CN118852410A - High-solubility humanized III type collagen, expression mode thereof and recombinant plasmid - Google Patents

Abstract

The invention discloses high-solubility humanized III type collagen, an expression mode and a recombinant plasmid thereof, belonging to the technical field of genetic engineering; according to the invention, the amino acid of a human III type collagen alpha 1 chain is subjected to hydrophilic analysis, a highly hydrophilic collagen gene fragment col3a is selected, a VNTR enhancer derived from a human STIN2 protein and a TES enhancer derived from a mouse Sox9 gene are introduced before the recombinant collagen protein, the selected col3a, VNTR enhancer fragment and TES enhancer fragment are optimized and synthesized, a PCR cloning is utilized to obtain a connecting fragment, and the collagen fragment (col 3 a) n, the VNTR enhancer fragment, the TES enhancer fragment and the pPIC9K vector skeleton fragment are connected through a Gibson Assembly seamless connection technology, so that the recombinant plasmid pPIC9K-VNTR-TES- (col 3 a) n is constructed.

Description

High-solubility humanized III type collagen, expression mode thereof and recombinant plasmid

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to high-solubility humanized III-type collagen, an expression mode thereof and a recombinant plasmid.

Background

Collagen is one of the most abundant functional structural proteins in the human body, widely distributed in the extracellular matrix (ECM) of connective tissue of humans and animals, and is involved in the composition of many human organ tissues, such as skin, cornea, neural retina, bone, muscle, etc. Accounting for 30% of the total protein of the whole body. Common collagens can be classified into type i, type ii, type III, type v and type xi, wherein type III collagen is one of the most abundant fibrillar collagens, and is a protein formed by winding 3 α1 chains into a right-handed triple helix structure with each other. Collagen plays an indispensable role in maintaining extracellular environment, maintaining normal physiological functions of tissues and organs, repairing damage, affecting cell attachment and proliferation and other various biological functions.

The III type collagen has rich contents in blood vessels, intestinal tracts and skin, plays an important role in the growth of organisms and the wound healing process, can be combined with receptors on the surfaces of cells in the wound healing process, promotes the adhesion and migration of cells, induces inflammatory cells and fibroblasts to migrate to wound sites, and promotes the formation and recovery of connective tissues. Type III collagen is commonly used as a dermal filler in cosmetic surgery to treat wrinkles and skin aging, as a rehabilitation aid for burn patients, and for bone reconstruction and various dental, orthopedic and surgical procedures to achieve artificial repair. The III type collagen has wide application in the skin care field, and a lot of research and study are also carried out, so that the application of the III type collagen at the front edge is hopeful to be expanded, such as: the biosynthesis cornea made of the human III type collagen is optically transparent, can replace a donor therapy or a transgenic pig therapy method, and does not need to use immunosuppressive drugs; the artificial synthesis of the heart valve and the endometrial perfusion repair are completed by using human III type collagen; the III type collagen is added into the tumor microenvironment, so that the tumor cells can be promoted to enter and maintain in a dormant state, and the proliferation of the tumor can be inhibited. Thus, collagen type III is potentially a huge market for applications.

The collagen products in the market at present are mostly extracted from tissues, organs and connective tissues of mammals, and animal-derived extraction methods are mainly prepared industrially by an acid method and an enzyme method, and the technology is mature but has the problems of animal-derived disease infection, immune rejection or allergic reaction, capacity limitation and the like, such as the risk of pathogen pollution of prion, HIV, foot-and-mouth disease viruses and the like. Along with the large-scale application of the genetic engineering technology, the generation of recombinant collagen through genetic engineering becomes the most potential method for solving the problem of collagen source limitation. Compared with animal-derived collagen, the recombinant collagen produced by the genetic engineering technology has the same properties as human-derived collagen, has the advantages of strong water solubility, high biological activity, low immunogenicity and high biocompatibility, has short production period and low cost, is suitable for large-scale production, and can also avoid the problems of animal protein immunogenicity, animal-derived diseases and the like. Thus, the preparation of recombinant collagen, particularly recombinant human collagen, is a hotspot in current collagen production research.

Since E.coli is a prokaryote, post-translational modification of proteins is not possible, and thus it is difficult for E.coli systems to express triple helix collagens having a natural higher structure. The human recombinant collagen produced by yeast fermentation overcomes the defects of animal sources and collagen expressed by escherichia coli. The yeast expression system has molecular chaperones and enzymes for post-translational modification of proteins (such as glycosylation, hydroxylation, acetylase and the like), and is very suitable for forming triple-helical collagen with a higher structure. At present, a lot of reports on the preparation of recombinant collagen are mainly focused on the expression of a human collagen gene, chinese patent publication No. CN111087463A, application day No. 2019, 12 month and 28 discloses recombinant human type III collagen and a prokaryotic expression method thereof, a gene sequence is obtained by codon optimization and design, the gene sequence is inserted between an NdeI and XhoI of an expression vector PET30a (+) to construct a recombinant expression vector pET301230, competent cells of escherichia coli BL211 (DE 3) are transformed, positive clones are selected, culture induction is conducted to perform efficient expression, the amino acid sequence and the spatial structure of protein of the obtained recombinant human type III collagen are closer to those of human type III collagen, and the recombinant human type III collagen has better bioactivity and the effects of promoting cell migration, adhesion and proliferation, but the human type III collagen does not have the technical effect of improving the solubility; the invention discloses a recombinant human type III collagen, a preparation method and application thereof, wherein the recombinant human type III collagen is prepared by connecting short repeated amino acid sequences in series (repeated 3-10 times) to form a target protein polypeptide sequence, and the recombinant human elastin obtained by in vitro expression of a prokaryotic expression system has higher superoxide radical scavenging activity, better stability and longer catalytic activity than that of a single original fragment, but the recombinant human elastin disclosed by the invention also cannot solve the technical problem of how to improve the yield and solubility of human type III collagen.

At present, research and report on the solubility modification of humanized type III collagen are not yet reported, so that a gene engineering technology is utilized to design an expression method of high-solubility humanized type III collagen, which is simple to operate and objective in yield, and has important research significance and practical value for the field of recombinant collagen.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the high-solubility humanized III type collagen, the expression mode and the recombinant plasmid thereof, the recombinant plasmid can efficiently express the high-solubility and stability recombinant humanized III type collagen through the Pichia pastoris cell GS115, and the protein degradation phenomenon in the later fermentation period is obviously reduced.

The technical scheme of the invention is as follows:

The invention aims at providing a construction method of recombinant plasmid for expressing high-solubility humanized III type collagen, which optimizes and synthesizes nucleotide sequences of a high-hydrophilicity collagen gene segment (col 3 a) n, a VNTR enhancer segment and a TES enhancer segment, obtains a connecting segment by PCR cloning and mixes the connecting segments, and connects the collagen segment (col 3 a) n, the VNTR enhancer segment, the TES enhancer segment and a pPIC9K carrier skeleton segment by a Gibson Assembly seamless connection technology to construct the recombinant plasmid, which is named as pPIC9K-VNTR-TES- (col 3 a) n.

Further, the collagen gene fragment (col 3 a) n is a sequence which is a percentage of the sequence derived from the human full-length III type collagen alpha 1 chain, and is positioned at the 908 th to 1137 th amino acid sequences of the human full-length III type collagen alpha 1 chain amino acid sequence.

Further, the collagen gene fragment (col 3 a) n is an amino acid sequence shown as SEQ ID NO.1, or an amino acid sequence modified by amino acid substitution, insertion, substitution, addition, deletion and the like to a certain extent on the basis of SEQ ID NO.1, or an amino acid sequence with more than 80% homology with the amino acid sequence of SEQ ID NO. 1; and n in the collagen gene fragment (col 3 a) n is an integer greater than or equal to 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 2.

Further, the collagen fragment (col 3 a) n NCBI sequence No. NP-000081.2.

Further, the VNTR enhancer fragment is derived from the VNTR of a human STIN2 protein; the TES enhancer fragment is derived from TES of a murine Sox9 gene.

Further, the nucleotide sequence corresponding to the VNTR enhancer after being modified is SEQ ID NO.3; the nucleotide sequence corresponding to the modified TES enhancer is SEQ ID NO.4.

Further, the NCBI sequence number of the VNTR is ng_055510.1; the NCBI sequence number of TES is NG_050881.

Further, the PCR cloning was performed on the ligation fragment using the primers pPIC9K-F/R, (col 3 a) n-F/R, VNTR-F/R, TES-F/R, respectively.

Further, the cloned collagen (col 3 a) n, VNTR enhancer fragment, TES enhancer fragment and expression vector pPIC9K fragment were mixed according to a concentration ratio of 3:4:4:1, respectively.

Further, the Gibson Assembly seamless connection and plasmid construction process is as follows:

Incubating the connection mixture collagen fragments (col 3 a) n, VNTR enhancer fragments, TES enhancer fragments and pPIC9K vector skeleton fragments for 0.5-1.5h in a metal bath at 45-55 ℃, then converting the connection mixture into competent cells of escherichia coli DH5 alpha by a heat shock method, after overnight incubation, picking positive monoclonal grown on ampicillin sodium and kanamycin plates for colony PCR preliminary verification, inoculating the single colony with successful verification, culturing in an LB test tube, and extracting plasmid sequencing confirmation to obtain the recombinant plasmid.

Further, the overnight incubation is at 36-38 ℃; single colonies that were verified to be successful were inoculated into 5mL LB tubes and incubated at 37℃for 16h.

The second object of the present invention is to provide a recombinant plasmid for expressing highly soluble humanized type III collagen.

The invention also provides a method for expressing high-solubility humanized III type collagen, which specifically comprises the following steps:

(1) Preparing competent cells according to a pichia pastoris sorbitol method, uniformly mixing the linearized plasmid pPIC9K-VNTR-TES- (col 3 a) n fragment and yeast competent P.pastoris GS115, rapidly transferring the mixture into an electric rotating cup, and enabling the recombinant plasmid fragment to enter the competent cells P.pastoris GS115 through electric shock transformation;

(2) Coating the transformed bacteria on an MD plate, culturing until single colonies appear on the plate, picking single colony culture and extracting genome for PCR verification, and sequencing and verification;

(3) Screening high-copy recombinant strains on recombinants with correct sequencing verification, and then transferring the recombinants into YPD liquid culture medium for overnight culture activation;

(4) Transferring the bacterial liquid activated overnight into BMGY liquid culture medium with an inoculum size of 1% for culturing for 24 hours, collecting bacterial bodies when OD ₆₀₀ reaches 1.0-1.2, suspending and centrifuging the collected bacterial bodies by using the BMMY liquid culture medium, fermenting and culturing, adding methanol into the culture liquid every 24 hours, and inducing the expression of the high-solubility humanized III type collagen.

Further, 1. Mu.g of the linearized plasmid pPIC9K-VNTR-TES- (col 3 a) n fragment was taken in the step (1), and mixed with 100. Mu.L of yeast competent GS115, and the mixture was rapidly transferred into an electrorotating cup for electric shock transformation to allow the recombinant plasmid fragment to enter competent cells GS 115.

Further, in the step (2), the transformed bacteria are coated on an MD plate and then are inverted and cultured in a constant temperature incubator at 30 ℃ for 3-4 days.

In the step (3), recombinant bacteria are respectively transfected on YPD plates with G418 concentration of 500-4mg/ml for screening, and the recombinant bacteria which can grow on the high-concentration G418 plates are activated for bacteria protection.

Further, the YPD liquid medium in the step (3) was cultured at 30℃and 220rpm overnight for 16 hours.

Further, in the step (4), the bacterial liquid activated overnight is transferred into BMGY liquid culture medium with the total system of 5mL at the inoculation amount of 1%, and the bacterial liquid is cultured for 24 hours at the temperature of 30 ℃ and at the speed of 220 rpm; the cells were collected by centrifugation at 5000rpm for 10min at 4℃and suspended in 50mL of BMMY liquid medium and cultured by fermentation at 30℃and 220rpm, and methanol was added to the culture medium at a final concentration of 0.5-1% every 24 hours for induction expression.

Further, after 72 hours of induction of expression, the culture solution was centrifuged at 12000rpm at 4℃for 10 minutes, and supernatants induced for 24 hours, 48 hours, 72 hours and 96 hours were collected, respectively, and subjected to SDS-PAGE protein electrophoresis to verify the protein expression.

The fourth object of the present invention is to provide a highly soluble humanized type III collagen.

Compared with the prior art, the invention has the beneficial effects that:

1. The invention designs the dissolubility of recombinant humanized III type collagen, and constructs a recombinant plasmid pPIC9K-VNTR-TES- (col 3 a) n which can be used for expressing high-dissolubility humanized III type collagen. According to the construction method, the amino acid of the alpha 1 chain of the human-source III-type collagen is subjected to hydrophilic analysis, and fragments with highest hydrophilicity are selected, so that the recombinant human-source III-type collagen has better solubility, and an enhancer of VNTR (human-source STIN 2) and a TES enhancer of murine Sox9 gene are introduced before the recombinant collagen, so that the expression quantity of the recombinant human-source III-type collagen is effectively improved.

2. The invention provides an expression method of high-solubility humanized type III collagen for the first time, which makes up that no research report about solubility modification of the humanized type III collagen exists at present. The expression method selects pichia pastoris GS115 formed by triple helix collagen with a high-level structure as an expression system, and the constructed recombinant plasmid pPIC9K-VNTR-TES- (col 3 a) n enters competent cells P.pastoris GS115 through electric shock transformation. The collagen produced by secretory expression of Pichia pastoris GS115 has higher yield, better solubility, obviously reduced protein degradation phenomenon in the later period of fermentation, and the like.

3. Compared with animal-derived collagen, the recombinant plasmid provided by the invention can efficiently express recombinant human-derived type III collagen with high solubility and stability through Pichia pastoris Pichia pastoris GS115,115, and the protein degradation phenomenon in the later fermentation period is obviously reduced; the high-solubility humanized III type collagen expressed by the method has the advantages of uniform molecular weight, high purity, no hidden danger of virus transmission and the like, has the inherent biocompatibility of natural collagen and the biological safety of low immune rejection reaction, is simple to operate, can realize the expression of recombinant humanized III type collagen with larger yield, and can be widely applied to the fields of biomedical materials, tissue engineering, cosmetics, foods and the like.

Drawings

FIG. 1 is a schematic diagram of the structure of the recombinant plasmid pPIC9K-VNTR-TES- (col 3 a) n;

FIG. 2 shows the results of electrophoresis of recombinant humanized type III collagen in performance test of the present invention, wherein M: marker lanes 1-4 are respectively schematic representations of supernatants of 24h, 48h, 72h and 96h fermentation of recombinant humanized type III collagen.

Detailed Description

The invention is further described below in connection with the preferred embodiments, and neither the endpoints of the ranges disclosed in the invention nor any of the values are limited to the precise range or value, and such range or value should be understood to include values near the range or value; for numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the following examples, the gene fragment col3a1, VNTR enhancer fragment and TES enhancer fragment were sent to Kirschner Biotechnology Inc. for full-sequence synthesis.

Example 1

The embodiment provides a construction method of recombinant plasmid for expressing high-solubility humanized III type collagen, which comprises the following steps:

S1, selecting 908 th to 1137 th amino acid sequences from a human full-length III type collagen alpha 1 chain (NCBI serial number: NP_ 000081.2), and repeating the steps once to obtain a collagen fragment col3a2;

Selecting a VNTR enhancer fragment derived from a human STIN2 protein; an enhancer fragment of TES derived from the murine Sox9 gene;

s2, optimizing the collagen amino acid sequence fragment col3a2, synthesizing the amino acid sequence fragment col after codons, modifying the VNTR enhancer sequence of the human STIN2 protein and the TES enhancer sequence of the murine Sox9 gene, synthesizing the amino acid sequence corresponding to the collagen by codons, wherein the amino acid sequence corresponding to the collagen is shown as SEQ ID NO.1, the nucleotide sequence corresponding to the collagen is shown as SEQ ID NO.2, and the nucleotide sequence corresponding to the modified VNTR enhancer of the human STIN2 protein is shown as SEQ ID NO. 3; the nucleotide sequence corresponding to the modified TES enhancer of the murine Sox9 gene is shown as SEQ ID NO. 4;

S3, performing PCR cloning on the expression vector pPIC9K, the collagen fragment col3a2, the VNTR enhancer fragment and the TES enhancer fragment by using primers pPIC9K-F/R, col3a2-F/R, VNTR-F/R, TES-F/R respectively to obtain a connecting fragment, and mixing the cloned collagen col3a2, the cloned VNTR enhancer fragment, the cloned TES enhancer fragment and the expression vector pPIC9K fragment according to a concentration ratio of 3:4:4:1 respectively;

further, the primer nucleotide sequence is as follows:

pPIC9K-F：tacgtagaattcgcggccgcgaattaattc；

pPIC9K-R：agcttcagcctctcttttctcgagagatacc；

(col3a)2-F:tcgagaaaagagaggctgaagctgcgggtaacactggtgctcctggc；

(col3a)2-R:tgcaggtcctggactgccgattgcaccctgctgac；

VNTR-F：attattcgaaggatccaaacgatgagactgaaaagacataat；

VNTR-R：taaaaattgaaggaaatctcatggggcaagaaggtggatttcc；

TES-F：accggtcttgctagattctaagcaaagtgtcagcagcc；

TES-R：ggcattctgacatcctcttgaggaagccaggaaggctgga。

S4, connecting a collagen fragment col3a2, a VNTR enhancer fragment, a TES enhancer fragment and a pPIC9K carrier skeleton fragment by using a Gibson Assembly seamless connection technology, incubating the connection mixture in a metal bath at 50 ℃ for 1h, then converting the connection mixture into E.coli DH5 alpha competent cells by a heat shock method, carrying out overnight culture at 37 ℃ and then picking up positive monoclonal grown on ampicillin sodium and kanamycin plates for colony PCR preliminary verification, verifying primers by using TES-F/R, inoculating the verified single colony into a 5mL LB test tube for 16h at 37 ℃, extracting plasmids, sequencing by a sequencing company to confirm whether the recombinant genes are consistent with the design, and obtaining the recombinant plasmid pPIC9K-VNTR-TES-col3a2 after confirmation, wherein the metal bath incubation conditions can be adjusted to 45-55 ℃ according to actual operation and incubated for 0.5-1.5h.

Example 2

The embodiment provides a method for expressing high-solubility humanized type III collagen, which comprises the following steps:

s1, streaking and activating a Pichia pastoris GS115 bacterial liquid on a YPD flat plate, culturing in a 30 ℃ incubator until single colonies grow out, picking the single colonies, inoculating the single colonies into 5mL YPD liquid culture medium, and culturing for 18h at 30 ℃ and 220 rpm; inoculating the seed solution into 50mL YPD with an inoculum size of 1%, culturing until OD 600 is 1.4, collecting bacterial liquid, centrifuging at 4 ℃ and 5000rpm for 5min, discarding supernatant, repeating for 2 times, re-suspending bacterial cells with 1M/L D-sorbitol solution, and sub-packaging into small parts according to 100 mu L/tube;

S2, taking 1 mug of the linearized plasmid pPIC9K-VNTR-TES-col3a2 fragment prepared in the embodiment 1, uniformly mixing with 100 mug pichia pastoris competent GS115 in the S1, quickly transferring the mixture into a precooled electric rotating cup with the thickness of 0.2cm, placing the mixture on ice for 5min, and then carrying out electric shock conversion under the conditions of electric shock voltage of 2kV, capacitance of 25 mug, resistance of 200 omega and electric shock time of 5 msec;

S3, immediately adding 650 mu L of precooled 1M sorbitol solution into the electric rotating cup after electric shock is finished, gently blowing and uniformly mixing, rapidly transferring into a 1.5mL centrifuge tube, and placing into a constant temperature incubator at 30 ℃ for static culture for 1.5h, so that the recombinant plasmid fragment enters competent cells P.pastoris GS 115;

S4, coating 100 mu L of recombinant strain on an MD plate, then inverting the plate and culturing the plate in a constant temperature incubator at 30 ℃ for 3d until single colony appears on the plate, picking single colony culture and extraction genome, carrying out PCR verification, and then sending to sequencing to verify whether the conversion is successful;

S5, respectively transfecting recombinant bacteria with correct sequencing verification on YPD plates with G418 concentration of 500 mug/ml-4 mg/ml, brushing and selecting, and preserving bacteria after activating the recombinant bacteria capable of growing on the high-concentration G418 plates;

S6, picking verified recombinant single colonies to be connected to a new 5mL YPD liquid culture medium, culturing and activating the recombinant single colonies at 30 ℃ at 220rpm overnight, and then transferring the bacterial liquid activated overnight to a BMGY liquid culture medium with a total system of 5mL at 30 ℃ at 1% of inoculum size, and culturing the bacterial liquid at 220rpm for 24 hours; centrifuging at 4 ℃ and 5000rpm for 10min to collect thalli;

s7, suspending and centrifuging the collected thalli by using 50mL of BMMY liquid culture medium, fermenting and culturing at 30 ℃ and 220rpm, and adding methanol with the final concentration of 0.7% into the culture solution every 24 hours for induction to express the high-solubility humanized type III collagen.

Performance testing

1. Expression test

The high-solubility humanized type III collagen was induced and expressed according to the method described in example 2,4 test groups were set, and induced and expressed for 24 hours, 48 hours, 72 hours and 96 hours, respectively, and then the culture solution was centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was collected for SDS-PAGE protein electrophoresis to verify the protein expression.

The experimental results are shown in fig. 2: m: marker lane 1 was 24h supernatant of recombinant collagen col3a2 fermentation, lane 2 was 48h supernatant of recombinant collagen col3a2 fermentation, lane 3 was 72h supernatant of recombinant collagen col3a2 fermentation, lane 4 was 96h supernatant of recombinant collagen col3a2 fermentation.

The molecular weight of the recombinant collagen col3a2 protein is between 40kDa and 50kDa, which is consistent with the theoretical molecular weight of the col3a2 protein of 44.8 kDa.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.

Claims (10)

1. The construction method of the recombinant plasmid for expressing the high-solubility humanized III type collagen is characterized by comprising the following steps: and (3) optimizing and synthesizing nucleotide sequences of a highly hydrophilic collagen gene fragment (col 3 a) n, a VNTR enhancer fragment and a TES enhancer fragment according to codon preference of pichia pastoris, obtaining and mixing connection fragments by utilizing PCR cloning, and connecting the collagen fragment (col 3 a) n, the VNTR enhancer fragment, the TES enhancer fragment and a pPIC9K carrier skeleton fragment by using a Gibson Assembly seamless connection technology to construct the recombinant plasmid, wherein the recombinant plasmid is named as pPIC9K-VNTR-TES- (col 3 a) n.

2. The method according to claim 1, wherein the collagen gene fragment (col 3 a) n is 100% of the sequence derived from the human full-length type III collagen alpha 1 chain, and is located between amino acid sequence 908 and amino acid sequence 1137 of the human full-length type III collagen alpha 1 chain amino acid sequence.

3. The method for constructing recombinant plasmid expressing high-solubility humanized type III collagen according to claim 2, wherein the collagen gene fragment (col 3 a) n is an amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence modified by amino acid substitution, insertion, substitution, addition, deletion or the like to a certain extent on the basis of SEQ ID NO.1, or an amino acid sequence having homology of more than 80% with the amino acid sequence of SEQ ID NO. 1;

And n in the collagen gene fragment (col 3 a) n is an integer greater than or equal to 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 2.

4. The method for constructing a recombinant plasmid expressing highly soluble humanized type III collagen according to claim 1, wherein the VNTR enhancer fragment is derived from VNTR of a human STIN2 protein; the TES enhancer fragment is derived from TES of a murine Sox9 gene.

5. The method for constructing a recombinant plasmid expressing high-solubility humanized type III collagen according to claim 4, wherein the nucleotide sequence corresponding to the modified VNTR enhancer is SEQ ID NO.3; the nucleotide sequence corresponding to the modified TES enhancer is SEQ ID NO.4.

6. The method for constructing a recombinant plasmid expressing highly soluble humanized type III collagen according to claim 1, wherein the PCR cloning of the connecting fragment is performed by using primers pPIC9K-F/R, (col 3 a) n-F/R, VNTR-F/R, TES-F/R, respectively; the cloned collagen (col 3 a) n, the VNTR enhancer fragment, the TES enhancer fragment and the expression vector pPIC9K fragment are respectively mixed according to the concentration ratio of 3:4:4:1.

7. The method for constructing the recombinant plasmid for expressing the high-solubility humanized type III collagen according to claim 1, wherein the Gibson Assembly seamless connection and plasmid construction process is as follows:

Incubating the connection mixture collagen fragments (col 3 a) n, VNTR enhancer fragments, TES enhancer fragments and pPIC9K vector skeleton fragments for 0.5-1.5h in a metal bath at 45-55 ℃, then converting the connection mixture into competent cells of escherichia coli DH5 alpha by a heat shock method, after overnight incubation, picking positive monoclonal grown on ampicillin sodium and kanamycin plates for colony PCR preliminary verification, inoculating the single colony with successful verification, culturing in an LB test tube, and extracting plasmid sequencing confirmation to obtain the recombinant plasmid.

8. A recombinant plasmid expressing highly soluble humanized type III collagen constructed according to the method of any one of claims 1 to 7.

9. A method for expressing high-solubility humanized type III collagen using the recombinant plasmid of claim 8, comprising the steps of:

(1) Preparing competent cells according to a pichia pastoris sorbitol method, uniformly mixing the linearized plasmid pPIC9K-VNTR-TES- (col 3 a) n fragment and yeast competent P.pastoris GS115, rapidly transferring the mixture into an electric rotating cup, and enabling the recombinant plasmid fragment to enter the competent cells P.pastoris GS115 through electric shock transformation;

(2) Coating the transformed bacteria on an MD plate, culturing until single colonies appear on the plate, picking single colony culture and extracting genome for PCR verification, and sequencing and verification;

(3) Screening high-copy recombinant strains on recombinants with correct sequencing verification, and then transferring the recombinants into YPD liquid culture medium for overnight culture activation;

(4) Transferring the bacterial liquid activated overnight into BMGY liquid culture medium with an inoculum size of 1% for culturing for 24 hours, collecting bacterial bodies when OD ₆₀₀ reaches 1.0-1.2, suspending and centrifuging the collected bacterial bodies by using the BMMY liquid culture medium, fermenting and culturing, adding methanol into the culture liquid every 24 hours, and inducing the expression of the high-solubility humanized III type collagen.

10. A highly soluble humanized type III collagen expressed by the method of claim 9.