A recombinant type III humanized collagen and its applications

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a recombinant type III humanized collagen and its application. In the present invention, regions with good hydrophilicity in the α-chain of human type III collagen are selected and connected in sequence as the expressed protein. The amino acid sequence is 100% identical to the corresponding part of natural human type III collagen and will not cause immune rejection when applied to the human body. The present invention transfers the newly screened type III humanized collagen sequence into Pichia pastoris for recombinant expression. The prepared recombinant type III humanized collagen has the advantages of strong stability, high expression level and good hydrophilicity. At the same time, the recombinant type III humanized collagen also has a significant effect on promoting cell proliferation and cell adhesion.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a recombinant type III humanized collagen and its application. Background Art

[0002] Collagen is a family of proteins with multiple types. It is the most abundant protein in the human body, accounting for 25-30% of the total protein. Collagen is distributed in various parts such as skin, tendon, cornea, cartilage, and blood vessels, and can play a certain role in structural support, thus maintaining the normal morphology of various organs and tissues. Common collagens include type I, type III, type XVII, etc. Among them, type III collagen is composed of three α1 chains and is a type of fibrous collagen, which has the highest content in infants and young children and then decreases continuously with age.

[0003] At present, the main sources of collagen include extraction from animal tissues and preparation by DNA recombination technology, etc. Collagen can be extracted from animal tissues by acid and alkali hydrolysis methods. However, the collagen extracted from animal tissues contains many collagen fibers that are insoluble in water, and inevitably, partial loss of the biological activity of collagen will occur during the extraction process. In addition, the collagen extracted from animal tissues is a heterologous protein, which may cause risks such as immune rejection, allergy, and diseases.

[0004] The development of recombinant technology enables the high-purity and high-specificity production of type III collagen without the use of animal sources, laying a solid foundation for its application in the fields of skin care products and medical devices. For example: the paper titled "Optimization of Recombinant Expression and Characterization of Human Type III Collagen in Pichia pastoris" published by Cai Size. This paper intercepted the amino acids at positions 908 to 1137 of the α1 chain of human type III collagen as the expressed protein and carried out recombinant expression in Pichia pastoris to produce recombinant humanized collagen. This method uses DNA recombination technology and selects the full-length or partial amino acid sequence fragments encoded by specific types of human collagen genes, or a combination of functional fragments of human collagen to prepare recombinant humanized collagen, which overcomes the disadvantages of heterologous proteins.

[0005] Patent document CN114085284A discloses a recombinant type III humanized collagen, nucleic acid, vector and implant. The recombinant type III humanized collagen selects the sequence peptide segments 549-560aa, 597-613aa, 881-902aa, 648-662aa with good stability obtained through long-term screening in the natural human type III collagen α1 chain. The four peptide chains are spliced in sequence to form a monomer, and then the monomer is expressed in series. Through fermentation and purification, the obtained recombinant type III humanized collagen has high stability, effectively solves the problem of degradation of recombinant type III humanized collagen during clinical use, and can be prepared into a liquid preparation for storage and use.

[0006] Patent document CN117264047A discloses a recombinant type III humanized collagen amino acid sequence, its preparation method and application. The amino acid sequence includes n repeated sequences, which are directly connected between each repeated sequence. The recombinant type III humanized collagen is highly expressed by using the engineered Escherichia coli high-density fermentation technology. Then, the recombinant type III humanized collagen is modified with PEG with a uniform molecular weight. The prepared recombinant type III humanized collagen has the advantages of good hydrophilicity, high stability and good biocompatibility.

[0007] However, currently, the recombinant collagen prepared by genetic engineering methods still has problems such as low expression level, low solubility or low activity of recombinant collagen. Summary of the Invention

[0008] In order to solve the defects of the prior art, the present invention provides a recombinant type III humanized collagen and its application. The present invention designs a new type III humanized collagen sequence to ensure that the recombinant type III humanized collagen product has high stability and good solubility. The present invention transfers the screened new type III humanized collagen sequence to a Pichia pastoris strain for recombinant expression. The prepared recombinant type III humanized collagen has the advantages of strong stability, high expression level and good hydrophilicity. At the same time, the recombinant type III humanized collagen also has a significant effect of promoting cell proliferation and cell adhesion.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The present invention provides a recombinant type III humanized collagen, and the amino acid sequence of the recombinant type III humanized collagen is as shown in SEQ ID NO:1.

[0011] Furthermore, the present invention provides a coding gene of the recombinant type III humanized collagen, and the nucleotide sequence of the coding gene of the recombinant type III humanized collagen is as shown in SEQ ID NO:2.

[0012] Furthermore, the present invention also provides a recombinant vector of recombinant type III humanized collagen, and the recombinant vector contains the coding gene of the above-mentioned recombinant type III humanized collagen.

[0013] Furthermore, the present invention also provides an engineered strain, and the engineered strain contains the coding gene of the above-mentioned recombinant type III humanized collagen or the above-mentioned recombinant vector of recombinant type III humanized collagen.

[0014] In addition, the present invention also provides the application of the recombinant type III humanized collagen in the preparation of cosmetic or medical device products.

[0015] Furthermore, the application of the recombinant type III humanized collagen in the preparation of anti-aging cosmetics, repair cosmetics or antioxidant cosmetics.

[0016] Furthermore, the application of the recombinant type III humanized collagen in the preparation of a collagen dressing with the functions of promoting cell proliferation, adhesion and migration.

[0017] In addition, the present invention also provides a composition, and the composition contains the above-mentioned recombinant type III humanized collagen and an acceptable excipient.

[0018] The recombinant type III humanized collagen provided by the present invention selects the hydrophilic region fragments in the α chain of human type III collagen and connects them in sequence to obtain the recombinant type III humanized collagen shown in SEQ ID NO.1. The recombinant type III humanized collagen prepared by the present invention has the advantages of strong stability, high expression level and good hydrophilicity. At the same time, it also has a significant effect of promoting cell proliferation and cell adhesion. In addition, the amino acid sequence of the recombinant type III humanized collagen provided by the present invention is 100% identical to the corresponding part of natural human type III collagen and will not cause immune rejection when applied to the human body.

[0019] In summary, compared with the prior art, the recombinant type III humanized collagen provided by the present invention has the following advantages:

[0020] (1) The amino acid sequence of the recombinant type III humanized collagen provided by the present invention is 100% identical to the corresponding part of natural human type III collagen and will not cause immune rejection when applied to the human body.

[0021] (2) The amino acid sequence of the recombinant type III humanized collagen provided by the present invention can be successfully expressed and secreted in the yeast expression system, and the expression level is relatively high, up to 12.04 g / L.

[0022] (3) The recombinant type III humanized collagen provided by the present invention has strong solubility, and its solubility in salt solution can reach 100 g / L.

[0023] (4) The recombinant type III humanized collagen provided by the present invention has a significant promoting effect on the proliferation of human embryonic skin fibroblasts (ESF). The cell proliferation rate of the recombinant type III humanized collagen at 0.0625 mg / mL is 122.01%, which is 1.17 times that of the bovine type I collagen standard at 1 mg / mL.

[0024] (5) The recombinant type III humanized collagen provided by the present invention has a significant promoting effect on the adhesion of L929 cells. The cell adhesion rate of the recombinant type III humanized collagen at 1 mg / mL is 82.01%, which is 2.80 times that of the bovine type I collagen standard at 2 mg / mL. Description of the Drawings

[0025] Figure 1 It is a result diagram of the hydrophobicity analysis of the recombinant type III humanized collagen.

[0026] Figure 2 It is a schematic diagram of the construction of the recombinant type III humanized collagen plasmid pPICZαA-hcc2.

[0027] Figure 3 It is an SDS-PAGE electrophoresis diagram of the shake flask expression of the recombinant type III humanized collagen, (M: Marker; 1: negative control, 2: verification of the shake flask expression of the recombinant type III humanized collagen Pichia pastoris genetic engineering bacteria).

[0028] Figure 4 It is a result diagram of the HPLC detection of the fermentation supernatant of the recombinant type III humanized collagen.

[0029] Figure 5 It is an SDS-PAGE electrophoresis result diagram of the purified recombinant type III humanized collagen, (1: recombinant type III humanized collagen; M: Marker).

[0030] Figure 6 It is a result diagram of the HPLC detection of the purified recombinant type III humanized collagen.

[0031] Figure 7 It is a result diagram of the proliferation rate of the recombinant type III humanized collagen on ESF cells, (NC is the blank control, PC is the bovine type I collagen standard).

[0032] Figure 8 It is a result diagram of the proliferation rate of the recombinant type III humanized collagen on ESF cells combined with the observation of the cell state.

[0033] Figure 9 Adhesion rate results of recombinant humanized type III collagen to L929 cells. (NC is the blank control, and the reference product is bovine type I collagen standard).

[0034] Figure 10 Relative adhesion results of recombinant humanized type III collagen to L929 cells. (NC is the blank control, and the reference product is bovine type I collagen standard).

[0035] Figure 11 Cell adhesion results of recombinant humanized type III collagen to L929 cells. (NC is the blank control, the reference product is bovine type I collagen standard, and PC is 0.1% gelatin). Detailed implementation manners

[0036] The present invention is further described below through the description of specific implementation manners. However, this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] Example 1: Design and synthesis of recombinant humanized type III collagen gene

[0038] 1. Amino acid sequence of recombinant humanized type III collagen:

[0039] The hydrophobicity of the α-chain of human type III collagen was analyzed, and a region fragment with a certain length and good hydrophilicity was selected and connected in sequence to obtain the amino acid sequence of recombinant humanized type III collagen shown in SEQ ID NO.1. The hydrophobicity analysis of SEQ ID NO.1 was performed, and the hydrophobicity analysis result of the recombinant humanized type III collagen prepared in the present invention is as Figure 1 shown. It can be seen from Figure 1 that the amino acid sequence of the recombinant humanized type III collagen prepared in the present invention has good hydrophilicity.

[0040] The amino acid sequence of the recombinant humanized type III collagen is (SEQ ID NO:1):

[0041] GARGNDGARGSDGQPGPPGPPGTAGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGGAGPPGPEGGKGAAGPPGPPGAAGTPGMP GERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGAPGQNGEPGGKGERGAPGEKGEGGPPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAP。

[0042] 2. Nucleotide sequence of recombinant type III humanized collagen:

[0043] The corresponding nucleotide sequence was designed according to the codon preference of Pichia pastoris for the amino acid sequence of the above recombinant type III humanized collagen, and the nucleotide sequence of the recombinant type III humanized collagen was optimized according to the codon preference of Pichia pastoris to obtain the nucleotide sequence of the recombinant type III humanized collagen shown in SEQ ID NO.2. It was commissioned to Nanjing Genscript Biotech Co., Ltd. for synthesis.

[0044] The nucleotide sequence of the recombinant type III humanized collagen is (SEQ ID NO:2):

[0045]

[0046] 3. Recombinant plasmid construction:

[0047] The nucleotide sequence of the synthesized recombinant type III humanized collagen was constructed into the pPICZαA vector to obtain a recombinant type III humanized collagen plasmid, named pPICZαA-hcc2. The schematic diagram of the recombinant type III humanized collagen plasmid pPICZαA-hcc2 constructed in the present invention is as Figure 2 shown.

[0048] Example 2. Preparation of recombinant type III humanized collagen gene engineering strain

[0049] 1. Linearized plasmid preparation:

[0050] The plasmid was linearized by single enzyme digestion. The recombinant type III humanized collagen plasmid pPICZαA-hcc2 prepared in Example 1 was extracted, digested with SacI enzyme, and then recovered by ethanol precipitation to obtain a recombinant type III humanized collagen linearized plasmid.

[0051] 2. Yeast competent preparation:

[0052] The Pichia pastoris strain X-33 was inoculated into YPD liquid medium and cultured overnight at 28°C and 220 rpm, and then the OD600 value of the bacterial solution was measured. It was inoculated into 50 mL of YPD liquid medium according to the initial OD600 = 0.5 and cultured until OD600 reached 1.3 - 1.5. After centrifugation at 4°C and 3000 g for 5 min, the medium was discarded. 50 mL of sterile water was added to resuspend the yeast cells, and after centrifugation at 4°C and 3000 g for 5 min, the supernatant was discarded. 25 mL of sterile water was added to resuspend the yeast cells, and after centrifugation at 4°C and 3000 g for 5 min, the supernatant was discarded. 1 mL of pre-cooled sorbitol was added to resuspend the yeast cells, and after centrifugation at 4°C and 3000 g for 5 min, the supernatant was discarded. After adding 100 μL of pre-cooled sorbitol to resuspend the yeast cells, the bacterial solution was aliquoted at 80 μL / tube.

[0053] 3. Transformation of Pichia pastoris:

[0054] Take 10 μL of the prepared recombinant type III humanized collagen linearized plasmid, add it to the Pichia pastoris competent cells, mix well, transfer it to an electroporation cup pre-cooled on ice, and incubate in ice for more than 5 min. Wipe the outer wall of the electroporation cup dry, put it into the electroporator for electroporation, then add 1 mL of sorbitol to suspend the cells, and then transfer the liquid to a 1.5 mL centrifuge tube. Incubate at 28°C for 3 - 12 h and then spread it on a YPD plate containing 0.1 mg / mL bleomycin, and incubate at 28°C until colonies grow.

[0055] 4. Expression verification of recombinant type III humanized collagen gene engineering strain

[0056] Pick a single colony and inoculate it into 10 mL of BMGY medium. Culture it at 28 °C and 220 rpm until OD600 = 2 - 6. Centrifuge at 3000 g for 5 min, resuspend the yeast cells with 10 mL of BMMY medium to make OD600 = 1, and continue to culture at 28 °C and 220 rpm. Add methanol every 24 hours to a final concentration of 0.5%. After induction for 72 h, centrifuge and take the supernatant, and send it for SDS-PAGE electrophoresis detection.

[0057] The SDS-PAGE electrophoresis detection results of the recombinant type III humanized collagen prepared by the present invention in shake flask expression are as Figure 3 shown. Figure 3 It is the SDS-PAGE electrophoresis diagram of the recombinant type III humanized collagen in shake flask expression. (M: Marker; 1: Negative control, 2: Verification of the shake flask expression of the recombinant type III humanized collagen Pichia pastoris genetic engineering bacteria). It can be seen from Figure 3 this that the new amino acid sequence of the recombinant type III humanized collagen provided by the present invention has been successfully expressed and secreted in the Pichia pastoris expression system.

[0058] Example 3. Preparation of recombinant type III humanized collagen

[0059] 1. Fermentation of recombinant type III humanized collagen:

[0060] Inoculate the recombinant type III humanized collagen expression strain prepared in Example 2 into YPD medium, and culture it at 28 °C and 220 rpm until the seed liquid concentration OD600 = 15 - 25, and there are no contaminants under microscopic examination. Inoculate the seed liquid into a 7.5 L fermenter (containing 3 L of BSM medium) at an inoculation amount of 5%. Initial culture conditions: rotation speed is 100 rpm, tank pressure is 0.05 MPa, control pH 5.2, and temperature 30 °C. Control the dissolved oxygen above 30% by adjusting the air flow rate and stirring speed.

[0061] When the carbon source in the initial fermentation medium is exhausted, the dissolved oxygen suddenly rises, and start to feed 50% glycerol to supplement the carbon source. When the carbon source (50% glycerol) is exhausted, stop glycerol feeding, start feeding methanol for induction, and maintain the dissolved oxygen (DO) above 5% by adjusting the rotation speed, air flow rate and methanol feeding rate. Take the fermentation supernatant for HPLC detection after induction for 88 h. The HPLC detection results of the fermentation supernatant of the recombinant type III humanized collagen prepared by the present invention are as Figure 4 shown.

[0062] 2. Purification of recombinant type III humanized collagen:

[0063] After 88 hours of induction, the fermentation was terminated to obtain the fermentation product. The fermentation product was centrifuged at 10 °C and 9000 rpm for 20 min to obtain the fermentation filtrate. The fermentation filtrate was taken and the protein was captured by a cation exchange medium, and the recombinant type III humanized collagen was eluted with an eluent (10 mmol / L PB buffer, pH 8.0). The purified recombinant type III humanized collagen was subjected to SDS-PAGE electrophoresis and HPLC detection.

[0064] The SDS-PAGE electrophoresis and HPLC detection results of the purified recombinant type III humanized collagen of the present invention are as Figure 5 and Figure 6 shown. Figure 5 Figure Figure 6 is the SDS-PAGE electrophoresis result diagram of the purified recombinant type III humanized collagen, (1: recombinant type III humanized collagen; M: Marker). Figure 5 and Figure 6 It can be seen from

[0065] Example 4, Solubility Detection of Recombinant Type III Humanized Collagen

[0066] 1. Experimental method:

[0067] The recombinant type III humanized collagen prepared in Example 3 was dissolved in 15 mL of PBS (pH 7.4) solvent to prepare recombinant type III humanized collagen solutions with concentrations of 10 g / L, 15 g / L, 25 g / L, 50 g / L, and 100 g / L, and the dissolution conditions within 30 min were observed.

[0068] 2. Experimental results:

[0069] The experimental results are shown in Table 1.

[0070] Table 1 Solubility Detection Results of Recombinant Type III Humanized Collagen

[0071] Serial number Concentration of recombinant type III humanized collagen (g / L) Whether completely dissolved 1 10 Yes 2 15 Yes 3 25 Yes 4 50 Yes 5 100 Yes

[0072] It can be seen from Table 1 that the recombinant type III humanized collagen prepared in the present invention has strong solubility, and the solubility in the salt solution can reach 100 g / L.

[0073] Example 5, Evaluation of Cell Proliferation Activity of Recombinant Type III Humanized Collagen

[0074] 1. Experimental method:

[0075] 1.1. Grouping situation:

[0076] The positive control group (PC) was the bovine type I collagen standard product from the National Institutes for Food and Drug Control, with a detection concentration of 1 mg / mL. The experimental sample group was the recombinant type III humanized collagen prepared in Example 3 of the present invention, and the detection concentrations included 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL.

[0077] 1.2 Detection of cell proliferation rate:

[0078] Prepare the samples of each group with DMEM medium, add them to a 96-well plate, and refrigerate and let stand for 24 h; discard the medium in the wells, take human embryonic skin fibroblasts (ESF) cells in good growth state, digest and count them, adjust the cell density, and then inoculate them into a 96-well plate and culture for 24 h; observe the cells under a microscope (100 μm) and take pictures; add 76 μL of CCK-8 working solution, culture in the dark for 1 h, and shake for 30 min; measure the absorbance value of each well at a wavelength of 450 nm in an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell proliferation rate.

[0079] The formula for calculating the cell proliferation rate is as follows:

[0080] Cell proliferation rate = OD 450e / OD 450b (OD 450e is the absorbance of the experimental sample group and the positive control group; OD 450b is the absorbance of the blank control group).

[0081] 2. Experimental results:

[0082] The experimental results are shown in Table 2 and Figures 7 - 8 as follows.

[0083] 2.1 The detection results of the proliferation rate of recombinant type III humanized collagen on ESF cells are shown in Table 2:

[0084] Table 2 Detection results of the proliferation rate of recombinant type III humanized collagen on ESF cells

[0085] Sample Proliferation rate (%) Remarks NC 100％ / PC (1mg / mL bovine type I collagen standard) 104.04％ / 0.015625mg / mL recombinant type III humanized collagen 110.28％ Proliferation rate is 1.06 times that of PC 0.03125mg / mL recombinant type III humanized collagen 111.23％ Proliferation rate is 1.07 times that of PC 0.0625mg / mL recombinant type III humanized collagen 122.01％ Proliferation rate is 1.17 times that of PC 0.125mg / mL recombinant type III humanized collagen 118.07％ Proliferation rate is 1.13 times that of PC 0.25mg / mL recombinant type III humanized collagen 117.13％ Proliferation rate is 1.13 times that of PC 0.5mg / mL recombinant type III humanized collagen 117.44％ Proliferation rate is 1.13 times that of PC 1mg / mL recombinant type III humanized collagen 116.36％ Proliferation rate is 1.12 times that of PC 2mg / mL recombinant type III humanized collagen 120.92％ Proliferation rate is 1.16 times that of PC

[0086] 2.2 The proliferation rate results of recombinant type III humanized collagen on ESF cells are as Figure 7 follows:

[0087] Figure 7 is the result graph of the proliferation rate of recombinant type III humanized collagen on ESF cells, (NC is the blank control, and PC is the bovine type I collagen standard product).

[0088] 2.3. The proliferation rate of recombinant type III humanized collagen on ESF cells and the results of observing cell status are as follows Figure 8 shown as

[0089] Figure 8 It is a graph showing the proliferation rate of recombinant type III humanized collagen on ESF cells and the results of observing cell status.

[0090] From Table 2 and Figure 7 it can be seen that the recombinant type III humanized collagen prepared by the present invention has a higher cell proliferation rate on ESF cells at concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL than the blank control group (NC) and the positive control group (PC). Moreover, the cell proliferation rate of 0.0625 mg / mL recombinant type III humanized collagen is 1.17 times that of the 1 mg / mL bovine type I collagen standard.

[0091] From Figure 8 it can be seen that from the cell number, it can be judged that the recombinant type III humanized collagen prepared by the present invention has a promoting effect on the proliferation of ESF cells at concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL.

[0092] Example 6. Evaluation of the cell adhesion activity of recombinant type III humanized collagen

[0093] 1. Experimental method:

[0094] 1.1. Grouping situation:

[0095] The reference product is the bovine type I collagen standard from the National Institutes for Food and Drug Control, and the sample concentration is 2 mg / mL; the positive control group is sigma gelatin, and the sample concentration is 0.1%; the experimental sample group is the recombinant type III humanized collagen prepared in Example 3 of the present invention, and the sample concentrations are 0.25 mg / mL, 1 mg / mL, and 4 mg / mL.

[0096] 1.2. Detection of cell adhesion rate:

[0097] Sample coating: Add the sample solution to a 96-well plate and incubate at 37°C for 1 h. Wash once with PBS. Incubate with 1% BSA at 37°C for 1 h for blocking, and wash twice with PBS.

[0098] Cell plating, centrifugation, and photography: Digest mouse skin fibroblasts (L929) cells; resuspend and count, and adjust the cell density to 1×105 Cells / mL, and Hoechst 33342 was added. Immediately, 100 μL was added to each well of a 96-well plate. The cells were incubated at 37 °C for 1 h. After filling up the culture medium, the plate was sealed with a sealing film, and the culture plate was inverted and centrifuged at 350 g for 5 min. Then, it was washed with PBS.

[0099] The formula for calculating the cell adhesion rate V is as follows:

[0100] Cell adhesion rate V: V = (number of cells after centrifugation / number of cells before centrifugation) × 100%

[0101] The formula for calculating the relative cell adhesiveness P is as follows:

[0102] Relative cell adhesiveness P: P = (average adhesion rate of each replicate well of the experimental group sample / average adhesion rate of each replicate well of the negative control) × 100%

[0103] 2. Experimental results:

[0104] The experimental results are shown in Table 3 and Figures 9 - 11 as follows.

[0105] 2.1. Detection results of the adhesion rate of recombinant humanized type III collagen to L929 cells are shown in Table 3:

[0106] Table 3 Detection results of the adhesion rate and relative adhesiveness of recombinant humanized type III collagen to L929 cells

[0107]

[0108] 2.2. Results of the adhesion rate and relative cell adhesiveness of recombinant humanized type III collagen to L929 cells are as Figures 9 - 10 follows:

[0109] Figure 9 is the graph of the adhesion rate of recombinant humanized type III collagen to L929 cells (NC is the blank control, and the reference product is bovine type I collagen standard). Figure 10 is the graph of the relative adhesiveness of recombinant humanized type III collagen to L929 cells (NC is the blank control, and the reference product is bovine type I collagen standard).

[0110] 2.3. Cell adhesion results of recombinant humanized type III collagen to L929 cells are as Figure 11 follows:

[0111] Figure 11 is the graph of the cell adhesion of recombinant humanized type III collagen to L929 cells (NC is the blank control, the reference product is bovine type I collagen standard, and PC is 0.1% gelatin).

[0112] As can be seen from Table 3 and Figures 9 - 10 it can be known that the adhesion rate of the recombinant type III humanized collagen prepared by the present invention to L929 cells at concentrations of 4 mg / mL, 1 mg / mL and 0.25 mg / mL is significantly different from that of the blank group, and the cell adhesion rate of 1 mg / mL recombinant type III humanized collagen is 2.80 times that of the 2 mg / mL bovine type I collagen standard product.

[0113] From Figure 11 it can be known that in the blank control group, some L929 cells adhered and spread; in the 1 mg / mL reference product (bovine type I collagen standard product) sample group, the cells did not adhere and spread; in the recombinant type III humanized collagen sample groups at 4 mg / mL, 1 mg / mL and 0.25 mg / mL, the cells all showed obvious adhesion and spread.

[0114] The above are only further embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, all belong to the protection scope 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:

1.

2. A gene encoding recombinant humanized type III collagen, characterized in that: The nucleotide sequence of the gene encoding the recombinant humanized type III collagen is shown in SEQ ID NO:

2.

3. A recombinant humanized type III collagen recombinant vector, characterized in that: The recombinant vector comprises the gene encoding the recombinant humanized type III collagen according to claim 2.

4. An engineered strain, characterized in that: The engineered strain comprises the gene encoding the recombinant humanized type III collagen according to claim 2 or the recombinant vector of the recombinant humanized type III collagen according to claim 3.

5. Use of the recombinant humanized type III collagen as claimed in claim 1 in the preparation of a collagen dressing for promoting cell proliferation, adhesion and migration.

6. A composition, characterized in that The composition comprises the recombinant humanized type III collagen according to claim 1 and an acceptable excipient.

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