Disclosure of Invention
The primary aim of the invention is to overcome the defects of the prior art and provide a recombinant human fusion collagen.
Another object of the present invention is to provide a method for efficient hydroxylation of the recombinant human fusion collagen.
It is still another object of the present invention to provide the use of the efficient hydroxylation method by recombinant human fusion collagen as described above.
The aim of the invention is achieved by the following technical scheme:
a recombinant human fusion collagen has an amino acid sequence shown in SEQ ID No. 1. The recombinant human fusion collagen is obtained by connecting a type I collagen peptide segment and a type III collagen peptide segment in series for 2 times, and has the structure of I-III-I-III.
SEQ ID No.1:
MGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGGGSGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGSEAAAKGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGGGSGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGERGRPGERGLPGENGVMGFPKPGEPGPKGMPGER。
The peptide of the type I collagen is (GERGDLGPQGIAGQRGVVGERGERGERGASGFPGER) 8 The amino acid sequence of the polypeptide is derived from a fibroblast adhesion promoting sequence (GERGDLGPQGIAGQRGVVGERGERGERGAS) and an integrin alpha 2 beta 1 recognition sequence (GFPGER) in type I collagen; III type collagen peptide fragment (GRPGERGLPGENGVMGFPKPGEPGPKGMPGER) 8 The amino acid sequence of the protein consists of a binding site GRPGER, GLPGEN, GMPGER with integrin alpha 2 beta 1 in III type collagen, a GVMGPP binding site and a sequence KPGEPGPK mediating platelet adhesion and signal transduction which are connected in series.
The recombinant humanized fusion collagen encoding nucleic acid can be obtained according to a codon rule; codon optimization can be performed according to the preference of the host cell; the preferred nucleic acid sequence is shown in SEQ ID No. 2.
SEQ ID No.2:
atgggcgagcgcggcgacttaggcccgcaaggcatcgcgggtcagcgtggcgtagttggtgaacggggtgaacggggtgagcgtggtgctagcggctttccgggtgaacgtggggaacgtggtgatctgggcccgcagggtatcgcgggtcagcgtggtgttgtgggcgaacgtggtgaacgtggtgagcgcggtgcatcgggctttccaggcgaacgcggggaacgcggtgatctggggccgcagggtattgccggccaacgtggcgtggttggtgagcggggtgagcgcggcgagcgcggtgcgagcggtttccccggcgaacgtggcgagcgtggcgacctgggcccgcaaggcatcgctggccagcgtggcgtggtcggtgaacgtggcgaacgtggggaacgtggcgcctctggcttccctggcgaacgaggtgaacgtggggatctgggcccgcaaggaattgcgggtcagcgcggcgttgttggcgaacgcggagagcgcggtgagcgtggcgcgagcggctttccgggcgaacgcggcgaacgcggtgatctgggtccgcagggcatcgctggtcaacgtggcgtagtcggtgagcgtggagaacgtggcgaacgtggcgcgtccggctttcccggtgaacgaggcgaacgtggggacctgggtccacagggtatcgctggccagcgcggcgttgtgggagagcgtggcgaacgcggcgaacgcggtgcatctggcttcccaggcgaacgtggagaacgcggtgacctgggtccgcagggcatcgcgggccagcgcggtgtggtcggcgagcgtggtgaacgtggtgaacggggcgcgtccggtttcccaggtgaaagaggtggcggttccggccgtcctggtgaacgcggcctgccgggtgaaaacggtgttatgggttttccgaaacctggcgagccgggccctaaaggcatgccaggtgaacgcggccgtccgggtgaacgcggtctgccgggcgaaaacggtgtgatgggctttccgaaaccaggtgagccgggcccgaagggtatgccgggggaacgtggtcgtccgggggaacgcggtctgccgggcgagaacggcgtaatgggcttccctaaaccgggcgaaccgggcccgaaaggtatgccgggcgagcgtggccgccctggtgagcgtggcctgcctggcgaaaacggtgtgatgggcttcccgaaaccgggtgaaccgggcccgaaaggcatgccgggtgaacgcgggcgtccgggcgagcgcggcctgccaggcgaaaatggtgtaatgggcttcccgaaaccaggtgaaccgggtccgaaaggtatgccgggtgagcgtggtcgtccgggcgaacgcgggctgccgggcgaaaacggcgttatgggtttcccgaaaccgggtgaaccaggcccgaaaggcatgccgggcgagcgtggtcgccccggcgaacgtggtttaccgggcgaaaatggcgttatgggtttcccgaagccgggtgaaccgggtccgaagggcatgccgggtgaacgtggccgtcctggtgaacgcggtttgccgggtgaaaacggcgtgatgggcttcccaaaaccgggcgaaccgggcccgaaaggcatgccgggcgagcgcggatccgaagcggcggcgaaaggcgagcgcggcgacttaggcccgcaaggcatcgcgggtcagcgtggcgtagttggtgaacggggtgaacggggtgagcgtggtgctagcggctttccgggtgaacgtggggaacgtggtgatctgggcccgcagggtatcgcgggtcagcgtggtgttgtgggcgaacgtggtgaacgtggtgagcgcggtgcatcgggctttccaggcgaacgcggggaacgcggtgatctggggccgcagggtattgccggccaacgtggcgtggttggtgagcggggtgagcgcggcgagcgcggtgcgagcggtttccccggcgaacgtggcgagcgtggcgacctgggcccgcaaggcatcgctggccagcgtggcgtggtcggtgaacgtggcgaacgtggggaacgtggcgcctctggcttccctggcgaacgaggtgaacgtggggatctgggcccgcaaggaattgcgggtcagcgcggcgttgttggcgaacgcggagagcgcggtgagcgtggcgcgagcggctttccgggcgaacgcggcgaacgcggtgatctgggtccgcagggcatcgctggtcaacgtggcgtagtcggtgagcgtggagaacgtggcgaacgtggcgcgtccggctttcccggtgaacgaggcgaacgtggggacctgggtccacagggtatcgctggccagcgcggcgttgtgggagagcgtggcgaacgcggcgaacgcggtgcatctggcttcccaggcgaacgtggagaacgcggtgacctgggtccgcagggcatcgcgggccagcgcggtgtggtcggcgagcgtggtgaacgtggtgaacggggcgcgtccggtttcccaggtgaaagaggtggcggttccggccgtcctggtgaacgcggcctgccgggtgaaaacggtgttatgggttttccgaaacctggcgagccgggccctaaaggcatgccaggtgaacgcggccgtccgggtgaacgcggtctgccgggcgaaaacggtgtgatgggctttccgaaaccaggtgagccgggcccgaagggtatgccgggggaacgtggtcgtccgggggaacgcggtctgccgggcgagaacggcgtaatgggcttccctaaaccgggcgaaccgggcccgaaaggtatgccgggcgagcgtggccgccctggtgagcgtggcctgcctggcgaaaacggtgtgatgggcttcccgaaaccgggtgaaccgggcccgaaaggcatgccgggtgaacgcgggcgtccgggcgagcgcggcctgccaggcgaaaatggtgtaatgggcttcccgaaaccaggtgaaccgggtccgaaaggtatgccgggtgagcgtggtcgtccgggcgaacgcgggctgccgggcgaaaacggcgttatgggtttcccgaaaccgggtgaaccaggcccgaaaggcatgccgggcgagcgtggtcgccccggcgaacgtggtttaccgggcgaaaatggcgttatgggtttcccgaagccgggtgaaccgggtccgaagggcatgccgggtgaacgtggccgtcctggtgaacgcggtttgccgggtgaaaacggcgtgatgggcttcccaaaaccgggcgaaccgggcccgaaaggcatgccgggcgagcgc。
A recombinant expression vector comprising the above recombinant human fusion collagen encoding nucleic acid.
A recombinant genetically engineered bacterium contains the recombinant expression vector.
The original strain of the recombinant genetically engineered bacterium is preferably escherichia coli; more preferably E.coli BL21 (DE 3).
The efficient hydroxylation method of the recombinant humanized fusion collagen comprises the following steps:
(1) Cloning the encoding nucleic acid of the recombinant humanized fusion collagen to an expression vector to obtain a collagen recombinant expression vector;
(2) Cloning coding nucleic acid of proline hydroxylase (BaP 4H) to an expression vector to obtain a hydroxylase recombinant expression vector;
(3) Co-transforming a collagen recombinant expression vector and a hydroxylase recombinant expression vector into an escherichia coli host cell to obtain recombinant genetically engineered bacteria, and then performing induced expression;
(4) Performing solid-liquid separation on the bacterial liquid induced to be expressed in the step (3), and taking and crushing bacterial cells;
(5) Performing solid-liquid separation on the crushed thalli, and taking a supernatant; and (3) purifying the supernatant to obtain the hydroxylated recombinant human fusion collagen.
The sequence of the coding nucleic acid described in step (1) is preferably as shown in SEQ ID NO. 2.
The expression vector in the step (1) is preferably an E.coli expression vector; more preferably an E.coli expression vector comprising a T7 promoter; more preferably pET series expression vectors; most preferred is the pET28a vector.
More preferably, step (1) is: the coding nucleic acid with the sequence shown as SEQ ID NO.2 is cloned between the digestion sites NdeI and BamHI of the pET28a vector, and the collagen recombinant expression vector is obtained.
The amino acid sequence of the proline hydroxylase (BaP 4H) described in step (2) is preferably as shown in SEQ ID No. 3.
SEQ ID No.3:
MTNNNQIGENKEQTIFDHKGNVIKTEDREIQIISKFEEPLIVVLGNVLSDEECDELIELSKSKLARSKVGSSRDVN DIRTSSGAFLDDNELTAKIEKRISSIMNVPASHGEGLHILNYEVDQQYKAHYDYFAEHSRSAANNRISTLVMYLN DVEEGGETFFPKLNLSVHPRKGMAVYFEYFYQDQSLNELTLHGGAPVTKGEKWIATQWVRRGTYK。
The sequence of the nucleic acid encoding the proline hydroxylase (BaP 4H) described in step (2) is preferably as shown in SEQ ID No. 4.
SEQ ID No.4:
atgactaataataaccaaataggtgaaaataaagagcagaccatctttgaccataaaggtaacgtaattaaaactgaagatcgtgaaatccagataattagtaaatttgaagaaccgctgatagtagttttaggtaacgtgttaagtgatgaagaatgcgatgaactgattgaactgagtaaaagtaaacttgcccgctctaaagtaggtagtagccgtgacgtgaatgacatccgcactagtagcggcgcattccttgatgataatgagttgacggctaaaatcgaaaaacgtatcagcagcatcatgaacgttccggcgagccacggtgaaggcctgcacatcctgaactacgaagttgatcagcagtacaaagcgcactacgattacttcgcggaacacagccgtagcgcggcgaacaaccgtatcagcaccctggttatgtacctgaacgatgttgaagaaggcggtgaaaccttcttcccgaaactgaacctgagcgttcacccgcgtaaaggcatggcggtttacttcgaatacttctaccaggatcagagcctgaacgaactgaccctgcacggtggtgcgccggttaccaaaggtgaaaaatggatcgcgacccagtgggttcgtcgtggtacctacaaa。
The expression vector in the step (2) is preferably an escherichia coli expression vector; which comprises a promoter weaker than the promoter in the expression vector described in step (1); more preferably an E.coli expression vector comprising an arabinose promoter; most preferred are pGro7 vectors.
The cloning method described in step (2) is preferably an RF (Restriction-free) cloning method.
The method of conversion described in step (3) is preferably the calcium chloride process.
The E.coli in the step (3) is preferably E.coli BL21 (DE 3).
The induction expression in the step (3) is preferably to induce and express proline hydroxylase for a period of time and then to induce and express recombinant human fusion collagen; the specific steps are preferably as follows:
A. inoculating recombinant genetically engineered bacteria into LB culture medium, culturing at 35-40 deg.C and 150-250 rpm;
B. inoculating the bacterial liquid obtained in the step A into LB culture medium containing 1.5-2.5 mg/mL L-arabinose (used for inducing hydroxylase expression), culturing for 2-4 hours at 35-40 ℃, and adding 0-1 mM IPTG to induce collagen gene expression.
The culture conditions described in step A are preferably those in which the temperature is 37℃and the rotational speed is 220rpm.
The culture time in the step A is the period from the culture to the late phase of the logarithmic phase or the stationary phase; preferably 8 hours or more; more preferably 10 to 12 hours.
The inoculation amount in the step B is 1-5% by volume; more preferably 1 to 3%.
The content of L-arabinose in the step B is preferably 2mg/mL.
The temperature of the culture described in step B is preferably 37 ℃.
The incubation time described in step B is preferably 3 hours.
Bacterial liquid OD before adding IPTG in step B 600 The value is preferably 0.7 to 0.9; more preferably 0.8.
The amount of IPTG added in step B is preferably 0.1mM in final concentration.
The time for inducing expression in the step B is preferably 4-6 hours; more preferably 5h.
The solid-liquid separation method in the step (4) is preferably centrifugation.
The centrifugation conditions are preferably 4 ℃ and 8000g centrifugation for 5-10 min.
The composition of the solution used in the disruption described in step (5) is as follows: 20mmol/L Tris-HCl, 20mmol/L imidazole, 500mmol/L NaCl, pH8.5.
The conditions for the disruption in step (5) are preferably disruption at a pressure of 20 to 25 kpsi; more preferably at a pressure of 22 kpsi.
The solid-liquid separation method in step (5) is preferably centrifugation.
The centrifugation conditions are preferably 4℃and 14000g for 40-50 min.
The purification method in step (5) is as follows: one or a combination of nickel affinity chromatography, cation exchange chromatography, gel filtration and membrane separation is adopted.
The efficient hydroxylation method of the recombinant human fusion collagen is applied to the preparation of the hydroxylated recombinant human fusion collagen.
The hydroxylation recombinant humanized fusion collagen is obtained by the efficient hydroxylation method.
Compared with the prior art, the invention has the following advantages and effects:
the recombinant collagen obtained by the invention is I-type and III-type fusion collagen, and integrates a plurality of site sequences of human I-type and III-type collagen and cell recognition and interaction activities. Under the same molecular weight condition, the cell activity of the type I and type III fusion collagen is higher than that of single protein. The proline hydroxylase from bacillus anthracis and the humanized collagen gene are constructed to co-express, the proline hydroxylase from bacillus anthracis is induced to be expressed, after a certain amount of intracellular hydroxylase is accumulated, the collagen under the control of a strong promoter is induced to be expressed rapidly, the expression and synchronous efficient hydroxylation of the large molecular weight fusion collagen are realized by regulating and controlling the expression time point of the enzyme and the collagen, the hydroxylation rate reaches 63%, and the collagen with higher hydroxylation rate has better stability and higher bioactivity.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
According to the recombinant humanized fusion collagen amino acid sequence SEQ ID No.1, aiming at codon preference of host escherichia coli expression, ndeI and BamHI enzyme cutting sites are avoided in the design process, the gene sequence is optimized, the optimized gene sequence is shown in SEQ ID No.2, the amino acid sequence of bacillus anthracis hydroxylase (BaP 4H) is reversely designed to obtain the gene sequence by the same way, and the two gene sequences are delivered to Shanghai biological limited company for complete gene synthesis. Wherein the recombinant collagen gene sequence is directly connected between the digestion site NdeI and BamHI of the expression vector pET28a to obtain a recombinant vector pET28a-rhCOL (I-III); the bacillus anthracis hydroxylase (BaP 4H) gene sequence is connected to the vector pUC57 to obtain a recombinant vector pUC57-BaP4H.
Construction of recombinant expression vector pGro7-BaP4H, inserting BaP4H into arabinose promoter of pGro7 vector, and replacing original molecular chaperone gene on the vector.
(1) Primer design: the amplification primers for the BaP4H fragment were designed according to the primer design principle of RF cloning (unrestricted cloning), as follows:
upstream primer F1:5'-TACGGACTTTCTCAAAGGAGAGTTATCAATGACTAATAATAACCAAATAGGTG-3';
the downstream primer R1:5'-GTTTATTTCTGCGAGGTGCAGGGCAATTATTTATCATCATCATCTTTATAATCTTTGTAGGTA-3'.
(2) Amplification of the BaP4H fragment: amplifying the BaP4H target gene by using the synthesized plasmid (pUC 57-BaP 4H) containing the BaP4H gene as a template and using the amplification primer, and recovering a PCR product, namely a BaP4H gene fragment, by using a DNA recovery and purification kit (TIANGEN); the result of the electrophoretic separation is shown in FIG. 1.
The PCR amplification system was as follows:
the PCR amplification procedure was as follows:
(3) RF linear amplification construction vector pGro7-BaP4H: the PCR linear amplification of the second round is carried out by taking pGro7 empty plasmid (TaKaRa) as a template and the recovery product as a primer, and the PCR system and the reaction program are as follows:
the PCR amplification system was as follows:
the PCR amplification procedure was as follows:
the amplified DNA fragment is subjected to DpnI digestion treatment to digest a small amount of template plasmid in the reaction system, and the digestion system and conditions are as follows:
the product after enzyme digestion is directly transformed into an escherichia coli DH5 alpha strain according to a calcium chloride method.
The positive transformants were picked for colony PCR verification (colony PCR primers were designed at 200bp upstream and downstream of the target gene), the electrophoresis results were as shown in FIG. 2, and the amplification primers were as follows:
the upstream primer F2:5'-TCGACCAGGACGACAGAGC-3';
downstream primer R2:5'-TGAAGTCAGCCCCATACGATA-3'.
And sending the obtained product to Shanghai biological limited company for sequencing and comparing, and extracting plasmids to obtain recombinant expression plasmids pGro7-BaP4H.
(4) Preparing coexpression engineering bacteria: the recombinant expression plasmids pGro7-BaP4H and pET28a-rhCOL (I-III) which are successfully constructed are mixed according to the mole ratio of 1:1, transforming into escherichia coli BL21 (DE 3) competence by a calcium chloride method, coating on LB plates containing kana and chloramphenicol resistance, and growing positive transformants. The final coexpression recombinant genetically engineered bacterium is obtained.
Example 2: co-expression and purification of recombinant human fusion collagen and proline hydroxylase in escherichia coli host
The co-expression conditions including culture medium composition, induction temperature, inducer concentration, IPTG addition time and the like are optimized, and a large amount of expression is carried out under the optimal conditions, and the optimal expression conditions are as follows:
(1) Seed liquid preparation: single colonies on the transformation plate of example 1 were picked up on an ultra clean bench and inoculated into 10mL of LB liquid medium (Cm) + And Kan + ) Shaking culture is carried out for 10-12 hours at a constant temperature of 37 ℃ and 220rpm to obtain seed liquid.
(2) Co-expression: the seed solution is transferred to 3 shaking flasks (Cm) with a liquid loading of 200mL medium/1000 mL according to a volume ratio of 1:100 + And Kan + ) Wherein, the No.1 shake flask is not added with any inducer, the No.3 shake flask is added with arabinose solution with the final concentration of 2mg/mL before inoculation, baP4H expression is induced in advance, and the mixture is placed at 37 ℃ and cultured for 2-3 hours by a constant temperature shaking table at 220rpm, and then bacterial liquid OD is measured 600 At a value of 0.8, IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to the respective shake flasks No.2 and No.3 at a final concentration of 0.1mM, to induce collagen expression, and culturing was continued for 5 hours. After the induction is finished, the bacterial liquid OD is measured 600 The cells were collected by centrifugation at 8000g for 7min at 4 ℃. After the cells were disrupted, the supernatant was subjected to SDS-PAGE, and the results were shown in FIG. 3, and when IPTG alone was added (shake flask No. 2), the cells were induced to express non-hydroxylated collagen; upon co-induction with IPTG and arabinose (shake flask No. 3), collagen and hydroxylase were expressed simultaneously. The cells in shake flasks No.2 and No.3 were collected and purified as follows to obtain non-hydroxylated and hydroxylated recombinant human fusion collagen.
1) Adding Buffer A solution according to the proportion of 10OD/mL to completely suspend the thalli, and placing the thalli after complete suspension under a high-pressure crusher to crush cells (temperature: 4 ℃, pressure: 22 kpsi); after crushing, obtaining a crushed supernatant and a sediment by centrifugation at 14000g at 4 ℃ for 45 min; collecting supernatant sample, namely crude protein expression liquid, and filtering the supernatant sample through a 0.22 μm filter membrane to obtain a pre-column sample.
2) By GE company (HisTrap) TM HP Column,5 mL) pre-packed ColumnFor purifying the medium, the AKTA chromatography system is washed with ultrapure water at a flow rate of 5mL/min until the UV baseline is stable, the column is packed, the nickel column is equilibrated with Buffer A, the sample is injected at a flow rate of 2mL/min, the sample is discharged after the column is collected, the sample is washed with Buffer A after the sample is loaded, and then is eluted linearly with (0-100%) Buffer B,20min,5mL/min, when the UV is applied 280 Collecting when the peak is generated, and stopping collecting after the peak is generated, namely obtaining a crude purified sample;
3) The crude purified sample is subjected to cation exchange chromatography, cation exchange is carried out by taking a pre-packed Column (SP HP Column,5 mL) of GE company as a purification medium, an AKTA chromatography system is washed by ultrapure water at a flow rate of 5mL/min until a UV baseline is stable, an initial buffer (Start buffer) is exchanged to the Column at a flow rate of 5mL/min, elution buffers (absorption buffers) with 5 Column volumes are used for washing, and a Start buffer balance system with 5-10 Column volumes is used. The sample is loaded at a speed of 5mL/min, and the sample is injected by using a sample injection ring when the sample amount is small. After the sample loading is finished, flushing by using a Start buffer with at least 5 column volumes; then starting the linear Elution, and using an Elution Buffer (0-100%) for 20min and 5mL/min for linear Elution when UV 280 And starting to collect when the peak is out, and stopping collecting after the peak is out, namely obtaining the sample after the cation exchange column. Ultrafiltration concentration can be performed using a 10kDa ultrafiltration tube (Millipore). The purified sample is subjected to SDS-PAGE electrophoresis to detect the size and purity of the protein (shown in figure 4), and the purity of the prepared recombinant human fusion collagen is 91%.
Buffer a configuration: 20mmol/L Tris-HCl, 20mmol/L imidazole, 500mmol/L NaCl, pH8.5. Filtering with 0.22 μm filter membrane to remove impurities, ultrasonically degassing (before use), and storing at normal temperature.
Buffer B configuration: 20mmol/L Tris-HCl, 500mmol/L imidazole, 500mmol/L NaCl, pH8.5. Filtering with 0.22 μm filter membrane to remove impurities, ultrasonically degassing (before use), and storing at normal temperature.
Start buffer: the pH was 8.0, 50mM bicine buffer, 0.22 μm filter removed impurities by suction filtration, and the air bubbles removed by sonication prior to use.
An execution buffer: adding sodium chloride with the final concentration of 1M into the Start buffer for dissolution, and carrying out suction filtration and ultrasonic treatment after preparation.
Example 3: structure detection of recombinant human fusion collagen
(1) Analysis of amino acid composition
Using BCA method, using protein concentration determination kit (Thermo Fisher) to determine protein concentration after ultrafiltration concentration, taking 10mg protein sample, adding 5mL hydrochloric acid with concentration of 6M into hydrolysis tube, vacuumizing and charging nitrogen gas, placing into 110 deg.C oven for hydrolysis for 24h, cooling hydrolysis tube to room temperature, and using ddH 2 O was set to 25mL, 2mL of nitrogen was taken and dried (small amounts of ddH were added) 2 O was repeatedly dried 2 times), and finally resuspended in 1ml of ph2.2, 0.02M hydrochloric acid buffer, filtered through a 0.45 μm filter, and detected using a irish L8900 amino acid autoanalyzer (hitachi, japan).
The amino acid analysis diagram is shown in figure 5, and the hydroxylation of the recombinant collagen is successfully realized by coexpression of a collagen gene and a bacillus anthracis proline hydroxylase gene; IPTG was added at various times after arabinose induction to achieve a maximum hydroxylation of 63.6% after co-expression condition optimization (table 1). The proline hydroxylation rate is calculated according to the following formula:
(2) Round two chromatographic detection
The recombinant human fusion collagen solution prepared in example 2 was concentrated to a final concentration of 0.5mg/mL in a 10kDa Spin-XR UF 500 centrifugal concentrator (millipore) in 20mM phosphate buffer, and stored at 4 ℃. And (3) carrying out spectrum scanning on the recombinant collagen solution by using a circular dichroscope, wherein the scanning wavelength is between 200nm and 260nm, the scanning temperature is 4 ℃, the wavelength step is 0.5nm, the average time is 0.5s, and the CD spectrum is the average value of three scans. As shown in FIG. 6, the maximum peak of the recombinant collagen prepared in example 2 of the present invention is a negative peak at 221nm and less than 200nm, which accords with the structural characteristics of triple helix of collagen and is a recombinant collagen with triple helix structure.
The solution of the non-hydroxylated recombinant human fusion collagen is treated according to the method, the temperature is raised at the speed of 1 ℃/min, and the relation between the molar ellipticity and the temperature of the hydroxylated recombinant collagen and the non-hydroxylated recombinant collagen is measured at 221 nm. As shown in FIG. 7, the denaturation temperature of the recombinant human fusion collagen prepared in the embodiment 2 of the invention is 38 ℃, and the denaturation temperature of the unhydroxylated recombinant collagen is 30.5 ℃, which indicates that the recombinant collagen has more stable structure due to the increased hydroxyproline content in the recombinant collagen after the bacillus anthracis proline hydroxylase is co-expressed.
Example 4: biological activity detection of recombinant human fusion collagen
The coding nucleotide sequence of the type I collagen peptide (i.e. the nucleotide sequence is obtained by repeating the nucleotide sequence of the type I collagen peptide derived from SEQ ID NO. 2) is constructed to an expression vector pET28a in series for 4 times (i.e. I-I-I-I), so as to obtain a recombinant vector pET28a-rhCOL I; the coding nucleotide sequence of the III type collagen peptide is connected in series for 4 times (namely III-III) (the nucleotide sequence is obtained by repeating the III type collagen peptide nucleotide sequence from SEQ ID NO. 2) and is constructed into an expression vector pET28a to obtain a recombinant vector pET28a-rhCOL III; and respectively converting the pET28a-rhCOL I and pET28a-rhCOL III vectors into an expression strain BL21 (DE 3) to obtain recombinant genetically engineered bacteria for expressing the type I and type III recombinant collagen.
The two recombinant genetically engineered bacteria single colonies were inoculated into 10mL LB medium (Kan + ) Shaking culture is carried out for 10-12 hours at a constant temperature of 37 ℃ and 220rpm to obtain seed liquid.
The seed solution was inoculated into LB medium at an inoculum size of 1% by volume (Kan + ) Culturing in shaking flask with liquid loading amount of 200mL culture medium/1000 mL at 37deg.C and 220rpm for 2-3 hr until OD600 value of the bacterial liquid reaches 0.8, adding 0.1mM IPTG to induce expression, culturing for 5 hr, and measuring OD of bacterial liquid 600 The cells were collected by centrifugation at 8000g for 7min at 4 ℃.
The cells were crushed, purified by a nickel column and purified by cation exchange in the same manner as in example 2 to obtain recombinant type I collagen and recombinant type III collagen having a molecular weight of about 120 kDa.
0.1% (w/v) gelatin solution, 0.1% (w/v) hydroxylated recombinant human fusion collagen solution prepared in example 2 of the present invention, 0.1% (w/v) non-hydroxylated recombinant human fusion collagen solution, 0.1% (w/v) recombinant type I collagen solution, 0.1% (w/v) recombinant type III collagen solution (PBS with pH7.4 as solvent) were coated on 96-well plates (4 complex wells were provided per group), 100. Mu.L of each well was added with an equal amount of PBS solution in a negative control group. Sealing and standing at 4deg.C overnight, collecting mouse fibroblast (NIH-3T 3) grown to 80% of culture dish area, and preparing into 1×10 cell density with high sugar DMEM medium containing 2% (v/v) FBS 5 Cell suspension of individual/mL; 100. Mu.L of cell suspension was added to each well and the wells were incubated at 37℃with 5% CO 2 After culturing in an incubator with saturated humidity for 24 hours, 10 mu L of CCK-8 reagent is added into each group, the mixture is placed in the cell incubator for incubation for 1-4 hours, and the absorbance value of each hole is measured at the wavelength of 450nm by using an enzyme-linked immunosorbent assay. Cell viability was calculated from the absorbance mean of each group according to the following formula:
the experimental results are shown in fig. 8, the activity of the recombinant human fusion type collagen is obviously higher than that of the recombinant type I collagen and the recombinant type III collagen which are expressed independently, and the activity is further obviously improved after the hydroxylation of proline. The hydroxylated recombinant human fusion collagen prepared in the embodiment 2 of the invention has good biocompatibility, no cytotoxicity and good safety.
Comparative example 1: proline hydroxylase of cyst RH1 for hydroxylation modification of recombinant collagen in the present invention
The difference between comparative example 1 and example 2 is that the recombinant expression vectors contained in the recombinant genetically engineered bacteria are pGro7-DsP H and pET28a-rhCOL (I-III), wherein the pGro7-DsP H vector is obtained by replacing the BaP4H gene fragment in the pGro7-BaP4H vector with the cyst RH1 proline hydroxylase DsP H gene (the amino acid sequence is shown as SEQ ID No.5, the nucleotide sequence is biosynthesized by Shanghai workers, and the sequence is shown as SEQ ID No. 6).
SEQ ID No.5:
MLTPTELKQYREAGYLLIEDGLGPREVDCLRRAAAALYAQDSPDRTLEKDGRTVRAVHGCHRRDPVCRDLVRHPRLLGPAMQILSGDVYVHQFKINAKAPMTGDVWPWHQDYIFWAREDGMDRPHVVNVAVLLDEATHLNGPLLFVPGTHELGLIDVERRAPAGDGDAQWLPQLSADLDYAIDADLLARLTAGRGIESATGPAGSILLFDSRIVHGSGTNMSPHPRGVVLVTYNRTDNALPAQAAPRPEFLAARDATPLVPLPAGFALAQPV。
SEQ ID No.6:
Atgctgaccccgactgaactgaaacagtaccgtgaagctggttatctgctgattgaagatggtctgggcccgcgtgaagttgattgcctgcgtcgcgcggcggcggcgctgtacgcgcaggatagcccggatcgtaccctggaaaaagatggccgtaccgttcgtgcggttcacggctgccaccgtcgtgatccggtttgccgcgacttagttcgtcacccgcgtctgctgggcccggctatgcagatcctgagcggcgatgtttacgttcaccagttcaaaatcaacgcgaaagcgccgatgaccggcgacgtgtggccgtggcaccaggattacatcttctgggcgcgtgaagatggtatggatcgtccgcacgttgtgaacgttgcggttctgctggatgaagcgacccacctgaacggcccgctgctgtttgttccgggtacccacgaactgggcctgatcgatgtggaacgtcgtgcgccggcaggcgatggtgatgctcagtggctgccgcagctgagcgcggacctggattacgctattgacgcagatctgctggcccgtctgactgcgggtcgtggcatcgaaagcgcgaccggcccggcgggttctatcctgctgttcgattctcgtattgtgcacggtagcggcaccaacatgagcccgcacccgcgcggcgttgttctggttacctacaaccgtaccgataacgcacttccggcgcaggcagccccgcgtcctgaattcttggcggctcgtgatgcgactccactcgttccgctgccggcgggctttgccctggctcagccggtttaa。
The construction of pGro7-DsP4H vector was similar to example 1, with DsP H gene amplification template pUC57-DsP4H, and the amplification primers were as follows:
upstream primer F3:5'-ACGGACTTTCTCAAAGGAGAGTTATCAATGCTGACCCCGACTGAACTGAAAC-3';
downstream primer R3:5'-TTTATTTCTGCGAGGTGCAGGGCAATTAAACCGGCTGAGCCAGGGCA-3'.
PCR amplification was performed as in example 1, colony PCR identification and sequencing verification were performed, and plasmids were extracted to obtain recombinant expression plasmid pGro7-DsP H. Recombinant plasmid pGro7-DsP H and pET28a-rhCOL (I-III) are mixed according to the mol ratio of 1:1, transforming into escherichia coli BL21 (DE 3) competence by a calcium chloride method, coating on an LB plate containing kana and chloramphenicol resistance, and growing positive transformants, namely the co-expression recombinant genetic engineering bacteria of the comparative example.
Expression purification was performed by the other procedure in example 2, and the hydroxyproline content was determined after acid hydrolysis.
Comparative example 2: the proline hydroxylase gene and the recombinant collagen gene are constructed into a single plasmid, and then co-expressed with two identical promoters (simultaneously expressed under the same promoters)
The difference between comparative example 2 and example 2 is that the recombinant expression vector contained in the recombinant genetically engineered bacterium was pETDute-rhCOL (I-III) -BaP4H, and the construction scheme of the vector is shown in FIG. 9, and the rhCOL (I-III) (labeled rhCOL in the figure) and the proline hydroxylase BaP4H genes were constructed downstream of two T7 promoters of pETDute-1 plasmid (Novagen), respectively. And the successfully constructed recombinant plasmid is transformed into escherichia coli BL21 (DE 3), thus obtaining the co-expression recombinant genetic engineering bacteria of the comparative example. The engineering bacteria single colony is inoculated into 10mL LB culture medium (Amp + ) Shaking culture is carried out for 10-12 hours at a constant temperature of 37 ℃ and 220rpm to obtain seed liquid.
The seed solution was inoculated into LB medium (Amp + ) The liquid loading amount is 200mL culture medium/1000 mL shaking flask, and the flask is placed at 37 ℃ and 220rpm for culturing for about 2 to 3 hours until the bacterial liquid OD 600 The value reaches 0.8, IPTG with the final concentration of 0.1mM is added for induction expression, the culture is continued for 5 hours, and the bacterial liquid OD is measured 600 The cells were collected by centrifugation at 8000g for 7min at 4 ℃.
The hydroxyproline content was determined after crushing, purification and acid hydrolysis as described in example 2.
Comparative example 3: simultaneous expression of enzymes and collagen under different promoters
Comparative example 3 differs from example 2 in that the inducers arabinose and IPTG are both at OD 600 At time=0.8, proline hydroxylase and recombinant human fusion collagen gene expression were simultaneously induced. And expression purification was performed by the other procedure of example 2, and hydroxyproline content was measured after acid hydrolysis.
As shown in the following table, baP4H has better hydroxylation effect on recombinant collagen rhCOL (I-III) than DsP H; under the three co-expression modes that the same promoter and different promoters induce and proline hydroxylase promoter induce earlier than the recombinant collagen promoter, the hydroxylation rate is higher when the adopted enzyme is induced first and then the collagen is induced to express.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> university of North China
<120> recombinant humanized fusion collagen and efficient hydroxylation method and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1104
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> recombinant human fusion collagen
<400> 1
Met Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg
1 5 10 15
Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly
20 25 30
Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile
35 40 45
Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg
50 55 60
Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly
65 70 75 80
Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu
85 90 95
Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg
100 105 110
Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly
115 120 125
Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu
130 135 140
Arg Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg
145 150 155 160
Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly
165 170 175
Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile
180 185 190
Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg
195 200 205
Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly
210 215 220
Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu
225 230 235 240
Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg
245 250 255
Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly
260 265 270
Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu
275 280 285
Arg Gly Gly Gly Ser Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
290 295 300
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
305 310 315 320
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
325 330 335
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
340 345 350
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
355 360 365
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
370 375 380
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
385 390 395 400
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
405 410 415
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
420 425 430
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
435 440 445
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
450 455 460
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
465 470 475 480
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
485 490 495
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
500 505 510
Met Pro Gly Glu Arg Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu
515 520 525
Asn Gly Val Met Gly Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly
530 535 540
Met Pro Gly Glu Arg Gly Ser Glu Ala Ala Ala Lys Gly Glu Arg Gly
545 550 555 560
Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu
565 570 575
Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg
580 585 590
Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
595 600 605
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe
610 615 620
Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala
625 630 635 640
Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly
645 650 655
Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro
660 665 670
Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg
675 680 685
Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg Gly
690 695 700
Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu
705 710 715 720
Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg
725 730 735
Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
740 745 750
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Phe
755 760 765
Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala
770 775 780
Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly
785 790 795 800
Ala Ser Gly Phe Pro Gly Glu Arg Gly Glu Arg Gly Asp Leu Gly Pro
805 810 815
Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg Gly Glu Arg
820 825 830
Gly Glu Arg Gly Ala Ser Gly Phe Pro Gly Glu Arg Gly Gly Gly Ser
835 840 845
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
850 855 860
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
865 870 875 880
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
885 890 895
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
900 905 910
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
915 920 925
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
930 935 940
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
945 950 955 960
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
965 970 975
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
980 985 990
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
995 1000 1005
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
1010 1015 1020
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
1025 1030 1035 1040
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
1045 1050 1055
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
1060 1065 1070
Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Glu Asn Gly Val Met Gly
1075 1080 1085
Phe Pro Lys Pro Gly Glu Pro Gly Pro Lys Gly Met Pro Gly Glu Arg
1090 1095 1100
<210> 2
<211> 3312
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> nucleic acid encoding recombinant human fusion collagen
<400> 2
atgggcgagc gcggcgactt aggcccgcaa ggcatcgcgg gtcagcgtgg cgtagttggt 60
gaacggggtg aacggggtga gcgtggtgct agcggctttc cgggtgaacg tggggaacgt 120
ggtgatctgg gcccgcaggg tatcgcgggt cagcgtggtg ttgtgggcga acgtggtgaa 180
cgtggtgagc gcggtgcatc gggctttcca ggcgaacgcg gggaacgcgg tgatctgggg 240
ccgcagggta ttgccggcca acgtggcgtg gttggtgagc ggggtgagcg cggcgagcgc 300
ggtgcgagcg gtttccccgg cgaacgtggc gagcgtggcg acctgggccc gcaaggcatc 360
gctggccagc gtggcgtggt cggtgaacgt ggcgaacgtg gggaacgtgg cgcctctggc 420
ttccctggcg aacgaggtga acgtggggat ctgggcccgc aaggaattgc gggtcagcgc 480
ggcgttgttg gcgaacgcgg agagcgcggt gagcgtggcg cgagcggctt tccgggcgaa 540
cgcggcgaac gcggtgatct gggtccgcag ggcatcgctg gtcaacgtgg cgtagtcggt 600
gagcgtggag aacgtggcga acgtggcgcg tccggctttc ccggtgaacg aggcgaacgt 660
ggggacctgg gtccacaggg tatcgctggc cagcgcggcg ttgtgggaga gcgtggcgaa 720
cgcggcgaac gcggtgcatc tggcttccca ggcgaacgtg gagaacgcgg tgacctgggt 780
ccgcagggca tcgcgggcca gcgcggtgtg gtcggcgagc gtggtgaacg tggtgaacgg 840
ggcgcgtccg gtttcccagg tgaaagaggt ggcggttccg gccgtcctgg tgaacgcggc 900
ctgccgggtg aaaacggtgt tatgggtttt ccgaaacctg gcgagccggg ccctaaaggc 960
atgccaggtg aacgcggccg tccgggtgaa cgcggtctgc cgggcgaaaa cggtgtgatg 1020
ggctttccga aaccaggtga gccgggcccg aagggtatgc cgggggaacg tggtcgtccg 1080
ggggaacgcg gtctgccggg cgagaacggc gtaatgggct tccctaaacc gggcgaaccg 1140
ggcccgaaag gtatgccggg cgagcgtggc cgccctggtg agcgtggcct gcctggcgaa 1200
aacggtgtga tgggcttccc gaaaccgggt gaaccgggcc cgaaaggcat gccgggtgaa 1260
cgcgggcgtc cgggcgagcg cggcctgcca ggcgaaaatg gtgtaatggg cttcccgaaa 1320
ccaggtgaac cgggtccgaa aggtatgccg ggtgagcgtg gtcgtccggg cgaacgcggg 1380
ctgccgggcg aaaacggcgt tatgggtttc ccgaaaccgg gtgaaccagg cccgaaaggc 1440
atgccgggcg agcgtggtcg ccccggcgaa cgtggtttac cgggcgaaaa tggcgttatg 1500
ggtttcccga agccgggtga accgggtccg aagggcatgc cgggtgaacg tggccgtcct 1560
ggtgaacgcg gtttgccggg tgaaaacggc gtgatgggct tcccaaaacc gggcgaaccg 1620
ggcccgaaag gcatgccggg cgagcgcgga tccgaagcgg cggcgaaagg cgagcgcggc 1680
gacttaggcc cgcaaggcat cgcgggtcag cgtggcgtag ttggtgaacg gggtgaacgg 1740
ggtgagcgtg gtgctagcgg ctttccgggt gaacgtgggg aacgtggtga tctgggcccg 1800
cagggtatcg cgggtcagcg tggtgttgtg ggcgaacgtg gtgaacgtgg tgagcgcggt 1860
gcatcgggct ttccaggcga acgcggggaa cgcggtgatc tggggccgca gggtattgcc 1920
ggccaacgtg gcgtggttgg tgagcggggt gagcgcggcg agcgcggtgc gagcggtttc 1980
cccggcgaac gtggcgagcg tggcgacctg ggcccgcaag gcatcgctgg ccagcgtggc 2040
gtggtcggtg aacgtggcga acgtggggaa cgtggcgcct ctggcttccc tggcgaacga 2100
ggtgaacgtg gggatctggg cccgcaagga attgcgggtc agcgcggcgt tgttggcgaa 2160
cgcggagagc gcggtgagcg tggcgcgagc ggctttccgg gcgaacgcgg cgaacgcggt 2220
gatctgggtc cgcagggcat cgctggtcaa cgtggcgtag tcggtgagcg tggagaacgt 2280
ggcgaacgtg gcgcgtccgg ctttcccggt gaacgaggcg aacgtgggga cctgggtcca 2340
cagggtatcg ctggccagcg cggcgttgtg ggagagcgtg gcgaacgcgg cgaacgcggt 2400
gcatctggct tcccaggcga acgtggagaa cgcggtgacc tgggtccgca gggcatcgcg 2460
ggccagcgcg gtgtggtcgg cgagcgtggt gaacgtggtg aacggggcgc gtccggtttc 2520
ccaggtgaaa gaggtggcgg ttccggccgt cctggtgaac gcggcctgcc gggtgaaaac 2580
ggtgttatgg gttttccgaa acctggcgag ccgggcccta aaggcatgcc aggtgaacgc 2640
ggccgtccgg gtgaacgcgg tctgccgggc gaaaacggtg tgatgggctt tccgaaacca 2700
ggtgagccgg gcccgaaggg tatgccgggg gaacgtggtc gtccggggga acgcggtctg 2760
ccgggcgaga acggcgtaat gggcttccct aaaccgggcg aaccgggccc gaaaggtatg 2820
ccgggcgagc gtggccgccc tggtgagcgt ggcctgcctg gcgaaaacgg tgtgatgggc 2880
ttcccgaaac cgggtgaacc gggcccgaaa ggcatgccgg gtgaacgcgg gcgtccgggc 2940
gagcgcggcc tgccaggcga aaatggtgta atgggcttcc cgaaaccagg tgaaccgggt 3000
ccgaaaggta tgccgggtga gcgtggtcgt ccgggcgaac gcgggctgcc gggcgaaaac 3060
ggcgttatgg gtttcccgaa accgggtgaa ccaggcccga aaggcatgcc gggcgagcgt 3120
ggtcgccccg gcgaacgtgg tttaccgggc gaaaatggcg ttatgggttt cccgaagccg 3180
ggtgaaccgg gtccgaaggg catgccgggt gaacgtggcc gtcctggtga acgcggtttg 3240
ccgggtgaaa acggcgtgat gggcttccca aaaccgggcg aaccgggccc gaaaggcatg 3300
ccgggcgagc gc 3312
<210> 3
<211> 216
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> proline hydroxylase (BaP 4H)
<400> 3
Met Thr Asn Asn Asn Gln Ile Gly Glu Asn Lys Glu Gln Thr Ile Phe
1 5 10 15
Asp His Lys Gly Asn Val Ile Lys Thr Glu Asp Arg Glu Ile Gln Ile
20 25 30
Ile Ser Lys Phe Glu Glu Pro Leu Ile Val Val Leu Gly Asn Val Leu
35 40 45
Ser Asp Glu Glu Cys Asp Glu Leu Ile Glu Leu Ser Lys Ser Lys Leu
50 55 60
Ala Arg Ser Lys Val Gly Ser Ser Arg Asp Val Asn Asp Ile Arg Thr
65 70 75 80
Ser Ser Gly Ala Phe Leu Asp Asp Asn Glu Leu Thr Ala Lys Ile Glu
85 90 95
Lys Arg Ile Ser Ser Ile Met Asn Val Pro Ala Ser His Gly Glu Gly
100 105 110
Leu His Ile Leu Asn Tyr Glu Val Asp Gln Gln Tyr Lys Ala His Tyr
115 120 125
Asp Tyr Phe Ala Glu His Ser Arg Ser Ala Ala Asn Asn Arg Ile Ser
130 135 140
Thr Leu Val Met Tyr Leu Asn Asp Val Glu Glu Gly Gly Glu Thr Phe
145 150 155 160
Phe Pro Lys Leu Asn Leu Ser Val His Pro Arg Lys Gly Met Ala Val
165 170 175
Tyr Phe Glu Tyr Phe Tyr Gln Asp Gln Ser Leu Asn Glu Leu Thr Leu
180 185 190
His Gly Gly Ala Pro Val Thr Lys Gly Glu Lys Trp Ile Ala Thr Gln
195 200 205
Trp Val Arg Arg Gly Thr Tyr Lys
210 215
<210> 4
<211> 648
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> nucleic acid encoding proline hydroxylase (BaP 4H)
<400> 4
atgactaata ataaccaaat aggtgaaaat aaagagcaga ccatctttga ccataaaggt 60
aacgtaatta aaactgaaga tcgtgaaatc cagataatta gtaaatttga agaaccgctg 120
atagtagttt taggtaacgt gttaagtgat gaagaatgcg atgaactgat tgaactgagt 180
aaaagtaaac ttgcccgctc taaagtaggt agtagccgtg acgtgaatga catccgcact 240
agtagcggcg cattccttga tgataatgag ttgacggcta aaatcgaaaa acgtatcagc 300
agcatcatga acgttccggc gagccacggt gaaggcctgc acatcctgaa ctacgaagtt 360
gatcagcagt acaaagcgca ctacgattac ttcgcggaac acagccgtag cgcggcgaac 420
aaccgtatca gcaccctggt tatgtacctg aacgatgttg aagaaggcgg tgaaaccttc 480
ttcccgaaac tgaacctgag cgttcacccg cgtaaaggca tggcggttta cttcgaatac 540
ttctaccagg atcagagcct gaacgaactg accctgcacg gtggtgcgcc ggttaccaaa 600
ggtgaaaaat ggatcgcgac ccagtgggtt cgtcgtggta cctacaaa 648
<210> 5
<211> 272
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> proline hydroxylase DsP H
<400> 5
Met Leu Thr Pro Thr Glu Leu Lys Gln Tyr Arg Glu Ala Gly Tyr Leu
1 5 10 15
Leu Ile Glu Asp Gly Leu Gly Pro Arg Glu Val Asp Cys Leu Arg Arg
20 25 30
Ala Ala Ala Ala Leu Tyr Ala Gln Asp Ser Pro Asp Arg Thr Leu Glu
35 40 45
Lys Asp Gly Arg Thr Val Arg Ala Val His Gly Cys His Arg Arg Asp
50 55 60
Pro Val Cys Arg Asp Leu Val Arg His Pro Arg Leu Leu Gly Pro Ala
65 70 75 80
Met Gln Ile Leu Ser Gly Asp Val Tyr Val His Gln Phe Lys Ile Asn
85 90 95
Ala Lys Ala Pro Met Thr Gly Asp Val Trp Pro Trp His Gln Asp Tyr
100 105 110
Ile Phe Trp Ala Arg Glu Asp Gly Met Asp Arg Pro His Val Val Asn
115 120 125
Val Ala Val Leu Leu Asp Glu Ala Thr His Leu Asn Gly Pro Leu Leu
130 135 140
Phe Val Pro Gly Thr His Glu Leu Gly Leu Ile Asp Val Glu Arg Arg
145 150 155 160
Ala Pro Ala Gly Asp Gly Asp Ala Gln Trp Leu Pro Gln Leu Ser Ala
165 170 175
Asp Leu Asp Tyr Ala Ile Asp Ala Asp Leu Leu Ala Arg Leu Thr Ala
180 185 190
Gly Arg Gly Ile Glu Ser Ala Thr Gly Pro Ala Gly Ser Ile Leu Leu
195 200 205
Phe Asp Ser Arg Ile Val His Gly Ser Gly Thr Asn Met Ser Pro His
210 215 220
Pro Arg Gly Val Val Leu Val Thr Tyr Asn Arg Thr Asp Asn Ala Leu
225 230 235 240
Pro Ala Gln Ala Ala Pro Arg Pro Glu Phe Leu Ala Ala Arg Asp Ala
245 250 255
Thr Pro Leu Val Pro Leu Pro Ala Gly Phe Ala Leu Ala Gln Pro Val
260 265 270
<210> 6
<211> 819
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> nucleic acid encoding proline hydroxylase DsP H
<400> 6
atgctgaccc cgactgaact gaaacagtac cgtgaagctg gttatctgct gattgaagat 60
ggtctgggcc cgcgtgaagt tgattgcctg cgtcgcgcgg cggcggcgct gtacgcgcag 120
gatagcccgg atcgtaccct ggaaaaagat ggccgtaccg ttcgtgcggt tcacggctgc 180
caccgtcgtg atccggtttg ccgcgactta gttcgtcacc cgcgtctgct gggcccggct 240
atgcagatcc tgagcggcga tgtttacgtt caccagttca aaatcaacgc gaaagcgccg 300
atgaccggcg acgtgtggcc gtggcaccag gattacatct tctgggcgcg tgaagatggt 360
atggatcgtc cgcacgttgt gaacgttgcg gttctgctgg atgaagcgac ccacctgaac 420
ggcccgctgc tgtttgttcc gggtacccac gaactgggcc tgatcgatgt ggaacgtcgt 480
gcgccggcag gcgatggtga tgctcagtgg ctgccgcagc tgagcgcgga cctggattac 540
gctattgacg cagatctgct ggcccgtctg actgcgggtc gtggcatcga aagcgcgacc 600
ggcccggcgg gttctatcct gctgttcgat tctcgtattg tgcacggtag cggcaccaac 660
atgagcccgc acccgcgcgg cgttgttctg gttacctaca accgtaccga taacgcactt 720
ccggcgcagg cagccccgcg tcctgaattc ttggcggctc gtgatgcgac tccactcgtt 780
ccgctgccgg cgggctttgc cctggctcag ccggtttaa 819
<210> 7
<211> 53
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer F1
<400> 7
tacggacttt ctcaaaggag agttatcaat gactaataat aaccaaatag gtg 53
<210> 8
<211> 63
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer R1
<400> 8
gtttatttct gcgaggtgca gggcaattat ttatcatcat catctttata atctttgtag 60
gta 63
<210> 9
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer F2
<400> 9
tcgaccagga cgacagagc 19
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer R2
<400> 10
tgaagtcagc cccatacgat a 21
<210> 11
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer F3
<400> 11
acggactttc tcaaaggaga gttatcaatg ctgaccccga ctgaactgaa ac 52
<210> 12
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> upstream primer R3
<400> 12
tttatttctg cgaggtgcag ggcaattaaa ccggctgagc cagggca 47