CN101928710B - Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application - Google Patents
Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application Download PDFInfo
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- CN101928710B CN101928710B CN 200910053692 CN200910053692A CN101928710B CN 101928710 B CN101928710 B CN 101928710B CN 200910053692 CN200910053692 CN 200910053692 CN 200910053692 A CN200910053692 A CN 200910053692A CN 101928710 B CN101928710 B CN 101928710B
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Abstract
The invention provides an anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides and a recombination expression method applied in escherichia coli. The method comprises the steps of cloning a full-length coding gene of an anticoagulation molecule Rhipilin-1 from the Rhipicephalus haemaphysaloides by creating a salivary glands subtractive library before and after bloodsucking of the Rhipicephalus haemaphysaloides and sequencing analysis, wherein the full length of the nucleotide is 620bp, open reading frame codes 164 amino acid residues, the prospected molecular weight is 18kDa, N-end is provided with a signal peptide sequence formed by 18 amino acids and contain a typical Kunitz functional domain, and the tissue channel inhibiting factor of one of the molecule and the anticoagulation molecule is obviously homologous. The gene is coned to a pGEX-4T-1 expression vector, converted to a BL21 (DE3) host fungus. Through IPTG induction, GST fusion protein is recombined so as to efficiently express in a soluble way. A test for plasma recalcification time and partial thrombin activating time by using purified recombined protein proves that the Rhipilin-1 molecule has anticoagulation bioactivity and can be used for preparing anticoagulation biologic preparation.
Description
Technical field
The present invention relates to biotechnology, be specifically related to blood biological gene sequence, recombinant expressed and the application in preparation anti-freezing preparation of Rhipilin-1 gene in intestinal bacteria of crescent fan head tick anticoagulation molecule Rhipilin-1.
Background technology
In the organic evolution process, the blood coagulation system that Mammals has formed a complicacy stops the loss of blood, and often has the coagulation function of a lot of anticoagulant proteins with the antagonism host in the organism of much making a living with blood.Tick is one type of vermin of making a living to suck blood.Compare with other HAEMATOPHAGOUS ARTHROPODS, the time of sucking blood of tick is long especially, as the time of sucking blood of hard tick 10-14 days (Sonenshine, 1993).The process of sucking blood for a long time of tick must cause a series of hemostasis reactions of host, like vasoconstriction, platelet aggregation and blood coagulation.Suck blood for what continue; Tick must overcome host's hemostasis reaction, and since Waxman (1990) identified the anticoagulation molecule of first tick, the researchist found that the sialisterium of tick secrete the multiple anti-hemostasis molecule of generation in the process of sucking blood; The PGD2 of tick salivary gland secretion and I2 can shrink and inhibition platelet aggregation (Bowman by induction of vascular; 1995), several kinds of platelet aggregation arrestin molecules are also identified (Mans, 2002) from hard tick and soft ticks; Anticoagulation molecule to blood coagulation; Like zymoplasm (thrombin) arrestin molecule, factor Xa (FXa) arrestin molecule and tissue factor path arrestin molecule (TFPI) in multiple external advantage tick bodies such as lone star tick, Dermacentor variabilis, a prominent hard tick, Rhipicephalus appendicularis, boophilus microplus, come to light (Maritz-Olivier, 2007).Although the tick anticoagulant molecule of having reported is kind more than 20 nearly, identify fully also seldom, the gene order that obtains these protein moleculars of coding is no more than 8.From present interpretation of result; Tick anticoagulant molecule majority belongs to Kunitz family serinase specificity and suppresses molecule; Characteristic contains the cysteine residues of 1 or 2 KU structure function territories and high conservative, maybe be from same ancestors (Lai, 2004) on evolving.
Because human health in the popular feeling, cerebrovascular disease serious threat that thrombus causes, the anti-bolt preparation of available is limited at present, and therefore, the research of medicine for treating thrombus thing is the field of very paying close attention to (Bates and Weitz, 2006) always.Hirudin has been applied to clinical medicine (Turk, 2006 according to leech (leech) anticoagulation molecule synthetic anticoagulant peptides; Bates and Weitz, 2006).In the tick anticoagulant molecule of having identified; Have 2 molecules in animal model experiment, to have the antithrombotic therapy effect, wherein the TAP molecule identified is factor Xa (FXa) suppressor factor nineteen ninety, and recombinant expressed TAP has shown good anti thrombotic action on a plurality of animal models; Owing to reasons such as antigenicity also fail on the human clinical, to use (Bates and Weitz at present; 2006), but utilize structure and the mechanism of action design of TAP molecule littler, do not have antigenic medicine for treating thrombus thing, perhaps make up novel reorganization medicine for treating thrombus thing molecule and study and make progress; For example; Use TAP functional zone sequence and new reorganization anticoagulant protein molecule of anchorin Annexin V sequence construct, its antithrombotic acitivity improves 10 times (Chen, 2005) than the activity of single anchorin Annexin V.Another tick anticoagulant molecule I xolaris is in recent years from a molecule that prominent hard tick is identified, with people's anatomic passages supressor (TFPI) homology; Anticoagulant factor VIIa/tissue factor complex body; In rat model, very strong anti thrombotic action (Monteiro, 2005 have been shown; Nazareth, 2006).These researchs show that the anticoagulation molecule of tick possibly be developed as novel antithrombotic reagent.
The crescent Rh mainly is distributed in South Asia and southern china in hard tick, is advantage tick kind, at multiple animals such as ox, sheep, dog, the rabbit parasitism of sucking blood, also can attack human body and bite and suck blood, but also not see the research report about its anticoagulation molecule.
Summary of the invention
Technical problem to be solved by this invention is to study crescent fan head tick anticoagulation molecule, development of new anticoagulation biotechnological formulation.
The invention provides the gene order of crescent fan head tick anticoagulation molecule Rhipilin-1, the nucleotide sequence and the corresponding amino acid sequence of its gene are following:
The nucleotide sequence of crescent fan head tick anticoagulation molecule Rhipilin-1:
aatattggaa aaacttcagc gagcagaaaa caaccaaagg caatgaagat tttacaatac 60
gcgctcttgg cttgtcttct cgtctcgata ttaggcgatg atgatgacga agaaaacgag 120
ggtgcggcgg ggagtgatga tgcgggcgca acgcctacag cactgtcgaa gccagcagcc 180
acttctgcgg aaggaaacac cgcagcaaaa aactcaggtg gtgacatcaa gcaaccgaaa 240
caagaactgc ccgaaccgtc ctcaaaaggc caaaagaaac cgagactacc caagagatgc 300
ttattcaagc cagaacaggg aaactgcgca ggttctcgtt tccttcctag atggtggtat 360
aacccagaaa ctgagagctg cgaaccattc acttatccag tgtgcaataa gaaaaacgaa 420
gcattcgtat catgtacact gtgcatgaac atgtgcatga ggaataagag gggcagggag 480
aaggctaaat ggattagaaa agtttgtaga aaaagtccga aagtaaaatc tggatgaatg 540
tagttggctc tgccacttct tctgccataa aatattcgaa aacatttgtt atgaataata 600
aaattatcaa aaaaaaaaaa 620
Crescent fan head tick tick anticoagulant molecule Rhipilin-1 aminoacid sequence
Met Lys Ile Leu Gln Tyr Ala Leu Leu Ala Cys Leu Leu Val Ser Ile
1 5 10 15
Leu Gly Asp Asp Asp Asp Glu Glu Asn Glu Gly Ala Ala Gly Ser Asp
20 25 30
Asp Ala Gly Ala Thr Pro Thr Ala Leu Ser Lys Pro Ala Ala Thr Ser
35 40 45
Ala Glu Gly Asn Thr Ala Ala Lys Asn Ser Gly Gly Asp Ile Lys Gln
50 55 60
Pro Lys Gln Glu Leu Pro Glu Pro Ser Ser Lys Gly Gln Lys Lys Pro
65 70 75 80
Arg Leu Pro Lys Arg Cys Leu Phe Lys Pro Glu Gln Gly Asn Cys Ala
85 90 95
Gly Ser Arg Phe Leu Pro Arg Trp Trp Tyr Asn Pro Glu Thr Glu Ser
100 105 110
Cys Glu Pro Phe Thr Tyr Pro Val Cys Asn Lys Lys Asn Glu Ala Phe
115 120 125
Val Ser Cys Thr Leu Cys Met Asn Met Cys Met Arg Asn Lys Arg Gly
130 135 140
Arg Glu Lys Ala Lys Trp Ile Arg Lys Val Cys Arg Lys Ser Pro Lys
145 150 155 160
Val Lys Ser Gly
Sialisterium subtractive library before and after the present invention sucks blood through the female tick of structure crescent fan head tick carries out the end sequencing analysis to all clones, obtains 247 effective est sequences altogether.To the clone of one of them anticoagulation molecule homologous genes, carried out complete sequence determination and bioinformatic analysis, find that it is complete encoding sox, called after Rhipilin-1 gene.This full length gene is 620bp, holds the signal sequence AATAAAA that polyA and polyA are arranged 3 ', and containing has a complete ORFs, from the 43rd base to 536 base, and 164 amino acid of encoding, predicted molecular weight is 18kDa.The protein signal peptide analysis, the signal peptide sequence that has 18 amino acid to constitute at the N-end; The protein function domain analysis is a typical serpin Kunitz domain at 139 amino acid of the 84th amino acid to the.Blast analyzes the remarkable homology of anatomic passages supressor of finding one of this molecule and anticoagulation molecule.
Another object of the present invention has provided the recombinant expression method of said crescent fan head tick anticoagulation molecule Rhipilin-1 in intestinal bacteria.
According to crescent fan head tick anticoagulation molecule Rhipilin-1 gene biological information science analytical results; With the actual coding district subclone that does not comprise signal peptide sequence in prokaryotic expression carrier pGEX-4T-1; Make up recombinant expression plasmid pGEX-4T-1/Rhipilin-1; After being transformed into BL21 (DE3) expression bacterium, use the IPTG abduction delivering.The SDS-PAGE electrophoretic analysis shows that the reorganization gst fusion protein obtains to efficiently express, and the GST-fusion rotein is 44kDa, big or small consistent with the expection recombinant protein, and further analysis revealed, the recombination fusion protein majority exists with the soluble proteins form.
Another purpose of the present invention has provided the application of said crescent fan head tick anticoagulation molecule Rhipilin-1 in preparation anticoagulation biotechnological formulation.
The present invention is with the crescent fan head tick anticoagulation molecule Rhipilin-1 recombinant protein of purifying; Carry out rabbit plasma recalcification time (Recalcification Time; RT) and partial prothrombinase activationary time (Activated PartialThromboplastin Time; APTT) experiment has confirmed that this molecule has anticoagulant active.
Description of drawings
The nucleotide sequence and the corresponding amino acid sequence analysis of Fig. 1 crescent fan head tick anticoagulation molecule Rhipilin-1 gene
Wherein ATG is an initiator codon; Under to draw the solid line sequence be signal peptide sequence; Arrow is the cleavage site of signal peptide; Under to draw the dotted line sequence be the Kunitz domain; TAG is a terminator codon; AATAAA is a tailing signal.
Expression and the purifying of Fig. 2 crescent fan head tick anticoagulation molecule reorganization Rhipilin-1
M is standard molecular weight albumen; 1 and 2 are respectively and contain before the empty carrier bacteria-induction and induce the back protein band, 3 and 4 be respectively contain protein band before the recombinant vectors bacteria-induction, induce after protein band; 5,6,7 be respectively insoluble protein, soluble proteins and the purified recombinant albumen of inducing bacterium
Fig. 3 crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 blood plasma recalcification time experimental result
A is contrast GST albumen (concentration 18.75～300nM); B is a saline water; C, d, e, f, g are respectively the reorganization Rhipilin-1 sample that concentration is 18.75nM, 37.5nM, 75nM, 150nM, 300nM
Fig. 4 crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 influences 1 for contrasting GST albumen (concentration 15.625～250nM) to rabbit plasma partial prothrombinase activationary time; 2 is saline water; 3,4,5,6,7 be respectively the reorganization Rhipilin-1 sample that concentration is 15.625nM, 31.25nM, 62.5nM, 125nM, 250nM
Embodiment
Gene clone and the sequential analysis of embodiment 1 crescent fan head tick anticoagulation molecule Rhipilin-1
1. materials and methods
1.1 laboratory animal: the crescent fan head tick picks up from the Hubei buffalo, is preserved by this laboratory breeding.New zealand white rabbit is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
1.2 test strain, plasmid and reagent: coli strain DH5 α is available from vast Imtech, and plasmid pGEM-TEasy, T4DNA ligase enzyme are available from Promega company, and plasmid pGEX-4T-1 is available from Amersham PharmaciaBiotech; TRIZOL reagent is available from Invitrogen company; DNA glue reclaims test kit available from match Parkson company; Taq archaeal dna polymerase, DNA Marker are available from TaKaRa company; PCR SelectTM cDNA SubtractionKit, SMART PCR cDNA Synthesis Kit, PCR Purification Kit is the Clontech Company products.
1.3 the preparation of crescent fan head tick sialisterium
The crescent fan head tick is not sucked blood into tick by 1: 1 combined inoculation rabbit ear of male and female, and the female tick of half-full blood is taken out in inoculation back 4-5 days, and anatomical lens separates sialisterium down, and-70 ℃ of preservations are subsequent use; Same tick crowd does not suck blood the sialisterium of female tick by separating with quadrat method.
1.4 the extraction of the total RNA of tick sialisterium
Adopt the Trizol reagent of Invitrogen company, press the description of test operation, extract crescent fan head tick do not suck blood female tick and the total RNA of the female tick of half-full blood respectively, be dissolved in the water of no RNase, the mensuration total rna concentration, it is subsequent use to be stored in-70 ℃ of refrigerators then.
1.5 double-stranded cDNA's is synthetic
Adopt the SMART PCR cDNA Synthesis Kit of Clontech company; Be template with total RNA respectively; With Olig (dT) 18 primers synthetic first chain cDNA (ss cDNA) under the effect of M MLV reversed transcriptive enzyme, and with long range PCR (LD-PCR) amplification cDNA second chain (ds cDNA);
1.6 inhibition subtractive hybridization
Use Clontech PCR SelectTM cDNA Subtraction Kit.This test is Tester with the female tick sialisterium of half-full blood, and female tick sialisterium is not Driver to suck blood; Step is following: respectively Adaptor1-Tester cDNA and Adaptor 2R-Tester cDNA are mixed with Driver cDNA, 68 ℃ of incubation 8h carry out the 1st and take turns hybridization; Above-mentioned two reaction product are mixed, and add the Driver cDNA of capacity once more, 68 ℃ are spent the night, and carry out the 2nd and take turns hybridization; Product to subtractive hybridization carries out the two-wheeled pcr amplification, does contrast with the cDNA that adds the female tick of half-full blood that joint not subdues respectively.The primer sequence of PCR is corresponding to the sequence (Clontech PCR SelectTM cDNA Subtraction Kit) of Adaptor 1 and Adaptor 2R.
1.7 the foundation of subtractive library, the screening of positive colony, est sequence are measured and are analyzed
Subtractive hybridization product behind the pcr amplification is through PCR Purification Kit purifying; Be connected with the pGEM-T-easy carrier; Be transformed into competent cell DH5 α and screen blue hickie, picking is the white clone all, increases with nest-type PRC primer Primer 1 and Primer 2R; Positive colony is served the order-checking of sea base health biotech company, and sequencing result carries out homology analysis (http://www.ncbi.nlm.nih.gov/BLAST) on GenBank;
1.8 the full-length gene order of anticoagulation molecule Rhipilin-1 is measured and is analyzed
According to est sequence information, select one and be numbered 48 est sequence, carry out complete sequence determination and bioinformatic analysis, the result is complete coding anticoagulation molecule homologous genes, called after Rhipilin-1 gene.
2. result and analysis
The sialisterium subtractive library successfully made up before and after the female tick of crescent fan head tick was sucked blood, and all clones are carried out the end sequencing analysis, obtained 247 effective est sequences altogether.To the clone of one of them anticoagulation molecule homologous genes, carried out complete sequence determination and bioinformatic analysis, be found to be complete encoding sox, called after Rhipilin-1 gene.This full length gene is 620bp; The signal sequence AATAAAA that polyA and polyA are arranged at 3 ' end; Contain a complete ORFs is arranged; From the 43rd base to 536 base, 164 amino acid of encoding, predicted molecular weight is 18kDa; Protein signal peptide analysis (http://www.cbs.dtu.dk/services/SignalP/) finds to have at the N-end signal peptide sequence of 18 an amino acid formation, and protein function domain analysis (http://smart.embl-heidlberg.de/smart/job_status.) finds at the 84th base to 139 a base place one typical serpin Kunitz domain is arranged.Blast analyzes the remarkable homology of anatomic passages supressor of finding one of this molecule and anticoagulation molecule.
Recombinant expressed and the anticoagulant active checking of embodiment 2 crescent fan head tick anticoagulation molecule Rhipilin-1 genes
1. materials and methods
1.1 laboratory animal: new zealand white rabbit is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
1.2 test strain, plasmid and reagent: coli strain DH5 α, BL21 (DE3) are available from vast Imtech, and plasmid pGEM-T Easy, T4DNA ligase enzyme are available from Promega company, and plasmid pGEX-4T-1 is available from Amersham Pharmacia Biotech; DNA glue reclaims test kit available from match Parkson company; Taq archaeal dna polymerase, DNA Marker, EcoRI and Xhol restriction endonuclease are available from TaKaRa company.GSTBind
TMTest kit is available from Novagen; The APTT test kit is purchased in sun Bioisystech Co., Ltd; BCA analysis of protein test kit is the PIERCE Company products; Primer is synthetic by match Parkson company.
1.3 the construction of recombinant expression plasmid of crescent fan head tick anticoagulation molecule Rhipilin-1 gene
According to crescent fan head tick anticoagulation molecule Rhipilin-1 gene biological information science analytical results, in prokaryotic expression carrier pGEX-4T-1, upstream primer is with the actual coding district subclone that does not comprise signal peptide sequence:
5 '
GATGATGATGACGAAGAAAACGAG3 ' downstream primer is:
5 '
TCATCCAGATTTTACTTTCGGACT3 '; 5 ' end and 3 ' end are introduced EcoRI and Xhol I restriction enzyme site respectively; Tilted letter is represented restriction enzyme site, is the protectiveness base in the square frame.With recombinant cloning vector pGEM/Rhipilin-1 is that template is carried out pcr amplification; This program is: behind 94 ℃ of 2min, and with 94 ℃ of 45s, 53 ℃ of 45s, after 72 ℃ of 60s circulation 30 times, last 72 ℃ are extended 10min.With the PCR product reclaim, enzyme is cut, purifying; Simultaneously carrier pGEX-4T-1 is made double digestion with corresponding enzyme; The PCR product of enzyme being cut the back purifying is connected with carrier; Transform DH5 α, cut the positive bacterium colony of evaluation, press the recombinant plasmid pGEX-4T-1/Rhipilin-1 of test kit specification sheets operation extracting positive bacteria through PCR and enzyme;
1.4 the recombinant expressed and purifying of crescent fan head tick anticoagulation molecule Rhipilin-1
To BL21 (DE3), 37 ℃, 200r/min shaking culture are as bacterium liquid OD600 during in 0.6～0.8 left and right sides with recombinant plasmid transformed; In substratum, add IPTG, making final concentration is 1mmol/L, after continuing to cultivate 4h; Centrifugal collection thalline; With 12%SDS-PAGE electrophoretic analysis protein expression situation, reorganization GST amalgamation and expression albumen is used GSTBind
TMThe test kit purifying, the BCA method is surveyed protein concentration.
1.5 crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 is to the influence of rabbit plasma recalcification time
Added 3.8% Trisodium Citrate with healthy experimental rabbit whole blood according to 9: 1, the centrifugal 5min of 3000r/min processes in the rabbit Trisodium Citrate blood plasma.In 96 orifice plates, add 0.1mL blood plasma to be measured respectively, add the reorganization Rhipilin-1 sample that 0.1mL is dissolved in the different concns of saline water then, sample and saline water carry out doubling dilution (final concentration is 0.15nm/mL-2.4nm/mL).37 ℃ of insulation 15min; The CaCl that adds 0.1mL 0.025mol/L
2Solution in the 405nm place, whenever in the 0-10min is measured an OD value at a distance from 25s, and the increase of OD value reflects the blood plasma degree of condensing.Contrast adds the saline water and the GST albumen of same volume respectively.Three multiple holes are done in each experiment.
1.6 crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 adds blood plasma 0.1mL to be measured to the influence test of rabbit partial prothrombinase activationary time the Rhipilin-1 of 0.1mL gallogen partial thromboplastin solution and 0.1mL different concns; Hatch 5min for 37 ℃, add 0.025mol/L CaCl immediately
2Solution 0.1mL picks up counting simultaneously, when solution becomes turbid, stops timing, and this time is gallogen partial thromboplastin activationary time (APTT).Adding 0.1mL saline water and GST albumen simultaneously respectively replaces Rhipilin-1 not add the gallogen partial prothrombinase activationary time (APTT) of RhAP as blank determination.
2. result and analysis
2.1 crescent fan head tick anticoagulation molecule Rhipilin-1 expression of gene and purifying
Crescent fan head tick anticoagulation molecule Rhipilin-1 recombinant plasmid transformed is induced successful expression through IPTG in intestinal bacteria E.coli BL21.SDS-PAGE result shows, the GST albumen that empty plasmid pGEX-4T-1 expresses is 26kDa, and the GST-fusion rotein of expression of recombinant plasmid is 44kDa, big or small consistent with the expection recombinant protein.Further analysis revealed, the recombination fusion protein majority exists with the soluble proteins form, through GSTBind
TMThe test kit purifying obtains purified recombinant Rhipilin-1 (Fig. 2).
2.2 reorganization Rhipilin-1 is to the influence of rabbit plasma recalcification time
The result is as shown in Figure 3; After adding crescent fan head tick anticoagulation molecule reorganization Rhipilin-1; The rabbit plasma recalcification time increases with concentration and prolongs; When crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 concentration reaches 75nM when above, for the restraining effect significant difference of blood plasma agglutination, contrast reorganization GST all unrestraint blood plasma agglutination effects under the different concns condition; Saline water contrasts also unrestraint blood plasma agglutination effect, and the result shows that reorganization Rhipilin-1 has anticoagulation to rabbit plasma, and has concentration dependence characteristic.
2.3 reorganization Rhipilin-1 is to the influence of rabbit plasma partial prothrombinase activationary time
The experimental result of rabbit plasma partial prothrombinase activationary time (APTT) is as shown in Figure 4; Compare with reorganization GST with contrast saline water; When crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 final concentration is that 15.625nM is when above; But significant prolongation rabbit plasma gallogen partial prothrombinase activationary time (APTT), and along with concentration to increase activationary time long more, explain that the anticoagulation of crescent fan head tick anticoagulation molecule reorganization Rhipilin-1 is fairly obvious and has concentration dependence characteristics.The experimental result of APTT shows that also reorganization Rhipilin-1 not only suppresses blood coagulation from endogenous blood coagulation system, also has the obvious suppression effect to the external source coagulation pathway.
Sequence table
< 110>China Agriculture Academe Shanghai Veterinary Institute
< 120>gene order of a kind of anticoagulation molecule Rhipilin-1 of hard tick and recombinant expressed
< 130>specification sheets, claims
<160>2
<170>PatentIn version 3.3
<210>1
<211>620
<212>DNA
< 213>nucleotide sequence of anticoagulation molecule Rhipilin-1
<400>1
aatattggaa aaacttcagc gagcagaaaa caaccaaagg caatgaagat tttacaatac 60
gcgctcttgg cttgtcttct cgtctcgata ttaggcgatg atgatgacga agaaaacgag 120
ggtgcggcgg ggagtgatga tgcgggcgca acgcctacag cactgtcgaa gccagcagcc 180
acttctgcgg aaggaaacac cgcagcaaaa aactcaggtg gtgacatcaa gcaaccgaaa 240
caagaactgc ccgaaccgtc ctcaaaaggc caaaagaaac cgagactacc caagagatgc 300
ttattcaagc cagaacaggg aaactgcgca ggttctcgtt tccttcctag atggtggtat 360
aacccagaaa ctgagagctg cgaaccattc acttatccag tgtgcaataa gaaaaacgaa 420
gcattcgtat catgtacact gtgcatgaac atgtgcatga ggaataagag gggcagggag 480
aaggctaaat ggattagaaa agtttgtaga aaaagtccga aagtaaaatc tggatgaatg 540
tagttggctc tgccacttct tctgccataa aatattcgaa aacatttgtt atgaataata 600
aaattatcaa aaaaaaaaaa 620
<210>2
<211>164
<212>PRT
< 213>tick anticoagulant molecule Rhipilin-1 aminoacid sequence
<400>2
Met Lys Ile Leu Gln Tyr Ala Leu Leu Ala Cys Leu Leu Val Ser Ile
1 5 10 15
Leu Gly Asp Asp Asp Asp Glu Glu Asn Glu Gly Ala Ala Gly Ser Asp
20 25 30
Asp Ala Gly Ala Thr Pro Thr Ala Leu Ser Lys Pro Ala Ala Thr Ser
35 40 45
Ala Glu Gly Asn Thr Ala Ala Lys Asn Ser Gly Gly Asp Ile Lys Gln
50 55 60
Pro Lys Gln Glu Leu Pro Glu Pro Ser Ser Lys Gly Gln Lys Lys Pro
65 70 75 80
Arg Leu Pro Lys Arg Cys Leu Phe Lys Pro Glu Gln Gly Asn Cys Ala
85 90 95
Gly Ser Arg Phe Leu Pro Arg Trp Trp Tyr Asn Pro Glu Thr Glu Ser
100 105 110
Cys Glu Pro Phe Thr Tyr Pro Val Cys Asn Lys Lys Asn Glu Ala Phe
115 120 125
Val Ser Cys Thr Leu Cys Met Asn Met Cys Met Arg Asn Lys Arg Gly
130 135 140
Arg Glu Lys Ala Lys Trp Ile Arg Lys Val Cys Arg Lys Ser Pro Lys
145 150 155 160
Val Lys Ser Gly
Claims (3)
1. crescent fan head tick anticoagulation molecule Rhipilin-1 gene order; It is characterized in that said full length gene is 620bp; Hold the signal sequence AATAAA that polyA and polyA are arranged 3 ', contain a complete ORFs, from the 43rd base to 536 base; 164 amino acid of encoding, its molecular weight is 18kDa; The signal peptide sequence that its proteins encoded has 18 amino acid to constitute at the N-end; Cut point is between the 18th and the 19th amino acid: from 139 amino acid of the 84th amino acid to the, and typical serpin Kunitz domain of predictive coding;
Nucleotide sequence and the corresponding amino acid sequence of said crescent fan head tick anticoagulation molecule Rhipilin-1 are following:
The nucleotide sequence of crescent fan head tick anticoagulation molecule Rhipilin-1:
aatattggaa aaacttcagc gagcagaaaa caaccaaagg caatgaagat tttacaatac 60
gcgctcttgg cttgtcttct cgtctcgata ttaggcgatg atgatgacga agaaaacgag 120
ggtgcggcgg ggagtgatga tgcgggcgca acgcctacag cactgtcgaa gccagcagcc 180
acttctgcgg aaggaaacac cgcagcaaaa aactcaggtg gtgacatcaa gcaaccgaaa 240
caagaactgc ccgaaccgtc ctcaaaaggc caaaagaaac cgagactacc caagagatgc 300
ttattcaagc cagaacaggg aaactgcgca ggttctcgtt tccttcctag atggtggtat 360
aacccagaaa ctgagagctg cgaaccattc acttatccag tgtgcaataa gaaaaacgaa 420
gcattcgtat catgtacact gtgcatgaac atgtgcatga ggaataagag gggcagggag 480
aaggctaaat ggattagaaa agtttgtaga aaaagtccga aagtaaaatc tggatgaatg 540
tagttggctc tgccacttct tctgccataa aatattcgaa aacatttgtt atgaataata 600
aaattatcaa aaaaaaaaaa 620
Crescent fan head tick tick anticoagulant molecule Rhipilin-1 aminoacid sequence:
Met Lys Ile Leu Gln Tyr Ala Leu Leu Ala Cys Leu Leu Val Ser Ile
1 5 10 15
Leu Gly Asp Asp Asp Asp Glu Glu Asn Glu Gly Ala Ala Gly Ser Asp
20 25 30
Asp Ala Gly Ala Thr Pro Thr Ala Leu Ser Lys Pro Ala Ala Thr Ser
35 40 45
Ala Glu Gly Asn Thr Ala Ala Lys Asn Ser Gly Gly Asp Ile Lys Gln
50 55 60
Pro Lys Gln Glu Leu Pro Glu Pro Ser Ser Lys Gly Gln Lys Lys Pro
65 70 75 80
Arg Leu Pro Lys Arg Cys Leu Phe Lys Pro Glu Gln Gly Asn Cys Ala
85 90 95
Gly Ser Arg Phe Leu Pro Arg Trp Trp Tyr Asn Pro Glu Thr Glu Ser
100 105 110
Cys Glu Pro Phe Thr Tyr Pro Val Cys Asn Lys Lys Asn Glu Ala Phe
115 120 125
Val Ser Cys Thr Leu Cys Met Asn Met Cys Met Arg Asn Lys Arg Gly
130 135 140
Arg Glu Lys Ala Lys Trp Ile Arg Lys Val Cys Arg Lys Ser Pro Lys
145 150 155 160
Val Lys Ser Gly。
2. the recombinant expression method of a kind of crescent fan head tick anticoagulation molecule Rhipilin-1 as claimed in claim 1 in intestinal bacteria; It is characterized in that; With the actual coding district subclone that does not comprise signal peptide sequence in prokaryotic expression carrier pGEX-4T-1; Make up recombinant expression plasmid pGEX-4T-1/Rhipilin-1, be transformed into after BL21 expresses bacterium, use the IPTG abduction delivering; The SDS-PAGE electrophoretic analysis shows that the reorganization gst fusion protein obtains to efficiently express, and the GST-fusion rotein is 44kDa, big or small consistent with the expection recombinant protein.
3. the application of a kind of crescent fan head tick anticoagulation molecule Rhipilin-1 as claimed in claim 1 in preparation anticoagulation biotechnological formulation.
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| CN 200910053692 CN101928710B (en) | 2009-06-24 | 2009-06-24 | Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application |
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| CN 200910053692 CN101928710B (en) | 2009-06-24 | 2009-06-24 | Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application |
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| CN101928710B true CN101928710B (en) | 2012-05-09 |
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| CN110790833B (en) * | 2018-08-01 | 2022-07-15 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Tick autophagy-associated protein molecule ATG5 and application thereof |
| CN112083172B (en) * | 2020-09-17 | 2024-01-26 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Method for screening protein with anticoagulant activity from tick cDNA library |
| CN114853883B (en) * | 2022-05-06 | 2023-05-16 | 青海大学 | Qinghai blood tick serine protease inhibitor and polyclonal antibody thereof |
| CN116410303B (en) * | 2023-06-07 | 2023-09-12 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Application of tick anticoagulant protein Qinghienin and encoding gene thereof in anticoagulation |
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| 向飞宇等.镰形扇头蜱唾液腺抑制消减杂交文库的构建和分析.《中国农业科学》.2006,第39卷(第11期),第2347－2353页. * |
| 恭海燕等.镰形扇头蟀半饱血雌蝉cDNA文库的构建.《中国人兽共患病杂志》.2004,第20卷(第6期),第485-488页. * |
| 王玉霞等.蜱功能基因研究进展.《动物医学进展》.2005,第26卷(第7期),第31－35页. * |
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