Flow; the equilibration solution C in step 2 is citric acid plus a low-concentration NaCl buffer solution; the buffer D is citric acid plus a high-concentration NaCl buffer solution; the elution method for protein elution in step 2 is constant-current gradient elution, the pH value is adjusted to 4.0-5.5, and the target protein is eluted using a mixture of equilibration solution C and buffer D, and the ratio of the concentration of equilibration solution C to the concentration of buffer D is 4:1-9:

1.

7. The method for preparing recombinant type III collagen according to claim 2, characterized in that: The ultrafiltration membrane in step 3 is specifically a 10kd hollow fiber membrane.

8. The recombinant type III collagen obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the recombinant type III collagen is shown in SEQ ID NO.

1.

9. The recombinant type III collagen according to claim 8, characterized in that The amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.

2.

10. Use of the recombinant type III collagen obtained according to the method for preparing recombinant type III collagen according to any one of claims 1 to 7 in the field of medicine or cosmetics.

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