CN116891525A

CN116891525A - Recombinant human III type collagen and expression method and application thereof

Info

Publication number: CN116891525A
Application number: CN202310864370.9A
Authority: CN
Inventors: 郑丽慧; 陈学军; 黄静; 高佳; 吴佳婧; 徐昊; 张政
Original assignee: Meichao Haining Medical Instrument Co ltd; East China Normal University
Current assignee: Meichao Haining Medical Instrument Co ltd; East China Normal University
Priority date: 2023-01-19
Filing date: 2023-07-14
Publication date: 2023-10-17

Abstract

The invention discloses a recombinant human III type collagen produced by using escherichia coli, and an expression method and application thereof, belonging to the technical fields of genetic engineering and biological engineering. According to the invention, the collagen structure prediction is carried out through alpha fold2, a peptide segment formed by 579-608 amino acid residues in a natural human III type collagen sequence is selected to form a collagen monomer, the collagen monomer is repeatedly connected in series with monomers with different expression numbers to enable the collagen monomer to have a stable triple helix structure, and then the monomers with different repetition numbers are transformed into escherichia coli host bacteria to prepare the recombinant human III type collagen, so that the recombinant human III type collagen with high purity and good biological activity is obtained, and the recombinant human III type collagen can be widely applied to biomedical appliances and cosmetics industries and has good application prospects.

Description

Recombinant human III type collagen and expression method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant human III type collagen produced by using escherichia coli, and an expression method and application thereof.

Background

Collagen is one of the most abundant proteins in the human body, accounts for about 30% of the total protein content of the human body, is widely present in extracellular matrixes of connective tissues such as skin, bones, tendons, blood vessels and the like in the human body, and has important influence on maintaining the normal operation of the human body and repairing injuries. It has been found that collagen has repeated tripeptide Gly-X-Y domains, which are the basis for maintaining the triple helix structure of collagen.

Because the collagen has better biological activity, can promote proliferation and differentiation of cells, endow the skin with proper elasticity and toughness, and has non-negligible biological significance in the aspects of wound healing, tissue repair, cellular immunity and the like. Based on this, collagen is widely used in the fields of biological medicine, food daily chemicals and the like. With rapid development of genetic engineering and synthetic biology in recent years, protein expression systems based on animals, plants, fungi and bacteria have been established in a dispute, and recombinant human collagen is synthesized by utilizing a genetic engineering means, so that the immunogenicity problem of the collagen can be effectively solved, and the collagen can meet more clinical requirements and has a wide application prospect.

In the preparation of recombinant proteins, prokaryotic expression systems are often employed. Coli is one of the most important prokaryotic expression systems, has the advantages of clear genetic background, simple operation and high expression level, and is often the first choice for expression of recombinant proteins. There has been a report of expressing recombinant collagen by using escherichia coli as a host, for example, CN103122027a discloses a recombinant human collagen and a production method thereof, wherein DNA fragments of human type II and type III collagen gene helical regions are selected, and recombinant is performed by using a PCR method, so that the complete recombinant collagen is obtained, and has good hydrophilicity and stability. However, the existing patent has little research on the triple helix structure and biological activity of recombinant human collagen. Therefore, in the technical field of preparing the human collagen, development of a method for efficiently expressing the human collagen with a correct triple helix structure and good biological activity by using an escherichia coli system is urgently needed, and the method has great practical significance.

The invention is based on the original gene sequence of III type humanized collagen, adopts computer-aided protein structure prediction, selects the parts with high water solubility and high biological activity to carry out multistage tandem, splicing and recombination, and obtains the brand new recombinant III type humanized collagen, which has high application value in the industries of biological medicine, food daily chemicals and the like.

Disclosure of Invention

The invention aims to provide recombinant human III type collagen, and an expression method and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the invention provides a recombinant human III type collagen, which is prepared by predicting solubility through Protein-sol (Protein-sol sequence solubility (manchester. Ac. Uk)) and predicting Protein structure through alpha fold2, has high water solubility and stable triple helix structure, and comprises monomers repeatedly connected in series. Preferably, the monomer is a peptide segment composed of 30 amino acids, and the peptide segment composed of 579 to 608 amino acid residues in the sequence is selected according to the amino acid sequence of the humanized III type collagen. Preferably, the coding gene of the monomer is rhIII-3, and the nucleotide sequence of rhIII-3 is SEQ ID NO.1;

wherein, the rhIII-3 gene sequence is obtained by extracting total RNA from human skin fibroblasts, reversing the total RNA into cDNA, and designing PCR primer for amplification;

in the recombinant human-derived III-type collagen protein, the number of monomer series is 1-16, such as monomer, repeated series monomer, quadruple repeated series monomer, eight repeated series monomer, sixteen repeated series monomer and the like, which are respectively marked as (rhIII-3) n, wherein n represents the number of repeated monomers. The molecular weight of the recombinant human III type collagen is about 3KD-60KD.

The invention also provides a preparation method of the gene recombinant engineering bacteria and recombinant human III type collagen, which comprises the following steps:

construction of recombinant expression vectors

Total RNA is extracted from human skin fibroblasts, inverted into cDNA, and PCR amplification primers F1 and R1 are designed according to the gene sequence of GeneBank human III type collagen COL3A 1:

F1:CCCAAGCTTATGATGAGCTTTGTGCAA(SEQ ID NO.2)

R1:TGCTCTAGATTATAAAAAGCAAACAGGGC(SEQ ID NO.3)

the cDNA is used as a template for PCR amplification to obtain the COL3A1 gene, the length of the product is 4419bp, the sequence is shown as SEQ ID NO.4, and the recovered specific product is connected to a pUC19 vector to obtain a recombinant expression vector pUC19-COL3A1 for preservation.

SEQ ID NO.4：

CCCAAGCTTATGATGAGCTTTGTGCAAaaggggagctggctacttctcgctctgcttcatcccactattattttggcacaacaggaagctgttgaaggaggatgttcccatcttggtcagtcctatgcggatagagatgtctggaagccagaaccatgccaaatatgtgtctgtgactcaggatccgttctctgcgatgacataatatgtgacgatcaagaattagactgccccaacccagaaattccatttggagaatgttgtgcagtttgcccacagcctccaactgctcctactcgccctcctaatggtcaaggacctcaaggccccaagggagatccaggccctcctggtattcctgggagaaatggtgaccctggtattccaggacaaccagggtcccctggttctcctggcccccctggaatctgtgaatcatgccctactggtcctcagaactattctccccagtatgattcatatgatgtcaagtctggagtagcagtaggaggactcgcaggctatcctggaccagctggccccccaggccctcccggtccccctggtacatctggtcatcctggttcccctggatctccaggataccaaggaccccctggtgaacctgggcaagctggtccttcaggccctccaggacctcctggtgctataggtccatctggtcctgctggaaaagatggagaatcaggtagacccggacgacctggagagcgaggattgcctggacctccaggtatcaaaggtccagctgggatacctggattccctggtatgaaaggacacagaggcttcgatggacgaaatggagaaaag

ggtgaaacaggtgctcctggattaaagggtgaaaatggtcttccaggcgaaaatggagctcctggacccatgggtccaagaggggctcctggt

gagcgaggacggccaggacttcctggggctgcaggtgctcggggtaatgacggtgctcgaggcagtgatggtcaaccaggccctcctggtc

ctcctggaactgccggattccctggatcccctggtgctaagggtgaagttggacctgcagggtctcctggttcaaatggtgcccctggacaaaga

ggagaacctggacctcagggacacgctggtgctcaaggtcctcctggccctcctgggattaatggtagtcctggtggtaaaggcgaaatgggt

cccgctggcattcctggagctcctggactgatgggagcccggggtcctccaggaccagccggtgctaatggtgctcctggactgcgaggtggt

gcaggtgagcctggtaagaatggtgccaaaggagagcccggaccacgtggtgaacgcggtgaggctggtattccaggtgttccaggagctaa

aggcgaagatggcaaggatggatcacctggagaacctggtgcaaatgggcttccaggagctgcaggagaaaggggtgcccctgggttccga

ggacctgctggaccaaatggcatcccaggagaaaagggtcctgctggagagcgtggtgctccaggccctgcagggcccagaggagctgctg

gagaacctggcagagatggcgtccctggaggtccaggaatgaggggcatgcccggaagtccaggaggaccaggaagtgatgggaaacca

gggcctcccggaagtcaaggagaaagtggtcgaccaggtcctcctgggccatctggtccccgaggtcagcctggtgtcatgggcttccccggt

cctaaaggaaatgatggtgctcctggtaagaatggagaacgaggtggccctggaggacctggccctcagggtcctcctggaaagaatggtga

aactggacctcagggacccccagggcctactgggcctggtggtgacaaaggagacacaggaccccctggtccacaaggattacaaggcttg

cctggtacaggtggtcctccaggagaaaatggaaaacctggggaaccaggtccaaagggtgatgccggtgcacctggagctccaggaggca

agggtgatgctggtgcccctggtgaacgtggacctcctggattggcaggggccccaggacttagaggtggagctggtccccctggtcccgaa

ggaggaaagggtgctgctggtcctcctgggccacctggtgctgctggtactcctggtctgcaaggaatgcctggagaaagaggaggtcttgga

agtcctggtccaaagggtgacaagggtgaaccaggcggtccaggtgctgatggtgtcccagggaaagatggcccaaggggtcctactggtcc

tattggtcctcctggcccagctggccagcctggagataagggtgaaggtggtgcccccggacttccaggtatagctggacctcgtggtagccct

ggtgagagaggtgaaactggccctccaggacctgctggtttccctggtgctcctggacagaatggtgaacctggtggtaaaggagaaagagg

ggctccgggtgagaaaggtgaaggaggccctcctggagttgcaggaccccctggaggttctggacctgctggtcctcctggtccccaaggtgt

caaaggtgaacgtggcagtcctggtggacctggtgctgctggcttccctggtgctcgtggtcttcctggtcctcctggtagtaatggtaacccagg

acccccaggtcccagcggttctccaggcaaggatgggcccccaggtcctgcgggtaacactggtgctcctggcagccctggagtgtctggac

caaaaggtgatgctggccaaccaggagagaagggatcgcctggtgcccagggcccaccaggagctccaggcccacttgggattgctgggat

cactggagcacggggtcttgcaggaccaccaggcatgccaggtcctaggggaagccctggccctcagggtgtcaagggtgaaagtgggaaa

ccaggagctaacggtctcagtggagaacgtggtccccctggaccccagggtcttcctggtctggctggtacagctggtgaacctggaagagat

ggaaaccctggatcagatggtcttccaggccgagatggatctcctggtggcaagggtgatcgtggtgaaaatggctctcctggtgcccctggcg

ctcctggtcatccaggcccacctggtcctgtcggtccagctggaaagagtggtgacagaggagaaagtggccctgctggccctgctggtgctc

ccggtcctgctggttcccgaggtgctcctggtcctcaaggcccacgtggtgacaaaggtgaaacaggtgaacgtggagctgctggcatcaaag

gacatcgaggattccctggtaatccaggtgccccaggttctccaggccctgctggtcagcagggtgcaatcggcagtccaggacctgcaggcc

ccagaggacctgttggacccagtggacctcctggcaaagatggaaccagtggacatccaggtcccattggaccaccagggcctcgaggtaac

agaggtgaaagaggatctgagggctccccaggccacccagggcaaccaggccctcctggacctcctggtgcccctggtccttgctgtggtgg

tgttggagccgctgccattgctgggattggaggtgaaaaagctggcggttttgccccgtattatggagatgaaccaatggatttcaaaatcaacac

cgatgagattatgacttcactcaagtctgttaatggacaaatagaaagcctcattagtcctgatggttctcgtaaaaaccccgctagaaactgcaga

gacctgaaattctgccatcctgaactcaagagtggagaatactgggttgaccctaaccaaggatgcaaattggatgctatcaaggtattctgtaata

tggaaactggggaaacatgcataagtgccaatcctttgaatgttccacggaaacactggtggacagattctagtgctgagaagaaacacgtttgg

tttggagagtccatggatggtggttttcagtttagctacggcaatcctgaacttcctgaagatgtccttgatgtgcatctggcattccttcgacttctct

ccagccgagcttcccagaacatcacatatcactgcaaaaatagcattgcatacatggatcaggccagtggaaatgtaaagaaggccctgaagct

gatggggtcaaatgaaggtgaattcaaggctgaaggaaatagcaaattcacctacacagttctggaggatggttgcacgaaacacactgggga

atggagcaaaacagtctttgaatatcgaacacgcaaggctgtgagactacctattgtagatattgcaccctatgacattggtggtcctgatcaagaatttggtgtggacgttgGCCCTGTTTGCTTTTTATAATCTAGAGCA。

Further, the plasmid pUC19-COL3A1 is used as a template, and the primers F3 and R2 are used for amplifying the rhIII-3 gene, so that a specific product rhIII-3 is recovered, the length of the specific product rhIII-3 is 106bp, and the primers are as follows:

F2：GCGTCGACGGCTTCCCCGGTCCTAAA(SEQ ID NO.5)

F3：GCGTCGACCTGGCTTCCCCGGTCCTAAA(SEQ ID NO.6)

R2：CCGCTCGAGAGGAGGACCCTGAGGGCC(SEQ ID NO.7)

the product rhIII-3 and the vector pET32a (+) are respectively subjected to Sal I and Xho I double digestion, and are connected into pET32a (+) - (rhIII-3) 1 through T4 ligase, so that a single-segment monomer expression vector is constructed.

Taking pET32a (+) - (rhIII-3) 1 as a template, carrying out single enzyme digestion by Xho I, respectively carrying out double enzyme digestion by Sal I and Xho I on the specific products rhIII-3 amplified by F2 and R2, and then connecting again by T4 ligase to form pET32a (+) - (rhIII-3) 2, and so on, so as to construct a recombinant expression vector pET32a (+) - (rhIII-3) n of repeated multi-segment monomers, wherein n represents the number of repeated monomers; for example, the repetitive tetrad recombinant expression vector is pET32a (+) - (rhIII-3) 4, the repetitive octad recombinant expression vector is pET32a (+) - (rhIII-3) 8, the repetitive hexadecd recombinant expression vector is pET32a (+) - (rhIII-3) 16, and the like.

Construction of Gene recombination engineering bacterium

And (3) converting the prepared multistage monomer repeated recombinant expression vector pET32a (+) - (rhIII-3) n into BL21 (DE 3) competent cells, picking single colonies, carrying out colony PCR identification, sequencing successfully, and carrying out amplification culture on the single colonies to obtain the gene recombinant engineering bacteria.

(III) preparation of Gene recombinant human III collagen

And (3) carrying out induced expression on the target protein by the obtained gene recombinant engineering bacteria, and further separating and purifying the target protein to obtain the high-purity water-soluble recombinant human III type collagen.

Further, the specific steps of the induced expression, separation and purification in the third step are as follows:

(3.1) inoculating the engineering bacteria obtained in the step II into 10ml of LB culture medium, culturing overnight at 37 ℃, transferring the engineering bacteria into the LB culture medium according to the volume ratio of 2%, culturing at 200rpm at 37 ℃, adding IPTG with the final concentration of 0.1mM when the recombinant strain is cultured until the OD600 is 0.6-0.8, culturing at 25 ℃ for 4-8 hours, centrifuging at 10000rpm for 10min, and collecting bacterial precipitate. Wherein, the fermentation medium LB medium comprises the following components: 10g/L of tryptone (tryptone), 5g/L of yeast extract, 10g/L of NaCl and an initial pH of 7.2-7.4.

(3.2) washing the bacterial precipitate with 20ml of a bacterial buffer solution, then resuspending the bacterial precipitate, adding PMSF with a final concentration of 1mM to prevent protease from degrading target protein, carrying out ultrasonic disruption in an ice bath, centrifuging at 8000rpm for 20min, collecting supernatant, and resuspending and collecting the precipitate by using sterile water; the sterile water was used in a weight-suspension amount of 20ml/200ml based on the volume of the fermented liquid. Wherein, the composition components of the bacteria-destroying buffer solution are as follows: 300mM NaCl,50mM NaH2PO4, 10mM imidazole, ph=6.5-6.8; the ultrasonic crushing condition is 350W,5s ultrasonic and 5s intermittent, the protection temperature is 25 ℃, and the ultrasonic time is 30min. And adding a proper amount of enterokinase into the obtained crushed supernatant, and performing enzyme digestion reaction at 4 ℃ overnight to remove thioredoxin tags.

(3.3) balancing the nickel ion affinity column with a protein washing solution with the volume of 5 times, loading the obtained solution for removing thioredoxin labels on the column, enabling the target protein to be fully combined with the nickel column at the flow rate of 4 s/drop, and collecting penetrating fluid; washing the nickel column with 4 times of pre-cooled protein washing liquid to remove non-specific binding protein, and collecting washing liquid; eluting target protein with 4 times of pre-cooled protein eluent, collecting eluent, dialyzing the obtained product overnight, ultrafiltering, concentrating, and lyophilizing to obtain purified recombinant human source III type collagen.

Wherein, the components of the protein washing liquid are as follows: 20mM Tris,500mM NaCl,pH is 6.5 to 6.8; the composition of the protein eluent is 20mM Tris,500mM NaCl, the concentration gradient of imidazole is 30-500mM, and the pH is 6.5-6.8.

The invention also provides a method for detecting the bioactivity of the recombinant human-derived type III collagen, wherein the bioactivity detection is mainly a cell adhesion test, and the bioactivity and the effect of the recombinant human-derived type III collagen are reflected.

The recombinant human III type collagen has good biological activity of cell adhesion, and can be applied to the fields of cosmetics and biomedical appliances.

The invention further provides a biological material for tissue engineering and cosmetic products, which comprises the recombinant human III type collagen.

The invention has the beneficial effects that: according to the invention, the optimal amino acid which possibly forms a triple helix structure is screened out through computer-aided prediction of the natural III type collagen sequence structure, the escherichia coli genetic engineering bacteria with high expression are obtained by using an escherichia coli expression system, and the high-purity recombinant human III type collagen is obtained through preliminary fermentation and purification, so that the natural amino acid sequence is retained to the greatest extent, the homology is higher, and the recombinant human III type collagen is applied to a human body without generating reactions such as immune rejection, allergy and the like. The recombinant human III type collagen with different molecular weights is obtained by serially expressing monomers of different repeated fragments. The recombinant human-derived type III collagen with small molecular weight can be used as a biological material for cosmetic products such as cosmetics, and the recombinant human-derived type III collagen with large molecular weight can be used as a biological material for medical appliances, so that the application range of the collagen is widened. The recombinant human-derived type III collagen produced by the invention has cell adhesion activity compared with a blank control PBS through biological function test, and can be used as a product in the biological medicine and cosmetic industries.

Drawings

FIG. 1 is a block diagram of the different number of monomers in tandem of rhIII-3 predicted by alpha Fold2 in the present invention.

FIG. 2 is a technical scheme for constructing a recombinant expression vector pET32a (+) - (rhIII-3) n of Escherichia coli.

FIG. 3 is a SDS-PAGE diagram induced by recombinant strains repeatedly tandem with different numbers of monomers according to the invention; wherein M is marker,1 is empty pET32a (+) induced 6h fermentation broth, 2, 4, 6, 8, 10, 12 are not induced 6h soluble protein for recombinant strain, 3, 5, 7, 9, 11, 13 are induced 6h soluble protein for recombinant strain. The arrows from left to right indicate single, two, three, four, eight and sixteen strings of repeating monomeric fusion protein bands.

FIG. 4 shows the fusion protein bands obtained by purification according to the present invention. The arrows from left to right indicate single, two, three, four, eight and sixteen strings of repeating monomeric fusion protein bands.

FIG. 5 shows recombinant human type III collagen obtained by purification after enterokinase cleavage. The arrows from left to right indicate single, two, three, four, eight and sixteen strings of repeating monomeric recombinant human type III collagen bands.

FIG. 6 is a graph showing the biological activity of recombinant human type III collagen obtained by purification according to the present invention compared with that of PBS blank.

Detailed Description

For a clearer description of a specific embodiment of the present invention, the present invention will be further described in detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.

The invention is further illustrated by the following non-limiting examples.

EXAMPLE 1 construction of recombinant expression vector for collagen rhIII-3 monomer

Because the biological function of the collagen depends on the triple helix structure of the collagen, according to the amino acid sequence of human III type collagen COL3A1 in the Gene Bank, the conserved region peptide of the human III type collagen COL3A1 is selected, 30 amino acids in the conserved region are further selected to carry out single-segment structure alpha fold2 prediction, and the amino acid sequences rhIII-1 to rhIII-6 with good structures are screened. The amino acid sequences of rhIII-1 to rhIII-6 are as follows:

rhIII-1:GPQGPKGDPGPPGIPGRNGDPGIPGQPGSP(SEQ ID NO.14)

rhIII-2:GDPGPPGIPGRNGDPGIPGQPGSPGSPGPP(SEQ ID NO.15)

rhIII-3:GFPGPKGNDGAPGKNGERGGPGGPGPQGPP(SEQ ID NO.16)

rhIII-4:GLQGLPGTGGPPGENGKPGEPGPKGDAGAP(SEQ ID NO.17)

rhIII-5:GPPGAAGTPGLQGMPGERGGLGSPGPKGDK(SEQ ID NO.18)

rhIII-6:GEPGPRGERGEAGIPGVPGAKGEDGKDGSP(SEQ ID NO.19)

and further, carrying out multi-segment repeated alpha Fold2 structure prediction on the candidate sequence, and finally selecting rhIII-3 peptide segments with good prediction results and triple helix structures.

The specific preparation method of the recombinant human-derived type III collagen is as follows:

the constructed monomeric recombinant expression protein is named (rhIII-3) 1, which is a fusion protein, has 205 amino acids in total length, contains thioredoxin tags to increase the soluble expression, is LD at the connection position, has 6 histidine residues at the C terminal, and has the amino acid sequence (SEQ ID NO. 8) as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

1.1RNA extraction (see MagZol Reagent one-step RNA extraction Reagent Specification)

50mg of human skin fibroblasts were weighed and added to 1ml MagZolTM Reagent, and immediately homogenized with a pestle or homogenizer. Standing at room temperature for 3-5 minutes, and adding 200 mu l of chloroform into the lysate according to 1ml MagZolTM Reagent; shaking vigorously for 15 seconds, and standing at room temperature for 3 minutes; the supernatant (. About.500. Mu.l) was carefully transferred to a new 1.5ml centrifuge tube. Adding isopropanol with the volume equal to that of the mixture, mixing the mixture uniformly by vortex, and standing the mixture at room temperature for 10 minutes; RNA was precipitated by centrifugation at 12000g for 10min at 4 ℃; the supernatant was discarded. 1ml of 75% ethanol is added and mixed by vortex; centrifuge at 7500g for 5 min at 4 ℃. The supernatant was discarded, and the centrifuge tube was back-snapped onto clean absorbent paper to remove residual liquid. Air drying for 10-15 min. Adding proper amount of buffer solution, 100% formamide, DEPC treated water or RNase-free water to RNA precipitation; the RNA precipitate is resuspended by vortexing and kept on ice for 10-30 minutes to allow the RNA to dissolve well and stored at-80℃after complete dissolution.

1.2 reverse transcription into cDNA (see the company next holy Corp.)II 1st Strand cDNA Synthesis SuperMix) the reverse transcription reaction system is as follows:

the PCR reaction system is as follows:

the reverse transcription product obtained above was stored at-20 ℃.

1.3 amplification of COL3A1 Gene

PCR amplification primers F1 and R1 are designed according to the III type collagen Gene sequence in the Gene Bank:

F1:CCCAAGCTTATGATGAGCTTTGTGCAA(SEQ ID NO.2)

R1:TGCTCTAGATTATAAAAAGCAAACAGGGC(SEQ ID NO.3)

the PCR reaction system is as follows:

the PCR reaction procedure was as follows:

the genes COL3A1 and pUC19 were digested with HindIII and XbaI, respectively, and then electrophoretically recovered, and ligated with T4 ligase to transform into E.coli DH 5. Alpha. And 1, selecting a monoclonal antibody 1 for bacterial liquid PCR verification. pUC19-COL3A1/DH 5. Alpha. Was subjected to PCR verification using pUC19 universal primer M13F, M R, and the expected size was 4400bp. Clones verified to be positive are sent to a sequencing company for sequencing, and conform to the expected sequence, and pUC19-COL3A1 is successfully constructed.

1.4 construction of recombinant expression vector for collagen rhIII-3 monomer

(1) Construction of monomeric recombinant expression vectors

The PCR primers F2, F3 and R2 were designed using pUC19-COL3A1 constructed as described above as a template:

F2：GCGTCGACGGCTTCCCCGGTCCTAAA(SEQ ID NO.5)

F3：GCGTCGACCTGGCTTCCCCGGTCCTAAA(SEQ ID NO.6)

R2：CCGCTCGAGAGGAGGACCCTGAGGGCC(SEQ ID NO.7)

the PCR reaction system is as follows:

the PCR reaction procedure was as follows:

the specific products obtained by using the primers F3 and R2 and pET32a (+) are respectively subjected to double digestion by Sal I and Xho I, then are electrophoretically recovered, are connected overnight at 16 ℃ by T4 ligase, and are transformed into host bacterium BL21 (DE 3) by a heat shock method. Randomly selected transformants were PCR verified using F3 and pET32a (+) universal primer T7T, expected to be 200bp in size. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 1 is successfully constructed.

EXAMPLE 2 construction of recombinant expression vector of collagen rhIII-3 two-segment repeat monomer

The recombinant expression protein of the two-segment repeated monomer constructed in the embodiment 1 of the invention is named as (rhIII-3) 2, is a fusion protein, has 237 amino acids in total length, contains thioredoxin tags to increase the soluble expression, is formed by connecting two identical human III-type collagen fragments in series, has LD at the connection part and 6 histidine residues at the C terminal, and has the amino acid sequence (SEQ ID NO. 9) as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID

QNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPR

GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

further, the vector pET32a (+) - (rhIII-3) 1 successfully constructed in example 1 was subjected to single cleavage with Xho I, the rhIII-3 amplified by F2 and R2 was recovered and subjected to double cleavage with Xho I and Sal I, and the single-cleaved vector was connected with the double-cleaved gene overnight with T4 ligase, and transformed into host BL21 (DE 3) by a heat shock method. Randomly selecting transformants, performing PCR verification by using F3 and pET32a (+) universal primer T7T, and then screening the transformants to have an electrophoresis size of 300bp. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 2 is successfully constructed.

EXAMPLE 3 recombinant expression vector construction of collagen rhIII-3 triple repeat monomer

The recombinant expression protein of the three-segment repeated monomer constructed in the embodiment 1 of the invention is named as (rhIII-3) 3, is a fusion protein, has the total length of 269 amino acids, contains thioredoxin tags to increase the soluble expression, is formed by connecting three identical human III-type collagen fragments in series, has LD at the connection part and 6 histidine residues at the C terminal, and has the amino acid sequence (SEQ ID NO. 10) as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID

QNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPR

GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNG

ERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

further, the vector pET32a (+) - (rhIII-3) 2 successfully constructed in example 2 was subjected to single cleavage with Xho I, the rhIII-3 amplified by F2 and R2 was recovered and subjected to double cleavage with Xho I and Sal I, and the single-cleaved vector was subjected to double cleavage with T4 ligase overnight, and was transformed into host BL21 (DE 3) by a heat shock method. Randomly selecting transformants, performing PCR verification by using F3 and pET32a (+) universal primer T7T, and then screening the transformants to have the electrophoresis size of 400bp. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 3 is successfully constructed.

EXAMPLE 4 recombinant expression vector construction of collagen rhIII-3 four-segment repeat monomer

The recombinant expression protein of the four-segment repeated monomer constructed in the embodiment 1 of the invention is recorded as (rhIII-3) 4, is a fusion protein, has 306 amino acids in total length, contains thioredoxin tags to increase the soluble expression, is formed by connecting four identical human III-type collagen fragments in series, has LD at the connection part and 6 histidine residues at the C terminal, and has the amino acid sequence (SEQ ID NO. 11) as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

further, the vector pET32a (+) - (rhIII-3) 3 successfully constructed in example 3 was subjected to single cleavage with Xho I, the rhIII-3 amplified by F2 and R2 was recovered and subjected to double cleavage with Xho I and Sal I, and the single-cleaved vector was connected with the double-cleaved gene overnight with T4 ligase, and transformed into host BL21 (DE 3) by a heat shock method. Randomly selecting transformants, performing PCR verification by using F3 and pET32a (+) universal primer T7T, and then screening the transformants to have an electrophoresis size of 500bp. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 4 is successfully constructed.

EXAMPLE 5 construction of recombinant expression vector of eight-segment repeated monomer of collagen rhIII-3

The recombinant expression protein of eight-segment repeated monomer constructed in the embodiment 1 of the invention is recorded as (rhIII-3) 8, is fusion protein, has 434 amino acids in total and contains thioredoxin label to increase the soluble expression, is formed by connecting eight segments of identical human III-type collagen fragments in series, wherein the connection part is LD, the C terminal is connected with 6 histidine residues, and the amino acid sequence (SEQ ID NO. 12) is shown as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

further, the vector pET32a (+) - (rhIII-3) 4 successfully constructed in example 4 above was subjected to Xho I single cleavage. The successfully constructed vector pET32a (+) - (rhIII-3) 4 is used as a template, the PCR primers F2 and R2 are used for amplifying a fragment (rhIII-3) 4, xho I and Sal I double digestion are carried out, the single digested vector and the double digested gene are connected overnight through T4 ligase, and the single digested vector and the double digested gene are transformed into host bacterium BL21 (DE 3) through a heat shock method. Randomly selecting transformants, performing PCR verification by using F3 and pET32a (+) universal primer T7T, and then screening the transformants to have the electrophoresis size of 900bp. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 8 is successfully constructed.

EXAMPLE 6 recombinant expression vector construction of collagen rhIII-3 sixteen-segment repeat monomer

The sixteen-segment repeated monomer recombinant expression protein constructed in the embodiment 1 of the invention is recorded as (rhIII-3) 16, is a fusion protein, has 690 amino acids in total length, contains thioredoxin tags to increase the soluble expression, is formed by serially connecting sixteen identical human III-type collagen fragments, is LD at the connection position, is connected with 6 histidine residues at the C terminal, and has the amino acid sequence (SEQ ID NO. 13) as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH。

further, the vector pET32a (+) - (rhIII-3) 8 successfully constructed in example 5 above was subjected to Xho I single cleavage. The successfully constructed vector pET32a (+) - (rhIII-3) 8 is used as a template, the PCR primers F2 and R2 are used for amplifying a fragment (rhIII-3) 8, xho I and Sal I double digestion are carried out, the single digested vector and the double digested gene are connected overnight through T4 ligase, and the single digested vector and the double digested gene are transformed into host bacterium BL21 (DE 3) through a heat shock method. Randomly selecting transformants, performing PCR verification by using F3 and pET32a (+) universal primer T7T, and then screening out the transformants with the electrophoresis size of 1600bp. The clone verified to be positive is sent to a sequencing company for sequencing, accords with the expected sequence, and the monomer expression vector pET32a (+) - (rhIII-3) 16 is successfully constructed.

EXAMPLE 7 fermentation and inducible expression of recombinant human type III collagen Strain

The single colony of the recombinant human source III type collagen strain which is successfully constructed is selected and inoculated into 10mL (50 mL triangular flask) LB culture medium containing 100mg/mL Amp, the culture is carried out at 37 ℃ for overnight, then 2 percent is transferred to two bottles of 200mL LB culture medium containing 100mg/mL Amp, the culture is carried out at 37 ℃ until the OD600 is 0.6-0.8, the IPTG with the final concentration of 0.1mM is added, one bottle is added with IPTG for induction, the other bottle is not added with IPTG, the culture is continued for 4-8 hours at 25 ℃, and the fermentation broth is collected. Centrifuging the fermentation broth in a refrigerated centrifuge at 4deg.C and 10000rpm for 10min, and collecting bacterial precipitate.

EXAMPLE 8 purification of recombinant human type III collagen

The bacterial pellet from example 7 was resuspended in 20mL of pre-chilled buffer and transferred to a 50mL centrifuge tube and centrifuged at 12000rpm for 20 minutes at 4deg.C; the supernatant was discarded, and after the bacterial cells were resuspended in 20mL of a lysis buffer (containing 1mM final concentration of PMSF), they were sonicated in an ice bath for 30min (350W, 5s sonication, 5s intervals, protection temperature 25 ℃); centrifugation at 8000rpm at 4℃for 20 minutes, collecting supernatant and precipitate, and SDS-PAGE to examine the solubility of the target protein expression, the examination results are shown in FIG. three, and the crushed supernatant contains recombinant collagen. The resulting disrupted supernatant was added to enterokinase and subjected to cleavage reaction at 4℃overnight to remove thioredoxin tags.

Balancing a nickel ion affinity column with a protein washing solution with the volume of 5 times, loading the obtained solution with the thioredoxin label removed on the column at the flow rate of 4 s/drop, fully combining target protein with the nickel column, and collecting penetrating fluid; washing the nickel column with 4 times of pre-cooled protein washing liquid to remove non-specific binding protein, and collecting washing liquid; eluting target protein with 4 times of pre-cooled protein eluent, collecting eluent, dialyzing the obtained product overnight, ultrafiltering and concentrating to obtain purified recombinant human III type collagen.

EXAMPLE 9 detection of biological Activity of recombinant human type III collagen

1. Collagen concentration was determined by SDS-PAGE and UV spectrophotometry.

The specific steps for determining the protein concentration are as follows:

the purified collagen solution was subjected to SDS-PAGE electrophoresis to verify that the protein concentration was estimated with reference to a known protein concentration (BSA protein) band. Simultaneously, ultraviolet spectrophotometry is used for measuring ultraviolet absorbance values of the collagen solution at 215nm and 225nm, and the ultraviolet absorbance values are calculated according to the following formula: c=144× (a 215-a 225), the calculated protein concentration units are μg/mL.

2. Cell adhesion of recombinant human type III collagen

Taking out the mouse embryo fibroblast 3T3 from the refrigerator at the temperature of minus 80 ℃, inoculating the mouse embryo fibroblast 3T3 into a cell culture medium, culturing the mouse embryo fibroblast 3T3 in a cell culture box at the temperature of 37 ℃, and carrying out cell passage when the cell grows to 80% -90% of a culture bottle. 2mL trypsin solution is added into a cell culture flask, and digestion is carried out for 2-3 min at 37 ℃. Cell digestions were observed under an inverted microscope. When the cells are rounded close to the wall, the trypsin solution is poured off. 10mL of the cell culture solution was added, and the cells were gently swirled with a pipette to remove the walls. Transferring the digested cells into a centrifuge tube, centrifuging at 1000rpm and 22 ℃ for 3min, discarding the supernatant, adding 1mL of cell culture solution to resuspend the cells, and preparing a cell suspension.

Preparing the purified recombinant human collagen III prepared in the step of the embodiment 8 into a protein solution of 0.5mg/ml, adding 100 mu L of recombinant human collagen into a 96-well plate to serve as a cell to be tested, and taking PBS as a blank controlIncubate for 1h at 37 ℃. The 96 Kong Bankong liquid was removed, 200 μlpbs solution was added, and the solution was washed 2-3 times. After removing the liquid in the wells, 100. Mu.L of a heat-inactivated 1% BSA-PBS solution (1 g BSA (CAS: 9048-46-8) powder was weighed, dissolved in 100mL PBS buffer solution, heat-inactivated at 56℃for 30 min) was added, and incubated in an incubator at 37℃for 1h. Removing liquid in the hole, adding 200 mu LPBS solution, cleaning for 2-3 times, and removing the hole for standby. Diluting the above cell suspension with cell culture solution to cell density of 1×10 ⁶ Each mL was added to a 96-well plate at 100. Mu.L/well, and cultured in a cell culture incubator at 37℃for 1 hour. After incubation for 1h, the 96-well plates were washed with 200 μlpbs added to each well, repeated 4 times, and the liquid was removed from the plates for use. The absorbance OD was measured at 450nm, following the instructions of the Cell Counting Kit-8 (CCK-8) detection kit.

The calculation formula of the relative adhesion rate of the recombinant collagen cells is as follows:

cell relative adhesion (%) = (test cell OD-blank OD)/blank od×100.

In conclusion, the invention screens out the optimal amino acid which can form a triple helix structure through the computer-aided prediction of the natural III type collagen sequence structure, obtains the highly expressed escherichia coli genetic engineering bacteria by using the escherichia coli expression system, obtains the recombinant humanized III type collagen through preliminary fermentation and purification, has high water solubility and stable triple helix structure, has good biological activity of cell adhesion, can be widely applied to the biological medicine and cosmetic industry, and lays a foundation for the application of the recombinant humanized collagen.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims

1. The recombinant human-derived type III collagen is characterized by comprising monomers repeatedly connected in series, wherein the monomers select peptide fragments consisting of 579-608 amino acid residues in a natural sequence of the human-derived type III collagen, the encoding gene of the monomers is rhIII-3, and the nucleotide sequence of the rhIII-3 is SEQ ID NO.1.

2. The recombinant human-derived type III collagen according to claim 1, wherein the rhIII-3 gene sequence is obtained by extracting total RNA from human skin fibroblasts, reversing the total RNA into cDNA, and designing PCR primers for amplification.

3. The recombinant human-derived type iii collagen according to claim 1, wherein the number of monomers in tandem in the recombinant human-derived type iii collagen is 1 to 16; the recombinant proteins of the repeated tandem monomers are respectively denoted as (rhIII-3) n, wherein n represents the number of repeated monomers.

4. The recombinant human type iii collagen of claim 1, wherein the recombinant human type iii collagen has a molecular weight of 3KD to 60KD.

5. A method of preparing recombinant human type iii collagen according to claim 2 or 3, comprising the steps of:

(1) Recombinant expression vector construction:

a) Total RNA is extracted from human skin fibroblasts, inverted into cDNA, PCR primers are designed to obtain human III type collagen COL3A1 genes, and the human III type collagen COL3A1 genes are connected with a cloning vector pUC19 to construct a recombinant vector pUC19-COL3A1. Taking pUC19-COL3A1 as a template, designing a PCR primer to obtain rhIII-3 genes, inserting the rhIII-3 genes between cleavage sites Sal I and Xho I of an expression plasmid pET32a (+) and transforming the rhIII-3 genes into host bacteria BL21 (DE 3) by a heat shock method, picking positive clones, and extracting plasmids by a plasmid extraction kit to obtain recombinant plasmid pET32a (+) - (rhIII-3) 1;

b) Taking plasmid pET32a (+) - (rhIII-3) 1 as a template, carrying out single enzyme digestion by Xho I, carrying out double enzyme digestion by Sal I and Xho I on a specific product rhIII-3 amplified by the PCR primer, and then connecting again by T4 ligase to form pET32a (+) - (rhIII-3) 2, and so on, so as to construct a recombinant expression vector pET32a (+) - (rhIII-3) n of a repeated multi-segment monomer, wherein n represents the number of repeated monomers;

(2) Construction of genetic recombinant engineering bacteria:

converting the prepared multistage monomer repeated recombinant expression vector pET32a (+) - (rhIII-3) n into BL21 (DE 3) competent cells, picking single bacterial colonies, carrying out colony PCR identification, sequencing successfully, and carrying out amplification culture to obtain the gene recombinant engineering bacteria;

(3) Preparation of Gene recombinant human source III collagen

6. The method for preparing recombinant human-derived type III collagen according to claim 5, wherein the specific method for separation and purification in the step (3) comprises the steps of:

inoculating the engineering bacteria obtained in the step (2) into 10ml of LB culture medium, culturing overnight at 37 ℃, transferring the engineering bacteria to the LB culture medium according to the volume ratio of 2%, culturing at 200rpm at 37 ℃, adding IPTG with the final concentration of 0.1mM when the recombinant strain is cultured to OD600 of 0.6-0.8, culturing at 25 ℃ for 4-8 hours, centrifuging at 10000rpm for 10min by using centrifugal force, and collecting bacterial precipitate;

(3.2) washing the bacterial precipitate with 20ml of a buffer solution for breaking bacteria, re-suspending the bacterial precipitate, adding PMSF with a final concentration of 1mM to prevent protease from degrading the target protein, performing ultrasonic disruption in an ice bath, centrifuging at 8000rpm for 20min, collecting supernatant,

resuspension and collection of the sediment are carried out by using sterile water; the sterile water was used in a weight-suspension amount of 20ml/200ml based on the volume of the fermented liquid.

Adding the obtained crushed supernatant into enterokinase, and performing enzyme digestion reaction at 4 ℃ overnight to remove thioredoxin tags;

(3.3) balancing the nickel ion affinity column with a protein washing solution with the volume of 5 times, loading the obtained solution for removing thioredoxin labels on the column, enabling the target protein to be fully combined with the nickel column at the flow rate of 4 s/drop, and collecting penetrating fluid; washing the nickel column with 4 times of pre-cooled protein washing liquid to remove non-specific binding protein, and collecting washing liquid; eluting target protein with 4 times of pre-cooled protein eluent, collecting eluent, dialyzing the obtained product overnight, ultrafiltering and concentrating to obtain purified recombinant human III type collagen.

7. The recombinant human type iii collagen according to any one of claims 1 to 4, or the recombinant human type iii collagen prepared according to claim 5 or 6, wherein the recombinant human type iii collagen has a biological activity of good cell adhesion.

8. Use of the recombinant human-derived type iii collagen according to any one of claims 1 to 4, or the recombinant human-derived type iii collagen prepared according to claim 5 or 6, in the fields of cosmetics and biomedical devices.

9. A biomaterial for tissue engineering and cosmetic products, characterized in that it comprises the recombinant human-derived type iii collagen according to any one of claims 1 to 4 or the recombinant human-derived type iii collagen obtained by the preparation method according to any one of claims 5 to 6.