A preparation method of a recombinant human collagen III fragment tandem

Abstract

The present invention relates to a method for preparing a recombinant human collagen III fragment tandem. The amino acid sequence of the recombinant human collagen III fragment tandem is shown as SEQ NO.2, containing 396 amino acids, with a monomer molecular weight of 46 kDa and a trimer molecular weight of 138 kDa. The preparation method is as follows: constructing a Pichia pastoris engineering bacterium for constitutive secretion and expression of the recombinant human collagen III fragment tandem, and screening to obtain a highly expressing Pichia pastoris engineering bacterium; performing high-density fermentation culture on the recombinant yeast to achieve high-efficiency expression of the recombinant human collagen III fragment tandem; separating and purifying the recombinant human collagen III fragment tandem protein from the fermentation broth. The recombinant human collagen III fragment tandem of the present invention is a completely new amino acid sequence composed entirely of human collagen III fragments, and its encoding gene is a completely new artificial gene sequence; the present invention easily realizes the fermentation production of this collagen engineering bacterium.

Description

Technical Field:

[0001] The present invention relates to the field of biotechnology, and specifically relates to a method for preparing a highly hydrophilic recombinant human collagen fragment tandem. Background Art:

[0002] Collagen III is an important component of various human tissues, and plays a key role in maintaining the structure and elasticity of organs, especially in tissues such as blood vessel walls, internal organs, and embryos. Collagen tubes are mainly used for artificial blood vessels, artificial blood vessel catheters for the treatment of blood vessel stenosis and embolism, nerve repair catheters, etc. Its medicinal value is mainly reflected in wound healing, joint health, and bone health; its aesthetic value is mainly reflected in beauty skin care, anti-aging, and filling and shaping.

[0003] Natural collagen itself has a hemostatic function and can promote platelet aggregation and plasma clotting. Collagen can be used as an attachment and scaffold for cell growth and can induce the proliferation of epithelial cells, etc. The biodegradability of collagen enables it to be used as a material for organ transplantation. Collagen has a certain mechanical strength and is currently widely used in beauty filling. At present, collagen is a new favorite in the field of biomedicine and biomedical materials. Among them, collagen solution is mainly used for repairing skin tissue, soft tissue filling materials, bone tissue regeneration fillers, etc.; collagen sponge is mainly used for surgical hemostasis, oral resorbable dressings, tissue filling, tissue engineering scaffolds, etc., collagen film is mainly used for promoting the regeneration of periodontal ligament, artificial skin, skin covering for burns and scalds, promoting the healing of skin corneal epithelial cells, etc.; collagen particles are mainly used for fracture and bone defect fillers, drug sustained release, etc.

[0004] At present, generating collagen from animal skin tissues (such as pig skin, fish skin, donkey skin, etc.) through protease hydrolysis is a method. The produced collagen has a stable composition and biological activity, but it often has compositional heterogeneity, obvious antigenicity, and the potential risk of zoonotic viruses, which limits their wide application in the pharmaceutical and medical aesthetic fields. Currently, genetic recombination technology is the main method for producing recombinant human collagen. When expressing collagen in Escherichia coli, the pyrogen produced easily makes the expression product difficult to be applied clinically; at the same time, the target protein is often expressed in the form of inclusion bodies, making the product purification difficult; the post-translational processing and modification system of the prokaryotic expression system is imperfect, and collagen itself has many disulfide bonds, which leads to low biological activity of the prokaryotic expression product. To obtain recombinant collagen with better performance, sufficient post-translational modification of eukaryotes is necessary, including hydroxylation and glycosylation, etc. In 1997, the Collagen Research Center of the University of Oulu in Finland first expressed human collagen using Pichia pastoris, but the expression level was too low. Through the analysis of the collagen structure and attempts at yeast expression, it is possible to successfully express collagen fragments efficiently. In 2012, Liu Bin et al. used Pichia pastoris and induced it with methanol to efficiently secrete a highly hydrophilic human-like recombinant collagen dimer. Judging from numerous reports, the recombinant expression of human collagen in yeast has become a research hotspot, making its industrialization possible. Due to the many medicinal values of collagen, the domestic and international market demand for collagen has always been large. Currently, recombinant collagen is expensive because of its low yield, high cost of purification and separation. Therefore, developing a method for preparing collagen that is efficient, provides a stable source, and can maintain the structural integrity of the protein has important application value. Summary of the Invention:

[0005] The object of the present invention is to provide a method for preparing a tandem body of recombinant human collagen III fragments, and this method for preparing a tandem body of recombinant human collagen III fragments is used to solve the problems of low yield of recombinant collagen, high cost of purification and separation, and high price in the prior art.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows: This method for preparing a tandem body of recombinant human collagen III fragments includes the following steps:

[0007] Step 1: Construct a Pichia pastoris engineering bacterium expressing a tandem body of recombinant human collagen III fragments, and screen to obtain a Pichia pastoris engineering bacterium with high expression:

[0008] (1) Based on the amino acid sequence of the tandem fragment of human collagen III and according to the codon preference of Pichia pastoris, optimize it using the high-frequency codons of Pichia pastoris, add the TGA stop codon, with a full length of 1191 bp, named 396C, and the gene optimization result is shown in SEQ NO.3;

[0009] (2) The optimized gene was inserted into the Pichia pastoris constitutive secretion expression vector pGAPZaA through seamless synthesis; then it was transformed into Escherichia coli to construct and screen positive strains containing pGAPZa-396; the linearized pGAPZa-396 cDNA was transformed into Pichia pastoris SDM1168 for the expression of the tandem of human collagen III fragments, and the genetically engineered Pichia pastoris strain was obtained by screening;

[0010] Step 2: The recombinant yeast was cultured by high-density fermentation to achieve the high-efficiency expression of the recombinant human collagen III fragment tandem.

[0011] Step 3: The recombinant human collagen III fragment tandem protein was isolated and purified from the fermentation broth.

[0012] In the above scheme, the recombinant human collagen III fragment tandem is based on the triple peptide repeat sequence characteristics of the collagen domain Gly-X-Y of the human type III collagen α1 chain and its strong hydrophilicity. 99 amino acid fragments from the 981st amino acid to the 1079th amino acid of human collagen III were selected and tandemly repeated 4 times to obtain a structure containing 396 amino acids, as shown in SEQ NO.2. The monomer molecular weight is 46 kDa, and the trimer molecular weight is 138 kDa; the 99 amino acid fragment from the 981st amino acid to the 1079th amino acid of human collagen III is shown in SEQ NO.1.

[0013] The specific content of Step 2 in the above scheme is as follows:

[0014] The high-density fermentation technology was adopted, with the YPD medium as the basic medium. Through indirect fed-batch culture, the cell density and protein expression level of the genetically engineered Pichia pastoris strain were increased; meanwhile, the seed medium was YPD, the loading volume of the Erlenmeyer flask was 1 / 5 of the capacity, and the culture conditions were 30 °C and 230 rmp. When OD 600 = 1.3 - 1.5, it was stored at 4 °C for standby; the fermentation medium formula was 2% peptone, 1% yeast extract, 2.5% glucose, 0.02% antifoam agent, the inoculation amount was 10%, and the optimal fermentation conditions were 30 °C, the aeration rate was 1.3 - 1.5 vvm, the tank pressure was 0.17 - 0.3 MPa, the stirring speed was 300 - 350 r / min, to make the dissolved oxygen > 30%, and 20% ammonia water was added dropwise to maintain the pH value at 7.2 - 7.5; after 18 hours of fermentation, 20% sterile glucose and a sterile mixed solution containing 20% peptone and 10% yeast extract were added dropwise for fed-batch feeding. The feeding time was 36 hours to maintain a 1% glucose concentration in the fermentation broth. When the cell mass concentration reached 320 g / L and OD 600 reached 300, the fermentation was terminated, and the fermentation time was 60 - 62 hours.

[0015] The specific content of Step 3 in the above scheme is as follows:

[0016] Centrifuge the fermentation broth at 4°C and 8000 - 10000g for 10 minutes, and take the supernatant; slowly add ammonium sulfate powder with stirring until the ammonium sulfate saturation of the solution reaches 50%, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, take the precipitate, wash the precipitate once with 50% ammonium sulfate solution, centrifuge at 9000 - 10000g for 15 minutes, dissolve the precipitate with distilled water, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, and take the supernatant; desalt the supernatant using Sephadex G-50 and 20 mM Tris-Cl pH 7.5 solution, and collect the solution with a UV detection peak at 280 nm; then perform column chromatography using Q Seplife FF ion exchange, equilibrate the column with 20 mM Tris-Cl pH 7.5 buffer, after loading the sample, elute linearly with pH 7.5, 20 mM Tris-Cl plus 1 M NaCl, and collect the elution peak detected by UV at 280 nm with 20 mM Tris-Cl plus 0.15 M NaCl; then separate and purify using Sephadex G-100 gel column chromatography, elute with PBS solution, and collect the liquid with the largest peak, which is the target protein. The solution is ultrafiltered and concentrated with a molecular weight cut-off of 30 kDa, and then lyophilized to obtain the recombinant human collagen III fragment tandem.

[0017] The yield of the recombinant human collagen III fragment tandem in the above scheme reaches 6 - 7 g / L.

[0018] The present invention has the following beneficial effects:

[0019] 1. The present invention provides a method for constitutive and highly efficient secretory expression of recombinant human collagen III fragment tandem using recombinant Pichia pastoris and its fermentation and extraction technology for the recombinant collagen. The amino acid structure and gene of the recombinant human collagen are different from those reported currently. Compared with methanol-induced Pichia pastoris, this high-density fermentation is simpler and easier to operate, has a shorter fermentation period, uses a simpler and cheaper culture medium formula, and the purification and extraction method is more efficient and convenient.

[0020] 2. The recombinant human collagen III fragment tandem of the present invention is a brand-new amino acid sequence, but is completely composed of human collagen III fragments, and its encoded gene is a brand-new artificial gene sequence; this method easily realizes the fermentation production of this collagen engineering bacterium, and the expressed recombinant protein has the characteristic of promoting the growth of fibroblasts.

[0021] 3. The present invention can efficiently and stably produce recombinant human collagen III fragment tandem with uniform structure and maintain the integrity of its protein structure. Compared with the traditional methanol induction method of Pichia pastoris, this method has better operability, lower culture medium cost, shorter fermentation time, and is more suitable for industrial production. Brief Description of Drawings:

[0022] Figure 1 It is a diagram of the insertion site of the tandem gene of human collagen III fragment in pGAPZaA.

[0023] Figure 2 It is a diagram for screening high-resistant recombinant strains.

[0024] Figure 3 It is SDS-PAGE of recombinant human collagen III fragment tandem protein secreted and expressed by different recombinant yeast strains. The figure shows significant differences in the expression levels of the target proteins of different recombinant yeast strains. The arrow indicates the target protein of the highly expressing strain.

[0025] Figure 4 It is an SDS-PAGE electrophoresis diagram of the target protein secreted and expressed by recombinant yeast during fermentation for 1 - 84 hours. From right to left in the lanes of the figure are the expression results of Marker, 24h, 48h, 60h, and 72h.

[0026] Figure 5 It is an SDS-PAGE electrophoresis diagram of the target protein precipitated by different ammonium sulfate saturation levels. From right to left in the lanes of the figure are the precipitation effects at 25%, 35%, 45%h, 55%, 60%, 65%, and 70% saturation levels.

[0027] Figure 6 It is an SDS-PAGE diagram of purified recombinant human collagen III fragment tandem.

[0028] Figure 7 It is a non-denaturing 6% polyacrylamide electrophoresis diagram of recombinant human collagen III fragment tandem. Detailed Implementation Manner:

[0029] The present invention will be further described below with reference to the drawings:

[0030] This recombinant human collagen III fragment tandem has an amino acid sequence as shown in SEQ NO.2, containing 396 amino acids. The monomer molecular weight is 46 kDa, and the trimer molecular weight is 138 kDa. Its preparation method includes: constructing a Pichia pastoris engineering bacterium for constitutive secretion and expression of the recombinant human collagen III fragment tandem, screening to obtain a highly expressing Pichia pastoris engineering bacterium; performing high-density fermentation culture on the recombinant yeast to achieve high-efficiency expression of the recombinant human collagen III fragment tandem; separating and purifying the recombinant human collagen III fragment tandem protein from the fermentation broth.

[0031] I. Design of Recombinant Human Collagen III Fragment Tandem and Its Amino Acid Structure

[0032] Based on the triple - peptide repeat sequence characteristics and strong hydrophilicity of the Gly - X - Y collagen domain of human type III collagen α1 chain, a 99 - amino - acid fragment from the 981st amino acid to the 1079th amino acid of human - sourced collagen III, such as SEQ NO.1, was selected. This fragment was concatenated 4 times to obtain the following 396 - amino - acid structure, as shown in SEQ NO.2.

[0033] II. Construction of the gene for expressing the recombinant human - sourced collagen III fragment tandem and the genetic engineering bacterium is as follows:

[0034] 1) Based on the codon preference of Pichia pastoris and the amino - acid sequence of the human collagen III tandem fragment, it was optimized using the high - frequency codons of Pichia pastoris, and a TGA stop codon was added. The full - length is 1191bp and it is named 396C. The gene optimization result is shown in SEQ NO.3 as follows.

[0035] 2) The optimized gene was inserted into the Pichia pastoris constitutive secretion expression vector pGAPZaA through seamless synthesis, as shown at the red arrow above; then it was transformed into Escherichia coli to construct and screen the positive strain containing pGAPZa - 396. The linearized pGAPZa - 396C DNA was transformed into Pichia pastoris SDM1168 for the expression of the human - sourced collagen III fragment tandem, and the Pichia pastoris genetic engineering bacterium was screened. Figure 1

[0036] III. The fermentation culture conditions of the genetic engineering bacterium and the constitutive secretion expression conditions of the recombinant human - sourced collagen III fragment tandem are as follows:

[0037] The high - density fermentation technology was adopted. Using the YPD medium as the basic medium, through indirect fed - batch culture, the cell density and protein expression level of the Pichia pastoris engineering strain were increased. Meanwhile, the seed medium was YPD, the loading volume in the Erlenmeyer flask was 1 / 5 of the capacity, and the culture conditions were 30°C and 230rmp. When OD 600 = 1.3 - 1.5, it was stored at 4°C for standby. The fermentation medium formula was 2% peptone, 1% yeast extract, 2.5% glucose, 0.02% antifoam agent, the inoculation amount was 10%, and the optimal fermentation conditions were 30°C, the ventilation rate was 1.3 - 1.5vvm, the tank pressure was 0.17 - 0.3MPa, the stirring speed was 300 - 350r / min, to make the dissolved oxygen > 30%, and 20% ammonia water was fed to maintain the pH value at 7.2 - 7.5; after 18 hours of fermentation, 20% sterile glucose and a sterile mixed solution containing 20% peptone and 10% yeast extract were respectively fed for feeding, the feeding time was 36 hours, maintaining a 1% glucose concentration in the fermentation broth. When the cell mass concentration reached 320g / L and OD 600 reached 300, the fermentation broth was discharged from the tank, and the fermentation time was 60 - 62 hours.​

[0038] IV. The extraction and purification of the recombinant human collagen III fragment tandem is as follows:

[0039] Centrifuge the fermentation broth at 4°C and 8000 - 10000g for 10 minutes, and take the supernatant; slowly add ammonium sulfate powder with stirring until the ammonium sulfate saturation of the solution reaches 50%, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, take the precipitate, wash the precipitate once with 50% ammonium sulfate solution, centrifuge at 9000 - 10000g for 15 minutes, dissolve the precipitate with distilled water, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, and take the supernatant; desalt the supernatant using Sephadex G-50 and 20 mM Tris-Cl pH 7.5 solution, and collect the solution with the ultraviolet detection peak at 280 nm; then perform column chromatography using Q Seplife FF ion exchange, equilibrate the column with 20 mM Tris-Cl pH 7.5 buffer, after loading the sample, perform linear elution with pH 7.5, 20 mM Tris-Cl plus 1 M NaCl, and collect the elution peak detected by ultraviolet at 280 nm with 20 mM Tris-Cl plus 0.15 M NaCl; then perform separation and purification using Sephadex G-100 gel column chromatography, elute with PBS solution, and collect the liquid with the largest peak as the target protein. The solution is ultrafiltered and concentrated with a molecular weight cut-off of 30 kDa, and then freeze-dried to obtain the finished product.

[0040] The preparation method of this recombinant human collagen III fragment tandem is described in detail as follows:

[0041] Selection of human collagen III fragment

[0042] Select the amino acids from positions 981 to 1079 from human collagen III, and use the 99-amino acid fragment containing three disulfide bonds as the tandem basis. Its sequence is the amino acid sequence of the basic fragment of human collagen III, as shown in SEQ NO.1:

[0043]

[0044] Amino acid sequence of the human collagen III tandem fragment:

[0045] Tandemly connect the above basic fragments of human collagen III four times to obtain the following structure of 396 amino acids, and the sequence is shown in SEQ NO.2:

[0046]

[0047] Codon-optimized gene construction:

[0048] Based on the codon preference of Pichia pastoris, taking the amino acid sequence of the tandem fragment of human collagen III as the basis, the high-frequency codons of Pichia pastoris were used to optimize it, avoiding the AvrII restriction site. The gene sequence synthesized artificially and chemically after optimization is as follows SEQ NO.3, is the stop codon, with a full length of 1191bp, named 396C, and the sequence is shown in SEQ NO.3:

[0049]

[0050] Construction of constitutive secretion-expressing recombinant yeast and screening of highly expressing recombinants:

[0051] The gene of the tandem fragment of human collagen III was inserted into the Pichia pastoris constitutive secretion expression vector pGAPZaA by seamless chemical synthesis technology, and the site is as Figure 1 shown by the red arrow; then it was transformed into DH5a, and recombinant Escherichia coli was screened using a low-salt LB plate containing 25 μg / mL bleomycin, and PCR identification was carried out.

[0052] The recombinant Escherichia coli was cultured in a low-salt LB liquid medium containing 25 μg / mL bleomycin, and the recombinant plasmid pGAPZa-396C was extracted using a plasmid extraction kit. Then, the plasmid was linearized using the AvrII rapid restriction enzyme, and the linearization result was detected by agarose electrophoresis; after complete linearization, it was extracted once with phenol-chloroform (1:1) to remove the restriction enzyme. Then, 1 / 10 volume of 3M sodium acetate and 2.5 - 3 volumes of absolute ethanol were added, and the precipitated linearized DNA was recovered by high-speed centrifugation. The precipitate was washed twice with 80% ethanol, dried, and 12 μL of deionized water was added, and the amount of DNA should reach 5 - 10 μg.

[0053] Prepare SMD1168 Pichia pastoris competent cells. Mix 10 μL of linearized pGAPZa-396C with 80 μL of SMD1168 competent cells, add them to a 0.2 cm electroporation cuvette, ice-bath for 5 min, then perform electroporation using an electroporator at 1500 v. After electroporation, quickly add 1 mL of 1M cold sorbitol, place it in a 15 mL sterile centrifuge tube, stand at 30 °C for 1 - 2 h, then mix well and take 150 μL of the solution and spread it on a YPDS plate containing 100 μg / mL bleomycin, and incubate it upside down at 30 °C for 3 - 4 days until colonies appear; then use a sterile pipette tip to pick the colonies and spot them onto a YPDS plate containing 500 μg / mL bleomycin, and incubate it upside down at 30 °C for 2 - 3 days, see Figure 2, Select recombinant yeast strains with large colonies, and inoculate them into YPD liquid medium containing 100 μg / mL bleomycin respectively. Culture them in a shaker at 30 °C and 240 rmp for 3 days, then centrifuge at 1500 g for 5 minutes, take the supernatant, perform SDS-PAGE electrophoresis and thin-layer scanning analysis, and screen out recombinant yeasts that highly secrete and express the tandem of human collagen III fragments by the thickness of the stained target band, such as Figure 3 The arrow indicates the highly expressed strain.

[0054] High-density fermentation method:

[0055] Use an automatic fermenter, with YPD medium as the basal medium, the liquid loading volume is 2 / 5. Adopt high-density fermentation technology, through indirect fed-batch culture, to improve the cell density and protein expression level of Pichia pastoris engineering strains. At the same time, the seed medium is YPD, the loading volume in the Erlenmeyer flask is 1 / 5 of the volume, and the culture conditions are 30 °C and 240 rmp. When OD600 = 1.3 - 1.5, store at 4 °C for standby. The fermentation medium formula is 2% peptone, 1% yeast extract, 2.5% glucose, and 0.02% antifoam agent. High-density fermentation conditions: the inoculation amount is 10%, the optimal fermentation temperature is 30 °C, the oxygen supply is 1.3 - 1.5 vvm, the stirring speed reaches 300 - 350 r / min, the tank pressure is 0.18 - 0.3 MPa, the dissolved oxygen > 30%, and ammonia water with a volume fraction of 25% is added dropwise to maintain the pH value at 7.2 - 7.5; after 18 hours of fermentation, a sterile mixed solution containing 20% sterile glucose, 20% peptone, and 10% yeast extract is added dropwise for feeding to maintain a glucose concentration of 1% in the fermentation broth. The feeding time is 36 hours. When the cell mass concentration reaches 320 g / L and OD 600 reaches 300, discharge the tank, and the fermentation time is 60 - 62 hours. The expression level of the recombinant tandem of human collagen III fragments protein gradually increases with the extension of fermentation time. As Figure 4 shown, the expression level of the target protein gradually increases, and in the fermentation broth, the recombinant tandem of human collagen III fragments protein can reach 6 - 7 g / L.

[0056] Extraction method:

[0057] Centrifuge the fermentation broth at 4 °C and 8000 - 10000 g for 10 minutes, take the supernatant; slowly add ammonium sulfate powder with stirring to make the ammonium sulfate saturation of the solution reach 50%, then centrifuge at 4 °C and 9000 - 10000 g for 15 minutes, take the precipitate, wash the precipitate once with 50% ammonium sulfate solution, centrifuge at 9000 - 10000 g for 15 minutes, collect the precipitate, and the ammonium sulfate precipitation effect is as Figure 5; The precipitate was dissolved in distilled water, centrifuged at 4°C and 9000 - 10000 g for 15 minutes, and the supernatant was taken; The supernatant was desalted with Sephadex G-50 and 20 mM Tris-Cl pH 7.5 solution, and the solution with a UV detection peak at 280 nm was collected; Then, column chromatography was performed using Q Seplife FF ion exchange. The column was equilibrated with 20 mM Tris-Cl pH 7.5 buffer. After sample loading, linear elution was performed with pH 7.5, 20 mM Tris-Cl plus 1 M NaCl, and the elution peak detected by UV at 280 nm with 20 mM Tris-Cl plus 0.15 M NaCl was collected; Then, separation and purification were performed using Sephadex G-100 gel column chromatography, eluted with PBS solution, and the liquid with the largest peak was collected as the target protein. The solution was ultrafiltered and concentrated with a molecular weight cut-off of 30 kDa, and then lyophilized to obtain the finished product. After lyophilization, it was a recombinant human-derived collagen III fragment tandem protein with a purity of ≥96%, as Figure 6 shown.

[0058] Protein structure identification:

[0059] Determined by SDS-PAGE, the monomer molecular weight of this recombinant human collagen III tandem is 46 KDa, as Figure 2 The theoretical prediction is 35208 Da, and the molecular weight measured by high-resolution Xevo G2-XS QTof (Waters) mass spectrometry is 35263.5 Da. Determined by non-denaturing PAGE electrophoresis (as Figure 7 analysis showed that this recombinant human collagen III fragment tandem is a trimer.

[0060] The predicted isoelectric point of this protein is 5.8. Measured by isoelectric focusing electrophoresis, the pI of this recombinant human collagen III tandem is 6.0 - 6.2, slightly larger than the prediction.

[0061] Analysis by infrared spectroscopy scanning, the wavenumbers of amide A, amide B, amide I, amide II, and amide III can determine that this recombinant protein has similar structural characteristics to natural collagen.

[0062] Determined by the MTT method, this recombinant collagen with a concentration greater than 0.1 mg / mL can significantly promote the growth of NIH3T3 cells.

[0063] Predicted by AlphaFold, the similarity between this recombinant human collagen III fragment tandem and human collagen I chain reaches 64%, and the structural correctness reaches 73%; The similarity between this recombinant human collagen III fragment tandem and human collagen a-1 chain reaches 64%, and the structural correctness reaches 72%. It indicates that this recombinant human collagen III fragment tandem has similar functionality to them.

Claims

1. A method for preparing a recombinant tandem of human collagen III fragments, characterized in that It includes the following steps: Step 1: Construct a Pichia pastoris engineering bacterium expressing a recombinant human collagen III fragment tandem, and screen to obtain a Pichia pastoris engineering bacterium with high expression: (1) Based on the codon preference of Pichia pastoris and the amino acid sequence of the recombinant human collagen III fragment tandem, optimize it using the high-frequency codons of Pichia pastoris, add a TGA stop codon, with a full length of 1191 bp, named 396C, and the gene optimization result is shown in SEQ ID NO.3; (2) Insert the optimized gene into the Pichia pastoris constitutive secretion expression vector pGAPZaA by seamless synthesis; then transform Escherichia coli to construct and screen positive strains containing pGAPZa-396C; transform the linearized pGAPZa-396C DNA into Pichia pastoris SDM1168 for the expression of the human collagen III fragment tandem, and screen to obtain the Pichia pastoris engineering bacterium; Step 2: Conduct high-density fermentation culture on the Pichia pastoris engineering bacterium to achieve high expression of the recombinant human collagen III fragment tandem; Step 3: Isolate and purify the recombinant human collagen III fragment tandem protein from the fermentation broth.

2. The preparation method of the recombinant human collagen III fragment tandem according to claim 1, characterized in that: The recombinant human collagen III fragment tandem is based on the triple peptide repeat sequence characteristics of the collagen domain Gly-X-Y of the human type III collagen α1 chain and its strong hydrophilicity. Select 99 amino acid fragments from the 981st to 1079th amino acids of human collagen III and tandem them 4 times to obtain a structure containing 396 amino acids, as shown in SEQ ID NO.2, with a monomer molecular weight of 46 kDa and a trimer molecular weight of 138 kDa; the 99 amino acid fragment from the 981st to 1079th amino acids of human collagen III is shown in SEQ ID NO.

1.

3. The preparation method of the recombinant human collagen III fragment tandem according to claim 2, characterized in that: The specific content of Step 2 is: Using high-density fermentation technology, with YPD medium as the basal medium, through indirect fed-batch culture to increase the cell density and protein expression level of the engineered Pichia pastoris strain; meanwhile, the seed medium is YPD, the loading volume in the Erlenmeyer flask is 1 / 5 of the capacity, the culture conditions are 30 °C and 230 rmp. When OD 600 = 1.3 - 1.5, it is stored at 4 °C for standby; the fermentation medium formula is 2% peptone, 1% yeast extract, 2.5% glucose, 0.02% antifoam agent, the inoculation amount is 10%, the optimal fermentation conditions are 30 °C, the aeration rate is 1.3 - 1.5 vvm, the tank pressure is 0.17 - 0.3 MPa, the stirring speed is 300 - 350 r / min to make the dissolved oxygen > 30%, and ammonia water with a volume fraction of 20% is fed to maintain the pH value at 7.2 - 7.5; after 18 hours of fermentation, sterile glucose with a concentration of 20% and a sterile mixed solution containing 20% peptone and 10% yeast extract are respectively used for fed-batch feeding, the feeding time is 36 hours, and the glucose concentration in the fermentation broth is maintained at 1%. When the cell mass concentration reaches 320 g / L and OD 600 reaches 300, the fermentation broth is discharged, and the fermentation time is 60 - 62 hours.

4. The preparation method of the recombinant human collagen III fragment tandem according to claim 3, characterized in that: The specific content of Step 3 is: Centrifuge the fermentation broth at 4°C and 8000 - 10000g for 10 minutes, and take the supernatant; slowly add ammonium sulfate powder with stirring until the ammonium sulfate saturation of the solution reaches 50%, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, take the precipitate, wash the precipitate once with 50% ammonium sulfate solution, centrifuge at 9000 - 10000g for 15 minutes, dissolve the precipitate in distilled water, then centrifuge at 4°C and 9000 - 10000g for 15 minutes, and take the supernatant; desalt the supernatant with Sephadex G - 50 and 20 mM Tris - Cl pH 7.5 solution, and collect the solution with a UV detection peak at 280 nm; then perform column chromatography using Q Seplife FF ion exchange, equilibrate the column with 20 mM Tris - Cl pH 7.5 buffer, after loading the sample, perform linear elution with pH 7.5, 20 mM Tris - Cl plus 1M NaCl, and collect the elution peak detected by UV at 280 nm with 20 mM Tris - Cl plus 0.15M NaCl; then separate and purify using Sephadex G - 100 gel column chromatography, elute with PBS solution, and collect the liquid with the largest peak, which is the target protein. The solution is ultrafiltered and concentrated with a molecular weight cut - off of 30 kDa, and then lyophilized to obtain the recombinant human - derived collagen III fragment tandem body.

5. The preparation method of the recombinant human collagen III fragment tandem according to claim 4, characterized in that: The yield of the recombinant human - derived collagen III fragment tandem body reaches 6 - 7 g / L.

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