CN102167733A - Construction, expression and application of acidly cleavable high-copy antihypertensive peptide tandem gene - Google Patents
Construction, expression and application of acidly cleavable high-copy antihypertensive peptide tandem gene Download PDFInfo
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- CN102167733A CN102167733A CN 201010547009 CN201010547009A CN102167733A CN 102167733 A CN102167733 A CN 102167733A CN 201010547009 CN201010547009 CN 201010547009 CN 201010547009 A CN201010547009 A CN 201010547009A CN 102167733 A CN102167733 A CN 102167733A
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Abstract
The invention discloses construction, expression and application of an acidly cleavable high-copy antihypertensive peptide tandem gene, and provides a method for constructing acidly cleavable antihypertensive peptide tandem and a method for connecting DNA (Deoxyribonucleic Acid) units of an antihypertensive peptide in tandem to form a high copy number. Meanwhile, the invention provides the high-copy antihypertensive peptide tandem DNA constructed by using the method; and the gene has a nucleotide sequence shown as SEQ ID NO. 2. The invention also provides an antihypertensive peptide tandem DNA-containing expression vector pET30a-32PD and converted recombinant escherichia coli thereof. A target polypeptide which is connected in tandem for 32 times is expressed in a fusion mode by using small His label fragments (about 4kDa) on the pET-30a, so that molecular weight of the polypeptide reaches about 26kDa (the weight molecular of total recombinant protein is about 30kDa), proportion of the target polypeptide in the recombinant protein is increased, expression efficiency is greatly increased and separation difficulty of a small peptide is reduced.
Description
Technical field
The present invention relates to the genetically engineered field, relate to preparation high copy number blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp more specifically
-Gly-Asp the DNA that connects utilizes its expression vector to prepare the method for described recombinant polypeptide and the medical usage of described recombinant polypeptide.
Background technology
Essential hypertension is to be caused by multiple pathogenic factors and complicated pathogenesis, and comprising heredity, food habits, the mental status etc., in the research of treatment hypertension drug, the research of blood pressure lowering peptide has become focus at present.Blood pressure lowering peptide is a kind of hypertensinase (Angiotensin I-Converting Enzyme, ACE) inhibitor, by suppressing the activity of blood plasma and vascular endothelial cell ACE, regulate renin-angiotensin system (Renin-Angiotensin System, RAS) and kallikrein kinin system (Kallikrein-Kinin System, KKS) (referring to: Yin Xiaofeng. wait the people, the Chinese Pharmacological circular, 19:971-975 (2003).) play hypotensive effect.
Now successively from multiple animal-plant material and tankage, as viper venom, sardines, cheese, soybean meal, fermenting bean milk, corn refuse lac, former albumen (referring to Huang Jiayin. wait the people, food and fermentation industries, 32:81-86(2006).) wait and isolated multiple active polypeptide with antihypertensive function, because of its blood pressure lowering effect is obvious, safe without toxic side effect, become the focus of bioactive peptide research.
Blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp be a kind of from bonita flesh of fish hydrolyzate (referring to Kohama, people such as Y., AgricBiol Chem., 55:2169-2170(1991); People such as Fida Hasan., Biochemistry, 41:505-511 (2006).) separate the bioactive peptide obtain, IC
50Value is about 0.9 μ M, but separates this bioactive peptide complex steps from flesh of fish hydrolyzate, yields poorly, and the industrial separation method of being fit to is not arranged at present as yet.
Development of biology provides a kind of new method for producing blood pressure lowering peptide at present, but blood pressure lowering peptide is owing to generally all be the small peptide that several amino acid is formed, and to realize the directly expression in the host bacterium and separate very difficulty of small peptide, and take the amalgamation and expression mode also very low because of adopting bigger fusion tag and low hypotensive gene copy to cause expressing output.(referring to: Zhang Lijun. wait the people, Chinese biochemical drug magazine, 27 (1): 19-21(2006).）
Therefore make up and contain gene, and adopt less fusion tag to come this gene pairs of amalgamation and expression to realize that efficiently expressing with suitability for industrialized production of this blood pressure lowering peptide has significance with suitable high copy number.
Summary of the invention
Provide the structure theobromine to cut the placed in-line method of blood pressure lowering peptide in a first aspect of the present invention.With blood pressure lowering peptide head and the tail linear series, the connection site of polypeptide is formic acid cracking peptide bond specificity site, can be cracked into the blood pressure lowering peptide monomer under the formic acid effect again.
Provide the method that blood pressure lowering peptide dna single unit is connected into high copy number in a second aspect of the present invention, utilize design in the isocaudarner site at blood pressure lowering peptide dna single unit two ends repeatedly enzyme cut to connect and be built into high copy number series connection DNA.
In a third aspect of the present invention, provide to make up high copy blood pressure lowering peptide series connection DNA according to the method described above, this gene has the nucleotide sequence shown in SEQ ID NO2.
The expression vector pET30a-32PD that contains above-mentioned blood pressure lowering peptide series connection DNA is provided in a fourth aspect of the present invention.
The recombination bacillus coli that utilizes above-mentioned carrier to transform is provided in a fifth aspect of the present invention.This bacterial classification is the Chinese typical culture collection center preservation in Chinese Wuhan on August 19th, 2010, and deposit number is intestinal bacteria
Escherichai coliBL21-M32PD CCTCC NO:M 2010205.
Advantage of the present invention is: utilize pET-30a to go up less His label segment (about 4kDa) 32 desired polypeptides of amalgamation and expression of series (Pro-Thr-His-Ile-Lys-Trp-Gly-Asp), make the molecular weight of polypeptide reach (about total recombinant protein molecular weight 30kDa) about 26kDa, increase the shared ratio of desired polypeptides in the recombinant protein, increased expression efficiency and the separating difficulty that has reduced little peptide greatly.
Description of drawings
Wherein Fig. 1 makes up synoptic diagram for multi-copy gene of the present invention;
Fig. 2 is for containing different copy gene plasmid carrier PCR electrophorograms;
Fig. 3 is different copy gene plasmid carrier double digestion electrophorograms;
Fig. 4 is a recombinant plasmid PET30a-32PD double digestion electrophorogram;
Fig. 5 is a BL21-M32PD engineering bacteria abduction delivering electrophorogram of the present invention, wherein annotates 1, protein Marker; 2: the BL21 bacterial strain (not inducing) 3 that contains PET-32PD: the BL21 bacterial strain (IPTG induces) that contains PET-32PD; 4: BL21 bacterial strain (IPTG induces) the ultrasonication precipitation 5 that contains PET-32P: BL21 bacterial strain (IPTG induces) the ultrasonication supernatant that contains PET-32PD.
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Embodiment 1Theobromine is cut blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp tandem polypeptide unit design
The N end of the blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp of the present invention's design is Asp for Pro, C end.The Asp-Pro sequence is the specificity cleavage site of formic acid, can specificity rupture at the peptide bond between the Asp-Pro sequence under 70% the formic acid effect, for obtaining the dna sequence dna of suitable size, so earlier blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp is connected 4 times from beginning to end, is Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp(SEQ ID NO3).
Embodiment 2Theobromine is cut the design of blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp series connection dna single unit
1, in order to obtain blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp high copy number series connection DNA, adds at series connection blood pressure lowering peptide (sequence is SEQ ID NO3) corresponding DNA two ends
BamThe H I and
BglII isocaudarner restriction enzyme site is used to make up different copy number series connection DNA.
BamThe H I and
BglThe amino acid of II isocaudarner restriction enzyme site GGATCC and AGATCA correspondence is respectively Gly-Ser and Arg-Ser, in order not introduce additional amino acid, with one of its reading frame displacement, filling Nucleotide is GGGGATCCG and GGAGATCAG, corresponding amino acid is Gly-Asp-Pro and Gly-Asp-Leu, and Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp peptide sequence is adjusted to Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Pro-Thr-His-Ile-Lys-Trp-Gly-Asp-Leu-Tyr(SEQ ID NO4).
2, the Gly-Asp-Pro with step 1 gained peptide sequence N end and C end becomes nucleotide sequence GGGGATCCG and GGAGATCAGTAT with the Gly-Asp-Leu-Tyr amino acid sequence translation, all the other press the optimization of e. coli codon frequency of utilization, and nucleotide sequence is shown in SEQ ID NO1:
GGGGATCCGACCCATATTAAATGGGGCGATCCGACCCACATCAAGTGGGGTGACCCGACGCATATTAAATGGGGCGATCCGACCCATATCAAATGGGGAG
3, add at 5 of sequence SEQ ID NO1 ' end then
KpnI restriction enzyme site, 3 ' end adds
HindThe III restriction enzyme site, nucleotide sequence is shown in SEQ ID NO2:
GGTACCGGGGATCCGACCCATATTAAATGGGGCGATCCGACCCACATCAAGTGGGGTGACCCGACGCATATTAAATGGGGCGATCCGACCCATATCAAAT
Embodiment 3The structure that contains the high copy series connection of blood pressure lowering peptide Pro-Thr-His-Ile-Lys-Trp-Gly-Asp DNA recombinant vectors
1, nucleotide sequence SEQ ID NO2 is synthetic by Shanghai rising sun hat biotech development company limited, is cloned into pUC18 carrier (available from Novagen company), and the restriction enzyme site of insertion is
KpnI and
HindIII is with this recombinant vectors called after pUC18-4PD.
2, gained pUC18-4PD is utilized pUC18 universal primer M13PrimerRV (CAGGAAACAGCTATGAC) and M13M13PrimerM3 (GTAAAACGACGGCCAGT) (available from TaKaRa company) carry out PCR and get the purpose segment, reclaim the purpose segment and carry out
BamThe H I and
HindIII double digestion (available from TaKaRa company) double digestion.
3, the pUC18-4PD carrier is carried out
BglII and
HindIII double digestion (available from TaKaRa company) reclaims the big segment of carrier.
4, because
BamThe H I and
BglII is an isocaudarner, utilizes the T4 ligase enzyme that purpose segment and the large stretch of disconnection of step 3 recovery carrier that step 2 reclaims connect, transformed into escherichia coli (
Escherichia coli) DH5 α, carrying out bacterium colony PCR, screening positive clone is cultivated positive colony, extracts plasmid and carries out
BamThe H I and
HindIII double digestion (available from TaKaRa company) is identified, obtains to contain the plasmid of 8 repetition blood pressure lowering peptide dna sequence dnas, called after pUC18-8PD.
5,, obtain to contain the plasmid of 16 repetition blood pressure lowering peptide dna sequence dnas, called after pUC18-16PD with pUC18-8PD repeating step 2-4 operation.PUC18-16PD is continued the plasmid that above step 2-4 can obtain to contain 32 repetition blood pressure lowering peptide dna sequence dnas, called after pUC18-32PD.
6, the DH5 α bacterial strain that will contain the pUC18-32PD plasmid is delivered to the order-checking of Shanghai living worker's biotechnology company limited, and sequencing result is consistent with design.(above operation as shown in Figure 1, the PCR of the plasmid that is obtained and
BamThe H I and
HindIII double digestion electrophoresis result is shown in Fig. 2 and 3.）
7, will be undertaken by 32 the multiple plasmid pUC18-32PD that contain of above operation gained
KpnI and
HindIII (available from TaKaRa company) double digestion reclaims the purpose segment about 750bp.
8, simultaneously pET30a carrier (available from Novagen company) is carried out same double digestion, reclaim big segment.
9, utilize the T4 ligase enzyme that the purpose segment and the large stretch of phase failure of pET30a carrier of reclaiming connected, transformed into escherichia coli DH5 α carries out bacterium colony PCR, and screening positive clone extracts plasmid and carries out
KpnI and
HindIII double digestion (available from TaKaRa company) is identified (as shown in Figure 4), obtains to contain the plasmid of 32 repetition blood pressure lowering peptide dna sequence dnas, called after pET30a-32PD.
10, the DH5 α bacterial strain that will contain the PET30a-32PD plasmid is delivered to the order-checking of Shanghai biotechnology company limited, and sequencing result is consistent with design.Extract the pET30a-32PD plasmid from containing the correct DH5 α bacterial strain of order-checking, transformed into escherichia coli (
Escherichia coli) BL21, with obtained strains called after BL21-M32PD, this bacterial classification in 2010 in China's typical culture collection center preservation, deposit number is
Escherichai coliBL21-M32PD CCTCC M 2010205.
Embodiment 3The expression and the detection of reorganization tandem polypeptide
E. coli bl21-M32PD that example 2 is obtained is that 1% inoculum size is inoculated in 50 mL and contains kantlex Kan(50 μ g/ml by volume) LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L, pH7.0) in, 37 ℃, 200 rmin
-1It is 1.5 o'clock that the constant temperature shaking table is cultured to OD600, adds isopropyl-(IPTG) to final concentration 1 mmolL
– 1, induce 12 h for 28 ℃, 5000 rmin
-1Centrifugal collection thalline freezes thalline molten 3 times repeatedly, carries out ultrasonication (300W, 2s/2s, 2 min), 8000 rmin at PBS buffered soln (pH7.0)
-1Centrifuging and taking precipitation and supernatant carry out the SDS-PAGE electrophoresis, and electrophorogram as shown in Figure 5.Electrophoresis shows intestinal bacteria induces the back target protein mainly to be present in the precipitation of intestinal bacteria ultrasonication liquid after centrifugal, and the molecular weight size of recombinant protein is about 30 kDa, coincide with the molecular weight size of design.
Embodiment 4Reorganization monomer polypeptide prepares purifying
The precipitation of the centrifugal gained of embodiment 3 described methods is dissolved in carries out 8ml upper prop buffered soln, carry out activatory Ni-post (Novagen, the U.S.) purifying obtains the recombinant protein of His mark, recombinant protein fully is dissolved in the 6M urea, add two volumes pH and be 8.0 0.1% enteropeptidase solution (available from Novagen company), 37 ℃ of temperature were bathed 8 hours, carry out activatory Ni-post (available from Novagen company) and remove His mark fusion tag, make the tandem polypeptide of fusion tag, 70% formic acid that the resultant tandem polypeptide solution that gets is added 1 times of volume, 50 ℃ of temperature were bathed 12 hours, CELLUFINE GH-25 post (available from CHISSO company) filtration chromatography gained purified polypeptide, detecting monomer polypeptide expression amount through bicinchoninic acid (BCA) method is 0.38gL
– 1
Embodiment 5Reorganization monomer polypeptide active detects
The used polypeptide of active detection is according to embodiment 3 and the preparation of embodiment 4 methods.
External activity detection employing Vermeirssen etc. are improved to be the spectrophotometry of angiotensin stand-in with FAPGG, as table 1, records IC
50Value is 1.8 mg/L, has good blood pressure lowering effect, than the activity value (IC of bibliographical information
500.86 mg/L) low (referring to Kohama, people such as Y., AgricBiol Chem., 55:2169-2170(1991).）。
Table 1 ACE suppresses active mensuration
| ? | Blank well (μ L) | Sample well (μ L) |
| ACE(0.1U/mL) | 10 | 10 |
| FAPGG(mmol/L) 1 | 50 | 50 |
| The matrix damping fluid 2 | 40 | 0 |
| Recombinant polypeptide | 0 | 40 |
Annotate: 1.FAPGG (1.0mmol/L): get 3.994mg FAPGG and add the matrix damping fluid, be settled to 10ml, dissolving mixes, and puts 4 ℃ of lucifuges and places; 2. matrix damping fluid: HEPES 1.910g, NaCl 1.755g after the bi-distilled water dissolving, is transferred to pH8.3 restock water to 100ml with NaOH, put 4 ℃ standby.
Claims (5)
1. a theobromine is cut the construction process of series connection blood pressure lowering peptide, and with blood pressure lowering peptide head and the tail linear series, the connection site of polypeptide is formic acid cracking peptide bond specificity site, can be cracked into the blood pressure lowering peptide monomer under the formic acid effect again.
2. blood pressure lowering peptide dna single unit is connected into the method for high copy number, utilize design in the isocaudarner site at blood pressure lowering peptide dna single unit two ends repeatedly enzyme cut to connect and be built into high copy number series connection DNA.
3. the height copy blood pressure lowering peptide series connection DNA that method according to claim 2 makes up, this gene has the nucleotide sequence shown in SEQ ID NO2.
4. the expression vector pET30a-32PD that contains the described blood pressure lowering peptide series connection of claim 3 DNA.
5. the recombination bacillus coli that utilizes the described carrier of claim 3 to transform, deposit number is
Escherichai coliBL21-M32PD CCTCC NO:M 2010205.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344900A (en) * | 2011-10-20 | 2012-02-08 | 江苏大学 | Engineering bacterium for expressing antihypertensive peptide and method for preparing antihypertensive peptide |
| CN104762354A (en) * | 2015-03-11 | 2015-07-08 | 江苏大学 | Method of increasing forming of recombinant protein inclusion body |
| CN105541969A (en) * | 2015-12-28 | 2016-05-04 | 合肥安德生制药有限公司 | Matrix metalloproteinase cleaved sequence peptide, expression vector, polynucleotide sequence and application |
| CN112080493A (en) * | 2020-09-07 | 2020-12-15 | 中国科学院天津工业生物技术研究所 | Method for preparing tandem repeat DNA, related biological material and application |
-
2010
- 2010-11-17 CN CN 201010547009 patent/CN102167733A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 《Agric. Biol. Chem.》 19911231 Yasuhiro Kohama et al Potent synthetic analogues of angiotensin-converting enzyme inhibitor derived from Tuna muscle 2169-2170 第55卷, 第8期 * |
| 《Process Biochemistry》 20061231 Fida Hasan et al Production kinetics of angiotensin-I converting enzyme inhibitory peptides from bonito meat in artificial gastric juice 505-511 第41卷, * |
| 《UniProtKB/Swiss-Prot》 20090728 Y.Kohama et al P18691 1 , * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344900A (en) * | 2011-10-20 | 2012-02-08 | 江苏大学 | Engineering bacterium for expressing antihypertensive peptide and method for preparing antihypertensive peptide |
| CN104762354A (en) * | 2015-03-11 | 2015-07-08 | 江苏大学 | Method of increasing forming of recombinant protein inclusion body |
| CN104762354B (en) * | 2015-03-11 | 2018-06-26 | 江苏大学 | A kind of method for improving recombinant protein inclusion body and being formed |
| CN105541969A (en) * | 2015-12-28 | 2016-05-04 | 合肥安德生制药有限公司 | Matrix metalloproteinase cleaved sequence peptide, expression vector, polynucleotide sequence and application |
| CN112080493A (en) * | 2020-09-07 | 2020-12-15 | 中国科学院天津工业生物技术研究所 | Method for preparing tandem repeat DNA, related biological material and application |
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Application publication date: 20110831 |