CN110903383A - Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof - Google Patents
Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof Download PDFInfo
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- CN110903383A CN110903383A CN201911145688.1A CN201911145688A CN110903383A CN 110903383 A CN110903383 A CN 110903383A CN 201911145688 A CN201911145688 A CN 201911145688A CN 110903383 A CN110903383 A CN 110903383A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a recombinant human type I collagen, a coding gene, an engineering bacterium and application thereof in preparing a collagen preparation, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2. The recombinant human type I collagen of the invention adopts an escherichia coli expression system, is suitable for large-scale amplification, can complete one round of fermentation within 18 hours, has very low production cost and short period, and improves the yield of target protein.
Description
(I) technical field
The invention relates to a recombinant human type I collagen and application thereof.
(II) background of the invention
Collagen provides a mechanical support for the organism, maintains the integrity of organs and tissues, and ensures the normal function of the organism, which is very important for parenchymal organs. Collagen is the major component of the parenchymal organ, basement membrane, and more than 80% of the organic matter in the skeleton is type I collagen, which plays a role in the skeleton and connective tissues primarily by forming and maintaining the integrity of the skeleton; and less than 20% of other collagens. The ratio of inorganic to organic components in bone changes with age. The synthesis of osteoblast type I collagen forming factors of old people is reduced, and the content of bone organic matters is reduced. The synthesis of collagen fibers is reduced, the mechanical support function for providing a framework for bone mineralization is reduced, inorganic substances such as calcium salt and the like cannot be deposited and mineralized, and finally, the bone absorption is increased. It can be said that the lack of bone mineralization is caused by the decrease in the synthesis of type I collagen and the insufficient arrangement of collagen fibers. The trend of the type I collagen can also influence the bone strength, more longitudinal collagen fibers can increase the tensile strength, and the mixed collagen can enhance the compressive strength. In addition, collagen plays a major role in the formation of specific extracellular microenvironments, which are critical for maintaining cellular integrity and for transmitting extracellular signals.
In addition to the above-mentioned biomechanical effects, type I collagen has some important roles, such as serving as a signal molecule for establishing cell shape and behavior, playing an important role in cell differentiation and development, and the like type I collagen can also transmit a constantly changing exogenous stimulus signal to cells through transmembrane receptors such as integrins, discoidin domain receptors, glycoproteins, and the like, thereby playing a role as a signal molecule, and at the same time, collagen has a function of signaling a "transfer site" which can trap, store and transport growth factors, thereby playing an important role in processes such as organ development, wound healing, tissue repair, and the like.
The role of collagen fibers is so important that the consequences of a mutation in the collagen gene are very severe, most commonly osteogenesis imperfecta, but also others, such as osteoporosis, Marfan's syndrome, Ehlers-Danlos syndrome, etc. More than 90% of children with osteogenesis imperfecta are caused by type i collagen mutations. Patients with severe osteogenesis imperfecta often die during childhood. More than 70 mutations have been found in collagen genes, including gene deletions, insertion mutations, mRNA cleavage mutations, and the expression of collagen molecules that do not form the correct conformation or that do not form a trimeric structure at all.
Collagen can be used not only for skin care, treated collagen such as gelatin, but also as a food additive. Collagen plays an important role in capturing, storing, transporting and the like of growth factors, and therefore, can be used as a carrier of some drugs, such as vaccines, interferons and other biological agents, and can be transported into a body and then slowly released. The collagen fiber can also be used as orthopedic operation material. Collagen has many advantages such as biodegradability, low antigenicity and easy availability, and thus collagen is increasingly used in the industries of cosmetics, foods, medicines and the like. However, the collagen is derived from the leather, horn, bone, tendon, and cartilage tissues of animals, and is obtained after slaughtering the animals, and may have pathogenic contamination. The production method is also unsatisfactory, and mainly comprises heat treatment in a membrane breaking reagent, so that the original structure and characteristics of collagen molecules are damaged. Therefore, the increasing demand for collagen is not satisfied, either quantitatively or qualitatively.
Disclosure of the invention
The invention aims to provide a recombinant human type I collagen with high solubility and high activity, a coding gene, an engineering bacterium and application. The invention utilizes the recombination technology, selects a gene sequence with the length of 99bp from the full-length type I collagen gene of a human through professional software analysis and experimental screening, repeats the gene sequence for 9 times, and then constructs the gene sequence into an expression vector pET22b through a traceless cloning method. And screening out a batch of successfully constructed plasmids through transformation and bacterial liquid identification, and finally selecting out the strain containing the recombinant humanized I-type collagen with good water solubility through water solubility screening. Due to different characteristics of the type I human collagen and the type III human collagen, the two types of collagen can be mixed according to a certain proportion to achieve a better effect, so that the mixed type collagen with more complete functions can be obtained and applied to medical cosmetology, biological materials, cosmetics and the like, and the application fields of the collagen can be widened, and a larger market can be brought.
The technical scheme adopted by the invention is as follows:
the invention provides a recombinant human type I collagen, and the amino acid sequence of the recombinant human type I collagen is shown in SEQ ID NO. 2.
The nucleotide sequence of the recombinant humanized type I collagen (RHIC9) coding gene is shown in SEQ ID NO. 1.
The invention also provides a recombinant vector and an engineering bacterium constructed by the recombinant humanized type I collagen coding gene, wherein the vector is pET-22b, and the engineering bacterium takes escherichia coli BL21-DE3-PLySs as a host.
Further, the recombinant human type I collagen is prepared by the following method: inoculating the engineering bacteria containing recombinant human type I collagen gene to LB culture medium, culturing at 37 deg.C overnight, transferring to new LB culture medium according to volume ratio of 1:100, culturing at 37 deg.C to OD600Adding IPTG (0.2 mM) to the mixture to culture the mixture at 37 ℃ for 6 hours, centrifuging the mixture for 5 minutes at 5000g, collecting thalli, and separating and extracting protein to obtain the human type I collagen.
Further, the engineering bacteria containing the recombinant human type I collagen gene is constructed by using pET-22b as a vector and using Escherichia coli BL21-DE3-PLySs as a host, wherein the construction method comprises the steps of converting the recombinant human type I collagen (RHIC9) gene into Escherichia coli DH5 α, screening and picking out white spot clones by using an ampicillin sodium-LB plate with the final concentration of 100ug/ml, identifying the positive clones by using a PCR (polymerase chain reaction) method to be correct, picking out the positive clones to carry out sequencing, determining that the recombinant plasmids contain correct gene sequence reading frames, successfully constructing a pET22b-RHIC9 recombinant expression vector, converting the correct pET22b-RHIC9 plasmid into Escherichia coli BL21-DE3-PLySs, screening and using an ampicillin sodium-LB plate with the final concentration of 100ug/ml to carry out single spot cloning, and obtaining the engineering bacteria containing the recombinant human type I collagen gene.
Further, the method for separating and extracting the protein comprises the following steps: (1) washing the thallus with PBS buffer solution, then resuspending the thallus with 1 XPBS buffer solution to prepare a thallus suspension, adding Triton X-100 cell lysate containing 1mg/ml lysozyme, ultrasonically breaking the thallus in an ice-water mixture environment, centrifuging at 12000r/min for 20min, and collecting supernatant; the volume consumption of the Triton X-100 cell lysate containing 1mg/ml of lysozyme is 1ml/g based on the weight of the bacteria; the volume dosage of the buffer solution for resuspension is 6ml/g based on the weight of the thalli; the ultrasonic bacteria breaking conditions are 400W and 2s ultrasonic, 5s intermittent, the total length is 45min, and the protection temperature is 40 ℃; (2) washing a nickel ion affinity column (Ni-NTA His-Bind Resin) column material by using PBS buffer solution, mixing the column material and supernatant in a volume ratio of 1:5, gently shaking on ice for 30min, then loading the column at a speed of 2ml/min, eluting 2 column volumes by using PBS solution containing 15mM imidazole to remove foreign proteins, adding PPase protease with His labels, gently shaking on ice for 2 hours, eluting 2 column volumes by using the PBS solution at a speed of 2ml/min, collecting all effluent liquid, adding a dialysis bag, carrying out warm dialysis on the PBS solution for overnight by using the PBS as a dialysate, taking the retentate which is purified RHIC9 recombinant protein, and freeze-drying to obtain purified RHIC9 recombinant protein dry powder; the cut-off molecular weight of the dialysis bag is 16 kDa;
the invention also relates to the application of the recombinant human type I collagen in preparing a collagen preparation, wherein the collagen preparation comprises 10-50% of the recombinant human type I collagen and 50-90% of type III human collagen by mass concentration, and the total amount is 100%.
Preferably, the collagen preparation comprises 20% of recombinant human type I collagen and 80% of type III human collagen by mass concentration.
Compared with the prior art, the invention has the following beneficial effects:
the recombinant human type I collagen is obtained by performing codon optimization expression on a human type I collagen conserved region on the premise of not influencing an amino acid sequence; the invention adopts an escherichia coli expression system, is suitable for large-scale amplification, can complete one round of fermentation within 18 hours, has very low production cost and short period, adopts BL21-DE3-PLySs expression strains, can reduce the endogenous protein expression of bacteria and further improves the yield of target protein;
the recombinant human type I collagen produced by the human type I collagen through sequence analysis and experimental screening has very good hydrophilicity and stability, the amino acid repetitive sequence of the recombinant human type I collagen is 100 percent identical to the corresponding part of the amino acid sequence of the natural collagen, and the recombinant human type I collagen can not generate immunological rejection and anaphylactic reaction when being applied to a human body and can be widely applied to the industries of biological medicine and cosmetics;
the product of the invention has biological activity reaching or even exceeding the biological activity of natural protein of human body through activity detection, is the optimized collagen design scheme reported at present, can perform the function of the natural protein in human body, and achieves the purpose of real product application.
The recombinant human type I collagen (RHIC9) can be mixed with type III human collagen (RHIC 8) for use, and better biological activity is generated by mixing different proportions, so that a stronger collagen function is exerted. The experimental results show that the best effect can be achieved by using the composition according to the proportion of 20% RHIC9+ 80% RHIC 8.
(IV) description of the drawings
FIG. 1 is a plasmid map of pET22b-RHIC9 vector construction.
FIG. 2 shows codon optimization of the RHIC9 gene.
FIG. 3 is an electrophoretogram of RHIC9 protein after induction and purification.
FIG. 4 shows the results of the measurement of the biological activities of RHIC9 protein in comparison with human collagen.
FIG. 5 shows the results of biological activity tests of different application ratios of RHIC9 protein and RHIC 8 protein.
FIG. 6 shows the expression of pET22b-RHIC9 vector in different competencies.
FIG. 7 shows the results of the stability test of RHIC9 protein.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1
1. Construction and expression of RHIC9 gene expression vector
Selecting a human type I collagen conserved region peptide segment GSKGDTGEPGPVGVQGPPGPAGEEGKRGARGEP, repeating the sequence for 9 times, optimizing codons, entrusting Shanghai Senno biotechnology limited to carry out whole gene synthesis (marked as RHIC9 gene, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO. 2), adding enzyme cutting sites of Xba I and Sal I at two ends of the gene, carrying out amplification culture on the synthetic gene, extracting a plasmid with a target gene, carrying out double enzyme cutting by using the Xba I and the Sal I, and recovering the target gene by agarose gel electrophoresis; and the recombinant plasmid is incubated with a pET22b expression vector which is treated by corresponding enzyme digestion to construct an RHIC9 gene expression vector, which is shown in figure 1.
Adding 1U T4 DNA ligase into an RHIC9 gene expression vector, connecting for 2 hours at 25 ℃, transforming escherichia coli DH5 α, screening out white spot clones by using an ampicillin sodium-LB plate with a final concentration of 100ug/ml, identifying the correctness of positive clones by using a PCR (polymerase chain reaction) method, selecting the positive clones for sequencing, determining that recombinant plasmids contain correct gene sequence reading frames, successfully constructing a pET22b-RHIC9 recombinant expression vector, carrying out amplification culture on the correct clones, and extracting plasmids by using a Kangshi plasmid extraction kit.
2. Constructing escherichia coli genetic engineering bacteria, and inducing and expressing recombinant human collagen.
Respectively transforming the correct pET22b-RHIC9 plasmid into Escherichia coli DH5 α 21-DE3, BL21-DE3-PLySs, screening single-spot clones by using an ampicillin sodium-LB plate with the final concentration of 100ug/ml, culturing in 5ml LB medium at 37 ℃ overnight, transferring to LB medium according to the volume ratio of 1:100, and culturing in a shaking flask at 37 ℃ until OD is achieved600Adding IPTG with the final concentration of 0.2mM, culturing for 6 hours at 37 ℃, centrifuging for 5 minutes at 5000g, collecting thalli escherichia coli DH5 α -pET22b-RHIC9, escherichia coli BL21-DE3-pET22b-RHIC9, escherichia coli BL21-DE3-PLySs-pET22b-RHIC9 respectively, preserving at-20 ℃ or immediately entering the next step for purification, wherein the electrophoresis chart of the escherichia coli BL21-DE3-PLySs-pET22b-RHIC9 is shown in a lane induction purified RHIC9 in a picture 3.
3. Purification of recombinant human type I collagen
Washing Escherichia coli BL21-DE3-PLySs-pET22b-RHIC9 thallus with PBS buffer solution, resuspending 5g of thallus with 30ml of 1 XPBS buffer solution to prepare a bacterial suspension, adding 5ml of Triton X-100 cell lysate containing 1mg/ml lysozyme, ultrasonically breaking the bacteria under the environment of ice-water mixture (400W and 2s ultrasound, 5s intermission, total length 45min, protection temperature 40 ℃), centrifuging at 12000r/min for 20min, collecting supernatant, and inducing RHIC9 by using a lane in an electrophoretogram shown in FIG. 3. In this case, the solution contained a large amount of His-RHIC9 recombinant protein.
Washing a nickel ion affinity column (Ni-NTA His-Bind Resin, 10mL) column material with PBS buffer solution, mixing the column material and the supernatant fluid according to the volume ratio of 1:5, shaking gently on ice for 30min, loading the column at the speed of 2mL/min, eluting 2 column volumes with PBS solution containing 15mM imidazole to remove impurity proteins, leaving pure His-RHIC9 on the column, adding 20 microliter of Prescission Protease (PPase for short) Protease with His label on the column, shaking gently on ice for 2 hours, releasing RHIC9 protein from the column, eluting 2 column volumes with PBS solution at the speed of 2mL/min, collecting all effluent, adding a dialysis bag (the dialyzate is 16kDa in size and purchased from Shanghai Min chemical Co., Ltd.), dialyzing at the temperature of a dialysis solution room with PBS as a dialysis solution overnight, taking purified RHIC9 recombinant protein 5mL, namely purified RHIC9 recombinant protein dry powder 0.1g, and (5) standby. A portion of the purified sample was run on PAGE gel to determine protein size and purity, RHIC9 purified in lane FIG. 3.
4. RHIC9 protein activity detection
Protein concentration was measured by BCA method using protein concentration measurement kit (pelagic day P0006). Principle of BCA method: under alkaline condition, peptide bond structure in protein molecule is connected with Cu2+Complexing and adding Cu2+Reduction to Cu1+. BCA is specific to Cu1+Binding forms a stable blue-violet complex with maximum absorbance at 562nm and is proportional to protein concentration. The method realizes the simplicity, high stability, high sensitivity and high compatibility of protein concentration determination, and comprises the following specific operations:
(1) reagent
BCA working solution: adding the BCA reagent A into the BCA reagent according to the volume ratio of 50:1, preparing a BCA working solution, and fully and uniformly mixing.
Protein standard solution: mu.L of 5mg/ml RHIC9 protein standard solution (PBS as solvent from standard protein solution in kit) was diluted to 100. mu.L with PBS to a final concentration of 0.5 mg/ml.
Sample preparation: and (3) intercepting liquid in the step 3.
(2) Standard curve: the protein standard solution was added to the standard wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μ L and made up to 20 μ L with PBS. 200. mu.L of BCA working solution was added to each well, and the mixture was left at 37 ℃ for 30 minutes. Measuring the wavelength between A562(540-595nm is also acceptable) by using a spectrophotometer, drawing a standard curve by taking the concentration (x) of the standard substance as an abscissa and the absorbance (Y) of the A562 as an ordinate, wherein the equation of the curve is that Y is 0.673x +0.004, and R is2Is 0.997.
(3) And (3) adding 10 mu L of sample (Escherichia coli DH5 α -pET22b-RHIC9, Escherichia coli BL21-DE3-pET22b-RHIC9, Escherichia coli BL21-DE3-PLySs-pET22b-RHIC9 obtained in the step 2) into sample wells of a 96-well plate, adding PBS into 20 mu L, adding 200 mu L of BCA working solution into each well, standing at 37 ℃ for 30 minutes, measuring A562 (the wavelength between 540 and 595nm is also acceptable) by using a spectrophotometer, and calculating the protein concentration in the sample according to a standard curve, wherein the result is shown in figure 6.
(4) Cell anchorage rate: after the protein concentration was determined, the human collagen RHIIC 8 (purchased from Bio-Rad A001654) and the purified RHIC9 recombinant protein prepared in step 3 were adjusted to a concentration of 0.5mg/ml using PBS. A blank control group, a PBS group, a human collagen (namely RHIIC 8) group, an RHIC9 group, an RHIC9+ RHIIC 8 group with a volume ratio of 1:9, an RHIC9+ RHIC 8 group with a volume ratio of 2:8, an RHIC9+ RHIC 8 group with a volume ratio of 3:7, an RHIC9+ RHIC 8 group with a volume ratio of 4:6 and an RHIC9+ RHIC 8 group with a volume ratio of 5:5 are set. Wherein the blank control group is not added with substances, the PBS group uses PBS to replace collagen, 100 μ L of each group is respectively added into a 96-well plate, and the mixture is kept standing for 60min at room temperature. 105 well-cultured 3T3 cells (purchased from Zygosaku organism JH-M2003) were added to each well and incubated at 37 ℃ for 60 min. Each well was washed 4 times with PBS. The absorbance of OD450nm was measured using a cell adhesion assay kit (Beboc BB-48120). According to the value of the blank control, the cell adhesion rate is calculated, and the cell adhesion rate reflects the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for the cells in a short time, and the cells are attached to the wall. Relative cell adhesion activity of the RHIC9 sample (fig. 4) and cell adhesion activity of the mixed sample of RHIC9 and RHIIIC8 (fig. 5) were measured with the adherence rate of human collagen (i.e., RHIIIC8) being 1.
Cell adhesion rate [% of cell adhesion (test OD-blank OD)/(control OD-blank OD) ] x100
FIG. 4 shows that the activity of RHIC9 itself is high, and FIG. 5 shows that the effect of RHIC9 and RHIC 8 in combination is more obvious.
5. RHIC9 protein stability
The purified RHIC9 recombinant protein dry powder prepared in step 3 and the human type I collagen (purchased from Sangon Biotech (Shanghai) co., Ltd) were placed at a temperature of 25 ℃ every two days, the cell adherence rate was detected by the method of step (4) for 20 days continuously, and the results are shown in fig. 7, and the recombinant human type I collagen RHIC9 had better stability than the purchased human type III collagen.
Sequence listing
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Claims (10)
1. A recombinant human type I collagen is characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 2.
2. The recombinant human type I collagen encoding gene of claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1.
3. A recombinant vector constructed from the recombinant human type I collagen-encoding gene of claim 2.
4. A genetically engineered bacterium constructed from the recombinant vector of claim 3.
5. The genetically engineered bacterium of claim 4, wherein said engineered bacterium is host Escherichia coli BL21-DE 3-PLySs.
6. The recombinant human type I collagen according to claim 1, which is prepared by the following method: inoculating the engineering bacteria containing recombinant human type I collagen gene to LB culture medium, culturing at 37 deg.C overnight, transferring to new LB culture medium according to volume ratio of 1:100, culturing at 37 deg.C to OD600Adding the mixture into the mixture with the final concentration of 0.2mMIPTG, culturing for 6 hours at 37 ℃, centrifuging for 5 minutes at 5000g, collecting thalli, separating and extracting protein to obtain the human source type I collagenA protein.
7. The recombinant human type I collagen according to claim 6, wherein said method for isolating and extracting said protein comprises: (1) washing the thallus with PBS buffer solution, then resuspending the thallus with 1 XPBS buffer solution to prepare a thallus suspension, adding Triton X-100 cell lysate containing 1mg/ml lysozyme, carrying out ultrasonic bacteria breaking in an ice water mixture environment, centrifuging, and collecting a supernatant; the ultrasonic bacteria breaking conditions are 400W and 2s ultrasonic, 5s intermittent, the total length is 45min, and the protection temperature is 40 ℃; (2) washing a nickel ion affinity column material by PBS buffer solution, mixing the column material and the supernatant according to the volume ratio of 1:5, gently shaking for 30min on ice, loading the column material on the speed of 2ml/min, eluting 2 column volumes by PBS solution containing 15mM imidazole to remove foreign proteins, adding PPase protease with His labels, gently shaking for 2 hours on ice, eluting 2 column volumes by PBS solution on the speed of 2ml/min, collecting all effluent liquid, adding into a dialysis bag, carrying out warm dialysis on the PBS solution serving as the dialysis solution for overnight, taking the trapped liquid which is purified RHIC9 recombinant protein, and freeze-drying to obtain purified RHIC9 recombinant protein dry powder.
8. Use of the recombinant human type I collagen of claim 1 in the preparation of a collagen preparation.
9. The use according to claim 8, wherein the collagen preparation comprises 10-50% recombinant human type I collagen and 50-90% type III human collagen, by mass, the total amount being 100%.
10. The use according to claim 9, wherein the collagen preparation has a composition of 20% recombinant human type I collagen and 80% type III human collagen by mass.
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CN113621053A (en) * | 2021-08-30 | 2021-11-09 | 江苏亨瑞生物医药科技有限公司 | Recombinant human collagen and preparation method and application thereof |
CN113788891A (en) * | 2021-09-15 | 2021-12-14 | 山西锦波生物医药股份有限公司 | Recombinant I-type humanized collagen C1L4T and preparation method and application thereof |
CN113788891B (en) * | 2021-09-15 | 2023-10-31 | 山西锦波生物医药股份有限公司 | Recombinant I-type humanized collagen C1L4T and preparation method and application thereof |
CN115521373A (en) * | 2022-06-06 | 2022-12-27 | 胶原蛋白(武汉)生物科技有限公司 | Triple-helix recombinant humanized type I collagen, preparation method and application thereof |
CN115521373B (en) * | 2022-06-06 | 2024-04-19 | 胶原蛋白(武汉)生物科技有限公司 | Triple helix recombinant humanized type I collagen, preparation method and application thereof |
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CN117510618B (en) * | 2023-11-06 | 2024-07-05 | 江西崇山生物制品有限公司 | Synthetic I-type humanized collagen and preparation method and application thereof |
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