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

Abstract

The invention discloses a recombinant humanized III-type collagen. The amino acid sequence of the recombinant humanized III-type collagen is shown as SEQ ID NO: 3. The recombinant humanized III-type collagen is an engineered humanized collagen, is derived from natural III-type collagen (NP000081.2), has better water solubility, and can avoid immunological rejection when being applied to a human body; the invention also provides a preparation method of the recombinant human III-type collagen, which comprises the following steps: carrying out high-density fermentation and purification on a recombinant strain recombined with a recombinant vector of which the nucleotide sequence is inserted into a codon optimization sequence as shown in SEQ ID NO: 2 between a BamHI site and an EcoRI site to obtain the recombinant humanized III-type collagen as shown in an amino acid sequence as shown in SEQ ID NO: 3.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant humanized type III collagen, a preparation method and an application thereof. Background Art

[0002] Human type III collagen is a fibrillar collagen with a triple-helical structure composed of three alpha 1 chains, COL3A1. It is found in extensible connective tissues such as the skin, lungs, uterus, intestines, and vascular system, and is typically associated with type I collagen. Human type III collagen hCOL3A1 has a large molecular weight. Its precursor consists of 1446 amino acids with a theoretical molecular weight of 138,564 Da, and the mature peptide also contains 1068 amino acids with a theoretical molecular weight of 95,288 Da. hCOL3A1 is highly repetitive (GXY) and glycine-rich (approximately 30%), resulting in a high GC content in its gene sequence. Post-translational modifications such as high hydroxylation (20%) and cross-linking are also required, making recombinant expression of hCOL3A1 extremely challenging.

[0003] With the widespread application of collagen, the disadvantages of animal-derived collagen have become increasingly prominent, and the demand for engineered humanized collagen / gelatin is becoming increasingly urgent. Currently, the collagen peptides expressed are mostly small fragment repetitions or amino acid substitutions. The expression of larger fragments that are relatively complete and completely consistent with the natural collagen sequence is still rare. At the same time, most of them use yeast expression systems for expression, which not only takes a long time (usually 7 days) but also requires methanol flow induction, and the expression process is complicated.

[0004] The prior art uses the pNCMO2 plasmid as a vector to express humanized type III collagen C3. However, the pNCMO2 plasmid is large in structure and contains a strong P2 promoter, resulting in poor plasmid stability and inability to sustain growth, making it unsuitable for high-density fermentation (https: / / www.takarabio.com / documents / User%20Manual / HB111 / HB111_DS.v1811.pdf).

[0005] Therefore, there is an urgent need for a recombinant humanized type III collagen, and a method for stably expressing the recombinant humanized type III collagen through high-density fermentation can be provided. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a recombinant humanized type III collagen and a preparation method and application thereof.

[0007] The first object of the present invention is to provide a recombinant humanized type III collagen.

[0008] The second object of the present invention is to provide the use of the above-mentioned recombinant humanized type III collagen in the preparation of artificial organs and / or wound dressings.

[0009] The third object of the present invention is to provide a recombinant vector for producing the above-mentioned recombinant humanized type III collagen.

[0010] The fourth object of the present invention is to provide a recombinant strain for producing the above-mentioned recombinant humanized type III collagen.

[0011] The fifth object of the present invention is to provide the use of the above-mentioned recombinant vector and / or the above-mentioned recombinant strain in the production of recombinant humanized type III collagen.

[0012] The sixth object of the present invention is to provide a method for producing recombinant humanized type III collagen.

[0013] In order to achieve the above object, the present invention is implemented through the following scheme:

[0014] A recombinant humanized type III collagen protein, the amino acid sequence of the recombinant humanized type III collagen protein is shown in SEQ ID NO: 3.

[0015] Preferably, the nucleotide sequence of the cDNA encoding the recombinant humanized type III collagen is shown in SEQ ID NO: 4.

[0016] The present invention also seeks to protect the use of any of the above-mentioned recombinant humanized type III collagen in the preparation of artificial organs and / or wound dressings.

[0017] The present invention also claims protection for a recombinant vector for producing any of the above-mentioned recombinant humanized type III collagen, wherein the recombinant vector is an optimized sequence such as SEQ ID NO: 2 inserted between the BamHI site and the EcoRI site of the plasmid.

[0018] The nucleotide sequence is shown as the optimized sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the optimized sequence is shown in SEQ ID NO: 1.

[0019] Preferably, the plasmid is pNY326 plasmid.

[0020] Among them, the pNY326 plasmid itself has a relatively simple structure, a size of 3.4kb, and contains a P5 weak promoter. Using it as a vector backbone to construct a recombinant vector can stably inherit in host cells and is suitable for high-density fermentation to synthesize the target protein.

[0021] The present invention also seeks to protect a recombinant strain for producing any of the above-mentioned recombinant humanized type III collagen, wherein the recombinant strain is a strain containing any of the above-mentioned recombinant vectors.

[0022] Preferably, the strain is Bacillus pumilus.

[0023] More preferably, the Bacillus pumilus is HPD31-SP3.

[0024] The present invention also claims the use of any of the above-mentioned recombinant vectors and / or any of the above-mentioned recombinant strains in the production of recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0025] The present invention also claims a method for producing recombinant humanized type III collagen, comprising the following steps:

[0026] S1. The recombinant strain was inoculated into T2 medium at a final volume concentration of 0.1 to 5%, and cultured for 10 to 24 hours to obtain a fermentation seed solution;

[0027] S2. The fermentation seed solution obtained in step S1 was inoculated into 2SLFC medium at a final volume concentration of 0.5 to 5%, and the dissolved oxygen level of the medium was controlled to be not less than 30%, and the pH value was 6.0 to 8.0 to obtain a fermentation product;

[0028] The 2SLFC culture medium contains 2-4% glucose, 2-5% neutral soy peptone, 0.2-2% yeast extract powder, 0.2-2% fish meal, 0.2-2% cottonseed meal, 0.001-0.002% MnSO4·5H2O, 0.001-0.002% FeSO4·7H2O and 0.0001-0.0002% ZnSO4·7H2O in a mass concentration of 2-4%.

[0029] S3. The fermentation product obtained in step S2 is separated into solid and liquid, and the liquid is collected and purified to obtain the recombinant humanized type III collagen with a nucleotide sequence as shown in SEQ ID NO: 3.

[0030] Preferably, step S1 is: inoculating the recombinant strain into T2 culture medium at a final volume concentration of 0.4-2%.

[0031] More preferably, the T2 culture medium is a T2 culture medium containing 50 μg / ml Neo.

[0032] Preferably, step S1 is: culturing at 28-37° C. for 10-24 hours.

[0033] More preferably, the culture is carried out at 30°C for 12 to 16 hours.

[0034] Preferably, step S2 comprises: inoculating the fermentation seed liquid obtained in step S1 into 2SLFC culture medium at a final volume concentration of 5%.

[0035] Preferably, the fermentation in step S2 is high-density fermentation.

[0036] More preferably, the temperature during the high-density fermentation process is 28-37°C.

[0037] Further preferably, the temperature during the high-density fermentation process is 30°C.

[0038] More preferably, the fermentation in step S2 is: feeding with a feed medium at the beginning after 24 hours of fermentation, with a feeding rate of 10-50 ml / h; the feed medium is a medium containing a mass concentration of 20-60% glucose and 0.001-0.002% of % FeSO4·7H2O.

[0039] Further preferably, the fermentation discharge time in step S2 is 52 hours.

[0040] More preferably, the 2SLFC culture medium contains 2% glucose, 4% neutral soy peptone, 0.5% yeast extract powder, 0.5% fish meal, 0.5% cottonseed meal, 0.001% MnSO4·5H2O, 0.001% FeSO4·7H2O and 0.0001% ZnSO4·7H2O in a mass concentration.

[0041] Further preferably, the 2SLFC culture medium uses SL culture medium as the basic culture medium.

[0042] Preferably, in step S2, the dissolved oxygen level of the culture medium is controlled to be 30% and the pH value is controlled to be 7.0 for fermentation.

[0043] Preferably, the solid-liquid separation method in step S3 is centrifugation.

[0044] Preferably, the purification in step S3 is specifically as follows: the collected liquid is ultrafiltered through a 10 kD hollow fiber column to replace it with PB buffer, then passed through an SP cation column equilibrated with PB buffer, washed with PB buffer to the SP cation column baseline, then eluted with PB buffer containing 0.5 mol / L sodium chloride and the sodium chloride is removed using a 10 kD hollow fiber column.

[0045] More preferably, the concentration of the PB buffer is 10 mmol / L, and contains 1.2 g / L anhydrous sodium dihydrogen phosphate and 0.156 g / L NaOH.

[0046] Furthermore, the recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID NO: 3 can also be expressed and purified using the Pichia pastoris expression system, specifically:

[0047] The cDNA sequence encoding the recombinant humanized type III collagen as shown in SEQ ID NO: 4 is optimized according to the Pichia pastoris host to obtain the Pichia pastoris optimized sequence as shown in SEQ ID NO: 5, and the Pichia pastoris optimized sequence is inserted downstream of the α-factor sequence of the pPICZaA vector to obtain a Pichia pastoris recombinant vector;

[0048] The Pichia pastoris recombinant vector was electroporated into GS115 competent cells, and then fermented and purified to obtain recombinant humanized type III collagen with an amino acid sequence as shown in SEQ ID NO: 3.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The present invention provides a recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO: 3. The recombinant humanized type III collagen is an engineered humanized collagen derived from natural type III collagen (NP_000081.2), has good water solubility, and can avoid immune rejection when applied to the human body. Furthermore, a 0.5% mass concentration of a recombinant humanized type III collagen aqueous solution can promote the growth of NIH3T3 cells, and a 1% mass concentration of a recombinant humanized type III collagen aqueous solution can promote the adhesion of NIH3T3 cells when coated on an ELISA plate. Furthermore, a 1% mass concentration of a recombinant humanized type III collagen aqueous solution has excellent film-forming properties, indicating that the recombinant humanized type III collagen can be used as a material for wound dressings.

[0051] The present invention also provides a method for preparing the recombinant humanized type III collagen, which utilizes a recombinant strain having a recombinant vector with a codon-optimized sequence such as SEQ ID NO: 2 inserted between the BamHI site and the EcoRI site, and high-density fermentation and purification to obtain recombinant humanized type III collagen with an amino acid sequence such as SEQ ID NO: 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The figure is the SDS-PAGE gel electrophoresis result in Example 1;

[0053] Figure 2Figure 2 shows the effect of pH on fermentation during high-density fermentation in Example 2; A is a curve showing the change of pH value over time during the fermentation of strain C54#; B is a curve showing the change of OD600 over time when strain C54# was fermented at different pH values;

[0054] Figure 3 This is a graph showing the effect of dissolved oxygen levels on fermentation during high-density fermentation in Example 2;

[0055] Figure 4 This is the gel electrophoresis result after fermentation culture of C54# using fermentation medium 1 to fermentation medium 8 in Example 2;

[0056] Figure 5 This is a graph showing changes in OD600 and wet weight of substances in the fermenter over time during the fermentation process of Example 3;

[0057] Figure 6 The SDS-PAGE gel electrophoresis result in Example 3 is shown;

[0058] Figure 7 Figures 4 are the results of SDS-PAGE gel electrophoresis; A is the result of gel electrophoresis on days 1 to 6 during the fermentation of the Pichia pastoris transformant; B is the result of gel electrophoresis during high-density fermentation of the Pichia pastoris transformant;

[0059] Figure 8 Figure 2 is a graph showing the results of sequencing the membrane-forming properties of recombinant humanized type III collagen; Figure A shows the observation results of a 5% mass concentration aqueous solution of recombinant humanized type III collagen in a culture dish; Figure B shows the observation results of a 1% mass concentration aqueous solution of recombinant humanized type III collagen in a culture dish; Figure C shows the observation results of a 1% mass concentration aqueous solution of recombinant humanized type III collagen applied to the skin;

[0060] Figure 9 Figure 2 is a graph showing the effect of recombinant humanized type III collagen on the growth of NIH3T3 cells; A is a graph showing the observation results of cells in each well; B is a bar graph showing the survival rate of cells in each well;

[0061] Figure 10 This is a test result diagram of the effect of recombinant humanized type III collagen on NTH3T3 cell adhesion. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0063] Example 1 Construction of recombinant vectors and recombinant strains

[0064] 1. Experimental Methods

[0065] 1. Construction of recombinant vector

[0066] Based on the human collagen III α1 chain sequence (NP_000081.2) in the NCBI (National Center for Biotechnology Information) protein sequence database, the optimized sequence shown in SEQ ID NO: 2 was synthesized, and the amino acid sequence of the protein encoded by it was shown in SEQ ID NO: 1.

[0067] A BamHI restriction site (GGATCC) was connected to the 5' end of the optimized sequence shown in SEQ ID NO: 2, and an EcoRI restriction site (GAATTC) was connected to the 3' end. Then, double digestion was performed with BamHI and EcoRI enzymes, and the optimized sequence after digestion was recovered from the gel. The pNY326 plasmid (Takara Bio, Cat HB111) was linearized with BamHI and EcoRI enzymes to obtain a linearized pNY326 plasmid.

[0068] The optimized sequence after enzyme digestion and the linearized pNY326 plasmid were ligated using T4 DNA ligase, and the ligation product was collected to obtain the recombinant vector.

[0069] 2. Construction of recombinant strains

[0070] 15 μL of the recombinant vector prepared in step 1 was mixed evenly with 400 μL of electroporation competent HPD31-SP3 (Takara Bio, Cat HB116), placed in a 2 mm ice bath electroporation cuvette for 5 min, and then electroporated at 1.5 kV, 25 μF, and 1000 Ω. After electroporation, the cells were transferred to T2MM medium (T2 medium containing 0.001% MnSO4·5H2O, 0.001% FeSO4·7H2O, 0.0001% ZnSO4·7H2O, and 20 mmol / LMgCl2, pH 7.5). 7.0) were allowed to stand for 3 h, followed by centrifugation at 7000 rpm for 5 min. The precipitate (bacterial mass) was collected and evenly spread on a T2M agar plate (T2 medium containing 1.5% agar powder and 40 μg / mL Neo) and incubated at 37°C for 12 h. A single clone of the recombinant strain was selected and inoculated (final inoculum concentration of 0.4-2%) into T2N liquid medium (T2 medium containing 40 μg / mL Neo) containing 50 μg / mL Neo and incubated at 30°C and 250 rpm for 12 h to obtain a seed fermentation broth.

[0071] The seed fermentation broth was transferred into 2SL medium (2SL medium, containing 2% glucose, 4% neutral soy peptone, 0.5% yeast extract, 0.001% MnSO4·5H2O, 0.001% FeSO4·7H2O and 0.0001% ZnSO4·7H2O, w / w) at a volume ratio of 1:100, cultured at 30°C and 250 rpm for 48 h, and the supernatant was collected by centrifugation for SDS-PAGE gel electrophoresis. After electrophoresis at 200 V for 42 minutes, the protein expression was identified by staining; the electrophoresis protein molecular weight standard M corresponds to the protein product number of Biyuntian: P0075.

[0072] The monoclonal strain that clearly expressed the exogenous proteins (25 kDa and 15 kDa) in the gel electrophoresis results was the recombinant strain. The plasmid of the recombinant strain was extracted using a plasmid extraction kit, and sequencing was performed using a sequencing primer with a nucleotide sequence as shown in SEQ ID NO: 6 to obtain the sequencing results.

[0073] 2. Experimental Results

[0074] The results of SDS-PAGE gel electrophoresis are shown in the figure Figure 1 As shown, the results showed that the monoclonal strain in lane 4 in the electrophoresis gel had obvious protein bands at 25 kDa and 15 kDa, and was a recombinant strain that highly expressed human type III collagen, marked as C54# clone.

[0075] The C54# clone was sequenced using a sequencing primer with a nucleotide sequence as shown in SEQ ID NO: 6, obtaining a cDNA sequence with a nucleotide sequence as shown in SEQ ID NO: 4. The amino acid sequence of the protein encoded thereby was shown in SEQ ID NO: 3. The protein with the amino acid sequence as shown in SEQ ID NO: 3 was designated as recombinant humanized type III collagen (hCOL3A1r), which consists of 229 amino acids, has a theoretical molecular weight of approximately 20.8 kDa, and a pI of 9.90.

[0076] Example 2 Investigation of a method for producing recombinant human type III collagen

[0077] After long-term research, the present invention has discovered a method suitable for high-density fermentation of the recombinant strain (C54# clone) prepared in Example 1 to produce recombinant humanized III collagen, and based on this, provides method conditions for producing recombinant humanized III collagen using high-density fermentation.

[0078] First, the pH value, dissolved oxygen level and nitrogen source composition during high-density fermentation were changed respectively to verify the optimal conditions for high-density fermentation protein production.

[0079] 1. Experimental Methods

[0080] 1. Effect of pH on fermentation during high-density fermentation

[0081] The C54# clone strain prepared in Example 1 was activated and inoculated into a 2SL culture medium using a 5 L fermentor at a final volume concentration of 5%. The aeration rate during fermentation was controlled at 3 lpm and the temperature was 30°C. The dissolved oxygen (DO) level during fermentation was controlled at 30% by changing the rotation speed of the 5 L fermentor in series, and the changes in pH over time during fermentation were recorded.

[0082] The C54# clone strain prepared in Example 1 was activated and inoculated into a 2SL culture medium using a 5 L fermentor at a final volume concentration of 5%. The aeration rate during fermentation was controlled at 3 lpm and the temperature was 30° C. The dissolved oxygen (DO) level during fermentation was controlled at 30% by changing the rotation speed of the 5 L fermentor in series. At the same time, 10% (w / w) ammonia water and 2 mol / L H2SO4 were used to control the pH values ​​during fermentation to 6.0, 7.0, and 8.0, respectively. The OD600 values ​​during fermentation at different pH values ​​were tested, and the changes in OD600 values ​​over time were recorded.

[0083] 2. Effect of dissolved oxygen level on fermentation during high-density fermentation

[0084] The C54# clone strain prepared in Example 1 was activated and inoculated into a 2SL culture medium using a 5 L fermentor at a final volume concentration of 5%. The initial aeration rate during the fermentation process was controlled to be 1 lpm, the temperature was 30° C., and the pH value during the fermentation process was controlled to be maintained at 7.0 using 10% (w / w) ammonia water and 2 mol / L H2SO4. The DO level during the fermentation process was controlled in series with the rotation speed of the 5 L fermentor to be 10%, 20%, 30% and 40%, respectively. The OD600 values ​​during fermentation at different dissolved oxygen levels were tested, and the changes in OD600 values ​​over time were recorded.

[0085] 3. Effects of different nitrogen sources on fermentation during high-density fermentation

[0086] 8 different fermentation media were set up respectively, and the mass concentration of nitrogen source in the fermentation media was as follows:

[0087] Fermentation medium 1: 4% fermentation-specific soy peptone (Solaibo, FA0030);

[0088] Fermentation medium 2: 4% soy peptone (Hongrun Baoshun, Y005C);

[0089] Fermentation medium 3: 4% neutral soy peptone (Soya Peptone Neutralised, OXOID, LP0044C);

[0090] Fermentation medium 4: 4% Angel Soy Peptone FP410 (Angel, CX142013);

[0091] Fermentation medium 5: 4% neutral soy peptone (Soya Peptone Neutralised, OXOID, LP0044C) + 0.5% fish meal (Solaibo, FA0080);

[0092] Fermentation medium 6: 4% neutral soy peptone (Soya Peptone Neutralised, OXOID, LP0044C) + 0.5% fish meal (Solybo, FA0080) + 0.5% cottonseed meal (Solybo, FA0250);

[0093] Fermentation medium 7: 4% neutral soybean peptone (Soya Peptone Neutralised, OXOID, LP0044C) + 0.5% cottonseed meal (Solaibo, FA0250);

[0094] Fermentation medium 8: 4% yeast peptone FP102 (Angel, CX142030);

[0095] The other basic components of fermentation medium 1 to fermentation medium 8 are: 2% glucose, 0.5% yeast extract powder (OXOID, LP0021), 0.001% MnSO4·5H2O, 0.001% FeSO4·7H2O and 0.0001% ZnSO4·7H2O with a mass concentration of 7.2.

[0096] The C54# clone strain prepared in Example 1 was activated and inoculated into fermentation medium 1 to fermentation medium 8 at a final volume concentration of 5% (the HPD-SP3 host bacteria was inoculated into 2SL medium as a control). The fermentation medium 1 to fermentation medium 8 inoculated with the C54# clone strain were transferred to different 5L fermentors, respectively. The aeration rate during the fermentation process was controlled to 3 lpm and the temperature was 30°C. The dissolved oxygen (DO) level during the fermentation process was controlled to 30% by changing the rotation speed of the 5L fermentor in series. The pH value during the fermentation process was controlled to be maintained at 7.0 using 10% (w / w) ammonia water and 2 mol / L H2SO4. The fermentation was carried out for 48 hours. The fermentation product was collected and the supernatant was collected by centrifugation and subjected to SDS-PAGE gel electrophoresis. After electrophoresis at 200 V for 42 minutes, the protein expression was identified by staining. The electrophoresis protein molecular weight standard M corresponds to the protein with the product number of Biyuntian: P0075.

[0097] 2. Experimental Results

[0098] 1. Effect of pH on fermentation during high-density fermentation

[0099] The results of the effect of pH on fermentation during high-density fermentation are shown in the figure below. Figure 2 As shown, Figure 2 A is the curve of pH value changing with time during the fermentation process of C54# clone strain. Figure 2 B is the curve of OD600 change over time when the C54# clone strain was fermented at different pH values.

[0100] The results showed that the pH of the C54# clone strain fluctuated dramatically during the fermentation process. During the fermentation process, it tended to secrete alkaline substances or consume acidic culture medium components, resulting in an alkaline extracellular environment of the strain, which had an adverse effect on the protein expression ability of the C54# strain. When fermenting at different pH values, the OD600 change curve showed that when the pH value was 7.0, the OD600 during the fermentation process was significantly higher than that at other pH values, indicating that a pH value of 7.0 during the fermentation process was most beneficial for the growth of the C54# clone strain.

[0101] 2. Effect of dissolved oxygen level on fermentation during high-density fermentation

[0102] The effect of dissolved oxygen level on fermentation during high-density fermentation is shown in the figure below. Figure 3 The results show that as dissolved oxygen levels increase, the C54# clone grows faster (higher OD600). When dissolved oxygen levels exceed 30%, the effect of dissolved oxygen on the growth of the C54# clone decreases. Therefore, when using the C54# clone to ferment recombinant humanized type III collagen, it is necessary to control the dissolved oxygen level to no less than 30% during fermentation.

[0103] 3. Effects of different nitrogen sources on fermentation during high-density fermentation

[0104] The gel electrophoresis results of the C54# clone after fermentation culture using fermentation medium 1 to fermentation medium 8 are shown in the figure below. Figure 4 As shown, lanes 1 to 8 are the electrophoresis results corresponding to fermentation medium 1 to fermentation medium 8, respectively, and lane 0 is the fermentation control of HPD31-SP3 host cells in 2SL medium.

[0105] The results showed that when the C54# clone was fermented using fermentation medium 6 corresponding to lane 6, the expression level of the target protein (at 25kDa and 15kDa) was higher than that in other lanes, indicating that the combination of neutral soy peptone (Soya Peptone Neutralised, OXOID, LP0044C) and yeast extract powder (OXOID, LP0021), and the addition of 0.5% fish meal and 0.5% cottonseed cake powder (Solaibo, FA0250) can promote the protein expression ability of the C54# clone.

[0106] Example 3 A method for producing and purifying recombinant humanized type III collagen

[0107] 1. Experimental Methods

[0108] 1. Fermentation production of recombinant humanized type III collagen

[0109] The C54# clone strain prepared in Example 1 was activated and inoculated into 2SLFC medium {(2SLFC medium, containing 2% glucose, 4% neutral soy peptone (Soya Peptone Neutralised, OXOID, LP0044C), 0.5% yeast extract powder (OXOID, LP0021), 0.5% fish meal (Solyabin, FA0080), 0.5% cottonseed meal (Solyabin, FA0250), 0.001% MnSO4·5H2O, 0.001% FeSO4·7H2O and 0.0001% ZnSO4·7H2O, w / w) at a final volume concentration of 5% in a 5 L fermenter to control the fermentation process. The aeration rate was 3 lpm, the temperature was 30°C, and the dissolved oxygen (DO) level during fermentation was controlled at 30% by varying the rotation speed of the 5-L fermentor. Simultaneously, the pH value during fermentation was controlled at 7.0 using 10% (w / w) ammonia and 2 mol / L H2SO4, respectively. After 24 h of fermentation, feeding was initiated using a feed medium containing 50% glucose and 0.001% FeSO4·7H2O at a rate of 20 ml / h.

[0110] The OD600 and wet weight of the substances in the fermentation tank were recorded over time during the fermentation process.

[0111] 2. Purification

[0112] When the fermentation time in step 1 reaches 52 hours, the fermentor material is centrifuged at 8000 rpm for 15 minutes, and the supernatant is collected. The supernatant is ultrafiltered through a 10 kD hollow fiber column and replaced with 10 mmol PB buffer (pH 7.0, containing 1.2 g / L anhydrous sodium dihydrogen phosphate and 0.156 g / L NaOH), and then passed through an SP cation column and washed with 10 mmol PB buffer (pH 7.0) to the SP cation column baseline. The SP cation column flow-through is collected, and then eluted with 10 mmol PB buffer (pH 7.0) containing 0.5 mol / L sodium chloride. The PB eluate with 0.5 mol / L sodium chloride is collected and the sodium chloride is removed using a 10 kD hollow fiber column to obtain the purified protein (recombinant humanized type III collagen hCOL3A1r with an amino acid sequence as shown in SEQ ID NO: 3).

[0113] 3. Protein electrophoresis

[0114] The SP cation column flow-through (F) obtained in step 2, 5 μL of PB eluate (rc5), and 10 μL of PB eluate (rc10) were respectively subjected to SDS-PAGE gel electrophoresis detection, and the supernatant (C54#) obtained in step 1 when the fermentation time was 40 h was collected for SDS-PAGE gel electrophoresis detection. The HPD31-SP3 blank strain (Takara Bio, Cat HB116) was used as a blank control (N) for SDS-PAGE gel electrophoresis detection, and the electrophoresis detection results were recorded.

[0115] 2. Experimental Results

[0116] The curve of OD600 and wet weight of the material in the fermentation tank during the fermentation process in step 1 is as follows: Figure 5 As shown in the figure, the results showed that: with the increase of fermentation time, OD600 gradually increased and tended to be flat, while the wet weight of the material in the fermenter gradually increased with the increase of fermentation time, reaching a maximum value of about 110g / L at 52h of fermentation, indicating that in the process of fermentation production of recombinant human type III collagen, the most suitable discharge time is 52h of fermentation.

[0117] The results of SDS-PAGE gel electrophoresis are shown in the figure Figure 6 As shown, the results showed that after the fermentation supernatant of C54# clone was purified by SP cation exchange column according to the purification method shown above, the target protein (recombinant humanized type III collagen hCOL3A1r with an amino acid sequence as shown in SEQ ID NO: 3) with a purity greater than 90% could be obtained.

[0118] Example 4 Expression of recombinant humanized type III collagen in yeast cells

[0119] 1. Experimental Methods

[0120] 1. Construction and fermentation of Pichia pastoris transformants

[0121] The cDNA sequence shown in SEQ ID NO: 4 was optimized according to the Pichia pastoris host to obtain the Pichia pastoris optimized sequence shown in SEQ ID NO: 5, and the Pichia pastoris optimized sequence was subcloned and inserted downstream of the α-factor sequence of the pPICZaA vector to obtain the pPICZaA-hCOL3A1r recombinant.

[0122] The pPICZaA-hCOL3A1r recombinant was linearized with SacI enzyme and then electroporated into GS115 competent cells. The cells were then plated onto YPDS plates containing 100 μg / mL Zeocin until clones emerged. The clones were transferred to YPDS plates containing 500 μg / mL Zeocin and 1000 μg / mL Zeocin, respectively, and cultured until new clones emerged, which were the Pichia pastoris transformants.

[0123] The Pichia pastoris transformant was transferred to GS115 pre-induction BMGY medium and cultured for 24 h. The culture was then centrifuged at 4000 g for 5 min and the supernatant was discarded. GS115 methanol-induced GMMY medium was added to an OD600 of 1.0-1.5. The culture was fermented at 30°C and 240 rpm for 6 days. 1% methanol (w / w) was added every 24 h during the fermentation process.

[0124] 1 ml of the liquid from day 1 to day 6 of the fermentation process was taken respectively, and the supernatant was collected by centrifugation at 13,000 rpm for 10 minutes. The supernatant was subjected to SDS-PAGE gel electrophoresis at 200 V for 42 minutes and then stained to identify the protein expression.

[0125] 2. High-density fermentation of Pichia pastoris transformants

[0126] The Pichia pastoris transformant obtained in step 1 was inoculated into 20 mL of YPD medium and cultured at 30° C. and 240 rpm for 20 h to obtain a seed solution.

[0127] The seed solution was inoculated into 500 mL of BMGY medium at a volume ratio of 1:50, and cultured at 30°C and 240 rpm until the OD600 absorbance value was 6 to obtain the amplified culture solution;

[0128] The amplified culture fluid was inoculated into 3 L inorganic salt basal medium BSM (containing 20 g / L (NH4)2SO4, 40 g / L glycerol, 12 g / L KH2PO4, 1 g / L CaSO4·2H2O, 0.4 g / L Histidine and 4.35 ml / L PMT1) at a volume ratio of 1:10 and fermented in a 7 L fermentor.

[0129] The fermentation process temperature was controlled at 30°C, ammonia water was used to maintain the pH at 6.0 during the fermentation process, the ventilation volume was maintained at 2 vvm, and the dissolved oxygen (DO) level was controlled at 20% by changing the rotation speed of the 7L fermenter. After the DO level in the 7L fermenter reached 100%, glycerol (containing 12 ml / L of PTM1) was added to a final mass concentration of 50%. After the DO level in the 7L fermenter reached 100%, 100% methanol (containing 12 ml / L of PTM1) was added and the fermentation product was collected after 120 hours.

[0130] The fermentation product was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected to obtain a fermentation supernatant. The fermentation supernatant was ultrafiltered through a 10 kD hollow fiber column and exchanged into 10 mmol PB buffer (pH 7.0). The supernatant was then passed through an SP cation column and washed with 10 mmol PB buffer (pH 7.0) to the SP cation column baseline. The SP cation column flow-through 1 was collected, and then eluted with PB buffer containing 0.5 mol / L sodium chloride. The PB eluate 1 containing 0.5 mol / L sodium chloride was collected and the sodium chloride was removed using a 10 kD hollow fiber column to obtain a purified product.

[0131] 5 μL of fermentation supernatant (F5d5), 8 μL of fermentation supernatant (F5d8), 1 μg of PB eluate 1 (P1) and 4 μg of PB eluate 1 (P4) were subjected to SDS-PAGE gel electrophoresis, and the protein expression was identified after electrophoresis at 200 V for 42 minutes.

[0132] 2. Experimental Results

[0133] The results of SDS-PAGE gel electrophoresis are shown in the figure Figure 7 As shown, Figure 7 A is the gel electrophoresis test result of the Pichia pastoris transformant on the 1st to 6th day during the fermentation process. Figure 7 B is the gel electrophoresis detection result of high-density fermentation of Pichia pastoris transformants.

[0134] The results showed that when hCOL3A1r with the amino acid sequence shown in SEQ ID NO: 3 was produced by fermentation using Pichia pastoris, a small amount of impurity protein was present in the supernatant after 5 days of fermentation. After purification by SP cation exchange column, the target protein (hCOL3A1r) was obtained with a purity greater than 90%, indicating that high-purity hCOL3A1r (SEQ ID NO: 3) can also be obtained using the Pichia pastoris expression system.

[0135] Example 5: Film-forming and bioactivity testing of recombinant humanized type III collagen solution

[0136] 1. Experimental Methods

[0137] According to the method shown in Example 3, recombinant humanized type III collagen with an amino acid sequence as shown in SEQ ID NO: 3 was prepared, and the membrane-forming property and biological activity of the recombinant humanized type III collagen were tested.

[0138] 1. Recombinant humanized type III collagen film-forming test

[0139] The recombinant humanized type III collagen with an amino acid sequence as shown in SEQ ID NO: 3 was prepared into aqueous recombinant humanized type III collagen solutions with mass concentrations of 5% and 1%, respectively. 1 mL of the aqueous recombinant humanized type III collagen solutions of different mass concentrations was added to different culture dishes, and the dishes were placed in a 37°C incubator for 2 days, and the culture dishes were observed.

[0140] At the same time, 1 mL of a 1% recombinant humanized type III collagen aqueous solution was evenly applied on the skin, and the results were observed after drying.

[0141] 2. Effect of recombinant humanized type III collagen on the growth of NIH3T3 cells

[0142] NIH3T3 cells in good growth condition were seeded into 96-well plates at 5000 cells / well and cultured for 12 h.

[0143] Cell growth medium (DMEM+8% NCS, v / v) containing recombinant humanized type III collagen (amino acid sequence as shown in SEQ ID NO: 3) at mass concentrations of 0.5%, 1%, 2%, 5% and 10% were prepared respectively. NIH3T3 cells were cultured in the cell growth medium containing different mass concentrations of recombinant humanized type III collagen for 24 h; and NIH3T3 cells were cultured in the cell growth medium without recombinant humanized type III collagen as a blank control group.

[0144] After the culture was completed, the cells in each well were observed and the cell survival rate of each well was calculated by combining CCK8 reagent and taking the survival rate of the cells in the blank control group as 100%.

[0145] 3. Effect of recombinant humanized type III collagen on NTH3T3 cell adhesion

[0146] A 1% recombinant humanized type III collagen PBS solution was prepared using PBS solution and added to the ELISA plate at a rate of 50 μL / well as the experimental wells (1% rC); the blank control wells (PBS) were added with PBS solution at a rate of 50 μL / well; the positive control wells (1% Pc) were added with 1% gelatin coating solution (R20873, diluted in PBS, w / w) at a rate of 50 μL / well; 4 wells were set for the experimental wells, blank control wells, and positive control wells.

[0147] The ELISA plate with the wells set was placed in a cell culture incubator at 37°C for 12 h. The solution was discarded and washed with PBS. 5 A serum-free NIH3T3 cell suspension of 50 cells / mL was added to the ELISA plate at 100 μL / well and incubated at 37°C in a 5% (v / v) CO2 incubator for 90 minutes. After incubation, each well was washed with PBS buffer, using 200 μL each time, for a total of 3 washes.

[0148] After washing, 50 μL of distilled water lysis buffer containing 2% Triton X-100 (v / v) was added to each well and the cells were allowed to stand at room temperature for 90 min. Based on the kit instructions (Cytotoxicity Detection Kit LDH, Roche, 11644793001), 50 μL of LDH detection substrate was added to each well and the cells were allowed to stand until color developed. The absorbance at A490 nm was read and the average value was calculated.

[0149] 2. Experimental Results

[0150] 1. The results of the membrane-forming property test of recombinant humanized type III collagen are shown in the figure below. Figure 8 As shown, Figure 8 A is the observation result of a 5% mass concentration recombinant humanized type III collagen aqueous solution in a culture dish; Figure 8 B is the observation result of a 1% mass concentration recombinant humanized type III collagen aqueous solution in a culture dish; Figure 8 C shows the observation result of applying a 1% mass concentration recombinant humanized type III collagen aqueous solution on the skin.

[0151] The results showed that the recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID NO: 3 had excellent film-forming properties, and did not feel sticky or stringy when applied to the skin, and exhibited a bright, light-shielding feel after drying.

[0152] 2. The effect of recombinant humanized type III collagen on the growth of NIH3T3 cells is shown in the figure below. Figure 9 As shown, Figure 9 A is the observation result of cells in each well. Figure 9 B is a bar graph of the cell survival rate in each well.

[0153] The results showed that recombinant humanized type III collagen at a mass concentration of 0.5% had a significant promoting effect on the growth of NIH3T3 cells, while recombinant humanized type III collagen at a mass concentration of 1% had the strongest promoting effect on the growth of NIH3T3 cells.

[0154] 3. The test results of the effect of recombinant humanized type III collagen on NTH3T3 cell adhesion are shown in the figure below. Figure 10 As shown, the results showed that: 1% recombinant humanized type III collagen PBS solution and 1% gelatin coating solution (R20873, PBS dilution, w / w) can significantly promote the adhesion and adherence of NIH3T3 cells, and there is no statistical difference, indicating that recombinant humanized type III collagen can promote the adhesion of NIH3T3 cells.

[0155] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant humanized type III collagen, characterized in that: The amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO:

3.

2. The recombinant humanized type III collagen according to claim 1, characterized in that The nucleotide sequence of the cDNA encoding the recombinant humanized type III collagen is shown in SEQ ID NO:

4.

3. Use of the recombinant humanized type III collagen according to any one of claims 1 to 2 in the preparation of artificial organs and / or wound dressings.

4. A recombinant vector for producing the recombinant humanized type III collagen according to any one of claims 1 to 2, characterized in that: The recombinant vector is an optimized sequence having a nucleotide sequence such as SEQ ID NO: 2 inserted between the BamHI site and the EcoRI site of the plasmid.

5. The recombinant vector according to claim 4, characterized in that The plasmid is pNY326 plasmid.

6. A recombinant strain for producing the recombinant humanized type III collagen according to any one of claims 1 to 2, characterized in that: The recombinant strain is a strain containing the recombinant vector according to any one of claims 4 to 5.

7. The recombinant strain according to claim 6, characterized in that The strain is Bacillus pumilus.

8. Use of the recombinant vector according to any one of claims 4 to 5 and / or the recombinant strain according to any one of claims 6 to 7 in producing recombinant humanized type III collagen, wherein the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO:

3.

9. A method for producing recombinant humanized type III collagen, characterized in that: The following steps are involved: S1. The recombinant strain according to any one of claims 6 to 7 is inoculated into T2 medium at a final volume concentration of 0.1 to 5%, and cultured for 10 to 24 hours to obtain a fermentation seed solution; S2. The fermentation seed solution obtained in step S1 was inoculated into 2SLFC medium at a final volume concentration of 0.5 to 5%, and the dissolved oxygen level of the medium was controlled to be not less than 30%, and the pH value was 6.0 to 8.0 to obtain a fermentation product; The 2SLFC culture medium contains 2-4% glucose, 2-5% neutral soy peptone, 0.2-2% yeast extract powder, 0.2-2% fish meal, 0.2-2% cottonseed meal, 0.001-0.002% MnSO4·5H2O, 0.001-0.002% FeSO4·7H2O and 0.0001-0.0002% ZnSO4·7H2O in a mass concentration of 2-4%. S3. The fermentation product obtained in step S2 is separated into solid and liquid, and the liquid is collected and purified to obtain the recombinant humanized type III collagen with a nucleotide sequence as shown in SEQ ID NO:

3.

10. The method according to claim 9, characterized in that Step S2 is: controlling the dissolved oxygen level of the culture medium to 30% and the pH value to 7.0 for fermentation.

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