CN120818547A - A recombinant humanized type A and type III collagen, its preparation method and recombinant bacteria - Google Patents
A recombinant humanized type A and type III collagen, its preparation method and recombinant bacteriaInfo
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Abstract
The invention discloses a recombinant humanized type A III collagen, a preparation method thereof and recombinant bacteria, and relates to the technical field of bioengineering, wherein the preparation method integrates 100% peptide fragments of the same sequence of human type III collagen into host bacteria to obtain a recombinant bacteria for efficiently synthesizing the recombinant humanized type A III collagen, the recombinant humanized collagen produced by utilizing the recombinant bacteria does not contain any gene sequence of non-human collagen sources, and the recombinant humanized type A III collagen has the characteristics of small molecular weight, high yield, high purity, easy water solubility and the like, and can be widely applied to the fields of skin care products, medical cosmetology, skin repair dressings and the like, so that the recombinant humanized type A III collagen and the preparation method thereof have better industrial application prospects.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to recombinant humanized A-type III collagen, a preparation method and application thereof, recombinant bacteria and a construction method of the recombinant bacteria.
Background
Collagen is an important protein constituting human body structural tissues, is widely distributed in animal connective tissues, and plays an important role in maintaining normal physiological functions and injury repair of cells, tissues and organs. The recombinant humanized collagen has good physical properties and biological characteristics, and has wide application in the fields of medicine, tissue engineering, food, cosmetics and the like.
Collagen is a biopolymer, and the main component in animal connective tissue can play a plurality of roles of supporting, protecting, connecting and the like. The currently known collagen types are more than 29, and the most widely distributed collagen types are I, II, III, IV and V, wherein the content of the type I collagen is the most, and the type I collagen accounts for 80-90% of the total amount of the collagen. Wherein, the type I collagen is mainly in adult skin, tendon and bone tissue, the type II collagen is mainly in cartilage, eye vitreous body and intervertebral disc, etc., and the type III collagen is mainly in infant skin or blood vessel intima and intestinal tract.
The III type collagen has a fine and loose silk-screen structure relative to the I type collagen, is scattered at the connection part of epidermis and dermis, and provides elasticity and stress resistance for the skin. Type III collagen occupies about 80% of the period of infants, and as the age increases, type III collagen is continuously lost, the skin is in a dull and matt state, and therefore, type III collagen is also called infant collagen.
Currently, there are two general sources of collagen, animal extraction and biosynthesis, respectively. The animal collagen extraction process is complex, the product stability is poor, and the risk of carrying viruses exists. The recombinant collagen obtained by the biosynthesis method has low cost, high purity and high biosafety, and the market growth speed is about twice that of the collagen extracted by animals, but the existing biosynthesis method has the problems of low purity and low yield.
The 2021, 3 and 15 release the "recombinant collagen biomaterial naming guidance principle" (hereinafter "principle") to solve the phenomenon of irregular expression in industry. The principle divides recombinant collagen into three types, recombinant human collagen, recombinant humanized collagen and recombinant collagenous protein. The recombinant humanized collagen is divided into A type and B type, wherein the A type is a material which does not contain non-human collagen amino acid sequence, and the B type is a material which adds non-human collagen amino acid sequence such as connecting amino acid, tag amino acid and the like on the basis of combining the functional fragments of the human collagen. Since type a is entirely human collagen sequence, the allergic response after use is greatly reduced.
The recombinant type III collagen is a full-length or partial amino acid sequence fragment containing the codes of collagen specific genes prepared by a DNA recombination technology, or a combination of collagen functional fragments, but the accurate screening of the amino acid sequence of a target functional region in a specific collagen complete gene is not easy, and the analysis of the full-chain length gene sequence of the protein is needed, so that the screening of the amino acid of a core functional region is ensured to have the expected efficacy.
There are two types of recombinant type III collagen. The recombinant type III collagen is prepared by a DNA recombination technology, and is designed and modified and has a specific gene coding amino acid sequence or a fragment thereof, and although the yield is higher, the amino acid sequence has low homology with the human collagen sequence, the activity is low, and the immunogenicity risk exists. The other type is recombinant humanized III type collagen, the full length or partial amino acid sequence of human collagen has lower yield, but has high homology with the amino acid sequence of human collagen, high activity and no immunogenicity. However, there is currently no solution for expressing humanized type III collagen that is effective in improving its yield.
Therefore, there is a need to provide a method for preparing recombinant humanized type a III collagen with high yield, so as to meet the rapidly growing market demands of high-purity humanized collagen in the fields of medical science, medical appliances and the like.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a recombinant humanized type A III collagen, a preparation method, application and recombinant bacteria thereof and a construction method thereof, wherein the method integrates 100 percent peptide fragments with the same sequence from human type III collagen (Homo Sapiens) into host bacteria to obtain a recombinant bacteria for efficiently synthesizing the recombinant humanized type A III collagen, the recombinant humanized collagen produced by utilizing the recombinant bacteria does not contain any gene sequence from non-human collagen sources, and the collagen has the characteristics of small molecular weight, high yield, high purity and easy dissolution in water, and can be widely applied to the fields of skin care products, medical cosmetology, skin repair dressings and the like.
In order to solve the problems, the invention adopts the following technical scheme.
A preparation method of recombinant humanized type A III collagen comprises the following preparation steps:
S1, constructing recombinant bacteria, namely connecting a target gene shown as SEQ ID NO. 2 to an expression vector through enzyme digestion to obtain a recombinant vector, amplifying the recombinant vector, then converting the amplified recombinant vector into an expression host, and carrying out positive cloning to obtain the recombinant bacteria;
S2, carrying out methanol induction on the recombinant bacteria obtained in the step S1, screening high-expression strains through detecting protein expression quantity, inoculating the high-expression strains into a BSM culture medium for fermentation after screening to obtain fermentation liquor, centrifugally collecting thalli, carrying out cell disruption on the thalli, and centrifuging to obtain a supernatant, wherein the supernatant is crude protein liquid;
And S3, purifying and dialyzing the crude protein liquid to obtain recombinant humanized A type III collagen liquid.
In the step S1, the enzymes used for enzyme digestion are EcoRI and NotI, the expression vector is pPICZa A, the amplification host is escherichia coli, the expression host is pichia pastoris, preferably pichia X33, and the positive cloning is carried out by coating the expression host transformed with the recombinant vector on a YPD plate added with bleomycin and culturing for 2-3 days at 28 ℃.
Further, in the step S2, the fermentation temperature is 30 ℃, the fermentation pH is 5.0, the fermentation time is 70h, the centrifugation time is 30min, and the centrifugation rotating speed is 10000rpm.
Further, in the step S3, the purification comprises sequentially passing the crude protein solution through a desalting column and a cation exchange chromatographic column, wherein the desalting column is HiPre TM/10 DESALTING, the cation exchange chromatographic column is SP-FF, the components of the dialyzate are 10 mM Tris and 5mM NaCl, and the pH value of the dialyzate is 8.0.
The invention also provides a recombinant bacterium for expressing the recombinant humanized A type III collagen, which comprises recombinant plasmids pPICZa A-colla III and an expression host, wherein the recombinant plasmids comprise a target gene and an expression vector, the nucleotide sequence of the target gene is shown as SEQ ID NO. 2, the molecular weight of a protein product is 12kDa, and the expression vector is pPICZa A.
Further, the expression host is pichia pastoris, preferably pichia pastoris X33.
The invention also provides a construction method of the recombinant bacterium for expressing the recombinant humanized A type III collagen, which comprises the steps of connecting a target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacterium.
Further, the nucleotide sequence of the target gene is shown as SEQ ID NO. 2, the expression vector is pPICZa A, the recombinant plasmid is pPICZa A-colla III, and the expression host is Pichia pastoris, preferably Pichia pastoris X33.
Further, the construction method of the recombinant bacterium is specifically as follows:
S1, performing enzyme digestion by using EcoRI enzyme and NotI enzyme, and connecting a target gene to pPICZa A to obtain a recombinant plasmid pPICZa A-colla III;
S2, transferring pPICZa A-colla III obtained in the step S1 into escherichia coli for amplification, screening, extracting plasmids, and linearizing;
And S3, transferring pPICZa A-colla III linearized in the step S2 into an expression host for positive cloning, wherein the cloned strain is recombinant strain.
In the step S2, the culture medium for amplification is an LB plate with zeocin (the host in the step S2 is escherichia coli, the used LB plate is selected), the culture temperature is 37 ℃, the linearization enzyme is Sac I enzyme, in the step S3, the culture dish for positive cloning is a YPD plate added with zeocin (bleomycin), the culture temperature for positive cloning is 28 ℃, and the cloning time is 2-3 d.
Further, the E.coli is preferably E.coli DH 5. Alpha.
The invention also provides a recombinant humanized A type III collagen, which is prepared by the preparation method of the recombinant humanized A type III collagen or is obtained by expression of the recombinant strain for expressing the recombinant humanized A type III collagen.
Furthermore, the amino acid sequence of the recombinant humanized type A III collagen is shown as SEQ ID NO. 1.
The invention also provides application of the recombinant humanized A-type III collagen to preparation of external auxiliary materials or biological materials for moisturizing, moisturizing or repairing skin.
The invention also provides application of the recombinant humanized A-type III collagen in preparing moisturizing skin care products.
The invention also provides a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1.
The invention also provides a DNA molecule for encoding the polypeptide or the recombinant humanized A-type III collagen, and the base sequence of the DNA molecule is shown as SEQ ID NO. 2.
Specifically, the invention is realized as follows:
In a first aspect, the invention provides a recombinant bacterium for efficiently synthesizing recombinant humanized type A III collagen, which comprises a recombinant plasmid and an expression host, wherein the recombinant plasmid comprises a target gene and an expression vector, and the target gene is a gene with the sequence 100% identical to that of human type III collagen.
In order to obtain recombinant humanized type-III collagen with higher yield, the inventor uses Kyte-Doolittle hydrophilicity index to screen a plurality of high-hydrophilicity regions, confirms collagen triple helix domain through InterPro or Pfam to obtain candidate molecules, then sends the candidate molecules to Shanghai Biotechnology limited company to synthesize encoding DNA of the candidate molecules, transfers the encoding DNA into cells to express, screens to obtain a plurality of candidate fragments with moisturizing and moisturizing functions, loads the encoding DNA of the candidate fragments into recombinant bacteria, and screens out functional fragments capable of being highly expressed in the recombinant bacteria.
Specifically, the amino acid sequence of the functional fragment obtained by screening is shown as SEQ ID NO. 1:
PGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGV.
Because different species and cells have codon bias, in order to enable the host cells to obtain stable and high-expression recombinant humanized type A III collagen by utilizing the bias of codons, the functional fragment gene obtained by screening is reversely designed into a gene sequence by utilizing an online design tool Jcat, and the gene sequence is subjected to codon optimization aiming at the expression of pichia pastoris, and in some embodiments, the nucleotide sequence of the optimized recombinant humanized type A III collagen is shown as SEQ ID NO. 2:
CCTGGTGCCGATGGACAACCTGGAGCAAAGGGTGAACCTG GAGACGCTGGTGCTAAAGGTGATGCTGGTCCACCTGGTCCAGCAGGTCCAGCTGGTCCACCTGGTCCTATCGGTAACGTGGGAGCCCCCG GTGCTAAAGGCGCACGTGGTTCTGCTGGACCCCCTGGAGCTACTGGATTTCCCGGTGCCGCAGGTAGAGTTGGACCACCTGGACCTTCTGGAAACGCCGGACCTCCTGGTCCACCAGGTCCTGCCGGTAAAGAGGGAGGA AAGGGCCCAAGAGGAGAAACGGGTCCTGCTGGCAGACCCGGAGAAGTTGGTCCTCCAGGACCACCAGGTCCAGCCGGAGAGAAAGGTTCCCCCGGAGCTGATGGTCCAGCTGGAGCACCTGGTACTCCCGGTCCCCAGGG TATAGCTGGTCAGCGTGGTGTT.
in some embodiments, the expression host is pichia, preferably pichia X33.
In some embodiments, the expression vector is pPICZa A.
In a second aspect, the invention provides a construction method of the recombinant bacterium, which comprises the steps of connecting a target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacterium.
In some embodiments, the recombinant plasmid is pPICZa A-colla III and the expression host is pichia pastoris, preferably pichia X33.
In a third aspect, the invention provides the use of the recombinant bacterium described above in recombinant humanized type A III collagen.
In a fourth aspect, the present invention also provides a method for producing recombinant humanized type a type III collagen, comprising constructing the recombinant bacterium, inoculating the recombinant bacterium into a BSM medium for fermentation to obtain a fermentation broth, centrifugally collecting thalli, disrupting cells of the thalli, and centrifuging to obtain a supernatant, wherein the supernatant is a crude protein solution.
In some embodiments, the recombinant bacteria are fermented in BSM medium at a ph=5.0 for a period of 70 hours.
In some embodiments, the method further comprises purifying and dialyzing the crude protein solution.
In some embodiments, the purifying comprises passing the crude protein solution sequentially through a desalting column and a cation exchange chromatography column.
In some embodiments, the desalting column is HiPre TM/10 DESALTING.
In some embodiments, the cation exchange chromatography column is SP-FF.
In some embodiments, the components of the dialysate for dialysis are 10mM Tris and 5mM NaCl, and the pH of the dialysate = 8.0.
Compared with the prior art, the invention has the advantages that:
The invention integrates 100 percent peptide fragments with the same sequence from human type III collagen (Homo Sapiens) into host bacteria to obtain a recombinant bacteria for efficiently synthesizing recombinant humanized type A type III collagen, the recombinant humanized collagen produced by the recombinant bacteria does not contain any gene sequence from non-human collagen, the collagen has the characteristics of small molecular weight, high yield, high purity, easy water dissolution and the like, and can be widely applied to the fields of skin care products, medical cosmetology, skin repair dressings and the like, so that the recombinant humanized A-type III collagen and the preparation method thereof provided by the invention have better industrial application prospects.
Drawings
FIG. 1 is a plasmid map pPICZa A-colla III of recombinant Pichia pastoris synthetic expression recombinant humanized type III collagen of example 1;
FIG. 2 is a diagram showing the construction of DNA for plasmid pPICZa A-colla III of example 1;
FIG. 3 is a SDS-PAGE diagram of recombinant Pichia pastoris pPICZa A-colla III expressed proteins of example 2;
FIG. 4 is a SDS-PAGE map of the cation exchange chromatography column purification of example 2;
FIG. 5 is a graph showing the results of the thermal stability study of the purified collagen of example 2;
FIG. 6 shows the results of the water-solubility test of the purified collagen of example 2.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Coli DH 5. Alpha. Used in the following examples was purchased from Biotechnology (Shanghai) Co., ltd. And Pichia X33 was purchased from vast Ling plasmid platform.
The culture medium referred to in the following examples was yeast extract peptone glucose medium (YPD) purchased from Bio/engineering (Shanghai) Inc., BMGY medium purchased from Bio/engineering (Shanghai) Inc., BMMY medium purchased from Bio/engineering (Shanghai) Inc., and BSM medium purchased from Bio/engineering (Shanghai) Inc. SOC media was purchased from Biotechnology (Shanghai) Inc.
Restriction enzymes, ligases are purchased from NEW ENGLAND Bioland (New England Biotechnology (Beijing) Inc.).
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Examples
The embodiment is a construction method for preparing recombinant bacteria of recombinant humanized A type III collagen, comprising the following steps:
(1) Ligating a synthetic gene (the gene fragment shown in SEQ ID NO. 2) to the vector pPICZa A by EcoRI and NotI cleavage (using T4 DNA ligase at 16 ℃ overnight), naming the ligation product as pPICZa A-colla III, then carrying out sequencing to verify the ligation correctness, and then sending the result of double cleavage of the complete vector to the Souzhou Jin Weizhi Biotechnology Co., ltd. Gene sequencing to verify that the construction is successful by sequence alignment;
(2) Transferring pPICZa A-colla III into Escherichia coli DH5 alpha (heat shock at 42 ℃ C. For 45s after ice bath for 30min, ice bath for 2min again, adding 800 μl of SOC culture medium and resuscitating for 1h at 37 ℃ C.), amplifying pPICZa A-colla III, then coating on LB plate containing 25 μg/mL Zeocin (bleomycin), culturing at 37 ℃ C. Overnight for screening, and extracting plasmid from positive transformant according to standard procedure (operation procedure is conventional operation and not detailed), and performing electrophoresis verification (electrophoresis result is shown in FIG. 2), wherein positive transformant shows white single colony;
(3) Performing enzyme digestion pPICZa A-colla III (37 ℃ for 2 h) by using Sac I to linearize the sample, and performing electrophoresis verification on the sample to confirm that linearization is completed;
(4) Linearized pPICZa A-colla III was electrotransferred into Pichia X33 (the electrotransfer procedure is conventional and will not be described in detail here), smeared onto YPD plates with 300. Mu.g/mL zeocin added, and positive cloned at 28℃for 2-3 days to give recombinant bacteria.
Single colonies were picked with sterile toothpicks or inoculating loops and inoculated into YPD+Zeocin broth for further validation. Subsequently 20% glycerol was added and stored frozen at-80 ℃.
Wherein FIG. 1 is a schematic diagram of construction of recombinant plasmids pPICZa A-colla III, and FIG. 2 is a verification diagram of construction results of recombinant plasmids pPICZa A-colla III. In FIG. 2, M is a marker representing DNA, 1, 2, 3 and 4 are all complete plasmids, namely target vectors, 5, 6, 7 and 8 are all double-enzyme cutting results of the complete vectors, the results show that the constructed vectors are as large as the target vectors, the size of the constructed vectors is consistent with that of the target vectors, and the double-enzyme cutting results are as expected.
Examples
The embodiment is a production and purification method of recombinant humanized A-type III collagen, which comprises the following steps:
(1) Ligating a synthetic gene (the gene fragment shown in SEQ ID NO. 2) to the vector pPICZa A by EcoRI and NotI cleavage (using T4 DNA ligase at 16 ℃ overnight), naming the ligation product as pPICZa A-colla III, then carrying out sequencing to verify the ligation correctness, and then sending the result of double cleavage of the complete vector to the Souzhou Jin Weizhi Biotechnology Co., ltd. Gene sequencing to verify that the construction is successful by sequence alignment;
(2) Transferring pPICZa A-colla III into Escherichia coli DH5 alpha (heat shock at 42 ℃ C. For 45s after ice bath for 30min, ice bath for 2min again, adding 800 μl of SOC culture medium and resuscitating for 1h at 37 ℃ C.), amplifying pPICZa A-colla III, then coating on LB plate containing 25 μg/mL Zeocin (bleomycin), culturing at 37 ℃ C. Overnight for screening, and extracting plasmid from positive transformant according to standard procedure (operation procedure is conventional operation and not detailed), and performing electrophoresis verification (electrophoresis result is shown in FIG. 2), wherein positive transformant shows white single colony;
(3) Performing enzyme digestion pPICZa A-colla III (37 ℃ for 2 h) by using Sac I to linearize the sample, and performing electrophoresis verification on the sample to confirm that linearization is completed;
(4) Carrying out electric transfer on the linearized pPICZa A-colla III into Pichia pastoris X33, coating the X33 on a YPD plate added with 300 mug/mL zeocin, and culturing for 2-3 days at 28 ℃ to carry out positive cloning to obtain recombinant bacteria;
(5) Colonies selected from YPD plates containing 300. Mu.g/mL zeocin were inoculated into BMGY medium for fermentation (30 ℃), cultured for 24 hours, transferred to BMMY medium, and added with 0.5% methanol for induction of expression;
(6) After methanol induction for 5 days, collecting the supernatant, detecting the protein expression level by SDS-PAGE, and selecting the strain corresponding to the protein band with the coarsest protein band (as the protein band with blue arrow in FIG. 3), thereby screening the strain with the highest expression level (number: A1);
(7) The strain A1 with the highest expression level is subjected to pilot fermentation (pH value is controlled to 5.0,30 ℃ C., 70 h) in BSM culture medium
(8) After fermentation, centrifuging the fermentation liquor, centrifuging 10000 r.min -1 for 30 min, and collecting the supernatant, wherein the supernatant is crude protein liquid;
(9) The purification mode is ion exchange column purification. Purifying the crude protein solution by HiPre TM/10 DESALTING and SP-FF respectively to obtain collagen solution;
(10) The recombinant humanized type A III collagen solution is obtained by using a dialysis solution (pH=8.0) composed of 10 mM Tris (tris) and 5mM NaCl to carry out dialysis (4 ℃ C., 12-16 h).
The obtained recombinant humanized type A III collagen is sent to a gene of Suzhou gold intelligence biotechnology limited company for LC-MS/MS analysis, and the sequence comparison proves that the amino acid sequence of the obtained recombinant humanized type A III collagen is consistent with that of SEQ ID NO. 1, and no gene sequence of non-human collagen source is detected.
FIG. 3 is a SDS-PAGE of recombinant Pichia X33/pPICZa A-colla III expression protein, in which NC is a blank, M represents markers of DNA, 1 to 10 represent 10 seed expression patterns selected, and blue arrows indicate seeds with high relative expression, i.e., colonies numbered A1 (seeds refer to colonies selected from YPD plates containing 300. Mu.g/mL zeocin), and the target protein is shown in a single band at 12 kDa.
FIG. 4 is a SDS-PAGE profile of recombinant humanized type A III collagen ion exchange column purification. Wherein M represents a marker of DNA, S represents a fermentation supernatant stock solution, FT represents a flow-through solution, E0 represents bufferA eluent, P1 represents 50mM NaCl eluent, P2 represents 100 mM NaCl eluent, P3 represents 200mM imidazole eluent, P4 represents 300 mM imidazole eluent, and P5 represents 500 mM imidazole eluent. In the figure, 65kDa is the brightest band in the protein marker, which facilitates comparison with the target protein. The figure shows that the target protein is mainly at the P3 peak, and the hybrid proteins are in P1 and P2.
Test example 1:
the purified recombinant humanized collagen solution of example 2 was subjected to a thermal stability test as follows:
The purified protein solution was placed in a4 degree refrigerator and samples were taken at different times and subjected to SDS-PAGE to detect whether the collagen solution was degraded.
Fig. 5 shows the results of the thermal stability study of purified collagen, wherein M is a protein marker,1 is a detection result of 0 day, and 2, 3 and 4 are detection results of 3 days, 8 days and 14 days respectively, and no degradation bands are seen from the figure, which indicates that the collagen has good thermal stability in a 4-degree refrigerator, namely, the recombinant humanized collagen has good thermal stability, namely, the purified collagen is placed for 3d,8d and 14d at 4 degrees and cannot be degraded.
Test example 2:
The purified recombinant humanized collagen solution of example 2 was subjected to a water solubility test as follows:
The lyophilized recombinant humanized collagen powder (150 mg) was weighed and dissolved in 10mL of pure water to observe the solubility.
As shown in FIG. 6, 150mg of the lyophilized powder was dissolved in 10mL of pure water, and the powder was completely dissolved, i.e., the recombinant humanized collagen had good water solubility.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (14)
1. A preparation method of recombinant humanized A type III collagen is characterized by comprising the following preparation steps:
S1, constructing recombinant bacteria, namely connecting a target gene shown as SEQ ID NO. 2 to an expression vector through enzyme digestion to obtain a recombinant vector, amplifying the recombinant vector, then converting the amplified recombinant vector into an expression host, and carrying out positive cloning to obtain the recombinant bacteria;
S2, carrying out methanol induction on the recombinant bacteria obtained in the step S1, screening high-expression strains through detecting protein expression quantity, inoculating the high-expression strains into a BSM culture medium for fermentation after screening to obtain fermentation liquor, centrifugally collecting thalli, carrying out cell disruption on the thalli, and centrifuging to obtain a supernatant, wherein the supernatant is crude protein liquid;
And S3, purifying and dialyzing the crude protein liquid to obtain recombinant humanized A type III collagen liquid.
2. The preparation method of the recombinant humanized type A III collagen according to claim 1 is characterized in that in the S1 step, enzymes used for enzyme digestion are EcoRI enzyme and NotI enzyme, the expression vector is pPICZa A, the amplification host is escherichia coli, the expression host is pichia pastoris, and the positive cloning is carried out by coating the expression host transformed with the recombinant vector on a YPD plate added with bleomycin and culturing for 2-3 days at 28 ℃.
3. The method for preparing recombinant humanized type A III collagen according to claim 1, wherein in the step S2, the fermentation temperature is 30 ℃, the fermentation pH is 5.0, the fermentation time is 70h, the centrifugation time is 30min, and the centrifugation speed is 10000rpm.
4. The method for preparing recombinant humanized type A III collagen according to claim 1, wherein in the step S3, the purification comprises sequentially passing crude protein solution through a desalting column and a cation exchange chromatographic column, wherein the desalting column is HiPre TM/10 DESALTING, the cation exchange chromatographic column is SP-FF, the components of dialyzate are 10 mM Tris and 5 mM NaCl, and the pH of dialyzate is 8.0.
5. A recombinant bacterium for expressing recombinant humanized type A III collagen is characterized by comprising recombinant plasmids pPICZa A-colla III and an expression host, wherein the recombinant plasmids comprise a target gene and an expression vector, the nucleotide sequence of the target gene is shown as SEQ ID NO. 2, the molecular weight of a protein product is 12kDa, and the expression vector is pPICZa A.
6. The recombinant bacterium for expressing recombinant humanized type A III collagen according to claim 5, wherein the expression host is Pichia pastoris.
7. The method for constructing recombinant strain expressing recombinant humanized type A III collagen according to any one of claims 5 to 6, wherein the recombinant strain is obtained by connecting a target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host.
8. The method for constructing recombinant bacteria expressing recombinant humanized type A III collagen according to claim 7, wherein the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the expression vector is pPICZa A, the recombinant plasmid is pPICZa A-colla III, and the expression host is Pichia pastoris.
9. A recombinant humanized type A III collagen is characterized by being prepared by the preparation method of the recombinant humanized type A III collagen according to any one of claims 1 to 4 or being expressed by a recombinant bacterium expressing the recombinant humanized type A III collagen according to any one of claims 5 to 6.
10. The recombinant humanized type A III collagen according to claim 9, wherein the amino acid sequence of the recombinant humanized type A III collagen is shown as SEQ ID NO. 1.
11. The method of claim 10, wherein the recombinant humanized type A III collagen is used for preparing an external auxiliary material or biological material for moisturizing, moisturizing or repairing skin.
12. Use of a recombinant humanized type a type III collagen according to claim 10 in the preparation of a moisturizing or moisturizing skin care product.
13. A polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
14. A DNA molecule encoding a polypeptide according to claim 13 or a recombinant humanized type A III collagen according to claim 10, wherein the base sequence is shown in SEQ ID NO. 2.
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