Strain capable of expressing exogenous protein, recombinant human-derived collagen, synthesis method, and use

Abstract

Disclosed are a strain capable of expressing an exogenous protein, a recombinant human-derived collagen, a synthesis method, and the use. The present invention first provides the strain capable of expressing the exogenous protein, and the strain is proline-auxotrophic Escherichia coli and can efficiently express the exogenous protein. Using the strain as a host to express the recombinant human-derived collagen with supplementation of exogenous proline and hydroxyproline can achieve accurate regulation and control on the collagen hydroxylation rate by means of regulating the ratio of proline to hydroxyproline in a culture medium. By means of the synthesis method, a recombinant collagen of which the hydroxylation rate is approximate to that of natural human collagen can be obtained and has better cell adhesion and protein stability.

Description

A strain capable of expressing exogenous proteins, recombinant human collagen and synthesis method and application TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a strain capable of expressing exogenous proteins, recombinant human collagen and synthesis method and application. BACKGROUND

[0002] Collagen is a protein widely present in the human body and other mammals, accounting for about 30% of the total protein. Its family members are numerous, and more than 30 have been found. The molecular structure of collagen contains a triple helix structure and a unique Gly-X-Y repeat sequence. Proline is usually found at the X position, and hydroxyproline is at the Y position. The presence of hydroxyproline at this position plays a decisive role in the stability of the triple helix structure of collagen. In the human body, types I, II and III collagen are the main components of collagen fibers. Type I collagen is widely distributed in the dermis, skeleton, tendons and ligaments, forming a highly ordered fibrous tissue that provides important support for the skin; type II collagen mainly exists in the fibrocartilage region of tendon and ligament attachment points, forming a fine network structure in cartilage; type III collagen is distributed in the placenta in addition to type I collagen, and is also present in connective tissue such as skin, tendon, ligament, blood vessels, etc., forming a network structure of extracellular matrix, which not only plays a key role in the support and protection of organs, but also is closely related to cell adhesion and migration.

[0003] At present, the industrial production of collagen mainly depends on the extraction from the tissues (skin or skeleton) of pigs, cows and other animals or the skin of deep-sea fish by acid, alkali or enzyme method. Although this extraction method is mature, its immunogenicity and limited source make it difficult to meet the growing market demand. With the wide application of genetic engineering technology, the production of recombinant collagen through genetic engineering means has become the most potential method to solve the source limitation of collagen. Compared with collagen derived from animals, recombinant collagen has the advantages of short production cycle and low cost, and is more suitable for large-scale production. Microorganisms used for fermentation include Escherichia coli, yeast, Bacillus subtilis, etc., but these microorganisms lack proline hydroxylase genes, so they cannot form collagen containing proline hydroxylation modification. However, animal-derived proline hydroxylase has a complex structure, and the presence of multimeric form limits its activity after expression in microorganisms, restricting its application.

[0004] Most of the existing methods for expressing recombinant hydroxylated collagen are co-expression of collagen genes and hydroxylase genes, which are derived from microorganisms or fragments of human hydroxylase, and the expression hosts include Escherichia coli, Pichia pastoris, Kluyveromyces and the like. For example, 202210567560.X discloses a kind of in vivo co-expression of proline hydroxylase BaP4H derived from Bacillus anthracis and a kind of recombinant human fusion I-III type collagen in Escherichia coli, two plasmids are induced by L-arabinose and IPTG respectively, and the time and conditions of the two inductions are optimized. CN111087464A discloses a kind of in vivo expression of giant virus proline hydroxylase Hy726 by pACYCDute-1 plasmid by IPTG induction to prepare hydroxylated recombinant human Ⅲ type collagen. CN111334512A discloses that Kluyveromyces is used as a chassis cell, and the constitutive expression of proline hydroxylase alpha subunit or lysine hydroxylase is realized, the secretory expression of human-like collagen is realized, and the synchronous hydroxylation of proline and lysine is realized. However, this method has certain uncontrollable hydroxylation efficiency.

[0005] SUMMARY

[0006] The primary purpose of the present application is to provide a strain capable of expressing exogenous proteins.

[0007] Another purpose of the present application is to provide the use of the above-mentioned strain capable of expressing exogenous proteins.

[0008] Still another purpose of the present application is to provide a method for synthesizing recombinant human collagen and the recombinant human collagen obtained therefrom.

[0009] The purposes of the present application are achieved by the following technical solutions:

[0010] A strain capable of expressing exogenous proteins is an Escherichia coli recombinant engineering strain in which the main enzyme genes in the proline synthetic metabolic pathway, the ompT protease (family outer membrane protease) gene and the Lon protease gene are knocked out; preferably prepared by the following steps:

[0011] (1) Taking an Escherichia coli engineering strain as the starting strain, knocking out the main enzyme genes in the proline synthetic metabolic pathway to obtain strain A;

[0012] (2) Knocking out the ompT protease gene based on strain A to obtain strain B;

[0013] (3) Knocking out the Lon protease gene based on strain B to obtain a strain capable of expressing exogenous proteins.

[0014] The starting strain of the E. coli recombinant engineering bacteria is preferably E. coli BL21(DE3), E. coli MG1655, E. coli DH5a, E. coli Top10, E. coli B0013, E. coli ATCC 8739, E. coli W3110(DE3) or E. coli JM109.

[0015] The E. coli engineering bacteria is preferably E. coli MG1655, E. coli DH5a, E. coli Top10, E. coli B0013, E. coli ATCC 8739, E. coli W3110(DE3) or E. coli JM109.

[0016] If the original E. coli strain genome contains the above three genes (knock out the major enzyme genes in the proline metabolic pathway, ompT protease gene and Lon protease gene), the strain capable of expressing exogenous proteins is obtained according to the above steps (1)-(3); if the strain has been previously metabolically engineered to knock out one or two genes, only the other gene needs to be knocked out, for example, if the E. coli BL21(DE3) genome has been knocked out of the ompT and Lon two protease genes, only step (1) is needed, that is, to knock out the major enzyme gene in the proline metabolic pathway.

[0017] The major enzyme gene in the proline metabolic pathway is preferably at least one of ProA, ProB and ProC (pyrroline-5-carboxylate reductase).

[0018] The knocking out method includes homologous arm recombination or gene editing; preferably gene editing.

[0019] The gene editing is preferably CRISPR-cas9 gene editing.

[0020] The vector targeting the major enzyme gene in the proline metabolic pathway is preferably obtained by the following steps: inserting the sgRNA targeting the major enzyme gene in the proline metabolic pathway and the knockout frame targeting the major enzyme gene in the proline metabolic pathway into the E. coli knockout plasmid.

[0021] The E. coli knockout plasmid is preferably pTargetF plasmid.

[0022] The nucleotide sequence of the sgRNA targeting the major enzyme gene in the proline metabolic pathway is shown in SEQ ID No. 1.

[0023] The nucleotide sequence of the knockout frame targeting the main enzyme gene in the proline anabolic pathway is shown in SEQ ID No. 2; it contains the homologous arms upstream and downstream of the ProC gene.

[0024] The step of knocking out the main enzyme gene in the proline anabolic pathway is as follows: in the cas9 plasmid-containing E. coli gene engineering strain competence, the vector targeting the main enzyme gene in the proline anabolic pathway is transformed, cultured, and the strain knocking out the main enzyme gene in the proline anabolic pathway, i.e. strain A, is obtained.

[0025] The vector targeting the ompT protease gene in the step of knocking out the ompT protease gene is preferably obtained by the following steps: inserting the sgRNA targeting the ompT protease gene and the knockout frame targeting the ompT protease gene on the E. coli knockout plasmid.

[0026] The E. coli knockout plasmid is preferably pTargetF plasmid.

[0027] The nucleotide sequence of the sgRNA targeting the ompT protease gene is shown in SEQ ID No. 3.

[0028] The nucleotide sequence of the knockout frame targeting the ompT protease gene is shown in SEQ ID No. 4; it contains the homologous arms upstream and downstream of the ompT protease gene.

[0029] The step of knocking out the ompT protease gene is as follows: in the strain A containing the cas9 plasmid, the vector targeting the ompT protease gene is transformed, cultured, and the strain knocking out the main enzyme gene in the proline anabolic pathway and knocking out the ompT protease gene, i.e. strain B, is obtained.

[0030] The vector targeting the Lon protease gene in the step of knocking out the Lon protease gene is preferably obtained by the following steps: inserting the sgRNA targeting the Lon protease gene and the knockout frame targeting the Lon protease gene on the E. coli knockout plasmid.

[0031] The E. coli knockout plasmid is preferably pTargetF plasmid.

[0032] The nucleotide sequence of the sgRNA targeting the Lon protease gene is shown in SEQ ID No. 5.

[0033] The nucleotide sequence of the knockout frame targeting the Lon protease gene is shown in SEQ ID No. 6; it contains the homologous arms upstream and downstream of the Lon protease gene.

[0034] The step of knocking out the Lon protease gene is as follows: in the cas9 plasmid-containing strain B competent cell, the vector targeting the Lon protease gene is introduced, cultured, and the strain in which the main enzyme gene in the proline synthesis metabolic pathway is knocked out, the ompT protease gene is knocked out, and the Lon protease gene is knocked out, i.e., the strain capable of expressing exogenous proteins, is obtained.

[0035] The above-mentioned strain capable of expressing exogenous proteins is a proline auxotrophic Escherichia coli, which is particularly suitable for use in exogenous protein expression containing proline and hydroxyproline. By introducing an exogenous protein coding gene rich in proline and hydroxyproline into the strain capable of expressing exogenous proteins and adjusting the ratio of proline and hydroxyproline in the culture medium, the hydroxylation rate in the exogenous protein can be accurately controlled to obtain the target protein.

[0036] The exogenous protein containing proline and hydroxyproline is preferably recombinant human collagen.

[0037] The amino acid sequence of the recombinant human collagen is preferably as shown in SEQ ID No. 7, SEQ ID No. 11 or SEQ ID No. 13.

[0038] The sequence of the coding nucleic acid of the recombinant human collagen is preferably as shown in SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 14.

[0039] A synthesis method of recombinant human collagen, comprising the following steps:

[0040] 1) introducing a recombinant expression vector capable of expressing recombinant human collagen and a recombinant vector capable of expressing T7 RNA polymerase into the above-mentioned strain capable of expressing exogenous proteins to obtain a recombinant genetically engineered bacterium;

[0041] 2) culturing the recombinant genetically engineered bacterium obtained in step 1), adding an inducer to induce the expression of T7 RNA polymerase;

[0042] 3) changing the liquid for starvation culture;

[0043] 4) adding IPTG, proline and hydroxyproline for culture to induce the expression of recombinant human collagen;

[0044] 5) performing solid-liquid separation on the culture solution obtained in step 4) to obtain the bacterial body;

[0045] 6) crushing the bacterial body, performing solid-liquid separation, purifying the obtained supernatant to obtain recombinant human collagen.

[0046] The amino acid sequence of the recombinant human-derived collagen protein described in step 1) is preferably that of a collagen protein as set forth in SEQ ID No. 7, SEQ ID No. 11, or SEQ ID No. 13.

[0047] The nucleic acid sequence of the recombinant human-derived collagen protein described in step 1) is preferably that as set forth in SEQ ID No. 8, SEQ ID No. 12, or SEQ ID No. 14.

[0048] The vector framework of the recombinant expression vector described in step 1) that is capable of expressing the recombinant human-derived collagen protein is an expression vector with a T7 promoter; preferably, it is a pET series plasmid; more preferably, it is pET28a.

[0049] The nucleic acid sequence of the recombinant human-derived collagen protein described in step 1) is preferably that as set forth in SEQ ID No. 8, SEQ ID No. 12, or SEQ ID No. 14.

[0050] The T7 RNA polymerase described in step 1) is derived from E. coli BL21(DE3), and its amino acid sequence is preferably that as set forth in SEQ ID No. 9.

[0051] The nucleotide sequence of the gene encoding the T7 RNA polymerase described is preferably that as set forth in SEQ ID No. 10.

[0052] The vector framework of the recombinant vector described in step 1) that is capable of expressing the T7 RNA polymerase is an expression vector with an arabinose promoter; more preferably, it is pBAD33.

[0053] The gene encoding the T7 RNA polymerase described in step 1) is disposed downstream of the arabinose promoter in the recombinant vector.

[0054] The recombinant expression vector that is capable of expressing the recombinant human-derived collagen protein and the recombinant vector that is capable of expressing the T7 RNA polymerase introduced in step 1) are preferably combined at a molar ratio of 1:1.

[0055] The method of introduction described in step 1) is preferably electroporation.

[0056] The medium used in the culturing described in step 2) is an LB medium containing antibiotics resistant to the recombinant expression vector that is capable of expressing the recombinant human-derived collagen protein and the recombinant vector that is capable of expressing the T7 RNA polymerase.

[0057] The medium described is preferably an LB medium containing 50 mg / L kanamycin and 25 mg / L chloramphenicol.

[0058] The culture condition in step 2) is preferably at 36-38°C, 150-250 rpm; more preferably at 37°C, 220 rpm.

[0059] The culture stage in step 2) is preferably at OD 600 2.5; more preferably at OD 600 2.5.

[0060] The inducer in step 2) is a substance that induces the activity of T7 RNA polymerase promoter; preferably arabinose.

[0061] The amount of inducer in step 2) is preferably 0.5-1.5 mg / mL in the culture system; more preferably 1 mg / mL in the culture system.

[0062] The time of induction in step 2) is preferably 5-40 min; more preferably 15 min.

[0063] The liquid exchange mode in step 3) can be centrifugation or sterile filtration; more preferably centrifugation.

[0064] The centrifugation condition is preferably at 2-8°C, 4000-8000 rpm for 3-8 min; more preferably at 4°C, 6000 rpm for 6 min.

[0065] The medium composition used in the starvation culture described in Step 3) is preferably as follows: M9 medium having a glucose concentration of 1 to 10 (w / v) % as a solvent, sodium chloride 300 to 600 mM, glycine 20 to 500 mg / L, alanine 20 to 500 mg / L, valine 20 to 500 mg / L, leucine 20 to 500 mg / L, isoleucine 20 to 500 mg / L, methionine 20 to 500 mg / L, tryptophan 20 to 500 mg / L, serine 20 to 500 mg / L, tyrosine 20 to 500 mg / L, cysteine 20 to 500 mg / L, phenylalanine 20 to 500 mg / L, asparagine 20 to 500 mg / L, glutamine 20 to 500 mg / L, threonine 20 to 500 mg / L, aspartic acid 20 to 500 mg / L, glutamic acid 20 to 500 mg / L, lysine 20 to 500 mg / L, arginine 20 to 500 mg / L, and histidine 20 to 500 mg / L; more preferably as follows: M9 medium having a glucose concentration of 1 to 10 (w / v) % as a solvent, sodium chloride 450 to 550 mM, glycine 90 to 110 mg / L, alanine 90 to 110 mg / L, valine 90 to 110 mg / L, leucine 90 to 110 mg / L, isoleucine 90 to 110 mg / L, methionine 90 to 110 mg / L, tryptophan 90 to 110 mg / L, serine 90 to 110 mg / L, tyrosine 90 to 110 mg / L, cysteine 90 to 110 mg / L, phenylalanine 90 to 110 mg / L, asparagine 90 to 110 mg / L, glutamine 90 to 110 mg / L, threonine 90 to 110 mg / L, aspartic acid 90 to 110 mg / L, glutamic acid 90 to 110 mg / L, lysine 90 to 110 mg / L, arginine 90 to 110 mg / L, and histidine 90 to 110 mg / L; and most preferably as follows: M9 medium having a glucose concentration of 10 (w / v) % as a solvent, sodium chloride 500 mM, glycine 100 mg / L, alanine 100 mg / L, valine 100 mg / L, leucine 100 mg / L, isoleucine 100 mg / L, methionine 100 mg / L, tryptophan 100 mg / L, serine 100 mg / L, tyrosine 100 mg / L, cysteine 100 mg / L, phenylalanine 100 mg / L, asparagine 100 mg / L, glutamine 100 mg / L, threonine 100 mg / L, aspartic acid 100 mg / L, glutamic acid 100 mg / L, lysine 100 mg / L, arginine 100 mg / L, and histidine 100 mg / L.

[0066] The starvation culture condition in step 3) is preferably 36-38℃, 150-250rpm for 30-90min; more preferably 37℃, 220rpm for 60min.

[0067] The IPTG concentration in step 4) is preferably 0.1-2mM; more preferably 0.8-1.2mM; most preferably 1mM.

[0068] The proline concentration in step 4) is preferably 0-40mM; more preferably 0-16mM; most preferably 10-12mM.

[0069] The hydroxyproline concentration in step 4) is preferably 0-40mM; more preferably 4-20mM; most preferably 8-10mM.

[0070] The proline and the hydroxyproline cannot be both 0; preferably in a molar ratio of 6:4-5; at this time the hydroxylation rate of the recombinant human collagen obtained is between 40-45%, which is similar to the hydroxylation rate of human natural collagen.

[0071] The culture condition in step 4) is preferably 36-38℃, 150-250rpm for 3-6h; more preferably 37℃, 220rpm for 5h.

[0072] The solid-liquid separation method in step 5) is preferably centrifugation.

[0073] The centrifugation condition is preferably 2-8℃, 6000-10000rpm for 3-8min; more preferably 4℃, 8000rpm for 5min.

[0074] The composition of the suspension used for cell disruption in step 6) is as follows: 15-25mmol / L Tris-HCl, 450-550mmol / L NaCl, pH 8.0-9.0; preferably as follows: 20mmol / L Tris-HCl, 500mmol / L NaCl, pH 8.5.

[0075] The cell disruption condition in step 6) is preferably 2-8℃, 25-35kpsi; more preferably 4℃, 30kpsi.

[0076] The solid-liquid separation method in step 6) is preferably centrifugation.

[0077] The centrifugation condition is preferably 2-8℃, 10000-15000rpm for 30-60min; more preferably 4℃, 12000rpm for 50min.

[0078] The purification described in step 6) is purification using a nickel affinity chromatography column.

[0079] The purification step described in step 6) is as follows: the supernatant of the broken liquid is loaded into a nickel affinity chromatography column, and the eluent is a solution containing 50-500 mM imidazole, and the eluate containing 100-150 mM imidazole is collected.

[0080] The synthesis method of the recombinant human collagen described above is applied to the preparation of recombinant human collagen with different hydroxylation rates.

[0081] The synthesis method of the recombinant human collagen described above is applied to the functional study of recombinant human collagen with different hydroxylation rates.

[0082] A recombinant human collagen obtained by the synthesis method described above; preferably, the recombinant human collagen has a hydroxylation rate of 40-50%; more preferably, the recombinant human collagen has a hydroxylation rate of 41-47%.

[0083] The recombinant human collagen described above is applied to the preparation of bioengineering materials and / or cosmetics.

[0084] The present application has the following advantages and effects relative to the prior art:

[0085] Most of the existing methods for expressing recombinant hydroxylated collagen are co-expression of collagen genes and hydroxylase genes. The hydroxylation rate of collagen obtained by these co-expression systems is unstable. The present application first provides a proline auxotrophic E. coli strain with a high expression amount of recombinant human collagen. Then, the present application uses a proline-deficient strain to culture a medium with exogenous proline and hydroxyproline, and by adjusting the ratio of proline to hydroxyproline in the medium, the hydroxylation rate of collagen can be precisely controlled, so that the recombinant collagen with a hydroxylation rate close to that of natural human collagen is obtained, and the obtained recombinant collagen has better cell adhesion and protein stability.

[0086] The synthesis method provided by the present application can precisely control the hydroxylation rate of collagen, thereby laying a foundation for the study of the function of hydroxylated collagen. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 is a colony verification result chart of the construction process of the proline auxotrophic E. coli △ProC△ompT△lon MG1655; wherein, lane 1 is the ProC gene with a length of 810 bp, lanes 4, 7 and 10 are ProC knockout verification, the verification primers of the ProC gene are the sequences at both ends thereof, and no band is amplified, indicating that the knockout is successful; lane 2 is the ompT protease gene with a length of 954 bp, lanes 5, 8 and 11 are ompT knockout verification, the verification primers are the sequences at 700 bp upstream and downstream of the gene on the genome, and a 1400 bp band is amplified, indicating that the knockout is successful; lane 3 is the lon gene with a length of 2355 bp, lanes 6, 9 and 12 are lon knockout verification, the verification primers are the sequences at 1000 bp upstream and downstream of the gene on the genome, and a 2000 bp band is amplified, indicating that the knockout is successful.

[0088] Figure 2 is a gel electrophoresis chart of recombinant human collagen expressed by different strains; wherein, lane M is a protein Marker (Thermo Fisher #26616), lane W is the whole liquid protein of cell crushing liquid, lane S is the supernatant protein of cell crushing liquid, lane P is the precipitated protein of cell crushing liquid, and the arrow is the collagen band.

[0089] Figure 3 is the hydroxylation rate of collagen prepared by adding different concentration ratios of proline and hydroxyproline in the culture medium.

[0090] Figure 4 is the hydroxylation rate of recombinant collagen with different molecular weights prepared by exogenous incorporation method.

[0091] Figure 5 is a purification result chart of human collagen prepared by different hydroxylation methods; wherein, lane M is a protein Marker, lane 1 is non-hydroxylated recombinant human collagen, lane 2 is hydroxylated recombinant human collagen prepared by exogenous incorporation method, and lane 3 is hydroxylated recombinant human collagen prepared by hydroxylase method.

[0092] Figure 6 is a circular dichroism chart of recombinant human collagen prepared by different methods.

[0093] Figure 7 is a comparison chart of thermal stability of recombinant human collagen prepared by different methods.

[0094] Figure 8 is a cell adhesion detection result chart of recombinant human collagen prepared by different methods and bovine collagen type I standard.

[0095] Figure 9 is a cell compatibility detection result chart of recombinant human collagen prepared by different methods and bovine collagen type I standard. DETAILED DESCRIPTION

[0096] The application will be further described in detail below in conjunction with the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0097] The primers used in the present application are shown in the following table:

[0098] Table 1 Primer sequence

[0099] Example 1: Proline auxotrophic E. coli construction

[0100] (1) The sgRNA targeting the ProC gene was designed using the gRNA online design website http: / / crispor.tefor.net / with the E. coli MG1655 genome ProC gene as the template, and the nucleotide sequence is shown as SEQ ID No. 1.

[0101] (2) The sgRNA fragment was inserted into the pTargetF plasmid by RF (restriction-free) cloning technology to obtain the sgRNA vector. The PCR reaction system is as follows: PrimeSTAR HS (premix) 5 μL, upstream primer F1 with a concentration of 10 μM 0.4 μL, downstream primer R1 with a concentration of 10 μM 0.4 μL, pTargetF plasmid with a concentration of 100 ng / μL 0.4 μL, and double distilled water to 10 μL. The reaction conditions of PCR are as follows: 98℃ 3min; 98℃ 10s, 55℃ 15s, 72℃ 1.5min, 30 cycles; 72℃ 5min; 4℃ storage.

[0102] (3) Construction of ProC knockout frame: The upstream and downstream homologous arm fragments of 500 bp of ProC were obtained by conventional molecular cloning method with E. coli MG1655 genome as the template. The PCR primers of the upstream homologous arm fragment were ProC-up-F and ProC-up-R, and the PCR primers of the downstream homologous arm fragment were ProC-dn-F and ProC-dn-R. The two fragments were inserted into the sgRNA vector obtained in step (2) using seamless cloning technology to form a knockout frame, and the nucleotide sequence is shown as SEQ ID No. 2. The obtained plasmid is named as ProC knockout plasmid.

[0103] The reaction system of seamless cloning is as follows: 3 μL of vector sgRNA double enzyme digestion (NaeI / SacI) product, 1 μL of upstream homologous arm fragment, 1 μL of downstream homologous arm fragment, and 5 μL of Seamless Mix Master.

[0104] The prepared system was incubated at 50℃ for 20 min.

[0105] (4) pCas9 plasmid (Thermo Fisher Scientific) was electro-transformed into E. coli MG1655 competent cells, which were induced using LB medium containing 50 mmol / L arabinose, and then prepared into competent cells. The plasmid successfully constructed in step (3) was electro-transformed into the competent cells, and then plated on LB plates containing kanamycin (25 mg / L) and spectinomycin (30 mg / L). Single colonies were selected and verified by PCR, and the proline-deficient E. coli △ProC MG1655 was screened. The verification primers were KZ-ProC-S and KZ-ProC-A.

[0106] (5) Steps (1)-(4) were repeated to construct an sgRNA vector containing an ompT knockout cassette, which was electro-transformed into the △ProC MG1655 competent cells containing the cas9 plasmid, and E. coli △ProC△ompT MG1655 was screened. The sgRNA nucleotide sequence is shown in SEQ ID No. 3, and the knockout cassette nucleotide sequence is shown in SEQ ID No. 4. The sgRNA fragment primers for RF cloning technology were the upstream primer F2 and the downstream primer R2. The verification primers were ompT-up700-S and ompT-dn700-A.

[0107] (6) Steps (1)-(4) were repeated to construct an sgRNA vector containing a lon knockout cassette, which was electro-transformed into the △ProC△ompT MG1655 competent cells containing the cas9 plasmid, and E. coli △ProC△ompT△lon MG1655 was screened. The sgRNA nucleotide sequence is shown in SEQ ID No. 5, and the knockout cassette nucleotide sequence is shown in SEQ ID No. 6. The sgRNA fragment primers for RF cloning technology were the upstream primer F3 and the downstream primer R3. The verification primers were lon-up1000-S and lon-dn1000-A.

[0108] The results of colony PCR verification are shown in FIG. 1, and the results of FIG. 1 show that the strains △ProC MG1655, △ProC△ompT MG1655 and △ProC△ompT△lon MG1655 were successfully obtained.

[0109] Example 2: Construction of recombinant collagen expression system

[0110] (1) According to the recombinant human collagen amino acid sequence SEQ ID No. 7, the codon preference of the host E. coli expression is designed in the process to avoid NdeI and BamHI restriction sites, and the optimized gene sequence is shown in SEQ ID No. 8. Then, the upstream primer F4 and the downstream primer R4 are used for amplification, and the fragment obtained by NdeI and BamHI double digestion is connected with the NdeI and BamHI double-digested expression vector pET28a to obtain the recombinant vector pET28a-rhCOL.

[0111] (2) Construction of recombinant expression vector pBAD33-T7RNAP:

[0112] 1) Primer design: According to the principle of primer design for seamless cloning, the T7RNAP fragment amplification primers are designed as the upstream primer F5 and the downstream primer R5.

[0113] 2) Amplification of T7RNAP gene fragment: Using E. coli BL21(DE3) genome as template, the upstream primer F5 and the downstream primer R5 are used for amplification, and the DNA recovery and purification kit (TIANGEN) is used to recover the PCR product T7RNAP gene fragment.

[0114] The PCR system is as follows: KOD One TM PCR Master Mix 25μL, upstream primer F5 with a concentration of 10μM 2μL, downstream primer R5 with a concentration of 10μM 2μL, E. coli BL21(DE3) genome 2μL, and double distilled water to 50μL. The PCR conditions are as follows: 98℃ 5min; 98℃ 10s, 55℃ 5s, 72℃ 15s, 30 cycles; 72℃ 5min; 4℃ storage.

[0115] 3) The T7RNAP gene fragment obtained in step 2) is inserted into the pBAD33 expression vector from the SalI and HindIII restriction sites by seamless cloning technology to obtain the recombinant vector pBAD33-T7RNAP.

[0116] (3) Preparation of co-expression engineering bacteria: The above successfully constructed recombinant plasmid pBAD33-T7RNAP and pET28a-rhCOL are mixed in a molar ratio of 1:1 to obtain a mixture, which is respectively transformed into the above constructed proline-deficient E. coli △ProC MG1655, △ProC△ompT MG1655 and △ProC△ompT△lon MG1655 competent cells, and coated on LB agar plates containing kanamycin Kan + (50mg / L) and chloramphenicol Cm +(25 mg / L) LB plates with antibiotic resistance (the concentration of antibiotics in the LB liquid medium below is the same as here) produce positive transformants, which are the final co-expressed recombinant genetically engineered bacteria, named strain rhCol.

[0117] (4) Inoculate the above-mentioned engineered bacteria into 10 mL of Cm + and Kan + The seed culture was prepared by incubating the culture in LB liquid medium at 37°C and 220 rpm for 10–12 h using a constant temperature shaker. The seed culture was then transferred at a volume ratio of 1:100 to a 50 mL culture medium containing Cm... + and Kan + LB medium was incubated with 1 mg / mL arabinose and cultured at 37°C and 220 rpm in a constant temperature shaker until the bacterial culture reached OD500. 600 When the concentration reaches approximately 0.6, add 1 mM IPTG, continue culturing for 5 hours, and then collect the bacterial cells.

[0118] (5) Add PBS buffer (0.01M, pH=7.4) at a ratio of 10 OD / mL to completely suspend the bacterial cells. Sonicate the cells to disrupt them, and take a sample as the whole-liquid protein W of the cell lysis buffer. Centrifuge to disrupt the cells, and take a sample from the supernatant as the supernatant protein S of the cell lysis buffer. Discard the supernatant and resuspend the precipitate with an equal volume of PBS buffer. Take a sample of the resulting suspension as the precipitated protein P of the cell lysis buffer. The uninduced protein band is the whole-liquid protein lysis buffer prepared without the addition of inducing agents (arabinose and IPTG) during the culture of engineered bacteria △ProC△ompT△lon MG1655. Add 40 μL of each of the above samples to 10 μL of 5×SDS-PAGE loading buffer and incubate in a boiling water bath for 10 min to denature the proteins. Transfer 10 μL of the sample to an SDS-PAGE gel for protein gel electrophoresis. Finally, stain with staining solution for 30 min and destain with destaining solution until the bands are clearly visible. The protein expression levels of different strains are shown in Figure 2. The results indicate that the production of the target recombinant protein was significantly increased after the protease genes ompT and lon were knocked out in the engineered bacteria.

[0119] Example 3: Preparation of hydroxylated collagen by exogenous incorporation of hydroxyproline and proline

[0120] (1) Seed culture preparation: In a clean bench, pick a single colony from the transformation plate in Example 2 and inoculate it into 10 mL of Cm solution. + and Kan + The seed culture was prepared by culturing in LB liquid medium at 37°C and 220 rpm for 10–12 h on a constant temperature shaker.

[0121] (2) Induction of T7 RNA polymerase: The above seed culture was transferred at a volume ratio of 1:100 to a 50 mL solution containing Cm+ and Kan + LB medium, and placed in a 37°C, 220 rpm constant temperature shaker for about 4.5 h. When the OD value of the bacterial solution was 2.5, 1 mg / mL of arabinose was added to induce the expression of T7 RNA polymerase, and the induction was carried out at 37°C, 220 rpm for 15 min. 600

[0122] (3) Centrifugal exchange: after the induction, the bacterial solution was centrifuged at 6000 rpm for 6 min at 4°C, and the supernatant was discarded by sterile operation in a clean bench. The bacterial solution was resuspended in a synthetic medium containing 25 mg / L Cm + and 50 mg / L Kan + , and starved at 37°C, 220 rpm for 1 h.

[0123] Synthetic medium: M9 medium (with 10% glucose) as solvent, 500 mM sodium chloride, 100 mg / L of 19 basic amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine) except proline.

[0124] (4) Inducing recombinant human collagen: after the starvation, 1 mM of IPTG was added, and different concentrations of proline and hydroxyproline were added, and the culture was continued for 5 h to induce the expression of recombinant protein. After the induction, the OD value of the bacterial solution was measured, and the bacterial solution was centrifuged at 8000 g for 5 min at 4°C to collect the bacterial body. 600

[0125] (5) The bacterial body was completely suspended by adding Buffer A solution at a ratio of 10 OD / mL, and the completely suspended bacterial body was subjected to cell disruption under a high-pressure disrupter (temperature: 4°C, pressure: 30 kpsi). After the cell disruption, the supernatant and the precipitate were obtained by centrifugation at 4°C, 12000 g for 50 min. The supernatant sample was collected, and the crude protein expression liquid was obtained by filtering the supernatant sample through a 0.22 μm filter membrane. The supernatant was added to a 5 mL Ni affinity resin gravity column, and the nickel column was washed with 25 mL of Buffer A solution containing 10 mM imidazole. Gradient elution was carried out using 15 mL of Buffer A solution containing 50 mM imidazole, 15 mL of Buffer A solution containing 100 mM imidazole, 15 mL of Buffer A solution containing 150 mM imidazole, 15 mL of Buffer A solution containing 250 mM imidazole, and 15 mL of Buffer A solution containing 500 mM imidazole, respectively. The protein was detected by SDS-PAGE, and the eluent (100-150 mM) containing the target protein and having good purity was combined to obtain a recombinant collagen sample.​​

[0126] Buffer A: 20 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 8.5, 0.22 μm filter impurity removal, room temperature storage.

[0127] (6) After purification, the sample is measured for hydroxylation rate, and the detection method is as follows:

[0128] 1) Hydrolysis of recombinant collagen

[0129] The above recombinant collagen sample is concentrated in a 10 kDa Spin-XR UF 500 centrifugal concentrator (Millipore), and the protein concentration is determined by a protein concentration determination kit (Thermo Fisher). Take 10 mg of protein sample, add 5 mL of 6M hydrochloric acid to the hydrolysis tube, vacuum and fill with nitrogen, and place in a 110°C oven for hydrolysis for 24h. After hydrolysis, cool the hydrolysis tube to room temperature, and dilute to 25 mL with ddH2O. Take 2 mL and dry with nitrogen (add a small amount of ddH2O and repeat drying twice), and finally resuspend with ddH2O.

[0130] 2) Detection of hydroxyproline concentration

[0131] Take 1 mL of the resuspension, add 0.5 mL of chloramine T solution, shake well and stand at room temperature for 20 min; add 0.5 mL of color developing agent, shake well, plug the stopper and heat in a 60°C test tube heater (or constant temperature water bath) for 20 min, then take out and cool quickly. Measure the absorbance at a wavelength of (558±2) nm.

[0132] Buffer solution (pH = 6.8): weigh 26.0 g of citric acid monohydrate, 14.0 g of sodium hydroxide, 78.0 g of anhydrous sodium acetate, dissolve the above reagents in 500 mL of water and transfer to a 1 L volumetric flask, add 250 mL of n-propanol, and dilute with water.

[0133] Chloramine T solution: weigh 1.41 g of N-chloro-p-toluenesulfonamide sodium salt (chloramine T), dissolve in 100 mL of buffer solution (pH = 6.8).

[0134] Color developing agent: weigh 10.0 g of p-dimethylaminobenzaldehyde, dissolve in 35 mL of perchloric acid solution (60%), slowly add 65 mL of isopropyl alcohol, and prepare just before use.

[0135] 3) Detection of proline concentration

[0136] Take 1 mL of the resuspension and mix with 0.5 mL of acidic ninhydrin reagent, 0.5 mL of glacial acetic acid. Mix the mixture at 100°C water bath for 1 h, immediately place on ice to terminate the reaction. Shake the mixture with 2 mL of dimethylbenzene and place at room temperature for 20 min. After the mixture is layered, take the upper liquid and measure the absorbance value at 520 nm in the ultraviolet spectrophotometer.

[0137] Acidic ninhydrin reagent: take 2.5 g of indole triketone, add 60 mL of glacial acetic acid and 40 mL of 6 mol / L phosphoric acid, heat to dissolve at 70°C, cool and store in a brown reagent bottle, store at 4°C, stable for two days.

[0138] 4) The hydroxylation rate of proline is calculated according to the following formula:

[0139] The hydroxylation rate of recombinant collagen obtained by infiltrating different concentrations of proline and hydroxyproline is shown in Figure 3. It can be seen that different hydroxylation rates of recombinant collagen can be prepared by infiltrating different concentrations of proline and hydroxyproline, and the highest hydroxylation rate can reach 88%.

[0140] Example 4 Hydroxylation of recombinant collagen with different molecular weights by exogenous incorporation method

[0141] (1) The recombinant human collagen rhColA has an amino acid sequence as shown in SEQ ID No. 11, and the optimized gene sequence is shown in SEQ ID No. 12. It is obtained by amplification with upstream primer F4 and downstream primer R6, the same as step (1) of Example 2, and the recombinant vector pET28a-rhCOLA is constructed.

[0142] (2) The recombinant human collagen rhColB has an amino acid sequence as shown in SEQ ID No. 13, and the optimized gene sequence is shown in SEQ ID No. 14. It is obtained by amplification with upstream primer F4 and downstream primer R4, the same as step (1) of Example 2, and the recombinant vector pET28a-rhCOLB is constructed.

[0143] (3) Preparation of recombinant collagen expression engineering bacteria with different molecular weights: the above successfully constructed recombinant plasmids are mixed with pBAD33-T7RNAP at a molar ratio of 1:1, respectively, and the obtained mixture is transformed into the above constructed proline-deficient Escherichia coli △ProC△ompT△lon MG1655 competent cells by electroporation, and plated on LB agar plates containing kanamycin Kan + (50 mg / L) and chloramphenicol Cm +(25mg / L) resistant LB plates, and the positive transformants were obtained, which were the final co-expression recombinant genetically engineered bacteria, and the engineered bacteria expressing rhColA were named as strain rhColA, and the engineered bacteria expressing rhColB were named as strain rhColB.

[0144] (4) Exogenous incorporation of hydroxyproline and proline to prepare hydroxylated collagen

[0145] The three strains of recombinant human collagen expression engineering bacteria constructed above were used to prepare hydroxylated recombinant collagen according to Example 3, and the hydroxylation rate was detected after purification, and the hydroxylation is shown in Figure 4, and the specific data is shown in Table 2. Even if the recombinant collagen of different types and different molecular weights, the hydroxylation rate obtained by exogenous incorporation method can still be controlled within a precise range.

[0146] Table 2 Hydroxylation rate of recombinant human collagen of different molecular weights obtained by exogenous incorporation method

[0147] Example 5 Recombinant collagen with different hydroxylation rates prepared by different methods

[0148] It is reported that the hydroxylation rate of naturally occurring collagen in the human body is more than 40%, in order to be closer to the hydroxylation rate of collagen in the human body, the hydroxylated recombinant human collagen is prepared according to Example 3 of the present application, that is, the exogenous incorporation method is used to prepare the hydroxylated recombinant human collagen; wherein, the step (4) induces the hydroxyproline in the recombinant human collagen to be 8mM, and the proline to be 12mM.

[0149] The non-hydroxylated collagen is prepared by the method of Example 2 step (4), and purified according to Example 3 step (5).

[0150] The control is the hydroxylated recombinant human collagen prepared by the hydroxylase method, which is obtained from the proline hydroxylase co-expression system constructed in the laboratory (according to the example in CN202210567560.X - a recombinant human fusion collagen and its efficient hydroxylation method and application, the recombinant collagen therein is replaced by the recombinant collagen rhCol of the present application), which is typical.

[0151] After purification, the protein size and purity of the sample were detected by SDS-PAGE electrophoresis, and the results are shown in Figure 5, and the purity of the protein sample is more than 95%.

[0152] The hydroxylation rate of the recombinant human collagen is shown in Table 3:

[0153] Table 3 Hydroxylation rate of recombinant human collagen prepared by different methods

[0154] Example 6: Performance characterization of recombinant human collagen

[0155] (1) Secondary structure determination of recombinant collagen with different hydroxylation rates

[0156] The recombinant human collagen solution prepared in Example 5 was concentrated to a final concentration of 1 mg / mL in a 10 kDa Spin-XR UF 500 centrifugal concentrator (millipore) with 20 mM phosphate buffer as the solvent and stored at 4°C. The above recombinant collagen solution was subjected to spectral scanning using a circular dichroism spectrometer, with a scanning wavelength of 190 nm to 260 nm, a 1 mm quartz cuvette, a scanning temperature of 4°C, a wavelength step of 1 nm, an average time of 1 s, and the CD spectrum being the average of three scans. The results are shown in Figure 6, which shows that the hydroxylated recombinant human collagen prepared by the two methods in Example 5 has a maximum absorption peak at 221 nm, and a negative peak at less than 200 nm, which is consistent with the structural characteristics of the triple helix of collagen, and the hydroxylated human collagen prepared by the exogenous incorporation method has a more obvious positive absorption peak.

[0157] (2) Thermal stability detection of recombinant collagen with different hydroxylation rates

[0158] The recombinant human collagen samples prepared above were subjected to thermal variation scanning using circular dichroism, with a temperature increase rate of 1°C / min, and the CD spectrum value at 221 nm was determined with respect to temperature. The results are shown in Figure 7, which shows that the denaturation temperature of the unhydroxylated recombinant human collagen is 22°C, the denaturation temperature of the hydroxylated recombinant human collagen prepared by the exogenous incorporation method is 27°C, and the denaturation temperature of the hydroxylated recombinant human collagen prepared by the hydroxylation enzyme method is 25°C, so the present method can significantly improve the thermal stability of the recombinant human collagen.

[0159] Example 7: Cell adhesion detection of recombinant collagen with different hydroxylation rates

[0160] (1) Cell adhesion experiment

[0161] The recombinant human collagen prepared in Example 5 was dissolved in PBS (0.01 M, pH = 7.4) to prepare a collagen solution of 0.2 mg / mL, and a 0.2 mg / mL bovine type I collagen standard was used as a positive control. 30 μL of each solution was added to each well of a 96-well plate and placed in a cell incubator for 1 h, with 3 replicate wells for each well. Mouse fibroblast (NIH3T3) cells were diluted to a density of 1 x 10 5The OD450nm absorbance value was detected by using the CCK-8 kit.

[0162] According to the value of the blank control, the relative absorbance of each well was calculated, and the calculated absorbance value was proportional to the number of adherent cells. The sample absorbance value = test well - blank well. The absorbance value of the unhydroxylated collagen was taken as 100%, and the adhesion activity of the hydroxylated recombinant human collagen and the bovine type I collagen standard prepared by the two methods was calculated.

[0163] The results are shown in Table 4 and Figure 8. Compared with the unhydroxylated and hydroxylated enzyme-prepared hydroxylated recombinant human collagen, the cell adhesion of the hydroxylated recombinant collagen was significantly improved.

[0164] Table 4 Cell adhesion of recombinant human collagen prepared by different methods

[0165] (2) Cell compatibility experiment

[0166] Mouse fibroblasts (NIH-3T3) growing to 70%-80% of the area of the culture dish were taken, and trypsin digestion was performed to prepare a cell suspension with a cell density of 5x10 4 μL of the cell suspension was inoculated in each well of a 96-well culture plate, and the plate was placed in a 37°C, 5% CO2 saturated humidity incubator for culture. After 12 hours of cell culture, the complete culture medium was aspirated. In the experimental group, 100 μg / mL of the hydroxylated recombinant human collagen prepared in Example 5 diluted in high-sugar DMEM medium was added to each well (3 replicates), and the positive control was the addition of the same concentration of bovine type I collagen standard in high-sugar DMEM medium to the cells. The plate was continued to be cultured in a 37°C, 5% CO2 saturated humidity incubator for 2 days, and the OD450nm absorbance value was detected by using the CCK-8 kit.

[0167] The absorbance value of the unhydroxylated collagen was taken as 100%, and the cell compatibility of the hydroxylated recombinant human collagen prepared by the two methods and the bovine type I collagen standard was calculated. The results are shown in Figure 9. Both hydroxylations had no effect on the cell compatibility of the recombinant human collagen.

[0168] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A bacterial strain capable of expressing a foreign protein, characterized in that: The strain capable of expressing exogenous proteins is an Escherichia coli recombinant engineering strain with knockout of main enzyme genes in proline synthetic metabolic pathway, knockout of ompT protease gene and knockout of Lon protease gene; The knockout includes: The nucleotide sequence of the knockout frame targeting the main enzyme genes in proline synthetic metabolic pathway is shown in SEQ ID No. 2; The nucleotide sequence of the knockout frame targeting the ompT protease gene is shown in SEQ ID No. 4; The nucleotide sequence of the knockout frame targeting the Lon protease gene is shown in SEQ ID No.

6.

2. The strain capable of expressing a foreign protein according to claim 1, characterized in that Preparation is obtained by the following steps: (1) Taking the Escherichia coli genetic engineering strain as the starting strain, the main enzyme genes in proline synthetic metabolic pathway are knocked out to obtain strain A; (2) On the basis of strain A, the ompT protease gene is knocked out to obtain strain B; (3) On the basis of strain B, the Lon protease gene is knocked out to obtain the strain capable of expressing exogenous proteins.

3. The strain capable of expressing exogenous proteins according to claim 1 or 2, characterized in that: The method for knocking out is CRISPR-cas9 gene editing knockout; The vector targeting the main enzyme genes in proline synthetic metabolic pathway in the knockout of the main enzyme genes in proline synthetic metabolic pathway is obtained by the following steps: inserting the sgRNA targeting the main enzyme genes in proline synthetic metabolic pathway and the knockout frame targeting the main enzyme genes in proline synthetic metabolic pathway on the Escherichia coli knockout plasmid; The vector targeting the ompT protease gene in the knockout of the ompT protease gene is obtained by the following steps: inserting the sgRNA targeting the ompT protease gene and the knockout frame targeting the ompT protease gene on the Escherichia coli knockout plasmid; The vector targeting the Lon protease gene in the knockout of the Lon protease gene is obtained by the following steps: inserting the sgRNA targeting the Lon protease gene and the knockout frame targeting the Lon protease gene on the Escherichia coli knockout plasmid.

4. The strain capable of expressing exogenous proteins according to claim 3, characterized in that: The Escherichia coli knockout plasmid is pTargetF plasmid; The nucleotide sequence of the sgRNA targeting the main enzyme genes in proline synthetic metabolic pathway is shown in SEQ ID No. 1; The nucleotide sequence of the sgRNA targeting the ompT protease gene is shown in SEQ ID No. 3; The nucleotide sequence of the sgRNA targeting the Lon protease gene is shown in SEQ ID No.

5.

5. The strain capable of expressing exogenous proteins according to any one of claims 1-4 is used in the expression of exogenous proteins containing proline and hydroxyproline.

6. Use according to claim 5, characterized in that: The exogenous protein containing proline and hydroxyproline is recombinant human collagen.

7. A method of synthesizing a recombinant human-derived collagen, characterized by Including the following steps: 1) Transferring the recombinant expression vector capable of expressing recombinant human collagen and the recombinant vector capable of expressing T7 RNA polymerase into the strain capable of expressing exogenous proteins according to any one of claims 1-4 to obtain a recombinant genetic engineering strain; 2) culturing the recombinant genetically engineered bacteria obtained in step 1), adding an inducer to induce expression of T7 RNA polymerase; 3) changing the medium and performing starvation culture; 4) adding IPTG, proline and hydroxyproline to perform culture to induce expression of recombinant human collagen; 5) performing solid-liquid separation on the culture solution obtained in step 4) to obtain bacterial cells; 6) crushing the bacterial cells, performing solid-liquid separation, purifying the obtained supernatant to obtain recombinant human collagen.

8. The synthesis method according to claim 7, characterized in that: the amino acid sequence of the recombinant human collagen in step 1) is as shown in SEQ ID No. 7, SEQ ID No. 11 or SEQ ID No. 13; the vector framework of the recombinant expression vector capable of expressing the recombinant human collagen in step 1) is an expression vector with a T7 promoter; the amino acid sequence of the T7 RNA polymerase in step 1) is as shown in SEQ ID No. 9; the vector framework of the recombinant vector capable of expressing the T7 RNA polymerase in step 1) is an expression vector containing an arabinose promoter; the medium used in the culture in step 2) is LB medium containing antibiotics resistant to the recombinant expression vector capable of expressing the recombinant human collagen and the recombinant vector capable of expressing the T7 RNA polymerase; the inducer in step 2) is a substance that enhances the promoter activity of the T7 RNA polymerase; the amount of the inducer added in step 2) is calculated according to its concentration in the culture system, which is 0.5-1.5 mg / mL; the time of the induction in step 2) is 5-40 min; the composition of the medium in the starvation culture in step 3) is as follows: M9 medium with a glucose concentration of 1-10 w / v% as a solvent, sodium chloride 300-600 mM, glycine 20-500 mg / L, alanine 20-500 mg / L, valine 20-500 mg / L, leucine 20-500 mg / L, isoleucine 20-500 mg / L, methionine 20-500 mg / L, tryptophan 20-500 mg / L, serine 20-500 mg / L, tyrosine 20-500 mg / L, cysteine 20-500 mg / L, phenylalanine 20-500 mg / L, asparagine 20-500 mg / L, glutamine 20-500 mg / L, threonine 20-500 mg / L, aspartic acid 20-500 mg / L, glutamic acid 20-500 mg / L, lysine 20-500 mg / L, arginine 20-500 mg / L and histidine 20-500 mg / L; the conditions of the starvation culture in step 3) are 36-38 °C, 150-250 rpm for 30-90 min; the concentration of the IPTG in step 4) is 0.1-2 mM; the concentration of the proline in step 4) is 0-40 mM; the concentration of the hydroxyproline in step 4) is 0-40 mM; the proline and the hydroxyproline cannot be both 0. ​ The composition of the bacterial suspension used in the disruption in step 6) is as follows: 15-25 mmol / L Tris-HCl, 450-550 mmol / L NaCl, pH 8.0-9.

0.

9. The synthetic method of claim 8, wherein: The sequence of the nucleic acid encoding the recombinant human-derived collagen in step 1) is as shown in SEQ ID No. 8, SEQ ID No. 12, or SEQ ID No. 14; The vector framework of the recombinant expression vector capable of expressing the recombinant human-derived collagen in step 1) is pET series plasmid; The nucleotide sequence of the gene encoding the T7 RNA polymerase is as shown in SEQ ID No. 10; The vector framework of the recombinant vector capable of expressing the T7 RNA polymerase in step 1) is pBAD33; The medium used in the culture in step 2) is LB medium containing 50 mg / L kanamycin and 25 mg / L chloramphenicol; The inducer in step 2) is arabinose; The induction time in step 2) is 15 min; The composition of the medium in the starvation culture in step 3) is as follows: M9 medium with a glucose concentration of 1-10 w / v% as a solvent, 450-550 mM sodium chloride, 90-110 mg / L glycine, 90-110 mg / L alanine, 90-110 mg / L valine, 90-110 mg / L leucine, 90-110 mg / L isoleucine, 90-110 mg / L methionine, 90-110 mg / L tryptophan, 90-110 mg / L serine, 90-110 mg / L tyrosine, 90-110 mg / L cysteine, 90-110 mg / L phenylalanine, 90-110 mg / L asparagine, 90-110 mg / L glutamine, 90-110 mg / L threonine, 90-110 mg / L aspartic acid, 90-110 mg / L glutamic acid, 90-110 mg / L lysine, 90-110 mg / L arginine, and 90-110 mg / L histidine; The conditions of the starvation culture in step 3) are 37°C, 220 rpm for 60 min; The concentration of IPTG in step 4) is 0.8-1.2 mM; The concentration of proline in step 4) is 0-16 mM; The concentration of hydroxyproline in step 4) is 4-20 mM; The composition of the bacterial suspension used in the disruption in step 6) is as follows: 20 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 8.5; The purification in step 6) is purification using a nickel affinity chromatography column; The purification steps in step 6) are as follows: the supernatant is loaded into a nickel affinity chromatography column, the eluent is a bacterial suspension containing 50-500 mM imidazole, and the eluate obtained by collecting the bacterial suspension containing 100-150 mM imidazole is collected.

10. The synthetic method of claim 7, wherein: The recombinant expression vector expressing recombinant human collagen and the recombinant expression vector expressing T7 RNA polymerase in step 1) are mixed at a molar ratio of 1:1; The culture condition in step 2) is 36-38℃, 150-250rpm; The degree of cultivation in step 2) is OD of the bacterial solution 600 is 1 to 4; The culture condition in step 4) is 36-38℃, 150-250rpm for 3-6h; The solid-liquid separation method in step 5) is centrifugation; The cell disruption condition in step 6) is 2-8℃, 25-35kpsi; The solid-liquid separation method in step 6) is centrifugation.

11. Use of the synthesis method of the recombinant human collagen according to any one of claims 7-10 in the preparation of recombinant human collagen with different hydroxylation rates.

12. A recombinant human-derived collagen, characterized in that: The recombinant human collagen is obtained by the synthesis method according to any one of claims 7-10.

13. The recombinant human-derived collagen of claim 12, wherein: The recombinant human collagen has a hydroxylation rate of 40-45%.

14. Use of the recombinant human collagen according to claim 13 in the preparation of bioengineering materials and / or cosmetics.

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