CN100404682C - Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application - Google Patents
Amphioxus dimethyl arginine hydrolase AmphiDDAH gene and its application Download PDFInfo
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- 101710085388 N(G),N(G)-dimethylarginine dimethylaminohydrolase Proteins 0.000 claims abstract description 25
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Abstract
The present invention relates to an amphioxus AmphiDDAH gene, a protein coded by the gene, and an application of the protein for preparing medicine for curing cardiovascular disease and arthritis. The amphioxus AmphiDDAH gene is separated from an amphioxus neurula by the cDNA library construction and large-scale sequence method, the DNA sequence of the thioredoxin peroxidase gene TPx is shown in a sequence <400>1 of a sequence table, and the amino acid sequence of the protein (P<DDAH>) coded by the gene is shown in a sequence <400>2 of the sequence table. The recombination amphioxus P<DDAH> protein has a function for adjusting NO generation, and can be used for preparing medicine for curing cardiovascular disease and arthritis.
Description
Technical field
The present invention relates to two methylarginine lytic enzyme (AmphiDDAH) genes of lancelet and encoded protein P thereof
DDAH, and the application of this albumen in preparation treatment cardiovascular disorder and arthritis drug.
Background technology
Lancelet (Branchiostoma belcheri tsingtauense), belong to Chordata (Chordata), Cephalochordata (cephalochordate), it is existing and the nearest invertebrates of vertebrates sibship, also being vertebrates ancestors' model, is than primary animal class in the ocean.In the lancelet embryo development procedure, for being differentiated to form of the immunizing power that improves self and tissue and organ, the lancelet embryo can produce DDAH, two methylarginines (ADMA) of hydrolysis asymmetry, strengthen the activity of nitric oxide synthetase (NOS), thereby regulate nitrogen protoxide (NO) content in vivo.
Ogawa in 1989 at first separates in the mouse liver with the biochemical method of extracting and obtains DDAH, this enzyme is the wall scroll peptide chain, molecular weight is about 33KDa, iso-electric point is 5.2, its its substrate A of hydrolysis specifically DMA generation guanidine propylhomoserin and methylamine, field of activity does not need cofactor between pH5.5-pH7.5.People such as calendar year 2001 Murray-Rust discover that to bacterium DDAH crystalline structure (Glu-114) three avtive spots combine with the inhibitor ADMA of its substrate NOS DDAH for C-249, His-162, and its substrate of hydrolysis by Cys-His-Glu.In the processing of myelin basic protein, heat shock protein, nucleus molecule protein, modification, discharge the arginine that methylates freely (ADMA), these molecular distribution have different concentration distribution in different tissues and organ in cytosol, blood plasma, tissue.Under pathological conditions, in the blood plasma as hyperlipidemia, hypertension, ephrosis change, atheroma patient, in the tissue juice at rheumatoid arthritis position,, cause ADMA concentration to rise because the activity of DDAH descends, the NO level descends, the generation of lemma pathology.Inflammatory cytokine IL-1 β can induce the expression of DDAH, reduces ADMA content, thereby increases the activity of NOS, increases the NO level, thus the growth of stimulated vascular smooth muscle cell.Vitamin A acid also can pass through to stimulate the expression of vascular endothelial cell DDAH, thereby regulates the maturation and the growth of cardiovascular systems.NO also can increase the content of cGMP by the activity of soluble guanylate cyclase in the stimulated vascular smooth muscle cell, causes smooth muscle loosening, is a kind of endogenic vasorelaxation thing.In sacroiliitis, the chondrocyte is insensitive to the relevant growth factors I (IGF-I) of Regular Insulin, and NO content raises, and is exposed among the NO when the chondrocyte is broken, and the phosphorylation of the IGF-I acceptor that IGF-I stimulates reduces, thereby influences chondrocyte's growth.Therefore, research and development lancelet DDAH is used for the treatment of cardiovascular disorder and osteoarthropathy, is a job highly significant.
At present, domestic research report to DDAH is less.NO content is had the DDAH of important regulating effect, carry out gene recombination, expression, tool has very important significance.
Summary of the invention
The object of the present invention is to provide a kind of new lancelet AmphiDDAH gene and encoded protein thereof.
Another object of the present invention is to provide the P of said gene
DDAHThe application of albumen in preparation treatment cardiovascular disorder and arthritis drug.
The present invention by the cDNA library structure and the method for large scale sequencing, from the lancelet neurula, separate obtaining neurula AmphiDDAH gene, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.
Albumen (recombinant protein DDAH, called after P that the invention described above gene A mphiDDAH is coded
DDAH), its aminoacid sequence as in the sequence table＜400〉2 sequences shown in; Iso-electric point is 4.12, and molecular weight is 30,649 dalton.
This albumen by expression vector pETTRX-AmphiDDAH in intestinal bacteria with soluble formal representation in the born of the same parents.
Described expression vector pETTRX-AmphiDDAH is that setting up with sulphur oxygen cyclase protein is the fusion companion body, the middle affinity labelling site of His and the efficient expression vector of protease 3 C recognition site of inserting, and this system makes P
DDAHIn intestinal bacteria with soluble formal representation in the born of the same parents.
The selected Qingdao branchiostoma of the present invention (Branchiostoma belcheri tsingtauense) is collected in sand saliva territory, Shandong Province.
The structure in Qingdao branchiostoma neurula cDNA library: extract the total RNA of neurula,, by the LD-PCR synthetic dsdna, after Sfi I enzyme is cut, be connected Transformed E .coli with pcDNA3.0, the construction cDNA library then by the synthetic chain DNA of inverse PCR.
The present invention has therefrom obtained the clone of 1 new coding lancelet DDAH by to the extensive sequencing of library recombinant clone, called after AmphiDDAH (its dna sequence dna as in the sequence table＜400〉1 sequences shown in).New 274 amino acid of genes encoding (its aminoacid sequence as in the sequence table＜400〉2 sequences shown in), iso-electric point is 4.12, molecular weight is 30,649 dalton, has conservative DDAH enzyme active sites.
The present invention is by having designed a pair of primer, and the new gene A mphiDDAH that will encode is cloned into prokaryotic fusion expression vector pETTRX and is gone up (this experiment structure), construction expression plasmid and with its transformed into escherichia coli BL21 (DE3).This expression vector (pETTRX-AmphiDDAH) is promotor with T7, TRX is that molecule merges the companion body, helps recombinant protein correctly folding, with soluble formal representation, the C end of TRX has gentle sequence and 6 * His structure, is convenient to utilize immobilization metal part affinity chromatography to carry out purifying.Designed upstream primer contains protease 3 C site, is beneficial to the monomeric acquisition of foreign protein.By to incubation time, induction time, the groping and optimize P of conditions such as temperature
DDAHThe Expression of Fusion Protein amount is higher, and major part is in solvable state.
The present invention also gropes and has optimized reorganization P
DDAHProteic purification condition, the ultrasonic degradation liquid of expression product is through Ni
2+Chelating Sepharose affinity chromatography obtains fusion rotein, and fusion rotein can obtain purity at the P more than 90% through cutting of 3C proteolytic enzyme and Q anion exchange chromatography
DDAHAlbumen.
Expression plasmid clone method of the present invention:, use CaCl with reference to Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Labroratory Press.USA) method
2Method with plasmid Transformed E .coli.DH5 α or BL21 (DE3) bacterial strain, transform bacterial strain with the LB culture medium culturing that contains penbritin (100 μ g/mL), alkaline process extracts plasmid.
Description of drawings
Fig. 1 is lancelet P of the present invention
DDAHAlbumen and the proteic comparative result of other species DDAH, wherein " * " expression is identical; ": " expression difference is less; ". " expression obvious difference; Complete difference is represented in the space.
Fig. 2 is the total RNA of lancelet neurula.
The double-stranded cDNA electrophoresis result of Fig. 3.
Fig. 4 is the recombinant expression plasmid pETTRX-AmphiDDAH design of graphics of gene A mphiDDAH.
Fig. 5 is reorganization P
DDAHProtein induced expression, affinity chromatography electrophorogram.1: lower molecular weight standard protein marker; 2: without inductive pETTRX-AmphiDDAH bacterial protein; 3: through IPTG inductive pETTRX-AmphiDDAH bacterial protein; 4: induce the ultrasonic supernatant total protein of bacterium; 5,6,7,8,9 are respectively 50mM, 100mM, 200mM, 300mM, 400mM imidazoles elution peak albumen;
Fig. 6 is reorganization P
DDAHThe G25 molecular sieve and the SDS-PAGE of Q post affinity chromatography analyze.1: lower molecular weight standard protein marker; 2: the TRX-P that the process affinity chromatography obtains
DDAHFusion rotein; 3:TRX-P
DDAHFusion rotein cuts through 3C proteolytic enzyme (Prescission proteolytic enzyme); The reorganization P of 4:Q column purification
DDAHAlbumen.
Embodiment
Below implement to help those of ordinary skill in the art further to understand the present invention, but do not limit the present invention in any form.
Embodiment 1: the extraction of the total RNA of lancelet neurula and cDNA's is synthetic
The extraction of total RNA and cDNA are synthetic: collect the lancelet neurula, adopt Trizol reagent method to extract the total RNA of neurula, protein is removed in phenol/chloroform extracting, obtains the total RNA of lancelet neurula, its A
260/ A
280=1.828, detect as seen 18s and 28s two bands clearly through 1% denaturing formaldehyde gel electrophoresis, ratio＞1 (see figure 2) shows that total RNA integrity is better, purity is also higher.Get the total RNA of 1ug, with SMART III olignuclotide (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ') and CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)
30N
-1N-3 ') carries out synthetic first chain of reverse transcription, obtain 10 μ l, one chain cDNA product.
Getting 2ulcDNA one chain product, is that primer carries out the synthetic two chain cDNA of LD-PCR with 5 ' PCR Primer (5 '-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3 ') and 3 ' PCR Primer (5 '-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3 ').
Embodiment 2: the mensuration and the analysis of lancelet neurula AmphiDDAH gene order
Two chain cDNA are connected to the pcDNA3.0 carrier, transform DH5 α intestinal bacteria, select the recombinant clone order-checking.Blast homology analysis revealed has obtained the est sequence of coding total length AmphiDDAH gene, and mrna length all is 822bp, and encode 274 amino acid whose P
DDAHAlbumen is 1 new DDAH albumen.
Use the ClustalW analysis software, to the DDAH protein sequence of the different plant species reported relatively, the result as shown in Figure 1.
As can be seen from Figure 1, three of DDAH avtive spots are comparatively conservative in different species.
Embodiment 3: reorganization P
DDAHThe structure of expression plasmid
According to the synthetic a pair of primer of two terminal sequences of AmphiDDAH gene, upstream primer contains Kpn I and Prescission proteolytic enzyme cutting site, and downstream primer contains Not I cleavage site.
Upstream primer (P1):
5’CGG?
GGT?ACC?
CTG?GAA?GTT?CTG?TTC?CAG?GGG?CCC?ATG?GCG
Kpn I Prescission proteolytic enzyme cutting site
GAT?TTC?TGT?TGG?AAT?TT3’；
Downstream primer (P2):
5’ATAAGAAT?
GCGGCCGC?TTA?CTA?GAA?CAG?CAG?AGA?TTG?GCA?CGT?C3’；
Not?I
With the pcDNA3.0 plasmid (available from Invitrogen company) that contains the AmphiDDAH gene is template, and P1, P2 are the primer PCR amplification, obtains the single band of specific amplified, and the product size is about 800bp.Pcr amplification product is cloned on the prokaryotic fusion expression vector pETTRX, obtains recombinant expression vector pETTRX-AmphiDDAH (its building process as shown in Figure 4).Foreign gene among the expression vector pETTRX-AmphiDDAH is identified correct through order-checking.Contain Prescission proteolytic enzyme cutting site in the designed upstream primer, so that excision fusion partner TRX.
Embodiment 4: lancelet P
DDAHExpression of Fusion Protein
With pETTRX-AmphiDDAH transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor shows that through the SDS-PAGE electrophoretic analysis bacterial strain has tangible specifically expressing product band after inducing, molecular weight conform to theoretical value 44kD with software DNATOOLS6.0 prediction (Fig. 5).
To incubation time, induced concentration, the groping of conditions such as temperature draws.The culture condition of genetic engineering bacterium is: the order bacterium colony is in 50ml ammonia benzyl resistance LB liquid nutrient medium, 37 ℃, the 250rpm overnight incubation, get in the ammonia benzyl resistance LB substratum that overnight culture 10ml is inoculated in 1L, 37 ℃, 250rpm is cultured to OD600=0.6 (the about 4h of enlarged culturing), adds 100mM IPTG and 20% glucose to final concentration and is respectively 0.1mM and 0.2%, 25 ℃, centrifugal collection thalline behind the 250rpm inducing culture 14h.Show through the SDS-PAGE electrophoretic analysis, with this understanding P
DDAHThe Expression of Fusion Protein amount accounts for more than 40% of bacterial protein, is in solvable state (Fig. 5).
Embodiment five: reorganization lancelet P
DDAHThe purifying of fusion rotein
With 50mM PB (pH6.5) washing, use an amount of 50mM PB (pH6.5) to suspend again total thalline, after the supersound process, the cracking supernatant liquor is through Ni
2+Chelating Sepharose affinity chromatography purifying, the expression of SDS-PAGE analysis fusioning protein and the result of chromatography (Fig. 5).Can draw from SDS-PAGE result: TRX-P
DDAHCan be by Ni
2+Post is adsorbed, washes Ni with the imidazoles of 50mM, 100mM
2+During post, can the foreign protein that major part is adsorbed on the post be washed, and can be target protein TRX-P with the imidazoles of 200mM, 300mM, 400mM concentration
DDAHWash.For yield and the concentration that improves recombinant protein, simplify experimental procedure, shorten treatment time to recombinant protein, with Ni2+ affinity chromatography purification of recombinant proteins the time, we adopt single stage method to carry out purifying.Foreign protein and the Ni expressed according to vector plasmid pETTRX
2+The characteristics that the binding ability of affinity column is stronger, we have selected the elution requirement of simplifying for use: wash Ni with the imidazoles of 100mM earlier
2+Post is abandoned elution peak, directly washes Ni with the imidazoles of 400mM then
2+Post, the elution peak of collection recombinant protein.
Elutriant is crossed the G25 gel column and is changed the 3C enzyme cutting buffering liquid, then, carries out TRX-P at 4 ℃ of refrigerators
DDAH3C enzyme (1: 1000) cut and spend the night, the protein solution after enzyme is cut changes the level pad of Q post into through G25, carries out the last column purification of Q post.Sample after enzyme is cut is when upper prop, and 3C, TRX major part are not adsorbed on the Q post, and percolation has fallen basically, when using the NaCl wash-out of 0.2M, and foreign protein under the wash-out, and when 0.3M, can wash our target protein P
DDAH(Fig. 6).
Embodiment 6:P
DDAHThe recombinant protein activation analysis
Add the enzymatic substrate A DMA 40ul of DDAH, the P of above-mentioned purifying
DDAHProtein 36 0ul after 37 ℃ of hydrolysis 1hr, carries out the colour developing of L-guanidine propylhomoserin according to people's such as Knipp method, surveys OD
230Value, this hydrolysis reaction repeats 3 times, is blank with PBS, measures Vm=0.015 ± 0.002mmolL
1Mg
1Min
1So resulting cDNA is the homologue of DDAH gene in lancelet, have the activity of hydrolysis substrate ADMA, can regulate the activity of NOS, thereby influence the generation of NO.
Sequence table
＜110〉Zhongshan University
＜120〉two methylarginine lytic enzyme (AmphiDDAH) genes of lancelet and application thereof
<160>2
<210>1
<211>822
<212>DNA
＜213〉lancelet (Branchiostoma belcheri tsingtauense)
<220>
<221>peptide
<222>(1)…(822)
<400>1
<210>2
<211>274
<212>PRT
＜213〉lancelet (Branchiostoma belcheri tsingtauense)
<220>
<221>peptide
<222>(1)…(274)
<400>2
1?MADFCWNFPE?FKHAVVREVS?DDVNNAVRDS?TSTETVDLEK?CRAEWELYVQ 50
51?ALRDLGLDVT?VIPQDPKLPD?CQFVEDPCIV?IGDTALITRP?ACGPRQGETT?100
101?VLEETMRNLG?LKVRVVESDT?ATLEGGDVIF?TGKEIFCGDS?VLSNEEGFKI?150
151?LEETFGDDYP?CHSIFVEYPE?FHLKGYAALA?APGIMAICDN?EWGHPAWKDI?200
201?CDTSNYPYKV?MWIPDDFGNN?CLYINGTIMH?APESQKPDTF?AVFQKDMADY?250
251?NLIPMSSEEV?SKVDAALTCQ?SLLF
＊ 274
Claims (6)
1. from the lancelet neurula, separate the neurula gene A mphiDDAH obtain, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.
2. the albumen P of the described genes encoding of claim 1
DDAH, its aminoacid sequence as in the sequence table＜400〉2 sequences shown in.
3. the described albumen P of claim 2
DDAHIn intestinal bacteria,, carry out according to following steps with the method for soluble formal representation in the born of the same parents:
(1) structure of recombinant expression vector pETTRX-DDAH shown in Figure 4;
(2) with pETTRX-DDAH transformed into escherichia coli BL21 (DE3);
(3) e. coli bl21 (DE3) that transforms is cultivated;
(4) reorganization lancelet P
DDAHThe purifying of fusion rotein.
4. albumen P according to claim 3
DDAHWith the method for soluble formal representation in the born of the same parents, it is characterized in that in intestinal bacteria: the structure of described recombinant expression vector pETTRX-DDAH may further comprise the steps:
(a) according to the described synthetic a pair of primer of two terminal sequences that separates the neurula gene A mphiDDAH that obtains from the lancelet neurula of claim 1, upstream primer contains Kpn I and Prescission proteolytic enzyme cutting site, and downstream primer contains Not I cleavage site,
Upstream primer (P1):
5’CGG?
GGT?ACC?
CTG?GAA?GTT?CTG?TTC?CAG?GGG?CCC?ATG?GCG?GATTTC?TGT?TGGAAT?TT3’；
Downstream primer (P2):
5’ATAAGAAT?
GCGGCCGC?TTA?CTA?GAA?CAG?CAG?AGA?TTG?GCA?CGT?C3’；
(b) be template with the pcDNA30 plasmid that contains the AmphiDDAH gene, P1, P2 are the primer PCR amplification;
(c) pcr amplification product is cloned on the prokaryotic fusion expression vector pETTRX, obtains recombinant expression vector pETTRX-DDAH.
5. albumen P according to claim 3
DDAHIn intestinal bacteria with the method for soluble formal representation in the born of the same parents, it is characterized in that: the culture condition of step (3) is: the order bacterium colony is in 50ml ammonia benzyl resistance LB liquid nutrient medium, 37 ℃, the 250rpm overnight incubation is got in the ammonia Bian resistance LB substratum that overnight culture 10ml is inoculated in 1L 37 ℃, 250rpm is cultured to OD600=0.6, add 100mM IPTG and 20% glucose to final concentration and be respectively 01mM and 02%, 25 ℃, centrifugal collection thalline behind the 250rpm inducing culture 14h.
6. albumen P according to claim 3
DDAHIn intestinal bacteria,, it is characterized in that: step (4) reorganization lancelet P with the method for soluble formal representation in the born of the same parents
DDAHThe purification process of fusion rotein is:
With the 50mMPB washing of total thalline with pH65, the 50mMPB with an amount of pH65 suspends again, and after the supersound process, the cracking supernatant liquor is through Ni
2+Chelating Sepharose affinity chromatography purifying, the elution peak of collection recombinant protein;
The elution peak liquid of recombinant protein is crossed the G25 gel column and is changed the 3C enzyme cutting buffering liquid, then, carries out TRX-P at 4 ℃
DDAH3C enzyme enzyme cut and spend the night, the protein solution after enzyme is cut changes the level pad of Q post into through G25, carries out the last column purification of Q post.
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| EP1356058A2 (en) * | 2000-08-18 | 2003-10-29 | University College London | Crystal structure of dimethylarginine dimethylaminohydrolase and arginine deiminase |
| WO2003089638A1 (en) * | 2002-04-19 | 2003-10-30 | Oy Jurilab Ltd | Nucleic acid molecule encoding a variant ddah 1 protein and uses thereof |
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| WO2003089638A1 (en) * | 2002-04-19 | 2003-10-30 | Oy Jurilab Ltd | Nucleic acid molecule encoding a variant ddah 1 protein and uses thereof |
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