CN112980865A - Construction method of recombinant human-like collagen engineering bacteria - Google Patents

Construction method of recombinant human-like collagen engineering bacteria Download PDF

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CN112980865A
CN112980865A CN202110299465.1A CN202110299465A CN112980865A CN 112980865 A CN112980865 A CN 112980865A CN 202110299465 A CN202110299465 A CN 202110299465A CN 112980865 A CN112980865 A CN 112980865A
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collagen
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张敏
景成宇
康俪义
刘丽霞
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Kefu Medical Technology Co ltd
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Abstract

The invention discloses a construction method of recombinant human-like collagen engineering bacteria, which specifically comprises the following steps: the design of the gene sequence of the recombinant human-like collagen specifically comprises the following steps: based on the amino acid sequence of the selected human-like collagen III; designing a gene sequence for coding the recombinant human-like collagen according to the preference codon of the escherichia coli; connecting the recombinant human-like collagen fragment with the enzyme digestion of the carrier to obtain an enzyme digestion product; carrying out electrophoresis on the enzyme digestion product by using agarose gel to obtain a target band; cutting the target strip into gel, purifying and recovering to obtain a human-like collagen fragment; connecting the human-like collagen fragment with a carrier to obtain a human-like collagen fragment connection product; and thermally converting the human-like collagen fragment connecting product into escherichia coli competent cells to obtain the recombinant human-like collagen engineering bacteria. The recombinant human-like collagen obtained by the method has the effect of inducing proliferation of human skin fibroblasts and human skin keratinocytes.

Description

Construction method of recombinant human-like collagen engineering bacteria
Technical Field
The invention relates to the technical field of biology, in particular to a construction method of a recombinant human-like collagen engineering bacterium.
Background
Collagen is an extremely important structural protein in connective tissues and plays an important role in maintaining normal physiological functions of cells, tissues and organs. Collagen is widely used in medical materials and pharmaceuticals, cosmetics, health products, and various industrial applications. However, animal-derived collagen prepared by the traditional method has certain virus hidden troubles such as mad cow disease, foot and mouth disease, swine plague and the like, and particularly easily causes xenogeneic rejection reaction when being applied to a human body, thereby limiting the application of the collagen in the aspect of medicine.
Human beings adopt genetic engineering means to produce recombinant collagen, and the production method has the advantages that the traditional extraction method is difficult to compare with: 1) the method has the advantages of safety and controllability, compared with the difficulty in tracking and uncertainty of raw material sources in the traditional collagen preparation method, the raw material components for producing the recombinant human-like collagen by using the genetic engineering technology are definite, the risk of virus infection is avoided, 2) the quality is controllable, the human-like collagen fragments with specific molecular weights can be expressed by using the genetic engineering technology, the repeatability among batches is good, the extracted collagen is usually a mixed product of different types of collagen, the difference among the batches is large, the quality control is not facilitated, 3) the lower rejection reaction is realized, the human-like collagen is constructed on the basis of human collagen genes, and the immune rejection reaction is smaller than that of collagen from animal sources.
Disclosure of Invention
The invention designs a recombinant human-like collagen with a unique gene sequence (shown as SEQ ID No. 1), combines with enzyme digestion connection of a pET24a carrier to obtain an enzyme digestion product, obtains a human-like collagen fragment based on the enzyme digestion product, connects the human-like collagen fragment with the pET-24a carrier to obtain a PET 24-human-like collagen fragment connection product, and finally obtains the recombinant human-like collagen engineering bacteria based on the PET 24-human-like collagen fragment connection product. The purified recombinant collagen obtained by culturing, protein induction expressing and purifying the recombinant human-like collagen engineering bacteria obtained by the method is simple in purification process and easy to control parameters, and the obtained purified recombinant collagen has a certain proliferation effect on human epidermal cells and human skin fibroblast primary cells within 72 hours and is high in biological activity. The specific technical scheme is as follows:
a construction method of recombinant human-like collagen engineering bacteria comprises the following steps:
the design of the gene sequence of the recombinant human-like collagen specifically comprises the following steps: based on the amino acid sequence of the selected human-like collagen III; designing a gene sequence for coding the recombinant human-like collagen according to the preference codon of the escherichia coli, and introducing a histidine purification tag into the 3' end of the sequence;
and (2) carrying out enzyme digestion connection on the recombinant human-like collagen fragment and a pET24a vector to obtain an enzyme digestion product, wherein: the gene sequence of the recombinant human-like collagen is shown as SEQ ID No. 1;
carrying out electrophoresis on the enzyme digestion product by using agarose gel to obtain a target band;
cutting the target strip into gel, purifying and recovering to obtain a human-like collagen fragment;
measuring the content of the human-like collagen fragment, and connecting the human-like collagen fragment with a pET-24a carrier to obtain a PET 24-human-like collagen fragment connection product;
and thermally transforming the PET 24-human-like collagen fragment connecting product into an escherichia coli competent cell BL21(DE3) to obtain the recombinant human-like collagen engineering bacterium.
Preferably, the amino acid sequence of the recombinant human-like collagen is shown in SEQ ID No. 2.
Preferably, the enzyme digestion reaction system for enzyme digestion and connection of the recombinant human-like collagen fragment and the pET24a vector is as follows:
plasmid containing human-like collagen fragment or pET24a plasmid: 10 mu l of the mixture;
10×Fastdigest buffer：10μl；
NdeI：5μl；
XhoI：5μl；
H2O：70μl；
37℃，30min。
preferably, the cleavage products are electrophoresed on a 1% agarose gel to obtain the desired band.
Preferably, the human-like collagen fragment is connected with the pET-24a vector, and the connection reaction system is as follows:
human-like collagen fragments: 4 mu l of the solution;
pET-24a vector: 1 mul;
T4 DNA Ligase：1μl；
10×T4 DNA Ligase buffer：1μl；
water: 3 mu l of the solution;
25℃，30min。
preferably, the method also comprises the steps of fermentation culture of escherichia coli, protein induction expression and purification, and specifically comprises the following steps:
and (3) fermentation culture of escherichia coli: inoculating the engineering cell strain into a centrifuge tube containing Kan LB culture solution, and culturing overnight; inoculating the strain cultured overnight into LB culture solution containing Kan, and shaking until thallus OD600 is 0.2-0.5;
protein induction expression: adding IPTG into the rest culture, shaking for 3-5h, and inducing protein expression;
protein purification: inoculating the overnight cultured bacterial liquid into a fresh LB culture medium containing Kan, culturing at 35-40 ℃, and shaking until the OD600 of the thallus is 0.6-0.8; adding IPTG; inducing expression at 35-40 deg.c for 3-5 hr; taking out the culture, centrifuging at room temperature, and discarding the supernatant; resuspending the thallus precipitate with Native Binding-Buffer, ultrasonically crushing, centrifuging at 3-8 ℃, and taking the supernatant; adding the supernatant into a Binding-Buffer pre-balanced Ni affinity chromatographic column, and uniformly rotating on a mute mixer; collecting the flow-through liquid; and eluting by using Native Wash Buffer to obtain the purified recombinant collagen.
Preferably, the fermentation culture of escherichia coli is specifically: inoculating the engineering cell strain into a centrifuge tube containing 3ml LB culture solution of 50 mu g/ml Kan, and culturing overnight at 37 ℃ and 220 rpm; the overnight cultured strains were mixed as 1: 100 was inoculated into 10ml of LB medium with 50. mu.g/ml Kan, and shaken at 37 ℃ and 220rpm until the OD600 of the cells became 0.4, the shaking time was 1.8 to 2.5 hours.
Preferably, the protein induces expression: adding IPTG into the rest culture until the final concentration is 0.5-0.8mmol/L, shaking at 35-40 deg.C and 220rpm for 3-6h, and inducing protein expression; the cells were collected by centrifugation, resuspended in 3ml Tris buffer, disrupted by sonication, and the supernatant and pellet were collected and subjected to SDS-PAGE.
Preferably, protein purification: the overnight culture broth was diluted with 1: 100 is transferred into fresh 1L LB culture medium containing 50 mug/ml Kan, mass culture is carried out at 37 ℃, OD600 is 0.6-0.8, shaking time is 1.8-2.2h, IPTG is supplemented until final concentration is 1mM, and induced expression is carried out for 4h at 37 ℃; taking out the culture, centrifuging at 12000g room temperature for 2min, and removing the supernatant; resuspending 1L of culture thallus precipitate induced and expressed by a nickel ion affinity chromatography column Ni-NTA Agarose with 30ml of Native Binding-Buffer, ultrasonically crushing, centrifuging 12000g at 4 ℃ for 20min, and taking supernatant; adding 10mL of the supernatant into a Binding-Buffer pre-balanced Ni affinity chromatographic column, and uniformly rotating on a mute mixer for 15 min; collecting the flow-through liquid; washing with 8mL Native Wash Buffer at flow rate of 0.5 mL/min; the column was washed with 20ml of Native Wash Buffer at a flow rate of 1 ml/min; the target protein was eluted at a flow rate of 1ml/min using 2ml of Native Elution-Buffer, 4ml of Native Elution-Buffer and 8ml of Native Elution-Buffer to obtain purified recombinant collagen.
Drawings
FIG. 1 is a schematic diagram showing the restriction enzyme digestion result of the expression vector detected by agarose gel electrophoresis in example 1;
FIG. 2 is a graph showing the results of induced expression of the protein in example 1;
FIG. 3 is a schematic diagram of the purified recombinant collagen of example 1;
FIG. 4 shows the effect of the recombinant collagen purified in example 1 on human keratinocyte HACAT for 48 hours;
FIG. 5 shows the effect of the recombinant collagen purified in example 1 on human keratinocyte HACAT for 72 hours;
FIG. 6 shows the effect of the purified recombinant collagen of example 1 on human skin fibroblasts primary cells (fibroplast) for 48 hours;
FIG. 7 shows the effect of the purified recombinant collagen of example 1 on human skin fibroblasts primary cells (fibroplast) for 72 hours;
FIG. 8 is a graph showing the effect of BSS protein in human keratinocyte HACAT in comparative example 1 at 48 hours;
FIG. 9 shows the effect of BSS protein in human keratinocyte HACAT for 72 hours in comparative example 1;
FIG. 10 shows the effect of BSS protein in comparative example 1 for 48 hours on human dermal fibroblast (fibroplast);
FIG. 11 shows the effect of BSS protein in comparative example 1 on human dermal fibroblast (fibroplast) cells for 72 hours.
Detailed Description
The embodiments of the present invention will be described in detail with reference to the accompanying drawings so that the advantages and features of the invention can be more easily understood by those skilled in the art, and the scope of the invention will be clearly and clearly defined.
Example 1:
a construction method of recombinant human-like collagen engineering bacteria comprises the following steps:
1. carrying out enzyme digestion connection on the recombinant human-like collagen gene fragment and a pET24a vector to obtain an enzyme digestion product, wherein: the gene sequence of the recombinant human collagen is shown as SEQ ID No.1, and specifically comprises the following steps:
ggtgaacgtggtgacctgggtccgcagggtattgcgggtcaacgtggtgtggttggtgaacgtggtgaacgtggcgagcgtggtgcgagcggtgaacgtggtgatctgggcccgcaaggcatcgcgggccagcgtggtgtggttggcgaacgtggtgagcgtggcgaacgtggtgcgagcggcgaacgtggtgacctgggcccgcaaggcattgctggccagcgtggtgtggtgggtgaacgtggtgaacgtggcgagcgtggtgcgtctggcgagcgtggtgatctgggtccgcaaggcatcgcgggtcagcgtggtgtggtgggcgagcgtggtgagcgtggcgaacgtggtgcgtctggcgaacgtggtgacttaggcccgcaaggcattgcgggccaacgtggtgtggtgggtgaacgtggtgaacgtggcgagcgtggtgcgagtggcgaacgtggtgatttaggcccgcaaggtatcgctggccaacgtggtgtggttggagagcgtggtgagcgtggcgaacgtggtgcgagtggcgaacgtggtgaccttggcccgcaaggtattgccggccagcgtggtgtggtcggtgaacgtggtgaacgtggcgagcgtggtgcgagtggggaacgtggtgatttaggaccgcaaggtattgctggacagcgtggtgtggttggtgagcgtggtgagcgtggtgaacgtggtgcgagcggtccgccgggtccgtgctgcggtggcggt。
the amino acid sequence is shown as SEQ ID No. 2:
GERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGERGDLGPQGIAGQRGVVGERGERGERGASGPPGPCCGGG。
the recombinant human-like collagen fragment is connected with pET24a carrier by enzyme digestion to obtain the enzyme digestion product, the enzyme digestion reaction system is as follows: plasmid containing human-like collagen fragment or pET24a plasmid: 10 mu l of the mixture;
10×Fastdigest buffer：10μl；
NdeI：5μl；
XhoI：5μl；
H2O：70μl；
37℃，30min。
2. the cleavage products were electrophoresed on 1% agarose gel to obtain the desired band, as shown in FIG. 1, wherein: m is DS5000marker (Dongsheng organism), wherein a size band of about 5000bp is a PET24 carrier band, and a size band of about 1000bp is a human-like collagen gene band.
3. And cutting the target band into gel, and purifying and recovering to obtain the human-like collagen fragment.
4. Measuring the content of the human-like collagen fragment, and connecting the human-like collagen fragment with a pET-24a carrier to obtain a PET 24-human-like collagen fragment connection product, wherein the connection reaction system is as follows:
human-like collagen fragments: 4 mu l of the solution;
pET-24a vector: 1 mul;
T4 DNA Ligase：1μl；
10×T4 DNA Ligase buffer：1μl；
water: 3 mu l of the solution;
25℃，30min。
5. and thermally transforming the PET 24-human-like collagen fragment connecting product into an escherichia coli competent cell BL21(DE3) to obtain the recombinant human-like collagen engineering bacterium.
10 single colonies were picked from the transformed plate, inoculated into 1ml of 50ug/ml Kan-LB liquid medium, cultured overnight at 37 ℃; randomly selecting 6 strains for plasmid extraction, numbering 1-6; the plasmid is subjected to enzyme digestion identification by NdeI and XhoI, wherein the enzyme digestion identification system comprises the following steps: plasmid: 3 μ g (about 3 μ l);
NdeI：0.5μl XhoI：
0.5μl H2O：6μl；
detecting the enzyme digestion result by adopting 0.8 percent agarose gel electrophoresis, and preliminarily identifying a positive result; and (4) sequencing the plasmid with the positive result detected by enzyme digestion. And if the sequencing result is in accordance with expectation, determining that the engineering cell strain is successfully constructed.
Fermentation culture, protein induction expression and purification of escherichia coli, and specifically comprises the following steps: selecting a single colony of the engineering bacteria, placing the single colony in an LB culture medium for activation culture, transferring the single colony to at least a fermentation culture medium, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, centrifugally collecting the bacteria, and respectively carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis on a supernatant and a cell precipitate to detect the expression condition of the collagen. After confirming that the protein is successfully expressed, culturing the engineering bacteria in a shake flask to induce the protein to express, breaking cells by ultrasonic wave, centrifuging to take supernatant, and purifying the supernatant by a nickel ion affinity chromatography column. The specific operation of this embodiment is as follows:
1. and (3) fermentation culture of escherichia coli: inoculating the engineering cell strain into a centrifuge tube containing 3ml LB culture solution of 50 mu g/ml Kan, and culturing overnight at 37 ℃ and 220 rpm; the overnight cultured strains were mixed as 1: 100 was inoculated into 10ml of LB medium with 50. mu.g/ml Kan, and shaken at 37 ℃ and 220rpm until the OD600 of the cells became 0.4, the shaking time was 1.8 to 2.5 hours.
2. Protein induction expression: protein expression was induced by adding IPTG to the remaining culture to a final concentration of 0.5mmol/L, shaking at 37 ℃ and 220rpm for 4h, as detailed in FIG. 2, FIG. 2: m is protein ladder, #26616, lane 1 is the supernatant before induction, lane 2 is the supernatant after induction, lane 3 is the precipitate after induction, and lanes 2 and 3 after induction express human-like collagen of the expected size of 35kDa compared with lane 1; the cells were collected by centrifugation, resuspended in 3ml Tris buffer, disrupted by sonication, and the supernatant and pellet were collected and subjected to SDS-PAGE.
3. Protein purification:
3.1, mixing the overnight culture broth with a ratio of 1: 100 is transferred into fresh 1L LB culture medium containing 50 mug/ml Kan, mass culture is carried out at 37 ℃, OD600 is 0.6, shaking time is 2.0h, IPTG is supplemented until final concentration is 1mM, and induced expression is carried out for 4h at 37 ℃;
3.2, taking out the culture, centrifuging at 12000g at room temperature for 2min, and removing the supernatant;
3.3, resuspending 1L of the culture thallus precipitate induced to express by a Ni-NTAAgarose (thermal fisher, K85001) through a nickel ion affinity chromatography column by using 30ml of Native Binding-Buffer, then carrying out ultrasonication (the power is 200W, the work time is 4sec, the pause time is 8sec, and 20min is totally spent), taking 12000g at the temperature of 4 ℃, centrifuging for 20min, and taking the supernatant;
3.4, adding 10mL of the supernatant into a Binding-Buffer pre-balanced Ni affinity chromatographic column, and uniformly rotating on a mute mixer for 15 min;
3.5, collecting flow-through liquid;
3.6, repeating the steps 3.4-3.5 to complete the purification of all the supernatant;
3.7, washing with 8mL Native Wash Buffer at the flow rate of 0.5 mL/min;
3.8, washing the column with 20ml of Native Wash Buffer at a flow rate of 1 ml/min;
3.9, sequentially eluting the target protein with 2ml of Native Elution-Buffer (imidazole concentration 20mM), 4ml of Native Elution-Buffer (imidazole concentration 100mM) and 8ml of Native Elution-Buffer (imidazole concentration 200mM) at a flow rate of 1ml/min, collecting the flow-through solution, 2 ml/tube;
3.10, sucking 12. mu.l from each tube, adding 3. mu.l of 5 × loading buffer, and performing 12% SDS-PAGE analysis to obtain purified recombinant collagen (calculated as COL protein (Col-2)), as shown in FIG. 3, wherein M is protein ladder (thermal fisher, #26616), lane 1 is purified recombinant human-like collagen, and as can be seen from FIG. 3, the recombinant human-like collagen with the purity of 85% is successfully obtained.
The purified recombinant collagen obtained in this example is induced to culture human skin Fibroblast (called Fibroblast, i.e., Fibroblast) and human epidermal cell (HACAT, i.e., human immortalized keratinocyte), and the CCK8 method is adopted to detect the proliferation effect of the recombinant collagen on human epidermal cell and human skin Fibroblast, specifically:
human skin fibroblast primary cell culture conditions: OPTI-MEM Medium, 15% serum, Triantibody 1%, 37 ℃, 5% CO2。
Human skin Fibroblast primary cells (fibroplast) and human epidermal cells (HACAT) were plated and seeded at 2X 103The cells were cultured in 96-well plates, 100ul per well, for 24 hours, and 5 96-well plates were plated each.
Recombinant collagen (i.e., COL protein) was diluted to final concentrations of 0 (negative control), 75ug/ml, 150ug/ml, 300ug/ml, 600ug/ml and 1200ug/ml with DMEM + 5% FBS (HACAT) and OPTI-MEM + 5% FBS (fibriplast).
The medium was discarded, and recombinant collagen (COL protein) and negative control (100 ul) were added to each concentration group for 48h and 72h, with 5 duplicate wells per group.
The reading is determined by CCK8, specifically according to the instruction of CCK8 kit, and is shown in figures 4-7, and can be seen from figures 4-7: FIG. 4 shows that the recombinant collagen of the present invention (Col-2) did not act on human epidermal cells (HACAT) at 48 hours; FIG. 5 shows that the recombinant collagen (Col-2) of the present invention acts on human epidermal cells (HACAT) at 72 hours; FIG. 6 shows that recombinant collagen of the invention (Col-2) acts on human skin Fibroblast primary cells (fibroplast) at 48 hours; FIG. 7 shows that the recombinant collagen of the invention (Col-2) acts on human skin Fibroblast primary cells (fibroplast) at 72 hours.
The purified recombinant collagen (i.e., COL protein) obtained in the embodiment acts on primary fibroblasts (fibroplasts) of human skin for 48 hours, and has the effect of remarkably promoting cell proliferation; has certain proliferation promoting effect after 72 hours of action.
The purified recombinant collagen (i.e., COL protein) obtained in this example acted on human epidermal HACAT cells for 72 hours, and had a certain proliferation-promoting effect.
As can be seen, the purified recombinant collagen (i.e., COL protein) obtained by the method of this example has excellent activity.
Comparative example 1:
BSS protein is purchased on the market, purchased from Jiangsu Jiangshan gathering source biotechnology limited company, the BSS protein is induced and cultured to human skin Fibroblast primary cell (named as Fibroplast) and human epidermal cell (HACAT), the BSS protein is diluted by DMEM + 5% FBS (HACAT) and OPTI-MEM + 5% FBS (fibroplast) to five concentrations of 0 (negative control), 75ug/ml, 150ug/ml, 300ug/ml and 600ug/ml, the result is detailed in figures 8-11, and specifically, the BSS protein comprises the following components:
FIG. 8 shows that BSS protein does not act on human epidermal cells (HACAT) at 48 hours; FIG. 9 shows that BSS protein does not act on human epidermal cells (HACAT) at 72 hours; FIG. 10 shows that BSS protein acts on human skin Fibroblast primary cells (fibroplast) at 48 hours; figure 11 shows that BSS protein acts on human skin fibroblasts (fibroplast) at 72 hours.
The principle of collagen in promoting cell proliferation is complex and can be mainly divided into physical support effect and specificity effect: the physical support effect means that the collagen can form a specific three-dimensional structure under physiological conditions and further self-assemble into fibers, and the fiber structure can play a role in supporting cell adhesion growth, protecting cells from physical damage and the like and promoting cell proliferation; the specific action means that cells such as epithelial cells, endothelial cells, macrophages and the like interact with collagen in the process of being retained in or on the collagen matrix, and directly or indirectly activate certain cell membrane receptors, so that the cell functions are regulated. Combining the cell assay data of example 1 (FIGS. 4-7) and comparative example 1 (FIGS. 8-11) shows: the commercial collagen BSS only acts on human skin Fibroblast primary cells (fibroplast), but both the human skin Fibroblast and the human skin keratinocyte obtained by the method have the effect of inducing proliferation, and the proliferation effect of the collagen on the human skin Fibroblast primary cells (fibroplast) is obviously better than that of the commercial collagen BSS in the prior art.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Kefu medical science and technology GmbH
<120> construction method of recombinant human-like collagen engineering bacteria
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 750
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ggtgaacgtg gtgacctggg tccgcagggt attgcgggtc aacgtggtgt ggttggtgaa 60
cgtggtgaac gtggcgagcg tggtgcgagc ggtgaacgtg gtgatctggg cccgcaaggc 120
atcgcgggcc agcgtggtgt ggttggcgaa cgtggtgagc gtggcgaacg tggtgcgagc 180
ggcgaacgtg gtgacctggg cccgcaaggc attgctggcc agcgtggtgt ggtgggtgaa 240
cgtggtgaac gtggcgagcg tggtgcgtct ggcgagcgtg gtgatctggg tccgcaaggc 300
atcgcgggtc agcgtggtgt ggtgggcgag cgtggtgagc gtggcgaacg tggtgcgtct 360
ggcgaacgtg gtgacttagg cccgcaaggc attgcgggcc aacgtggtgt ggtgggtgaa 420
cgtggtgaac gtggcgagcg tggtgcgagt ggcgaacgtg gtgatttagg cccgcaaggt 480
atcgctggcc aacgtggtgt ggttggagag cgtggtgagc gtggcgaacg tggtgcgagt 540
ggcgaacgtg gtgaccttgg cccgcaaggt attgccggcc agcgtggtgt ggtcggtgaa 600
cgtggtgaac gtggcgagcg tggtgcgagt ggggaacgtg gtgatttagg accgcaaggt 660
attgctggac agcgtggtgt ggttggtgag cgtggtgagc gtggtgaacg tggtgcgagc 720
ggtccgccgg gtccgtgctg cggtggcggt 750
<210> 2
<211> 250
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly
195 200 205
Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln
210 215 220
Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser
225 230 235 240
Gly Pro Pro Gly Pro Cys Cys Gly Gly Gly
245 250

Claims (9)

1. A construction method of recombinant human-like collagen engineering bacteria is characterized by comprising the following steps:
the design of the gene sequence of the recombinant human-like collagen specifically comprises the following steps: based on the amino acid sequence of the selected human-like collagen III; designing a gene sequence for coding the recombinant human-like collagen according to the preference codon of the escherichia coli, and introducing a histidine purification tag into the 3' end of the sequence;
and (2) carrying out enzyme digestion connection on the recombinant human-like collagen fragment and a pET24a vector to obtain an enzyme digestion product, wherein: the gene sequence of the recombinant human-like collagen is shown as SEQ ID No. 1;
carrying out electrophoresis on the enzyme digestion product by using agarose gel to obtain a target band;
cutting the target strip into gel, purifying and recovering to obtain a human-like collagen fragment;
measuring the content of the human-like collagen fragment, and connecting the human-like collagen fragment with a pET-24a carrier to obtain a PET 24-human-like collagen fragment connection product;
and thermally transforming the PET 24-human-like collagen fragment connecting product into an escherichia coli competent cell BL21(DE3) to obtain the recombinant human-like collagen engineering bacterium.
2. The method for constructing recombinant human-like collagen engineering bacteria according to claim 1, wherein the amino acid sequence of the recombinant human-like collagen is shown in SEQ ID No. 2.
3. The method for constructing the recombinant human-like collagen engineering bacteria according to claim 1, wherein the enzyme digestion reaction system for the enzyme digestion connection of the recombinant human-like collagen fragment and the pET24a vector is as follows:
plasmid containing human-like collagen fragment or pET24a plasmid: 10 mu l of the mixture;
10×Fastdigest buffer：10μl；
NdeI：5μl；
XhoI：5μl；
H2O：70μl；
37℃，30min。
4. the method for constructing recombinant human-like collagen engineering bacteria according to claim 1, wherein the enzyme-cleaved product is electrophoresed with 1% agarose gel to obtain a target band.
5. The method for constructing recombinant human-like collagen engineering bacteria according to claim 1, wherein the human-like collagen fragment is ligated to pET-24a vector in the following reaction system:
human-like collagen fragments: 4 mu l of the solution;
pET-24a vector: 1 mul;
T4 DNALigase：1μl；
10×T4 DNALigase buffer：1μl；
water: 3 mu l of the solution;
25℃，30min。
6. the method for constructing recombinant human-like collagen engineering bacteria according to any one of claims 1 to 5, further comprising fermentation culture, protein induction expression and purification of Escherichia coli, and specifically comprising the following steps:
and (3) fermentation culture of escherichia coli: inoculating the engineering cell strain into a centrifuge tube containing Kan LB culture solution, and culturing overnight; inoculating the strain cultured overnight into LB culture solution containing Kan, and shaking until thallus OD600 is 0.2-0.5;
protein induction expression: adding IPTG into the rest culture, shaking for 3-5h, and inducing protein expression;
protein purification: inoculating the overnight cultured bacterial liquid into a fresh LB culture medium containing Kan, culturing at 35-40 ℃, and shaking until the OD600 of the thallus is 0.6-0.8; adding IPTG; inducing expression at 35-40 deg.c for 3-5 hr; taking out the culture, centrifuging at room temperature, and discarding the supernatant; resuspending the thallus precipitate with Native Binding-Buffer, ultrasonically crushing, centrifuging at 3-8 ℃, and taking the supernatant; adding the supernatant into a Binding-Buffer pre-balanced Ni affinity chromatographic column, and uniformly rotating on a mute mixer; collecting the flow-through liquid; and eluting by using Native Wash Buffer to obtain the purified recombinant collagen.
7. The method for constructing recombinant human-like collagen engineering bacteria according to claim 6, wherein the fermentation culture of Escherichia coli is specifically: inoculating the engineering cell strain into a centrifuge tube containing 3ml LB culture solution of 50 mu g/ml Kan, and culturing overnight at 37 ℃ and 220 rpm; the overnight cultured strains were mixed as 1: 100 was inoculated into 10ml of LB medium with 50. mu.g/ml Kan, and shaken at 37 ℃ and 220rpm until the OD600 of the cells became 0.4, the shaking time was 1.8 to 2.5 hours.
8. The method for constructing recombinant human-like collagen engineering bacteria according to claim 7, wherein the protein is induced to express: adding IPTG into the rest culture until the final concentration is 0.5-0.8mmol/L, shaking at 35-40 deg.C and 220rpm for 3-6h, and inducing protein expression; the cells were collected by centrifugation, resuspended in 3ml Tris buffer, disrupted by sonication, and the supernatant and pellet were collected and subjected to SDS-PAGE.
9. The method for constructing recombinant human-like collagen engineering bacteria according to claim 8, wherein the protein purification comprises: the overnight culture broth was diluted with 1: 100 is transferred into fresh 1L LB culture medium containing 50 mug/ml Kan, mass culture is carried out at 37 ℃, OD600 is 0.6-0.8, shaking time is 1.8-2.2h, IPTG is supplemented until final concentration is 1mM, and induced expression is carried out for 4h at 37 ℃; taking out the culture, centrifuging at 12000g room temperature for 2min, and removing the supernatant; resuspending 1L of cultured thallus precipitate induced and expressed by a nickel ion affinity chromatography column Ni-NTAAgarose with 30ml of Native Binding-Buffer, ultrasonically crushing, centrifuging 12000g at 4 ℃ for 20min, and taking supernatant; adding 10mL of the supernatant into a Binding-Buffer pre-balanced Ni affinity chromatographic column, and uniformly rotating on a mute mixer for 15 min; collecting the flow-through liquid; washing with 8mL Native Wash Buffer at flow rate of 0.5 mL/min; the column was washed with 20ml of Native Wash Buffer at a flow rate of 1 ml/min; the target protein was eluted at a flow rate of 1ml/min using 2ml of Native Elution-Buffer, 4ml of Native Elution-Buffer and 8ml of Native Elution-Buffer to obtain purified recombinant collagen.
CN202110299465.1A 2021-03-22 2021-03-22 Construction method of recombinant human-like collagen engineering bacteria Pending CN112980865A (en)

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