CN116555320A - Recombinant human-derived III-type triple helix collagen engineering bacterium, and construction method and application thereof - Google Patents
Recombinant human-derived III-type triple helix collagen engineering bacterium, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant human-derived III-type triple helix collagen engineering bacterium, and a construction method and application thereof, wherein the construction method comprises the following steps: s1, inserting the optimized proline hydroxylase gene into a shuttle expression vector pPIC9k plasmid to obtain a recombinant vector A; s2, inserting the optimized human source III type triple helix collagen alpha 1 chain gene fragment into a pPICZ alpha A plasmid to obtain a recombinant vector B; s3, converting the recombinant vector A into a host strain Pichia pastoris, screening, converting the recombinant vector B into the host strain Pichia pastoris, and screening to obtain the recombinant human source III type triple helix collagen engineering bacteria. The triple helix conformation prepared by the engineering bacteria can realize various biological functions such as cell adhesion, migration, proliferation and the like, and meets the requirement of downstream products on maximization of biological activity.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a preparation method and application of recombinant human-derived III-type triple helix collagen.
Background
Collagen is the most abundant protein in extracellular matrix in animals and human bodies, widely exists in skin bones and the like, and plays roles of supporting stability and supporting extension. Particularly has important roles in wound recovery and healing. The main structure of collagen which plays its biological function is its triple helix structure, and type III collagen is formed from three identical alpha 1 peptide chains. Collagen is very complex in the in vivo synthesis route, and proline hydroxylase plays a vital role in the synthesis process. While ensuring that it can form an active triple helix structure, a hydroxylation modification of proline on the alpha chain is required. This process requires proline hydroxylase catalysis to be accomplished.
Because of the good healing promoting capability, the collagen is widely applied to the fields of cosmetic materials, medical materials and the like. The collagen used mainly at present is still natural collagen extracted from animals, the three-helix structure of the collagen is difficult and easily destroyed in the extraction process, and non-human sources possibly bring about allergy and virus hidden trouble. Meanwhile, natural collagen of animal origin is often not easily dissolved in water, which causes difficulties in subsequent processing and use.
Pichia pastoris (Pichia pastoris) is used as an exogenous protein expression system, so that the method is simple and easy to culture, and has low cost and high expression quantity. In addition, eukaryotic expression systems may be modified for foreign proteins such as glycosylation, protein phosphorylation, etc., and do not contain bacterial endotoxins. Therefore, the production of collagen by taking pichia pastoris as engineering bacteria becomes possible. Since pichia cells do not express proline hydroxylase, additional introduction of the gene for this enzyme is required.
Therefore, there is a need in the art to develop an engineering bacterium capable of expressing recombinant type iii triple helix collagen with good safety to solve the above problems.
Disclosure of Invention
In view of the above, the invention aims to overcome the defects in the prior art, and provides a recombinant human-derived type III triple helix collagen engineering bacterium, and a construction method and application thereof.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the first aspect of the invention provides a construction method of recombinant human-derived type III triple helix collagen engineering bacteria, which comprises the following steps:
s1, inserting the optimized proline hydroxylase gene into a shuttle expression vector pPIC9k plasmid to obtain a recombinant vector A;
s2, inserting the optimized human source III type triple helix collagen alpha 1 chain gene fragment into a pPICZ alpha A plasmid to obtain a recombinant vector B;
s3, converting the recombinant vector A into a host strain Pichia pastoris, screening, converting the recombinant vector B into the host strain Pichia pastoris, and screening to obtain the recombinant human source III type triple helix collagen engineering bacteria.
Preferably, the nucleotide sequence of the optimized proline hydroxylase gene is shown in SEQ ID NO: 1.
Preferably, the nucleotide sequence of the optimized human-derived type III triple helix collagen alpha 1 chain gene fragment is shown as SEQ ID NO: shown at 5.
Preferably, the step S1 includes the steps of:
s11, synthesizing a DNA fragment 1 of the proline hydroxylase of the micrococcus virus containing the fluorescent EGFP label;
s12, designing an amplification primer, and amplifying the DNA fragment A to obtain an amplified fragment with EcoRI and NotI restriction sites at two ends;
s13, performing enzyme digestion on the amplified fragment and the pPIC9k plasmid by using EcoRI and NotI restriction enzymes;
s14, connecting the amplified fragment and the pPIC9k plasmid by using ligase, and culturing overnight to obtain a recombinant vector A containing a proline hydroxylase gene;
s15, transforming the recombinant vector A into escherichia coli for plasmid cloning to obtain a large number of target recombinant vectors A.
Preferably, the step S2 includes the steps of:
s21, synthesizing a DNA fragment sequence B containing an alpha 1 chain of the human-derived III type triple helix collagen;
s22, designing an amplification primer containing a His affinity tag, and amplifying a DNA fragment sequence B to obtain an amplification fragment with EcoRI and NotI enzyme cutting sites at two ends;
s23, performing enzyme digestion on the amplified fragment and the pPICZ alpha A plasmid by using EcoRI and NotI restriction enzymes;
s24, connecting the amplified fragment and the pPICZ alpha A plasmid by using ligase to obtain the recombinant vector B containing the human III type triple helix collagen gene.
Preferably, step S3 comprises the steps of:
preparing competent host strain Pichia pastoris, linearizing recombinant vector A containing proline hydroxylase gene by using restriction enzyme, then performing electric shock transformation to host strain Pichia pastoris, screening positive cloned seeds, and culturing and selecting engineering bacteria producing fluorescence.
Preferably, step S4 comprises the steps of:
selecting fluorescent engineering bacteria, preparing competent host strain Pichia pastoris, electrically transferring the linearized recombinant vector B containing the human III type triple helix collagen gene to the host strain Pichia pastoris, and screening to obtain the recombinant human III type triple helix collagen engineering bacteria.
Preferably, the host strain pichia pastoris is GS115 pichia pastoris.
The invention also provides a recombinant human triple helix collagen engineering bacterium obtained by the construction method.
The third aspect of the invention also provides an application of the recombinant human type III triple helix collagen engineering bacteria in preparing human type III triple helix collagen.
Type III triple helix collagen is a triple helix structure consisting of three identical alpha 1 peptide chains. To ensure that it forms an active triple helix structure, a hydroxylation modification of proline on the alpha chain is required, which is catalysed by a proline hydroxylase. Pichia cells do not express such hydroxylase, so additional introduction of the gene for such enzyme is required. The proline hydroxylase gene of Chlorella virus 1 (PBCV-1) is similar to the human proline hydroxylase gene and is easy to express.
The invention selects human III type collagen alpha chain gene and chlorella virus 1 (PBCV-1) proline hydroxylase gene as target genes. According to the human type III collagen alpha chain gene, 599 amino acids 908 to 1446 in the sequence are selected, and a 6his purification tag is added at the tail end. According to the proline hydroxylase gene of Chlorella virus 1 (PBCV-1), 208 amino acids (100-726 genes) at positions 34 to 242 thereof were selected, the N-terminus was removed, and a fluorescent label was added. Then optimizing two groups of target genes according to codons of pichia pastoris, and avoiding possible enzyme cutting sites used in genetic engineering operation. Adding the genes of the enzyme cutting sites required by the genetic engineering before and after the two sections of target genes.
The engineering bacteria selected by the invention are pichia pastoris GS115 (high-yield type), and the plasmid is pPIC9k/pPICZ alpha A. Both plasmids were secretion expression vectors. Pichia pastoris GS115 is histidine-deficient and lacks the His4 gene. Transformants are conveniently screened.
The invention selects a method for constructing engineering bacteria expressing proline hydroxylase and then expressing human recombinant type III triple helix collagen. The method is simple Yi Ju, the subsequent production cost is low, plasmids can be cloned in batches through escherichia coli, and the acquisition path is simple. The recombinant protein is secreted extracellularly, and is convenient to collect and purify. The obtained recombinant protein can exert the biological function of the recombinant protein as III type triple helix collagen.
Compared with the prior art, the invention has the following advantages:
(1) According to the engineering bacterium construction method, proline hydroxylase similar to human sources and simple in structure and easy to express is selected to be connected with the shuttle plasmid, so that the cost is reduced, and the engineering bacterium construction method is simple and convenient to produce.
(2) According to the invention, the humanized triple helix collagen III fragment with good expression is selected and optimized, so that the problem that the collagen III is not well expressed is solved, the success rate of selecting the fragment expression is high, and the biological function of the collagen III is realized.
(3) The invention can ensure that proline sites in collagen molecular sequences are hydroxylated through the yeast fine engineering bacteria for expressing proline hydroxylase, and effectively improves the interaction force, the thermal stability and the enzymolysis resistance level between collagen molecules.
(4) The triple helix conformation prepared by the engineering bacteria can realize various biological functions such as cell adhesion, migration, proliferation and the like, and meets the requirement of downstream products on maximization of biological activity.
Drawings
FIG. 1 is a schematic diagram of the construction of a recombinant plasmid containing an optimized proline hydroxylase gene of the examples;
FIG. 2 is a schematic diagram of the construction of a recombinant plasmid containing an optimized triple helix collagen III gene according to the example;
FIG. 3 is a diagram of plasmid enzyme digestion electrophoresis of pPICZ alpha A containing triple helix collagen III gene;
FIG. 4 is a western blot of expression of human type III triple helix collagen in GS115 strain;
FIG. 5 is a solution of triple helix collagen type III prepared as described in the example.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention will be described in detail with reference to examples.
Examples
1. Construction of vectors
1.2 construction of plasmid containing proline hydroxylase Gene
Recombinant pichia pastoris strains capable of expressing proline hydroxylase are constructed. Plasmid pPIC9k was selected and the optimized PBCV-1 proline hydroxylase gene (shown in SEQ ID NO: 1) and EGFP fluorescent label (N-terminal) were inserted (shown in FIG. 1). The target gene fragment is obtained by total gene synthesis, and PCR is performed by using primers containing enzyme cutting sites.
Optimized proline hydroxylase gene sequence:
AGAAGAGAAGGTTTTGAAACTTCTGATAGACCTGGTGTTTGTGATGGTAAATACTACGAAAAAATCGATGGTTTTCTGTCTGATATTGAATGTGATGTTTTGATTAACGCTGCTATTAAGAAAGGTTTGATTAAGTCTGAAGTCGGTGGTGCTACTGAAAATGATCCTATTAAGTTGGATCCTAAGTCTCGTAACTCTGAACAAACTTGGTTCATGCCTGGTGAACATGAAGTTATTGATAAGATTCAAAAGAAGACCAGAGAATTTTTGAACTCTAAAAAGCATTGCATCGATAAGTACAACTTTGAAGATGTTCAAGTTGCTAGATACAAGCCAGGTCAATACTATTACCATCATTACGATGGTGACGATTGTGATGATGCTTGTCCTAAAGATCAAAGATTGGCTACTTTGATGGTTTACTTGAAAGCTCCTGAAGAAGGTGGTGGTGGTGAAACTGATTTTCCAACTTTGAAGACTAAGATTAAGCCTAAGAAAGGTACTTCTATTTTCTTCTGGGTTGCTGATCCAGTTACTAGAAAATTGTATAAGGAAACTCTGCATGCTGGTTTGCCTGTTAAATCTGGTGAAAAGATTATTGCTAACCAATGGATTAGAGCTGTTAAA（SEQ ID NO:1）。
upstream and downstream amplification primers:
upstream 5'-3': CCGGAATTCCACCATCATCATCATCACGTGAGCAAGGGCG (SEQ ID NO: 2);
downstream 5'-3': GCAAATGGCATTCTGACATCC (SEQ ID NO: 3);
and (3) connecting a primer: TTGCGGCCGCTTAGCTTTGAAAATACAGATTTTCCTTGTACAGCTCGTC (SEQ ID NO: 4);
cleavage site: ecoRI (GAATTC), notI (GCGGCCGC).
The whole gene synthesis was performed in two steps of PCR. In the first step, 50. Mu.L of the system was added with 0.5-1 pmol/oligo and DNA polymerase, and 15 cycles were performed at about 65℃to obtain a full-length fragment. In the second round, the target gene was obtained by adding 20pmol of full-length primer and high-fidelity DNA polymerase for 20-25 cycles using 1. Mu. LPCR product as a template. Finally, the gene of interest and the plasmid after cleavage were ligated with T4-DNA ligase (16 ℃ overnight).
1.2. Plasmid cloning
Plasmid pPIC9k is a shuttle plasmid, which is treated with CaCl 2 Transferring to Escherichia coli by heat shock method, culturing and cloning, crushing thallus after 24 hr to extract plasmid, and performing enzyme cutting SDS electrophoresis or colony PCR detection. Obtaining a large amount of plasmids containing the target.
1.3. Construction of plasmid containing III-type triple helix collagen Gene
Plasmid pPICZ alpha A was selected to construct a plasmid containing the optimized triple helix collagen III gene and a 6X his tag (C-terminus) (shown in SEQ ID NO: 2), and the optimized triple helix collagen III gene and 6X his tag (C-terminus) were inserted (shown in FIG. 2). The target gene fragment was obtained by total gene synthesis, and PCR was performed using primers containing cleavage sites. About 65 ℃,15 cycles were performed to obtain full length fragments.
Optimized three-helix collagen gene sequence (containing 6×his tag):
GCTGGTAACACTGGTGCTCCTGGTTCTCCAGGTGTTTCTGGTCCAAAGGGTGACGCTGGTCAACCAGGTGAAAAAGGTTCTCCAGGAGCCCAAGGTCCACCAGGTGCTCCAGGTCCATTGGGTATTGCTGGTATTACTGGTGCTAGAGGTTTGGCTGGTCCTCCAGGTATGCCTGGTCCTAGAGGTTCTCCAGGTCCACAAGGTGTTAAGGGTGAATCTGGTAAACCAGGTGCTAACGGTTTGTCTGGTGAAAGAGGTCCTCCAGGACCACAAGGTTTGCCTGGTTTGGCTGGAACTGCTGGTGAACCAGGTAGAGATGGTAACCCAGGTTCTGATGGTTTGCCTGGAAGAGATGGTTCTCCTGGTGGTAAAGGTGACAGAGGTGAAAACGGTTCTCCAGGTGCTCCTGGAGCCCCTGGTCATCCTGGTCCTCCAGGTCCAGTTGGTCCAGCTGGTAAATCTGGTGACAGAGGAGAATCTGGTCCAGCTGGACCTGCTGGTGCTCCTGGTCCAGCTGGTTCTCGTGGTGCTCCAGGACCTCAAGGTCCTAGAGGAGATAAAGGTGAAACTGGTGAAAGAGGAGCTGCTGGTATTAAGGGTCATAGAGGTTTTCCAGGTAACCCTGGTGCTCCTGGTTCCCCTGGTCCAGCCGGTCAACAAGGTGCTATTGGTTCTCCAGGTCCTGCTGGTCCTAGAGGTCCTGTTGGTCCATCTGGTCCACCAGGAAAGGATGGTACTTCTGGTCATCCTGGACCTATTGGTCCACCAGGTCCTAGAGGTAACAGAGGTGAAAGAGGTTCTGAAGGTTCTCCAGGTCATCCAGGTCAACCTGGTCCTCCTGGTCCACCTGGTGCTCCAGGTCCTTGTTGTGGTGGTGTTGGTGCTGCTGCTATTGCTGGTATCGGTGGTGAAAAGGCTGGTGGTTTCGCTCCTTATTACGGTGACGAACCAATGGATTTTAAGATTAACACTGATGAAATCATGACCTCTTTGAAATCTGTTAATGGTCAAATTGAGTCTTTGATTTCTCCTGATGGTTCTCGTAAAAATCCAGCTAGAAACTGTAGAGATTTGAAATTTTGTCACCCAGAATTGAAATCTGGTGAATATTGGGTTGATCCAAATCAAGGTTGTAAATTGGATGCTATTAAGGTTTTCTGTAACATGGAAACTGGTGAGACTTGTATTTCTGCTAACCCATTGAATGTTCCTAGAAAACATTGGTGGACTGATTCTTCTGCTGAAAAGAAACATGTTTGGTTTGGTGAATCTATGGATGGTGGTTTCCAATTCTCTTATGGTAACCCTGAATTGCCAGAAGATGTTTTGGATGTTCATTTGGCTTTCTTGAGATTGTTGTCTTCTCGTGCTTCTCAAAATATTACTTATCATTGTAAGAACTCCATCGCTTATATGGATCAAGCTTCTGGTAACGTTAAGAAGGCTTTGAAGTTGATGGGTTCTAACGAAGGTGAGTTTAAAGCTGAAGGTAATTCTAAATTCACCTATACTGTTTTGGAGGATGGTTGTACTAAGCATACTGGTGAATGGTCTAAGACTGTTTTCGAATACAGAACTAGAAAGGCTGTTAGATTGCCTATTGTTGATATTGCTCCATATGATATTGGTGGTCCAGATCAAGAATTTGGTGTTGATGTTGGTCCAGTTTGTTTTCTTCATCATCATCATCACCATTAA（SEQ ID NO:5）。
upstream and downstream amplification primers:
upstream: TACTATTGCCAGCATTGCTGC (SEQ ID NO: 6);
downstream: GCAAATGGCATTCTGACATCC (SEQ ID NO: 7);
cleavage site: ecoRI (GAATTC), notI (GCGGCCGC).
Finally, the gene of interest was ligated to the plasmid using T4-DNA ligase (16 ℃ C., overnight).
1.4. Plasmid cloning
Obtaining a large number of pPICZ alpha A plasmids containing target genes, inserting the target genes into plasmids of escherichia coli, performing cloning culture, crushing thalli to extract plasmids, and performing enzyme digestion electrophoresis on plasmids to recover the target genes. Then the target gene is linked to the digested plasmid. The plasmids were selected for sequencing enzyme digestion electrophoresis validation as shown in FIG. 3.
2. Bacteria transfer
And (5) preparing competent pichia pastoris. Taking 100 mu L of yeast liquid, adding YPD liquid culture medium, and culturing overnight until OD value is 1.3-1.5. After the supernatant was discarded by two centrifugation and resuspended in sterile water, the supernatant was discarded by two centrifugation and resuspended in sorbitol in ice bath 1M.
After verification, the plasmid containing the hydroxylase gene was linearized using SalI restriction enzyme, and then shocked to yeast at 1700V for 8mS for 2 times. Positive clonal selection was performed and cultured at 30 ℃ for 3 days. Selecting yeast producing fluorescence. Then, competent pichia pastoris is prepared again, linearized plasmids containing the humanized collagen gene are subjected to electrotransformation and then are screened, mixed solutions with different amounts are coated on a 100ug/ml Zeocin antibiotic YPD plate for 30 ℃ for 48 hours, after colonies grow out on the plate, single bacteria growing on the plate are picked up by an inoculating loop, and the single bacteria are inoculated into a test tube filled with 10ml YPD liquid medium (the concentration of the antibiotic Zeocin is 100 ug/ml), and cultured at 30 ℃ and 180rpm overnight.
3. Culture of small test
Positive strains (10 strains) were subjected to pilot culture, and were first inoculated into test tubes containing 10ml of YPD, respectively, and after 24 hours, 500uL was inoculated into 50ml of YPD liquid medium. Zeocin concentration was 200ug/mL,30 ℃. Samples were taken from the medium for 48h and the supernatant was collected by centrifugation. The supernatant was detected using the corresponding reagent, carrying a 6 XHis tag protein, and the Dot-Bot of the corresponding anti-His antibody was selected. And (3) analyzing results, selecting a strain with the strongest positive signal, and performing amplification culture by using the strain.
4. Fermentation purification
Shake flask induction expression of 1L was performed, inoculated into BMGY liquid medium containing bleomycin, shake-cultured for 24 hours, and then added into BMMY liquid medium containing 1% methanol for induction expression for 72 hours. The supernatant was collected.
His-tag fusion proteins were purified by Ni column affinity, by low pressure chromatography, and supernatant was loaded onto a Ni-IDA-Besps 6FF affinity column pre-equilibrated with binding buffer (50 mM NaH2PO4, 10 mM imidazole, 0.3M NaCl, pH 8.0) at a flow rate of 0.5 mL/min. Then washed with binding buffer at a flow rate of 0.5 mL/min until the effluent OD280 reached baseline. Then washed with buffer (50 mM NaH) 2 PO 4 20. 20 mM imidazole, 0.3M NaCl, pH 8.0) was flushed at a flow rate of 1 mL/min until the effluent OD280 reached baseline. Finally, the elution buffer (50 mM NaH) 2 PO 4 250. 250 mM imidazole, 0.3M NaCl, pH 8.0) was eluted at a flow rate of 1 mL/min, and the effluent was collected. The collected protein solution was added to a dialysis bag and dialyzed overnight using binding buffer. The recovery rate is more than 70%, the purity is more than 95%, and the water solubility is good, as shown in figure 5. And freeze-drying the collagen obtained by dialysis (-80 ℃ for 24 hours) to obtain collagen powder.
5. Verification
5.1. SDS-PAGE electrophoresis: molecular weight of the protein of interest.
10% SDS polyacrylamide gel is selected, three groups of 10 mul recombinant protein solutions are loaded, electrophoresis is started, gel concentration is 100V,15-20min, gel separation is 200V, and 50-50min.
Immunoblotting: and verifying that the recombinant protein is III type triple helix collagen.
Transferring the gel after electrophoresis to a nitrocellulose membrane soaked in advance, adding ice cubes for 60V2h, and dyeing for later use after transferring the membrane. Put into TBS to be blocked for 1h. After incubation of the primary antibody diluted with PBST for 1.5h at room temperature, PBST was washed 4 times for 10min each. And (3) performing color development treatment after the secondary antibody step. When the color development was clearly visible, the color was washed with distilled water 3 times for 10 minutes each. The results are shown in FIG. 4, where a distinct band can be seen at about 55 kDa.
5.2. Coating a cell culture plate: protein biological function and cytotoxicity.
Human skin fibroblasts were seeded and cultured at 37 ℃ for 3 to 7 days, using cell plates without recombinant protein coating as controls. Proliferation rate was calculated by observing and measuring its light absorbance at 450nm using CCK8 kit and microplate reader.
The absorption value of the experimental group/the absorption value of the control group is multiplied by 100%, and the results are shown in the following table, so that the cell proliferation rates of the groups with different concentrations are all more than 100%, the cytotoxicity is proved to be 0, and the skin cell proliferation can be promoted.
TABLE 1 light absorbance and cell proliferation Rate for different concentration groups
The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A construction method of recombinant human III type triple helix collagen engineering bacteria is characterized in that: the method comprises the following steps:
s1, inserting the optimized proline hydroxylase gene into a shuttle expression vector pPIC9k plasmid to obtain a recombinant vector A; the nucleotide sequence of the optimized proline hydroxylase gene is shown as SEQ ID NO:1 is shown in the specification;
s2, inserting the optimized human source III type triple helix collagen alpha 1 chain gene fragment into a pPICZ alpha A plasmid to obtain a recombinant vector B;
s3, converting the recombinant vector A into a host strain Pichia pastoris, screening, converting the recombinant vector B into the host strain Pichia pastoris, and screening to obtain the recombinant human source III type triple helix collagen engineering bacteria.
2. The method for constructing recombinant human-derived type III triple helix collagen engineering bacteria according to claim 1, wherein the method comprises the following steps: the nucleotide sequence of the optimized human source III type triple helix collagen alpha 1 chain gene fragment is shown as SEQ ID NO: shown at 5.
3. The method for constructing recombinant human-derived type III triple helix collagen engineering bacteria according to claim 1, wherein the method comprises the following steps: the step S1 includes the steps of:
s11, synthesizing a DNA fragment 1 of the proline hydroxylase of the micrococcus virus containing the fluorescent EGFP label;
s12, designing an amplification primer, and amplifying the DNA fragment A to obtain an amplified fragment with EcoRI and NotI restriction sites at two ends;
s13, performing enzyme digestion on the amplified fragment and the pPIC9k plasmid by using EcoRI and NotI restriction enzymes;
s14, connecting the amplified fragment and the pPIC9k plasmid by using ligase, and culturing overnight to obtain a recombinant vector A containing a proline hydroxylase gene;
s15, transforming the recombinant vector A into escherichia coli for plasmid cloning to obtain a large number of target recombinant vectors A.
4. The method for constructing recombinant human-derived type III triple helix collagen engineering bacteria according to claim 1, wherein the method comprises the following steps: the step S2 includes the steps of:
s21, synthesizing a DNA fragment sequence B containing an alpha 1 chain of the human-derived III type triple helix collagen;
s22, designing an amplification primer containing a His affinity tag, and amplifying a DNA fragment sequence B to obtain an amplification fragment with EcoRI and NotI enzyme cutting sites at two ends;
s23, performing enzyme digestion on the amplified fragment and the pPICZ alpha A plasmid by using EcoRI and NotI restriction enzymes;
s24, connecting the amplified fragment and the pPICZ alpha A plasmid by using ligase to obtain the recombinant vector B containing the human III type triple helix collagen gene.
5. The method for constructing recombinant human-derived type III triple helix collagen engineering bacteria according to claim 1, wherein the method comprises the following steps: step S3 includes the steps of:
preparing competent host strain Pichia pastoris, linearizing recombinant vector A containing proline hydroxylase gene by using restriction enzyme, then performing electric shock transformation to host strain Pichia pastoris, screening positive cloned seeds, and culturing and selecting engineering bacteria producing fluorescence.
6. The method for constructing recombinant human-derived type III triple helix collagen engineering bacteria according to claim 1, wherein the method comprises the following steps: step S4 includes the steps of:
selecting fluorescent engineering bacteria, preparing competent host strain Pichia pastoris, electrically transferring the linearized recombinant vector B containing the human III type triple helix collagen gene to the host strain Pichia pastoris, and screening to obtain the recombinant human III type triple helix collagen engineering bacteria.
7. The method for constructing recombinant human-derived triple helix collagen engineering bacteria according to claim 5, wherein the method comprises the following steps: the host strain pichia pastoris is GS115 pichia pastoris.
8. A recombinant human triple helix collagen engineering bacterium of type iii obtained by the construction method of any one of claims 1 to 7.
9. Use of the recombinant human triple helix collagen III engineering bacteria according to claim 8 in preparing human triple helix collagen III.
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| CN117430687A (en) * | 2023-10-24 | 2024-01-23 | 苏州猫尔科技有限公司 | Recombinant keratin with triple-helix structure, and preparation method and application thereof |
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| CN118271425B (en) * | 2024-03-29 | 2024-10-18 | 湖南丽赛药业有限公司 | Recombinant III type humanized collagen for wound repair and application thereof |
| CN118185979A (en) * | 2024-05-16 | 2024-06-14 | 山东美瑞生物技术有限公司 | Method for improving hydroxylation rate of recombinant human collagen |
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