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

Abstract

The invention discloses a method for producing recombinant human type III collagen using Escherichia coli and its expression method and application, and belongs to the technical fields of genetic engineering and bioengineering. The present invention uses AlphaFold2 to predict the structure of collagen, selects a peptide segment composed of amino acid residues from 579 to 608 in the natural human type III collagen sequence to form a collagen monomer, and repeatedly connects monomers with different expression numbers in series to make it It has a stable triple helix structure, and then the monomers with different repeat numbers are transformed into E. coli host bacteria to prepare recombinant human type III collagen, and obtain high purity and good biological activity of recombinant human type III collagen, which can be widely used. It is used in biomedical equipment and cosmetics industries and has good application prospects.

Description

A recombinant human type III collagen and its expression method and application

Technical field

The invention belongs to the field of genetic engineering technology, and specifically relates to a production of recombinant human type III collagen using Escherichia coli and its expression method and application.

Background technique

Collagen is one of the most abundant proteins in the human body, accounting for about 30% of the total protein content of the body. It is widely found in the extracellular matrix of connective tissues such as skin, bones, tendons, and blood vessels in the human body. It plays an important role in maintaining the normal operation of the body and preventing damage. Restoration has an important impact. Research has found that collagen has repeated tripeptide Gly-X-Y domains. These domains are the basis for maintaining the triple helix structure of collagen.

Because collagen has good biological activity, it can promote cell proliferation and differentiation, give the skin appropriate elasticity and toughness, and plays an important biological significance in wound healing, tissue repair, cellular immunity, etc. Based on this, collagen has been widely used in biomedicine, food, daily chemicals and other fields. In recent years, with the rapid development of genetic engineering and synthetic biology, protein expression systems based on animals, plants, fungi and bacteria have been established. The use of genetic engineering methods to synthesize recombinant human collagen can effectively solve the problem of collagen immunity. It can meet more clinical needs and has broad application prospects.

In the preparation of recombinant proteins, prokaryotic expression systems are often used. As one of the most important prokaryotic expression systems, E. coli 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 have been reports of using Escherichia coli as a host to express recombinant collagen. For example, CN103122027A discloses a recombinant human collagen and its production method, which selects DNA fragments from the helical region of human type II and type III collagen genes. , using PCR method to recombine, obtain complete recombinant collagen, which has good hydrophilicity and stability. However, there are few studies on the triple helix structure and biological activity of recombinant human collagen in existing patents. Therefore, in the technical field of preparing human collagen, there is an urgent need to develop a method that can use the E. coli system to efficiently express human collagen with a correct triple helix structure and good biological activity, which is of great practical significance.

This invention is based on the original gene sequence of type III human collagen, uses computer-aided protein structure prediction, selects highly water-soluble and highly biologically active parts for multi-segment series connection, splicing and recombination, and obtains a brand new recombinant type III humanized collagen. Protein has high application value in biomedicine, food and daily chemical industries.

Contents of the invention

The object of the present invention is to provide a recombinant human type III collagen and its expression method and application.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

The invention provides a recombinant human type III collagen, which has high water solubility and stability through Protein-sol (Protein-solsequence solubility (manchester.ac.uk)) for solubility prediction and AlphaFold2 for protein structure prediction. Triple helix structure, this recombinant human type III collagen contains repeating tandem monomers. Preferably, the monomer is a peptide segment consisting of 30 amino acids. According to the amino acid sequence of human type III collagen, a peptide segment consisting of amino acid residues 579 to 608 in the sequence is selected. 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, inverting it into cDNA, and designing PCR primers for amplification;

Wherein, in the recombinant human type III collagen, the number of monomers in series is 1 to 16, such as monomers, two-repeat series monomers, four-repeat series monomers, eight-repeat series monomers, and sixteen-repeat series monomers. The monomers, etc. are respectively recorded as (rhIII-3)n, where n represents the number of repeating monomers. The molecular weight of the recombinant human type III collagen is about 3KD-60KD.

The invention also provides a method for preparing genetically recombinant engineering bacteria and recombinant human type III collagen. The method includes the following steps:

(1) Construction of recombinant expression vector

Total RNA was extracted from human skin fibroblasts and reversed into cDNA. Based on the GeneBank human type III collagen COL3A1 gene sequence, PCR amplification primers F1 and R1 were designed:

F1:CCCAAGCTTATGATGAGCTTTGTGCAA(SEQ ID NO.2)

R1:TGCTCTAGATTATAAAAAGCAAACAGGGC(SEQ ID NO.3)

The COL3A1 gene was amplified by PCR using cDNA as a template. The length of the product was 4419 bp. The sequence was shown in SEQ ID NO. 4. The specific product was recovered and connected to the pUC19 vector to obtain the recombinant expression vector pUC19-COL3A1 for storage.

SEQ ID NO.4:

CCCAAGCTTATGATGAGCTTTGTGCAAaaggggagctggctacttctcgctctgcttcatcccactattattttggcacaacaggaagctgttgaaggaggatgttcccatcttggtcagtcctatgcggatagagatgtctggaagccagaaccatgccaaatatgtgtctgtgactcaggatccgttctctgcgatgacataatatgtg acgatcaagaattagactgccccaacccagaaattccatttggagaatgttgtgcagtttgcccacagcctccaactgctcctactcgccctcctaatggtcaaggacctcaaggccccaagggagatccaggccctcctggtattcctgggagaaatggtgaccctggtattccaggacaaccagggtcccctggttctcctggcccccctgga atctgtgaatcatgccctactggtcctcagaactattctccccagtatgattcatatgatgtcaagtctggagtagcagtaggaggactcgcaggctatcctggaccagctggccccccaggccctcccggtccccctggtacatctggtcatcctggttcccctggatctccaggataccaaggaccccctggtgaacctgggcaagctggtccttcaggccctcc aggacctcctggtgctataggtccatctggtcctgctggaaaagatggagaatcaggtagacccggacgacctggagagcgaggattgcctggacctccaggtatcaaaggtccagctgggatacctggattccctggtatgaaaggacacagaggcttcgatggacgaaatggagaaaag

ggtgaaacaggtgctcctggattaaagggtgaaaatggtcttccaggcgaaaatggagctcctggacccatgggtccaagaggggctcctggt

gagcgaggacggccaggacttcctggggctgcaggtgctcggggtaatgacggtgctcgaggcagtgatggtcaaccaggccctcctggtc

ctcctggaactgccggattccctggatcccctggtgctaagggtgaagttggacctgcagggtctcctggttcaaatggtgcccctggacaaaga

ggagaacctggacctcagggacacgctggtgctcaaggtcctcctggccctcctgggattaatggtagtcctggtggtaaaggcgaaatgggt

cccgctggcattcctggagctcctggactgatggggagcccggggtcctccaggaccagccggtgctaatggtgctcctggactgcgaggtggt

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

tggaaactggggaaacatgcataagtgccaatcctttgaatgttccacggaaacactggtggacagatctagtgctgagaagaaacacgtttgg

tttggagagtccatggatggtggttttcagtttagctacggcaatcctgaacttcctgaagatgtccttgatgtgcatctggcattccttcgacttctct

ccagccgagcttcccagaacatcacatatcactgcaaaaatagcattgcatacatggatcaggccagtggaaatgtaaagaaggccctgaagct

gatggggtcaaatgaaggtgaattcaaggctgaaggaaatagcaaattcacctacacagttctggaggatggttgcacgaaacacactgggga

atggagcaaaacagtctttgaatatcgaacacgcaaggctgtgagactacctattgtagatattgcaccctatgacattggtggtcctgatcaagaatttggtgtggacgttgGCCCTGTTTGCTTTTTATAATCTAGAGCA.

Further, using plasmid pUC19-COL3A1 as a template, primers F3 and R2 were used to amplify the rhIII-3 gene, and the specific product rhIII-3 was recovered. Its length is 106 bp. The primers are:

F2：GCGTCGACGGCTTCCCCGGTCCTAAA(SEQ ID NO.5)

F3: GCGTCGACCTGGCTTTCCCCGGTCCTAAA (SEQ ID NO.6)

R2: CCGCTCGAGAGGAGGACCCTGAGGGCC (SEQ ID NO.7)

The product rhIII-3 and the vector pET32a(+) were double-digested by Sal I and Xho I respectively, and ligated with T4 ligase to form pET32a(+)-(rhIII-3)1. What was constructed was a single-segment monomer. Expression vector.

Using pET32a(+)-(rhIII-3)1 as the template, it was digested with Xho I single enzyme, and then the specific product rhIII-3 amplified by F2 and R2 was double digested with Sal I and Xho I respectively. Then it is connected again with T4 ligase to form pET32a(+)-(rhIII-3)2, and so on to construct the recombinant expression vector pET32a(+)-(rhIII-3)n with repeated multi-segment monomers, where n represents the repeated monomer. times; for example, the repeating quadruplex recombinant expression vector is pET32a(+)-(rhIII-3)4, the repeating octet recombinant expression vector is pET32a(+)-(rhIII-3)8, and the repeating sixteenth recombinant expression vector is pET32a(+)-(rhIII-3)8. The vector is pET32a(+)-(rhIII-3)16, etc.

(2) Construction of genetically recombinant engineering bacteria

The multi-segment monomeric repeat recombinant expression vector pET32a(+)-(rhIII-3)n prepared above was transformed into BL21(DE3) competent cells, single colonies were picked and identified by colony PCR. After sequencing, the Expand the culture and obtain genetically recombinant engineering bacteria.

(3) Preparation of genetically recombinant human type III collagen

The genetically recombinant engineering bacteria obtained above are induced to express the target protein, and the target protein is further separated and purified to obtain high-purity water-soluble recombinant human type III collagen.

Further, the specific steps for induced expression, isolation and purification in step three are as follows:

(3.1) Inoculate the engineering bacteria obtained in step 2 into 10 ml of LB medium, culture it at 37°C overnight, transfer it to the LB medium at a volume ratio of 2%, and culture it at 37°C and 200rpm until the recombinant strain is cultured until the OD600 is 0.6 When ~0.8, add IPTG with a final concentration of 0.1mM. After culturing at 25°C for 4 to 8 hours, centrifuge at 10,000rpm for 10min to collect the bacterial sediment. Wherein, the composition of the fermentation medium LB medium is: tryptone (tryptone) 10g/L, yeast extract 5g/L, NaCl 10g/L, and the initial pH is 7.2-7.4.

(3.2) Wash the bacterial pellet with 20ml of sterilization buffer and resuspend the bacterial pellet. Add PMSF with a final concentration of 1mM to prevent protease from degrading the target protein. Ultrasonically disrupt it in an ice bath. Centrifuge at 8000rpm for 20min. Collect the supernatant and precipitate. Use sterile water to resuspend and collect; the resuspension volume of sterile water used is 20ml/200ml of the volume of the fermented bacterial liquid. Among them, the composition of the above-mentioned bacteriolysis buffer is: 300mM NaCl, 50mM NaH2PO4, 10mM imidazole, pH=6.5-6.8; the ultrasonic breaking conditions are 350W, 5s ultrasonic, 5s interval, the protection temperature is 25°C, and the ultrasonic time is 30 minutes. Add an appropriate amount of enterokinase to the fragmented supernatant obtained above, and perform an enzyme digestion reaction at 4°C overnight to remove the thioredoxin tag.

(3.3) Equilibrate the nickel ion affinity column with 5 times the volume of protein washing solution, and put the solution obtained above to remove the thioredoxin tag onto the column at a flow rate of 4 s/drop to fully combine the target protein with the nickel column, and collect the penetration solution; wash the nickel column with 4 times the volume of pre-cooled protein washing solution to remove non-specific binding proteins, and collect the washing solution; use 4 times the volume of pre-cooled protein eluent to elute the target protein, collect the eluate, and dialyze the resulting product After overnight, ultrafiltration and concentration, and freeze-drying, purified recombinant human type III collagen was obtained.

Among them, the composition of the protein washing liquid is: 20mM Tris, 500mM NaCl, pH is 6.5-6.8; the composition of the protein eluate is 20mM Tris, 500mM NaCl, the imidazole concentration gradient is 30-500mM, and the pH is 6.5-6.8.

The present invention also proposes a biological activity detection method of the recombinant human type III collagen. The biological activity detection is mainly a cell adhesion test, which reflects the biological activity and effect of the recombinant human type III collagen.

The recombinant human type III collagen of the present invention has good cell adhesion and biological activity, and can be used in the fields of cosmetics and biomedical devices.

The present invention further proposes a biological material used for tissue engineering and beauty products, which biological material contains the above-mentioned recombinant human type III collagen.

The beneficial effects of the present invention are: the present invention uses computer-aided prediction of the sequence structure of natural type III collagen, screens out the optimal amino acids that may form a triple helix structure, and uses the Escherichia coli expression system to obtain highly expressed Escherichia coli genetically engineered bacteria. After fermentation and purification, high-purity recombinant human type III collagen is obtained, which retains the natural amino acid sequence to the greatest extent and has high homology. It will not cause immune rejection or allergic reactions when applied to the human body. Recombinant human type III collagen of different molecular weights is obtained by expressing monomers of different repeating segments in series. Among them, the small molecular weight recombinant human type III collagen can be used as a biomaterial in cosmetics and other beauty products, and the large molecular weight recombinant human type III collagen can be used as a biomaterial in medical devices, broadening the application range of collagen. The recombinant human type III collagen produced by the present invention has been tested for biological functions. Compared with the blank control PBS, it has cell adhesion activity and meets the requirements for product use in the biomedicine and cosmetics industries.

Description of the drawings

Figure 1 is a structural diagram of rhIII-3 repeated in series with different numbers of monomers predicted by AlphaFold2 in the present invention.

Figure 2 is a technical roadmap for the construction of E. coli recombinant expression vector pET32a(+)-(rhIII-3)n.

Figure 3 is an SDS-PAGE diagram of the recombinant strain induced by repeated tandem connection of different numbers of monomers of the present invention; where M is a marker, 1 is the fermentation broth induced by empty pET32a(+) for 6 hours, 2, 4, 6, 8, 10 , 12 represents the soluble protein that was not induced by the recombinant strain for 6 hours, and 3, 5, 7, 9, 11, and 13 represents the soluble protein that was induced by the recombinant strain for 6 hours. The arrows from left to right point to the fusion protein bands of single, double, triple, quadruple, eight and sixteen repeating monomers.

Figure 4 shows the fusion protein band obtained by purification of the present invention. The arrows from left to right point to the fusion protein bands of single, double, triple, quadruple, eight and sixteen repeating monomers.

Figure 5 shows the recombinant human type III collagen purified by enterokinase digestion according to the present invention. The arrows from left to right point to the recombinant human type III collagen bands of single, double, triple, quadruple, eight and sixteen repeating monomers.

Figure 6 is a biological activity detection chart of the recombinant human type III collagen purified by the present invention compared with the blank control PBS.

Detailed ways

In order to illustrate the specific embodiments of the present invention more clearly, the present invention will be further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except those specifically mentioned below, are common knowledge and common sense in the field, and the present invention has no special limitations.

The present invention is further explained below through non-limiting examples.

Example 1 Construction of recombinant expression vector of collagen rhIII-3 monomer

Since the biological function of collagen depends on its triple helix structure, based on the amino acid sequence of human type III collagen COL3A1 in Gene Bank, the conserved region peptide of human type III collagen COL3A1 was selected, and 30 amino acids in the conserved region were further selected. The single-segment structure AlphaFold2 was predicted and the amino acid sequences rhIII-1~rhIII-6 with good structures were screened out. The amino acid sequences of rhIII-1 to rhIII-6 are as follows:

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

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

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

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

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

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

Furthermore, the above candidate sequences were subjected to multiple repeated AlphaFold2 structure predictions, and finally the rhIII-3 peptide with good prediction results and a triple helix structure was selected.

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

The constructed monomeric recombinant expression protein, designated as (rhIII-3)1, is a fusion protein with a full length of 205 amino acids and contains a thioredoxin tag to increase its soluble expression. The junction is LD and the C-terminal band There are 6 histidine residues, and their amino acid sequence (SEQ ID NO. 8) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

1.1 RNA extraction (refer to MagZol Reagent one-step RNA extraction reagent instructions)

Weigh 50 mg of human skin fibroblasts, add 1 ml of MagZolTM Reagent, and immediately homogenize thoroughly with a pestle or homogenizer. Leave at room temperature for 3 to 5 minutes. Add 200 μl of chloroform per 1 ml of MagZolTM Reagent to the lysis solution; shake vigorously for 15 seconds and leave at room temperature for 3 minutes; carefully transfer the supernatant (~500 μl) to a new 1.5 ml centrifuge tube. Add an equal volume of isopropanol, vortex to mix, and let stand at room temperature for 10 minutes; centrifuge at 12,000g for 10 minutes at 4°C to precipitate RNA; discard the supernatant. Add 1ml of 75% ethanol, vortex and mix; centrifuge at 7500g for 5 minutes at 4°C. Discard the supernatant and place the centrifuge tube on clean absorbent paper to absorb the remaining liquid. Air dry for 10 to 15 minutes. Add an appropriate amount of buffer, 100% formamide, DEPC-treated water, or RNase-free water to the RNA pellet; vortex to resuspend the RNA pellet, and place it on ice for 10-30 minutes to allow the RNA to fully dissolve. After complete dissolution -80 Store at ℃.

1.2 Reverse transcription into cDNA (refer to Yisheng Company Ⅱ1st Strand cDNA SynthesisSuperMix Instructions) 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°C.

1.3 Amplification of COL3A1 gene

Design PCR amplification primers F1 and R1 based on the type III collagen gene sequence in 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 is as follows:

Genes COL3A1 and pUC19 were digested with Hind III and Xba I respectively, recovered by electrophoresis, ligated with T4 ligase and transformed into E. coli DH5α. Pick 1 single clone for bacterial liquid PCR verification. pUC19-COL3A1/DH5α was verified by PCR using pUC19 universal primers M13F and M13R, and the expected size is 4400bp. The clone that was verified to be positive was sent to a sequencing company for sequencing. It matched the expected sequence and pUC19-COL3A1 was successfully constructed.

1.4 Construction of recombinant expression vector of collagen rhIII-3 monomer

(1) Construction of monomeric recombinant expression vector

Using the successfully constructed pUC19-COL3A1 above as a template, design PCR primers F2, F3, and R2:

F2：GCGTCGACGGCTTCCCCGGTCCTAAA(SEQ ID NO.5)

F3: GCGTCGACCTGGCTTTCCCCGGTCCTAAA (SEQ ID NO.6)

R2: CCGCTCGAGAGGAGGACCCTGAGGGCC (SEQ ID NO.7)

The PCR reaction system is as follows:

The PCR reaction procedure is as follows:

The above-mentioned specific products obtained using primers F3 and R2 and pET32a(+) were double-digested with Sal I and Xho I respectively, recovered by electrophoresis, ligated with T4 ligase at 16°C overnight, and transformed into the host strain BL21(+) by heat shock method. DE3). Randomly select transformants for PCR verification using F3 and pET32a(+) universal primer T7t, with an expected size of 200 bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)1 was successfully constructed.

Example 2 Construction of recombinant expression vector of two repeat monomers of collagen rhIII-3

The recombinant expression protein of two repeating monomers constructed in Example 1 of the present invention is designated as (rhIII-3)2. It is a fusion protein with a full length of 237 amino acids and contains a thioredoxin tag to increase its soluble expression. Two identical human type III collagen fragments are connected in series. The junction is LD and the C-terminus has 6 histidine residues. Its amino acid sequence (SEQ ID NO. 9) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID

QNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPR

GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

Furthermore, the vector pET32a(+)-(rhIII-3)1 successfully constructed in the above Example 1 was digested with Xho I single enzyme, and the rhIII-3 amplified by F2 and R2 was recovered and subjected to Xho I and Sal I double digestion. After enzyme digestion, the single-enzyme-digested vector and the double-enzyme-digested gene were ligated overnight using T4 ligase, and then transformed into the host strain BL21 (DE3) using the heat shock method. Randomly select transformants and use F3 and pET32a(+) universal primer T7t for PCR verification. By analogy, the electrophoresis size is selected to be 300 bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)2 was successfully constructed.

Example 3 Construction of recombinant expression vector for collagen rhIII-3 triple repeat monomer

The recombinant expression protein of three repeat monomers constructed in Example 1 of the present invention is designated as (rhIII-3)3. It is a fusion protein with a full length of 269 amino acids and contains a thioredoxin tag to increase its soluble expression. Three identical human type III collagen fragments are connected in series. The junction is LD and the C-terminus has 6 histidine residues. Its amino acid sequence (SEQ ID NO. 10) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID

QNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPR

GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNG

ERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

Furthermore, the vector pET32a(+)-(rhIII-3)2 successfully constructed in the above Example 2 was digested with Xho I single enzyme, and the rhIII-3 amplified by F2 and R2 was recovered and subjected to Xho I and Sal I double digestion. After enzyme digestion, the single-enzyme-digested vector and the double-enzyme-digested gene were ligated overnight using T4 ligase, and then transformed into the host strain BL21 (DE3) using the heat shock method. Randomly select transformants and use F3 and pET32a(+) universal primer T7t for PCR verification. By analogy, the electrophoresis size is 400bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)3 was successfully constructed.

Example 4 Construction of recombinant expression vector for collagen rhIII-3 four-segment repeat monomer

The recombinant expression protein of four repeat monomers constructed in Example 1 of the present invention is designated as (rhIII-3)4. It is a fusion protein with a full length of 306 amino acids and contains a thioredoxin tag to increase its soluble expression. Four identical human type III collagen fragments are connected in series. The junction is LD and the C-terminus has 6 histidine residues. Its amino acid sequence (SEQ ID NO. 11) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNG ERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

Furthermore, the vector pET32a(+)-(rhIII-3)3 successfully constructed in the above Example 3 was digested with Xho I single enzyme, and the rhIII-3 amplified by F2 and R2 was recovered and subjected to Xho I and Sal I double digestion. After enzyme digestion, the single-enzyme-digested vector and the double-enzyme-digested gene were ligated overnight using T4 ligase, and then transformed into the host strain BL21 (DE3) using the heat shock method. Randomly select transformants and use F3 and pET32a(+) universal primer T7t for PCR verification. By analogy, the electrophoresis size is 500bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)4 was successfully constructed.

Example 5 Construction of recombinant expression vector for collagen rhIII-3 eight-segment repeat monomer

The recombinant expression protein of eight repeat monomers constructed in Example 1 of the present invention is designated as (rhIII-3)8. It is a fusion protein with a full length of 434 amino acids and contains a thioredoxin tag to increase its soluble expression. Eight identical human type III collagen fragments are connected in series. The junction is LD, and the C-terminus is connected to 6 histidine residues. Its amino acid sequence (SEQ ID NO. 12) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNG ERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

Furthermore, the vector pET32a(+)-(rhIII-3)4 successfully constructed in the above Example 4 was digested with Xho I single enzyme. Using the successfully constructed vector pET32a(+)-(rhIII-3)4 as a template, the fragment (rhIII-3)4 was amplified using the above-mentioned PCR primers F2 and R2 and subjected to Xho I and Sal I double enzyme digestion. The enzyme-digested vector and the double-digested gene were ligated overnight using T4 ligase, and then transformed into the host strain BL21 (DE3) using the heat shock method. Randomly select transformants and use F3 and pET32a(+) universal primer T7t for PCR verification. By analogy, the electrophoresis size is 900bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)8 was successfully constructed.

Example 6 Construction of recombinant expression vector for collagen rhIII-3 sixteen-segment repeat monomer

The recombinant expression protein of sixteen repeat monomers constructed in Example 1 of the present invention is designated as (rhIII-3)16. It is a fusion protein with a full length of 690 amino acids and contains a thioredoxin tag to increase its soluble expression. It is composed of sixteen identical human type III collagen fragments connected in series. The junction is LD, and the C-terminus is connected to 6 histidine residues. Its amino acid sequence (SEQ ID NO. 13) is as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNG ERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGA PGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPK GNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

Furthermore, the vector pET32a(+)-(rhIII-3)8 successfully constructed in Example 5 above was digested with Xho I single enzyme. Using the above-mentioned successfully constructed vector pET32a(+)-(rhIII-3)8 as a template, the above-mentioned PCR primers F2 and R2 were used to amplify the fragment (rhIII-3)8 and perform double digestion with Xho I and Sal I. The enzyme-digested vector and the double-digested gene were ligated overnight using T4 ligase, and then transformed into the host strain BL21 (DE3) using the heat shock method. Randomly select transformants and use F3 and pET32a(+) universal primer T7t for PCR verification. By analogy, the electrophoresis size is 1600bp. The clones that were verified to be positive were sent to a sequencing company for sequencing, which matched the expected sequence. The monomeric expression vector pET32a(+)-(rhIII-3)16 was successfully constructed.

Example 7 Fermentation and induced expression of recombinant human type III collagen strain

Pick a single colony of the successfully constructed recombinant human type III collagen strain and inoculate it into 10 ml (50 mL Erlenmeyer flask) LB medium containing 100 mg/ml Amp, culture it at 37°C overnight, and then transfer it to two bottles containing 2% Amp. 200ml LB medium with 100mg/mlAmp, culture at 37℃ until OD600 is 0.6~0.8, add IPTG with a final concentration of 0.1mM, add IPTG to one bottle for induction, and add no IPTG to the other bottle, continue culturing at 25℃ for 4~8 hours. Collect the fermentation broth. Centrifuge the fermentation broth in a refrigerated centrifuge at 4°C and 10,000 rpm for 10 min. Discard the supernatant to collect the bacterial sediment.

Example 8 Purification of recombinant human type III collagen

Add 20 mL of pre-cooled sterilization buffer to resuspend the bacterial pellet in the above Example 7 and transfer it to a 50 mL centrifuge tube. Centrifuge at 12000 rpm for 20 minutes at 4°C; discard the supernatant and add 20 mL of sterilization buffer. After resuspending the bacteria in liquid (containing PMSF with a final concentration of 1mM), the bacteria were crushed by ultrasonic in an ice bath for 30 minutes (350W, 5s ultrasound, 5s interval, protection temperature is 25℃); centrifuge at 4℃, 8000rpm for 20 minutes, and collect the supernatant and precipitate respectively. SDS-PAGE detects the solubility of the target protein expression. The test results are shown in Figure 3. The supernatant after fragmentation contains recombinant collagen. Enterokinase was added to the fragmented supernatant obtained above, and the enzymatic digestion reaction was carried out at 4°C overnight to remove the thioredoxin tag.

Equilibrate the nickel ion affinity column with 5 times the volume of protein washing solution, and put the solution obtained above to remove the thioredoxin tag onto the column at a flow rate of 4 s/drop to fully combine the target protein with the nickel column, and collect the penetration fluid; use Wash the nickel column with 4 times the volume of pre-cooled protein wash solution to remove non-specific binding proteins and collect the wash solution; use 4 times the volume of pre-cooled protein eluent to elute the target protein and collect the eluate. The resulting product is dialyzed overnight and ultrasonic. Filter and concentrate to obtain purified recombinant human type III collagen.

Example 9 Biological activity detection of recombinant human type III collagen

1. Determine collagen concentration using SDS-PAGE and UV spectrophotometer.

The specific steps to determine protein concentration are as follows:

The purified collagen solution was verified by SDS-PAGE electrophoresis, and its protein concentration was estimated with reference to the known protein concentration (BSA protein) band. At the same time, use a UV spectrophotometer to measure the UV absorbance of the collagen solution at 215nm and 225nm, and calculate it according to the following formula: C=144×(A215-A225). The calculated protein concentration unit is μg/mL.

2. Cell adhesion of recombinant human type III collagen

Take out the mouse embryonic fibroblast 3T3 cells from the -80°C refrigerator, inoculate them in the cell culture medium, and culture them in a 37°C cell culture incubator. When the cells grow to 80% to 90% of the culture flask, the cells are passaged. Add 2 mL of trypsin solution to the cell culture flask and digest at 37°C for 2 to 3 minutes. Observe cell digestion under an inverted microscope. When the cells become round and close to detachment, the trypsin solution is discarded. Add 10 mL of cell culture medium and gently pipet with a pipette to detach the cells. Transfer the digested cells to a centrifuge tube, centrifuge at 1000 rpm and 22°C for 3 minutes, discard the supernatant, and add 1 mL of cell culture medium to resuspend the cells to make a cell suspension.

The purified recombinant human type III collagen prepared in step 8 of the present invention was prepared into a 0.5 mg/ml protein solution, and 100 μL of recombinant human collagen was added to a 96-well plate as cells to be tested, and PBS was used as a blank control. , incubate for 1 hour in a 37°C incubator. Remove the liquid from the wells of the 96-well plate, add 200 μL PBS solution, and wash 2-3 times. After removing the liquid in the well, add 100 μL of heat-inactivated 1% BSA-PBS solution (weigh 1g of BSA (CAS: 9048-46-8) powder and dissolve it in 100 mL of PBS buffer solution. Heat inactivate at 56°C for 30 minutes) , incubate for 1 hour in a 37°C incubator. Remove the liquid in the well, add 200 μL PBS solution, wash 2-3 times, remove the well and set aside for use. Take the above cell suspension, dilute it with cell culture medium to a cell density of 1×10 ⁶ cells/mL, add it to a 96-well plate at 100 μL/well, and culture it in a 37°C cell culture incubator for 1 hour. After incubating the 96-well plate for 1 hour, add 200 μL PBS to each well for cleaning. Repeat 4 times. Remove the liquid in the plate and set aside for use. Follow the Cell Counting Kit-8 (CCK-8) detection kit instructions and measure the absorbance OD value at 450nm.

Among them, the calculation formula for the relative adhesion rate of recombinant collagen cells is as follows:

Relative cell adhesion rate (%) = (OD of cells to be tested-Blank OD)/Blank OD×100.

In summary, the present invention uses computer-aided prediction of the sequence structure of natural type III collagen, screens out the optimal amino acids that may form a triple helix structure, and uses the E. coli expression system to obtain highly expressed E. coli genetically engineered bacteria. After preliminary fermentation and After purification, recombinant human type III collagen is obtained, which has high water solubility, stable triple helical structure, and good cell adhesion biological activity. It can be widely used in the biomedicine and cosmetics industries and provides recombinant human collagen. Lay the foundation for the application of collagen.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the concept of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and are protected by the appended claims.

Claims (9)

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

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.

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.