A recombinant collagen with high temperature resistance and its preparation method and application

Abstract

The present invention provides a recombinant collagen with high temperature resistance, a preparation method and application thereof, and screens a region in a human collagen sequence that has 50-80% homology with a fish collagen, and replaces the region with a fish collagen sequence, and then expresses it. The amino acid sequence of human collagen includes that shown in SEQ ID NO.1, and the fish collagen comes from African lungfish. The amino acid sequence of the fish-human chimeric recombinant collagen obtained by replacement includes that shown in SEQ ID NO.2. The preparation method provided by the present invention can effectively transform the characteristics of the original collagen molecular structure, improve its high temperature tolerance, and enhance the stability of the original human collagen molecule.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant proteins, and in particular to a recombinant collagen with high temperature resistance, a preparation method and an application thereof. Background Art

[0002] Protein is the main bearer of human life activities, an important component of human tissue, and the main raw material for human tissue metabolism, renewal and repair. The functions of protein in the human body also include transporting substances, participating in the body's immunity, providing energy and regulating hormones, etc. Among them, collagen is the most abundant protein in the human body, accounting for about 25-33% of the total protein in the body. Its function is to maintain the shape and structure of the skin and tissues and organs, and it plays an indispensable role in the normal functioning of the human body.

[0003] Collagen has excellent water-retaining properties and is a key component in maintaining skin elasticity and firmness. It can repair damaged skin tissue, reduce skin inflammation and sensitivity, accelerate the repair and regeneration of the stratum corneum, and improve skin texture. Therefore, collagen is widely used in many types of cosmetics such as facial masks, essences, lotions, and creams to provide all-round care for the skin.

[0004] African lungfish is a fish of the genus African lungfish of the family African lungfish of the order Diplodocus, also known as the proto-fin fish. African lungfish is distributed in freshwater areas inland Africa. During the long dry season, the protein of African lungfish can withstand the test of high temperature through special molecular structure or biological mechanism. It is "temporarily inactivated" under the condition of long-term high temperature and dehydration. When the rainy season comes, these proteins can "restore" their original biological activity and meet the needs of normal life activities.

[0005] Compared with the traditional collagen extraction method, recombinant human collagen is a product of genetic engineering. Human protein avoids immune rejection, greatly improves biocompatibility and biosafety, lacks animal-derived pathogen microbial contamination, and avoids strong acid, strong alkali, high temperature and other drastic measures during the production process, which not only reduces the degree of environmental pollution during the production process, but also greatly guarantees the activity of collagen in a more gentle production method. However, during the long-term storage of recombinant collagen, due to its fragile molecular structure, easy to be attacked by proteases, and intolerance to high temperatures, it is easy to aggregate and precipitate under room temperature storage conditions, resulting in the loss of protein biological activity.

[0006] Therefore, how to screen, transform and express collagen with high stability and high temperature resistance, and provide a method for preparing recombinant human collagen that is conducive to maintaining its activity has become an urgent problem that needs to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a recombinant collagen with high temperature resistance, and its preparation method and application. Human and fish collagen fragments are chimerically expressed. The recombinant collagen obtained after expression and purification by an Escherichia coli host has stronger high temperature resistance than human collagen, and the product can be widely used in medicines, oral care products, and cosmetics industries.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a recombinant collagen with high temperature resistance, wherein the recombinant collagen is obtained by replacing a homologous conservative sequence in a human collagen with a fish collagen fragment;

[0010] The fish collagen fragment has 50-80% homology with the homologous conservative sequence;

[0011] The amino acid sequence of the human collagen protein includes that shown in SEQ ID NO.1;

[0012] The fish-derived collagen fragment comes from the collagen of African lungfish.

[0013] In the present invention, it is creatively discovered that the recombinant collagen obtained by chimeric expression of human collagen and fish collagen fragments has a strong ability to withstand high temperatures, effectively transforming the characteristics of the molecular structure of human collagen, improving its high temperature tolerance, and enhancing the stability of the original human collagen molecule. In production practice, the storage and transportation conditions of recombinant collagen are reduced, and the application range of collagen is expanded.

[0014] In the present invention, the amino acid sequence of the human collagen is shown in SEQ ID NO.1.

[0015] SEQ ID NO.1:

[0016]

[0017] In the present invention, it is creatively discovered that the protein of African lungfish can withstand the test of high temperature through a special molecular structure or biological mechanism. The structural stability of the recombinant collagen can be greatly improved, and its heat resistance can be enhanced by replacing the human collagen fragments that have 50-80% homology with the fish collagen fragments and then expressing them.

[0018] Preferably, the fish-derived collagen fragment is selected from SEQ ID NO.3 and / or SEQ ID NO.4.

[0019] In the present invention, the amino acid sequence of the fish-derived collagen fragment is as follows:

[0020] SEQ ID NO.3:GPPGAVGRKGEKGDQGPKGERG;

[0021] SEQ ID NO. 4: GPRGPKGDIGFPGIHGPKG.

[0022] Preferably, the amino acid sequence of the recombinant collagen with high temperature resistance includes that shown in SEQ ID NO.2.

[0023] SEQ ID NO.2:

[0024]

[0025] In the present invention, a fragment of human collagen having 50-80% homology with fish collagen is replaced with a fragment of fish collagen and then expressed. After testing, the recombinant collagen obtained by chimerizing the above fragments retains the ability of human collagen to promote repair and anti-aging, and has further improved heat resistance, and has extremely excellent high temperature resistance.

[0026] In a second aspect, the present invention provides use of the recombinant collagen with high temperature resistance described in the first aspect in the preparation of medicines, oral care products and / or cosmetics.

[0027] In the present invention, the prepared recombinant collagen is largely consistent with the original amino acid sequence, so the ability of human collagen to promote repair and anti-aging is still retained. The heat resistance of the recombinant collagen is further improved, which can not only meet the required functions of drugs and / or cosmetics, but also reduce the storage conditions and transportation conditions, which is more conducive to expanding the application range of drugs, oral care products and / or cosmetics.

[0028] In a third aspect, the present invention provides a nucleic acid molecule encoding the recombinant collagen with high temperature resistance described in the first aspect.

[0029] In a fourth aspect, the present invention provides an expression vector comprising at least one copy of the nucleic acid molecule described in the third aspect.

[0030] In a fifth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the recombinant collagen with high temperature resistance described in the first aspect.

[0031] Preferably, the recombinant cell contains the expression vector described in the fourth aspect, or the nucleic acid molecule described in the third aspect is integrated into the genome of the recombinant cell.

[0032] In a sixth aspect, the present invention provides a method for preparing the recombinant collagen with high temperature resistance according to the first aspect, the steps of the preparation method comprising:

[0033] Synthesize a nucleic acid molecule encoding the recombinant collagen with high temperature resistance described in the first aspect, construct an expression vector, transfer the expression vector into a recipient cell, and screen;

[0034] The engineered strain obtained by screening is cultured, induced to express, purified and desalted to obtain the recombinant collagen with high temperature resistance.

[0035] In the present invention, the prepared recombinant collagen often exists in a soluble form in Escherichia coli.

[0036] Preferably, the recipient cell comprises an E. coli expression strain.

[0037] Preferably, the Escherichia coli expression strain is BL21 STAR (DE3).

[0038] Preferably, the expression vector is selected from any one of pET23c vector, pET24b vector, pET28a vector or pET32b vector; preferably pET28a vector.

[0039] Preferably, the method of inducing expression comprises IPTG induction.

[0040] Preferably, the IPTG concentration for IPTG induction is 0.5-1.5 mM, for example, 0.5 mM, 0.7 mM, 1.0 mM, 1.3 mM or 1.5 mM.

[0041] Preferably, the IPTG induction time is 4-6 h, for example, 4 h, 4.5 h, 5 h, 5.5 h or 6 h.

[0042] Preferably, the purification further includes a step of disrupting the bacterial cells.

[0043] Preferably, the purification method comprises metal nickel chelate affinity column chromatography.

[0044] Preferably, the purification method comprises desalting using a dextran gel column.

[0045] Preferably, the preparation method comprises:

[0046] (1) connecting the nucleic acid molecule encoding the recombinant humanized collagen into a plasmid vector to construct an expression vector, and transferring the expression vector into an Escherichia coli expression strain, and screening to obtain an Escherichia coli genetically engineered strain;

[0047] (2) fermenting and culturing the genetically engineered Escherichia coli strain, inducing protein expression with IPTG, and collecting the cells by centrifugation;

[0048] (3) subjecting the collected bacterial cells to ultrasonic disruption and collecting the supernatant by centrifugation;

[0049] (4) The supernatant is purified by metal nickel chelate affinity column chromatography and desalted by dextran gel column to obtain the recombinant collagen with high temperature resistance.

[0050] Compared with the prior art, the present invention has at least the following beneficial effects:

[0051] 1. The preparation method of the present invention is suitable for the expression and production of a high-temperature resistant recombinant collagen, which can effectively transform the characteristics of the original collagen molecular structure, improve its high-temperature tolerance, and enhance the stability of the original collagen molecules.

[0052] 2. The target product expressed by the preparation method of the present invention is often present in a soluble form in Escherichia coli with high yield, and the modified product can withstand a high temperature of 48°C, and its stability and heat resistance are improved compared with the original collagen.

[0053] 3. The present invention utilizes the gene modification method to replace the human and fish collagen fragments for chimeric expression. After expression and purification by the E. coli host, the product can be widely used in the pharmaceutical, oral care products, and cosmetics industries. DETAILED DESCRIPTION

[0054] For ease of understanding of the present invention, the present invention lists the following embodiments. Those skilled in the art should understand that the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] Various instruments, consumables and reagents not specifically described in the examples of the present invention are conventional commercial products in the field and can be obtained through commercial channels. The specific experimental materials and their sources listed in the following examples are merely exemplary and are not intended to limit the present invention. Materials with the same or similar types, models, qualities, properties or functions as the following tissues, cells, reagents and instruments can be used to implement the present invention.

[0056] The sources of reagents used in the following examples are:

[0057] The source of Escherichia coli host BL21 STAR (DE3) was Suzhou Hongxun Biotechnology Co., Ltd.; the source of pET28a vector was Suzhou Hongxun Biotechnology Co., Ltd.;

[0058] The nickel chelate affinity chromatography filler was from Changzhou Tiandirenhe Biotechnology Co., Ltd.

[0059] The source of dextran gel filler is Changzhou Tiandirenhe Biotechnology Co., Ltd.

[0060] Example 1

[0061] (1) Construction of host bacteria

[0062] Constructing human-fish chimeric collagen amino acid sequence, constructing recombinant vector, and transforming host cells, the specific method is as follows:

[0063] The collagen sequences related to African lungfish were retrieved from NCBI, multiple sequence alignment was performed through clustal omega, and the data of its evolutionary tree were analyzed to find the homologous conserved sequence regions between species. The fish collagen fragments obtained by screening were SEQ ID NO.3 and SEQ ID NO.4. The fragments of human collagen with 50-80% homology with the fish collagen fragments were screened, and these human regions were completely replaced with fish collagen fragments in situ, and finally a complete human-fish chimeric collagen amino acid sequence was obtained.

[0064] The amino acid sequence of fish collagen fragment is:

[0065] SEQ ID NO.3:GPPGAVGRKGEKGDQGPKGERG;

[0066] SEQ ID NO. 4: GPRGPKGDIGFPGIHGPKG.

[0067] The obtained complete human-fish chimeric collagen amino acid sequence includes: SEQ ID NO.2, wherein the number of sequence replacements with 50-80% homology to SEQ ID NO.3 and SEQ ID NO.4 in situ are 21 and 4, respectively. The above replacement method is to replace all or part of the bases of SEQ ID NO.3 and SEQ ID NO.4.

[0068] A 6*HIS tag was added to the N-terminus of the human-fish chimeric collagen fragment, and after E. coli codon optimization, it was constructed into a pET28a vector, and the constructed vector was introduced into the E. coli host BL21 STAR (DE3) cells.

[0069] (2) Strain expression

[0070] Induce E. coli to express recombinant collagen with high temperature resistance, the specific method is as follows:

[0071] Pick a single recombinant E. coli clone, transfer it to 50mL LB medium, add kanamycin with a final concentration of 50μg / mL, and culture overnight at 37℃ and shaking speed of 220rpm. The next day, take the bacterial liquid from the seed liquid and inoculate it into fresh LB medium at a ratio of 1:100. Culture it under the same conditions for 3h and wait for the growth OD 600 When the length grew to 0.6-0.8, IPTG was added at a final concentration of 1 mM to induce the product. After the E. coli was induced to express for 6 hours, the cells were collected by centrifugation.

[0072] (3) Strain crushing

[0073] The bacterial cells are broken to obtain a mixed solution containing recombinant collagen with high temperature resistance. The specific method is as follows:

[0074] Take an appropriate amount of bacteria, resuspend them in an aqueous solution containing 20mM PB and 200mM NaCl, pH7.4 at a ratio of 1:10, and use a high-pressure homogenizer to break the bacteria three times at 800bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 14000rpm for 40-60min, collect the supernatant as the bacterial cell breaking supernatant, and filter the bacterial solution with a 0.45μm filter membrane. The filtrate is a mixed solution containing recombinant collagen with high temperature resistance.

[0075] (4) Purification

[0076] The mixed solution is purified to obtain recombinant collagen with high temperature resistance, which is specifically obtained as follows:

[0077] The mixed solution was purified by Ni-NTA column chromatography, washed with an aqueous solution containing 20mM PB, 200mM NaCl and 50mM imidazole, pH 7.4 and an aqueous solution containing 20mM PB, 200mM NaCl and 100mM imidazole, pH 7.4, and then eluted with an aqueous solution containing 20mM PB, 200mM NaCl and 200mM imidazole, pH 7.4 and an aqueous solution containing 20mM PB, 200mM NaCl and 500mM imidazole, pH 7.4. The eluate was further desalted with a G25 dextran gel column to obtain a recombinant collagen product.

[0078] Embodiment 2-4

[0079] This example is used to verify the influence of fish collagen fragments of different homology on the effect.

[0080] The difference between this embodiment and embodiment 1 is that the percentage of homology sequences replaced in human collagen is different. Embodiments 2-4 respectively replace sequences with 80-100% homology to sequences SEQ ID NO.3 and SEQ ID NO.4 in human collagen, and replace sequences with fish collagen fragments with 50-80% homology to SEQ ID NO.3 or SEQ ID NO.4. In embodiments 2-4, all sequences that meet the homology requirements are replaced. When the similarity of the sequences is less than 50%, it is considered that there is no homology and no replacement is meaningful, so no embodiment is set. In embodiment 2, the number of sequences replaced with 80-100% homology to SEQ ID NO.3 and SEQ ID NO.4 are 2 and 3, respectively; in embodiments 3-4, the number of sequences replaced with 50-80% homology to SEQ ID NO.3 or SEQ ID NO.4 are 21 and 4, respectively.

[0081] Table 1

[0082] Example Homology Number of replacement sequences (and replacement sequences) Example 2 80％-100％ 2 (SEQ ID NO.3), 3 (SEQ ID NO.4) Example 3 50％-80％ 21 items (SEQ ID NO.3) Example 4 50％-80％ 4(SEQ ID NO.4)

[0083] Embodiment 5-6

[0084] This example is used to verify the effect of replacing different amounts of fish-derived collagen fragments.

[0085] This embodiment provides a method for preparing recombinant collagen with high temperature resistance. The specific operation is different from that of Example 1 only in that different amounts of fish collagen fragments are used to replace human collagen.

[0086] Table 2

[0087] Example Replacement quantity Replacement sequence Example 5 2 1 fragment each of SEQ ID NO.3 and SEQ ID NO.4 Example 6 10 SEQ ID NO.3, SEQ ID NO.4 fragments 8, 2 respectively

[0088] Example 5, the sequence of the recombinant collagen after replacing one fragment of SEQ ID NO.3 and one fragment of SEQ ID NO.4 is:

[0089] SEQ ID NO.5:

[0090]

[0091] Example 6, the sequence of the recombinant collagen after replacing 8 fragments of SEQ ID NO.3 and 2 fragments of SEQ ID NO.4 is:

[0092] SEQ ID NO.6:

[0093]

[0094] Comparative Example 1

[0095] This comparative example is used to verify the effects of original human collagen with and without replacement.

[0096] The difference between this comparative example and Example 1 is that the human collagen sequence is not replaced during the construction of the host bacteria, and the original human collagen amino acid sequence is directly used.

[0097] Test Case

[0098] Test Example 1: Protein Purity Detection

[0099] This test example is used to verify the purity of the recombinant collagen prepared in the above examples and comparative examples.

[0100] The collagen samples prepared in each embodiment and comparative example were taken, and protein separation was performed using SDS-PAGE electrophoresis, and then the protein gel was scanned using the grayscale scanning method. The proportion of collagen in the entire lane was the purity of the recombinant collagen. As shown in Table 3, the purity of the recombinant collagen prepared by the present invention is high and can be used for subsequent detection.

[0101] Table 3

[0102]

[0103]

[0104] Test Example 2: Thermal stability test of recombinant collagen

[0105] The collagen prepared in each embodiment and comparative example was diluted to 1 mg / mL with 20 mM PBS solution at pH 7.4 and dispensed into 5 mL vials, 2 mL in each bottle. The vials were placed in a constant temperature and humidity incubator at 48°C for accelerated destruction test, and the effects of samples placed for 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, 2 d, 5 d, and 7 d on the proliferation and migration of HaCaT cells (laboratory preserved cells) were detected.

[0106] (1) Effect on cell proliferation

[0107] HaCaT cells with good growth status were plated on 96-well plates and cultured for 17 hours. After replacing the serum-free basal medium and culturing for 7 hours, the old medium was discarded and the medium containing the recombinant collagen sample was added (the concentration of recombinant collagen in the medium was 50 μg / mL). The recombinant collagen in the medium of the blank group was replaced with 20 mM, pH 7.4 PBS solution. After culturing for 48 hours, the OD was detected by CCK8 method. 450 and OD 630The value (630 nm as the reference wavelength) was used to obtain the effect of the test samples on the viability of HaCaT cells, thereby obtaining the effect of the test samples on cell proliferation, which can be specifically seen from the cell viability test results in Table 4.

[0108] The calculation method of cell viability (referring to the activity of cell proliferation) is:

[0109]

[0110] A 1 : absorbance of wells with cells, culture medium, CCK8 solution, and recombinant collagen;

[0111] A 2 : Absorbance of wells with cells, culture medium, CCK8 solution, and no recombinant collagen;

[0112] A 0 : Absorbance of wells with culture medium and CCK8 solution but without cells and recombinant collagen.

[0113] Table 4

[0114]

[0115] By comparing Example 1 to Example 2, it can be seen that the present invention can significantly improve the thermal stability of recombinant collagen by limiting the homology to 50-80%. The cell viability of Example 2 drops to a lower point after heating for 12 hours, while the cells of Example 1 still have high activity after heating for 7 days.

[0116] By comparing Example 1 with Examples 3-4, it can be seen that the present invention replaces two sequences at the same time, which can achieve a better improvement in the thermal stability of the recombinant collagen. The technical effect obtained by only replacing a single fragment in Example 3-4 is far lower than that in Example 1.

[0117] By comparing Example 1 with Examples 5-6, it can be seen that the present invention can achieve better improvement in the thermal stability of recombinant collagen by further replacing all of the homology of 50-80%. The technical effect obtained by only replacing some fragments in Examples 5-6 is far lower than that in Example 1.

[0118] It can be seen from Example 1 and Comparative Example 1 that the present invention can achieve the technical effect of improving the thermal stability of recombinant protein by constructing recombinant collagen, while when only human collagen is used, it cannot achieve the technical effect of improving thermal stability.

[0119] (2) Effects on cell migration

[0120] HaCaT cells in good growth state were plated on 24-well plates and cultured for 17 hours. After replacing serum-free basal medium and culturing for 7 hours, the old medium was discarded, and a 10μL pipette tip was used to draw a line. Then, a medium containing recombinant collagen was added (the concentration of recombinant collagen in the medium was 2μg / mL). The medium of the blank group did not contain recombinant collagen, but contained 20mM PBS solution. Photos were taken and counted at the beginning of culture and 48 hours after culture to obtain the effect of the test samples on the migration of HaCaT cells. The results are shown in Table 5 Cell migration rate experimental results.

[0121] Determination of cell migration distance: Image J software was used to measure the scratch area at each fixed point.

[0122]

[0123] Table 5

[0124]

[0125] The above results show that the recombinant collagen prepared by the present invention can significantly improve the thermal stability of collagen, and still maintain a high cell viability after heating for 7 days.

[0126] Test Example 3: Skin Barrier Repair Test

[0127] The recombinant collagen prepared in Example 1 and Comparative Example 1 was prepared into lyophilized powder, which was prepared by lyophilizing the following raw materials: 2 parts of recombinant collagen, 10 parts of trehalose, 40 parts of mannitol, 0.8 parts of xanthan gum, and 50 parts of buffer solution (0.05wt% bovine serum albumin, 2mM EDTA-2Na, 0.2M NaCl, 0.05wt% Tween-80, 20mM PBS, pH 6.0).

[0128] The blank control group differs from the freeze-dried powder prepared in Example 1 and Comparative Example 1 in that no recombinant collagen is added, and other components and preparation processes are the same.

[0129] Thirty volunteers, aged between 30 and 50, were recruited. Three experimental sites were selected from the inner arm of each subject, and the skin was repeatedly smeared with 0.5% sodium dodecyl sulfate (SLS) solution to form a chemical damage model. Then, recombinant collagen freeze-dried powder (dissolved in physiological saline to 5 mg / mL) was smeared on the damaged skin for 28 consecutive days. The transepidermal water loss values ​​(TEWL values) of the skin were tested and recorded on the 0th day, the 14th day and the 18th day. The results are shown in Table 6 Skin Moisture Change Experiment Table.

[0130] The calculation formula of the rate of change is:

[0131] Rate of change = (TEWL(第n天) -TEWL (第0天) ) / TEWL (第0天) ×100%

[0132] Table 6

[0133]

[0134] The results showed that the recombinant collagen freeze-dried powder of Example 1 and the recombinant human collagen freeze-dried powder of Comparative Example 1 had almost the same skin barrier repair ability.

[0135] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A recombinant collagen with high temperature resistance, characterized in that: The recombinant collagen is obtained by replacing the homologous conservative sequence in human collagen with fish collagen fragments; The fish-derived collagen fragment has 50-80% homology with the homologous conservative sequence; The amino acid sequence of the human collagen protein includes that shown in SEQ ID NO.1; The fish-derived collagen fragment is derived from the collagen of African lungfish; The fish-derived collagen fragment is selected from SEQ ID NO.3 and / or SEQ ID NO.4; The amino acid sequence of the recombinant collagen is shown in SEQ ID NO.

2.

2. Use of the high temperature resistant recombinant collagen according to claim 1 in the preparation of oral care products and / or cosmetics.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant collagen with high temperature resistance as claimed in claim 1.

4. An expression vector, characterized in that: The expression vector contains at least one copy of the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that The recombinant cell expresses the recombinant collagen with high temperature resistance as claimed in claim 1.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell contains the expression vector according to claim 4, or the nucleic acid molecule according to claim 3 is integrated into the genome of the recombinant cell.

7. A method for preparing the high temperature resistant recombinant collagen according to claim 1, characterized in that: The steps of the preparation method include: Synthesize a nucleic acid molecule encoding the recombinant collagen with high temperature resistance as claimed in claim 1, construct an expression vector, transfer the expression vector into a recipient cell, and screen; The engineered strain obtained by screening is cultured, induced to express, purified and desalted to obtain the recombinant collagen with high temperature resistance.

8. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The recipient cells include E. coli expression strains.

9. The method for preparing recombinant collagen with high temperature resistance according to claim 8, characterized in that: The E. coli expression strain is BL21 STAR (DE3).

10. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The expression vector is selected from any one of pET23c vector, pET24b vector, pET28a vector or pET32b vector.

11. The method for preparing recombinant collagen with high temperature resistance according to claim 10, characterized in that: The expression vector is pET28a vector.

12. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The method of inducing expression includes IPTG induction.

13. The method for preparing recombinant collagen with high temperature resistance according to claim 12, characterized in that: The IPTG concentration for IPTG induction is 0.5-1.5 mM.

14. The method for preparing recombinant collagen with high temperature resistance according to claim 12, characterized in that: The IPTG induction time is 4-6h.

15. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The purification also includes a step of disrupting the bacteria.

16. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The purification method comprises metal nickel chelate affinity column chromatography.

17. The method for preparing recombinant collagen with high temperature resistance according to claim 7, characterized in that: The desalting method comprises desalting by using a dextran gel column.

18. The method for preparing a recombinant collagen with high temperature resistance according to any one of claims 8 to 17, characterized in that: The preparation method comprises: (1) connecting the nucleic acid molecule encoding the recombinant high temperature resistant collagen into a plasmid vector to construct an expression vector, transferring the expression vector into an Escherichia coli expression strain, and screening to obtain an Escherichia coli genetically engineered strain; (2) fermenting and culturing the genetically engineered Escherichia coli strain, inducing protein expression with IPTG, and collecting the cells by centrifugation; (3) subjecting the collected bacterial cells to ultrasonic disruption and collecting the supernatant by centrifugation; (4) The supernatant is purified by metal nickel chelate affinity column chromatography and desalted by dextran gel column to obtain the recombinant collagen with high temperature resistance.

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