CN102532297B - Ancylostoma caninum anticoagulant peptide and preparation and application thereof - Google Patents
Ancylostoma caninum anticoagulant peptide and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a branch scheme of an invention patent application with the application number as 200710079673.0 and discloses a new ancylostoma caninum anticoagulant peptide and a coding sequence thereof. The obtained anticoagulant peptide has anticoagulant activity, can remarkably prolong human plasma prothrombin time (PT) and activated partial thromboplastin time (aPTT), and has a function of remarkably resisting thrombopoiesis. The invention further relates to application of the anticoagulant peptide in aspects of anticoagulant medicines and anti-thrombosis medicines.
Description
The application is application number 200710079673.0, the applying date to be dividing an application of on March 5th, 2007, denomination of invention " Ancylostoma caninum anticoagulant peptide and preparation thereof and application ".
Technical field
The present invention relates to medicine and biological technical field.Specifically, the present invention relates to the new gene of Ancylostoma caninum anticoagulant peptide that is separated to and the protein of coding thereof from the dog hookworm.The invention still further relates to preparation and the application of these new Ancylostoma caninum anticoagulant peptides.
Technical background
Thrombotic diseases is a kind of common disease, often shows as myocardial infarction, Ischemic Cerebral Infarction, venous thromboembolism.In annual each thousand people, there is 1～3 people that multi-form thrombotic diseases occurs.In the current mankind disease, thrombotic disease has become the principal element of harm humans health, by its death such as the myocardial infarction due to thromboembolism, cerebral infarction, pulmonary infarctions, is add up to first of the various causes of disease.How preventing and treating such disease is focus at present clinical and medical science research, although the history that has the medicines such as heparin, oral tonka bean camphor, streptokinase and urokinase to apply for many years, but the side effects limit such as hemorrhage, the thrombopenia of these medicines its application, also need special monitoring during some medications.Therefore develop good effect, extremely scientific research personnel's concern of thrombotic diseases control medicine that toxic side effect is low.The drug main for the treatment of clinically thrombotic diseases at present will be divided into anti-platelet drug, anticoagulation medicine and thrombolytic agent three major types.Wherein, anticoagulant is that a class is disturbed thrombin, is the medicine that stops blood coagulation, is mainly used in prevention and the treatment of thrombotic disease, is used in especially the prevention of embolism class diseases.Heparin class (as low molecular weight heparin LMWH) medicine is anticoagulation medicine in the highest flight in the market, as 6,000 ten thousand patients are arranged in the world every year, accepts heparin therapy with various thrombotic diseases such as control acute coronary artery syndromes.
Hookworm parasitizes human or animal host's enteron aisle, causes host's chronic blood loss, thereby causes host's anaemia.Wherein one of reason is that hookworm can secrete anticoagulant substances, makes the host be stung attached wound by hookworm and is difficult for blood coagulation, and blood is also discharged through the hookworm digestive tube rapidly.As far back as 20 beginnings of the century, just there is the scholar to confirm that dog hookworm (Ancylostoma caninum) extract has anticoagulation, but until just isolate first anticoagulant peptide from the dog hookworm in 1993, but thereafter and confirm this anticoagulant peptide significant prolongation prothrombin time (PT) and activated partial thrombozyme time (aPTT).This anticoagulant peptide is named as Ancylostoma caninum anticoagulant peptide (Ancylostoma caninum anticoagulant peptide, AcAP), also claim nematode anticoagulant peptide (nematode anticoagulant peptide, NAP) or nematode anticoagulant protein (nematode anticoagulant protein, NAP).1996, the bases such as Stassens record anticoagulant peptide partial amino-acid series designing nucleic acid probe, screen three anticoagulant peptide genes from dog hookworm cDNA library, their expression product rAcAP5 (rNAP5), rAcAP6 (rNAP6) in Pichia pastoris and rAcAPc2 (rNAPc2) be significant prolongation PT all, and becomes dose-dependence.Wherein, extend the PT activity and take rAcAPc2 (rNAPc2) as best, extend the aPTT activity and take rAcAP5 (rNAP5) as best, rAcAP6 (rNAP6) anticoagulating active slightly is weaker than rAcAP5 (rNAP5) (Stanssens P, P W Bergum, Y Gansemans et al.Anticoagulant repertoire of the hookworm Ancylostoma caninum.Proc Natl Acad Sci USA.1996; 93:2149～2154.).2002, Stassens etc. have reported employing cDNA end rapid amplifying technology, obtained anticoagulant peptide AceAP1 gene from Ancylostoma ceylanicum (Ancylostoma ceylanicum), and obtained rAceAP1 at expression in escherichia coli, its anticoagulating active is far beyond rAcAP5 (rNAP5), rAcAP6 (rNAP6) and the low (Harssion of rAcAPc2 (rNAPc2), L.M., Nerlinger, r.D.Bungiro et al.Molecular characterization of Ancylostoma inhibitors of coagulation factor Xa.Hookworm anticoagulant activity in vitro predicts parasite bloodfeeding in vivo[J] .J.Biol.Chem, 2002, 277 (8): 6223-29).2004, the people such as Juliusz have reported according to AcAPc2 signal peptide amino acid conserved sequence design primer, adopt RT-PCR to obtain two kinds of homeopeptides from the dog hookworm, and difference called after AcAPc3, AcAPc4, recombination expression product rAcAPc3, the rAcAPc4 anticoagulating active is obviously not as good as rAcAPc2 (rNAPc2) (Mieszczanek J, Harrison L M, Vlasuk G P and Cappello M.Anticoagulant peptides from Ancylostoma caninum are immunologically distinct and localize to separate structures within the adult hookworm.Mol Biochem Parasitol.2004, 133 (2), 319-323).Recently, the inventor also discloses (the GenBank typing number: DQ435781), (GenBank typing number: DQ435782), and experiment confirms that their recombination expression product has fine anticoagulating active (but result that the applicant is to be delivered also unexposed) to AcaNAP8 of Ancylostoma caninum anticoagulant peptide AcaNAP7 gene order.Hookworm coagulate peptide resistant and recombinant products thereof have clear and definite blood coagulation resisting function, its anticoagulation mechanism caused people's attention and by approach and application in clinical practice.Clinical or the preclinical test of rAcAPc2 (rNAPc2) and rAcAP5 (rNAP5) shows that they have broad application prospects, and is the anti-freezing antithrombotic reagent of developing of current extremely medical Experts ' Attention.
The inventor is through extensive and deep research, new Ancylostoma caninum anticoagulant peptide AcaNAP9, AcaNAP10 and AcaNAP11 gene have been separated to dog hookworm in the host dog enteron aisle available from Zhanjiang area and Guangxi, and expression and purification AcaNAP9, AcaNAP10 and AcaNAP11, and further disclosed these anticoagulant peptides and there is good anticoagulating active and the application aspect anticoagulant, antithrombotic reagent and anti-freezing preparation thereof.
Summary of the invention
One object of the present invention is to provide a kind of new Ancylostoma caninum anticoagulant peptide AcaNAP9 (SEQ ID NO.1), or its conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue.
One object of the present invention is to provide a kind of new Ancylostoma caninum anticoagulant peptide AcaNAP10 (SEQ ID NO.2), or its conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue.
One object of the present invention is to provide a kind of new Ancylostoma caninum anticoagulant peptide AcaNAP11 (SEQ ID NO.3), or its conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue.
One object of the present invention is to provide in the class of amino acid sequence and includes: the peptide sequence with anticoagulation of Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 characteristic sequence forms.
One object of the present invention is to provide the DNA (SEQ ID NO.11) of coding anticoagulant peptide AcaNAP9 or the DNA of its active fragments.
One object of the present invention is to provide the DNA (SEQ ID NO.12) of coding anticoagulant peptide AcaNAP10 or the DNA of its active fragments.
One object of the present invention is to provide the DNA (SEQ ID NO.13) of coding anticoagulant peptide AcaNAP11 or the DNA of its active fragments.
One object of the present invention is to provide the conservative property variation polypeptide of coding anticoagulant peptide AcaNAP9, AcaNAP10, AcaNAP11 or the DNA of its active fragments or its reactive derivative or its analogue.
In a first aspect of the present invention, the invention provides a kind of new polypeptide---AcaNAP9, it is comprised of the aminoacid sequence shown in SEQ ID NO.1 basically, preferably not containing signal peptide sequence (SEQ ID NO.4), or not containing 3 amino acid of carboxyl terminal (COO-) (SEQ ID NO.5), or not containing 3 amino acid of carboxyl terminal (COO-) and 3 amino acid of aminoterminal (NH-) (SEQ ID NO.6).The present invention also provides conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue of AcaNAP9.Polypeptide of the present invention can be natural polypeptides, recombinant polypeptide, synthetic polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast, insect and mammalian cell).
In a second aspect of the present invention, the invention provides a kind of new polypeptide---AcaNAP10, it is comprised of the aminoacid sequence shown in SEQ ID NO.2 basically, preferably not containing 3 amino acid of carboxyl terminal (COO-) (SEQ ID NO.7), or not containing 3 amino acid of carboxyl terminal (COO-) and 3 amino acid of aminoterminal (NH-) (SEQ ID NO.8).The present invention also provides conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue of AcaNAP10.Polypeptide of the present invention can be natural polypeptides, recombinant polypeptide, synthetic polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast, insect and mammalian cell).
In a third aspect of the present invention, the invention provides a kind of new polypeptide---AcaNAP11, it is comprised of the aminoacid sequence shown in SEQ ID NO.3 basically, preferably not containing 3 amino acid of carboxyl terminal (COO-) (SEQ ID NO.9), or not containing 3 amino acid of carboxyl terminal (COO-) and 3 amino acid of aminoterminal (NH-) (SEQ ID NO.10).The present invention also provides conservative property variation polypeptide or its active fragments or its reactive derivative or its analogue of AcaNAP11.Polypeptide of the present invention can be natural polypeptides, recombinant polypeptide, synthetic polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast, insect and mammalian cell).
In a fourth aspect of the present invention, the invention provides in aminoacid sequence the polypeptide with anticoagulation that includes Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 characteristic sequence.Wherein, 1. A1 is one section and includes aminoacid sequence Gly-Glu-Asn-Glu-Glu-Tyr-Asp-Val (SEQ ID NO.14), or Gly-Glu-Asn-Glu-Arg-His-Asp-Glu (SEQ ID NO.15), or sequence or their conservative variations aminoacid sequence or their analogue of one of Gly-Glu-Asn-Glu-Arg-Tyr-Asp-Asp (SEQ ID NO.16); 2. A2 is one section and includes aminoacid sequence Gly-Asn-Arg-Thr (SEQ ID NO.17), or Ser-Arg-Lys-Glu (SEQ ID NO.18), or Asn-Arg-Lys-Glu (SEQ ID NO.19). one of sequence or their conservative variations aminoacid sequence or their analogue; 3. A3 is one section and includes aminoacid sequence Asp-Leu-Lys (SEQ ID NO.20), or sequence or their conservative variations aminoacid sequence or their analogue of one of Asp-Pro-Lys (SEQ ID NO.21); 4. A4 is one section and includes aminoacid sequence Gln-Tyr-Asp-Gly-Ala-Glu-Lys-Lys-Asp-Glu-Glu-Arg-Asn-Ala-Glu (SEQ ID NO.22), or Lys-Tyr-Asp-Gly-Thr-Glu-Glu-Lys-Asp-Asp-Glu-Lys-Pro-Val-Val (SEQ ID NO.23), or sequence or their conservative variations aminoacid sequence or their analogue of one of Lys-Tyr-Asp-Gly-Thr-Glu-Glu-Lys-Asp-Asp-Glu-Lys-Pro-Val-Glu (SEQ ID NO.24); 5. A5 is the sequence that one section 4 amino-acid residue forms; 6. A6 is one section and includes aminoacid sequence Tyr-Asp-Gly-Asp (SEQ ID NO.25), or Tyr-Gly-Asp (SEQ ID NO.26), or sequence or their conservative variations aminoacid sequence or their analogue of one of His-Gly-Asp (SEQ ID NO.27); 7. A7 is Val (α-amino-isovaleric acid) or Ile (Isoleucine), or its conservative variations amino acid; 8. A8 is one section and includes aminoacid sequence Arg-Lys-Gly-Phe-Tyr-Arg-Asn-Asn-Asn-Gly-Arg (SEQ ID NO.28), or Arg-Asp-Gly-Phe-Leu-Arg-Lys-Asn-Asn-Gly-Ala (SEQ ID NO.29). one of sequence or their conservative variations aminoacid sequence or their analogue; 9. A9 is one section and includes aminoacid sequence Val-Thr-Ala-Glu-Asp (SEQ ID NO.30), or sequence or their conservative variations aminoacid sequence or their analogue of one of Val-Lys-Ala-Glu-Asp (SEQ ID NO.31); 10. A10 is one section sequence or its conservative variations aminoacid sequence or its analogue that includes aminoacid sequence Asn-Met-Glu-Phe-Ile-Tyr-Pro (SEQ ID NO.32).
In a fifth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP9 gene, these polynucleotide derive from the dog hookworm, it comprises: coding has SEQ ID NO.1, or SEQ ID NO.4, or SEQ ID NO.5, or the polynucleotide of the polypeptide of the aminoacid sequence of SEQ ID NO.6.
In a sixth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP10 gene, these polynucleotide derive from the dog hookworm, and it comprises: basic coding has SEQ ID NO.2, or SEQ ID NO.7, or the polynucleotide of the polypeptide of the aminoacid sequence of SEQ ID NO.8.
In a seventh aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP11 gene, these polynucleotide derive from the dog hookworm, and it comprises: basic coding has SEQ ID NO.3, or SEQ ID NO.9, or the polynucleotide of the polypeptide of the aminoacid sequence of SEQ ID NO.10.
In a eighth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation, the conservative property variation polypeptide that it comprises encoding sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 or the polynucleotide of their active fragments or their reactive derivative or their analogue.
In a ninth aspect of the present invention, the invention provides the application aspect anticoagulant or anti-freezing preparation of anticoagulant peptide AcaNAP9 or AcaNAP10 or AcaNAP11 and their conservative property variation polypeptide or their active fragments or their reactive derivative or their analogue.
Other side of the present invention, because this paper technology contents discloses, is easy to understand and implements those skilled in the art.For example, utilize the present invention can relate to a kind of carrier that contains the DNA of anticoagulant peptide in the code book invention; The genetically engineered host cell of expression vector that a kind of use contains the DNA of anticoagulant peptide in the code book invention; A kind of method of cultivating genetically engineered host cell and preparing polypeptide of the present invention that comprises.
The following terms in the specification and claims have following implication unless stated otherwise:
" amino acid " refers to natural L-type amino acid and has maintained bioactive D type amino acid.Natural L-type amino acid comprises L-Ala (Ala, A), arginine (Arg, R), l-asparagine (Asn, N), aspartic acid (Asp, D), halfcystine (Cys, C), glutamine (Gln, Q), L-glutamic acid (Glu, E), glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), leucine (Leu, L), Methionin (Lys, K), methionine(Met) (methionine(Met)) (Met, M), phenylalanine (Phe, F), proline(Pro) (Pro, P), Serine (Ser, S), Threonine (Thr, T), tryptophane (Trp, W), tyrosine (Tyr, Y) and α-amino-isovaleric acid (Val, V).
" aminoacid sequence " refers to oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " in the present invention relates to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " do not mean that this is restricted to the complete natural amino acid relevant to described protein molecule by aminoacid sequence.Similarly, term " nucleotide sequence " refers to oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA.
Protein or polynucleotide " variant " refer to a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and the amino acid of wherein replacing has the structure similar with original acid or chemical property, as with leucine, replaced Isoleucine.Variant also can have non-conservation and change, as with tryptophane, replaced glycine.
For example, in the original environment (, spontaneous its natural surroundings that just refers to) that " separation " word refers to material is existed from it, separate.For example, spontaneous polynucleotide or polypeptide are exactly not to be separated in being present in organism, but separately separation of the material that same polynucleotide sequence or polypeptide coexist with it in natural system (as aforementioned organism) with some or all.
" cDNA " refers to complementary DNA.
" mRNA " refers to messenger RNA(mRNA).
" anti-freezing " refers to have the effect that inhibition blood (comprising blood plasma) solidifies.
In a first aspect of the present invention, the invention provides a kind of new polypeptide-AcaNAP9 of separation, it is undertaken by the total RNA from dog hookworm adult that amplification obtains reverse transcription nucleotide sequence coded.The peptide total length is comprised of 94 amino-acid residues, and wherein, mature peptide is comprised of 81 amino-acid residues, the signal peptide be comprised of 13 amino-acid residues in addition.According to amino acid identity relatively, (GenBank typing number: AAP82926) have maximum homology, their consistence is 65% (62/94) for this polypeptide total length and Ancylostoma caninum anticoagulant peptide AcAPc4; Mature peptide and AcAPc3 (GenBank typing number: AAP57305) there is maximum homology, secondly their consistence is 68% (52/76), with AcAPc2 (GenBank typing number: AAC47080) and AcaNAP7 (GenBank typing number: ABD98795) 65% consistence is also arranged.
In a second aspect of the present invention, the invention provides a kind of new polypeptide-AcaNAP10 of separation, it is undertaken by the total RNA from dog hookworm adult that amplification obtains reverse transcription nucleotide sequence coded.This peptide is comprised of 80 amino-acid residues.According to amino acid identity relatively, this polypeptide and Ancylostoma caninum anticoagulant peptide AcAPc4 (GenBank typing number: AAP82926) there is maximum homology, their consistence is 87%, with Ancylostoma caninum anticoagulant peptide AcAPc3 (GenBank typing number: AAP57305), AcaNAP7 (GenBank typing number: ABD98795), (GenBank typing number: consistence AAC47080) is followed successively by 76%, 70%, 66% to AcAPc2.
In a third aspect of the present invention, the invention provides a kind of new polypeptide-AcaNAP11 of separation, it is undertaken by the total RNA from dog hookworm adult that amplification obtains reverse transcription nucleotide sequence coded.This peptide is comprised of 80 amino-acid residues.According to amino acid identity relatively, this polypeptide and Ancylostoma caninum anticoagulant peptide AcAPc4 (GenBank typing number: AAP82926) there is maximum homology, their consistence is 92%, with Ancylostoma caninum anticoagulant peptide AcAPc3 (GenBank typing number: AAP57305), AcaNAP7 (GenBank typing number: ABD98795), (GenBank typing number: consistence AAC47080) is followed successively by 76%, 73%, 69% to AcAPc2.
Anticoagulant peptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, first-selected recombinant polypeptide.Polypeptide of the present invention can be the natural product of separation and purification, or chemosynthesis product, or the product that uses recombinant technology for example, to express in protokaryon or eucaryon host (, bacterium, yeast, insect and mammalian cell).
In the present invention, the conservative property variation polypeptide, active fragments, derivative and the analogue that also comprise Ancylostoma caninum anticoagulant peptide AcaNAP9, AcaNAP10, AcaNAP11.In the present invention, term " conservative property variation polypeptide ", " fragment ", " derivative " and " analogue " refer to and basically keep Ancylostoma caninum anticoagulant peptide AcaNAP9 of the present invention, biological function or active polypeptide that AcaNAP10, AcaNAP11 are identical.These polypeptide can be: 1., in aminoacid sequence, the polypeptide of the formation that amino acid is replaced that one or more amino acid is similar or close by character is arranged; 2. in aminoacid sequence, several aminoacid deletion, insertion are arranged or replace formed polypeptide; 3. the allelic variant of polypeptide, natural variation body, natural mutation, induced mutation body; 4. the fusion of aforementioned anticoagulant peptide and another compound (as polyoxyethylene glycol); 5. the chemically derived form of aforementioned anticoagulant peptide is as acetylize, carboxylated or glycosylation etc.; 6. the peptide sequence that adds one section aminoacid sequence to be integrated into aforementioned polypeptide and form.By the elaboration of this paper, such " conservative property variation polypeptide ", " fragment ", " derivative " and " analogue " are considered within those skilled in the art's ken.Should be appreciated that polypeptide of the present invention is not limited to the above-mentioned representative polypeptide of enumerating.
In a fourth aspect of the present invention, the invention provides in aminoacid sequence the polypeptide with anticoagulation that includes Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 sequence signature.Indication " A1 ", " A2 ", " A3 ", " A4 ", " A5 ", " A6 ", " A7 ", " A8 ", " A9 " are followed successively by one section aminoacid sequence between adjacent two halfcystines (Cys) in sequence, and " A10 " is one section aminoacid sequence after last halfcystine (Cys) carboxyl terminal.In the present invention, 1. A1 is one section and includes aminoacid sequence Gly-Glu-Asn-Glu-Glu-Tyr-Asp-Val (SEQ ID NO.14), or Gly-Glu-Asn-Glu-Arg-His-Asp-Glu (SEQ ID NO.15), or sequence or their conservative variations aminoacid sequence or their analogue of one of Gly-Glu-Asn-Glu-Arg-Tyr-Asp-Asp (SEQ ID NO.16), preferably this sequence is that 8 amino-acid residues form; 2. A2 is one section and includes aminoacid sequence Gly-Asn-Arg-Thr (SEQ ID NO.17), or Ser-Arg-Lys-Glu (SEQ ID NO.18), or sequence or their conservative variations aminoacid sequence or their analogue of one of Asn-Arg-Lys-Glu. (SEQ ID NO.19), preferably this sequence is that 4 amino-acid residues form; 3. A3 is one section and includes aminoacid sequence Asp-Leu-Lys (SEQ ID NO.20), or sequence or their conservative variations aminoacid sequence or their analogue of one of Asp-Pro-Lys (SEQ ID NO.21), preferably this sequence is that 3 amino-acid residues form; 4. A4 is one section and includes aminoacid sequence Gln-Tyr-Asp-Gly-Ala-Glu-Lys-Lys-Asp-Glu-Glu-Arg-Asn-Ala-Glu (SEQ ID NO.22), or Lys-Tyr-Asp-Gly-Thr-Glu-Glu-Lys-Asp-Asp-Glu-Lys-Pro-Val-Val (SEQ ID NO.23), or sequence or their conservative variations aminoacid sequence or their analogue of one of Lys-Tyr-Asp-Gly-Thr-Glu-Glu-Lys-Asp-Asp-Glu-Lys-Pro-Val-Glu (SEQ ID NO.24), preferably this sequence is that 15 amino-acid residues form; 5. A5 is the sequence that one section 4 amino-acid residue forms; 6. A6 is one section and includes aminoacid sequence Tyr-Asp-Gly-Asp (SEQ ID NO.25), or Tyr-Gly-Asp (SEQ ID NO.26), or sequence or their conservative variations aminoacid sequence or their analogue of one of His-Gly-Asp (SEQ ID NO.27), preferably this sequence is that 3-4 amino-acid residue forms; 7. A7 is Val (α-amino-isovaleric acid) or Ile (Isoleucine), or its conservative variations amino acid; 8. A8 is one section and includes aminoacid sequence Arg-Lys-Gly-Phe-Tyr-Arg-Asn-Asn-Asn-Gly-Arg (SEQ ID NO.28), or sequence or their conservative variations aminoacid sequence or their analogue of one of Arg-Asp-Gly-Phe-Leu-Arg-Lys-Asn-Asn-Gly-Ala. (SEQ ID NO.29), preferably this sequence is that 11 amino-acid residues form; 9. A9 is one section and includes aminoacid sequence Val-Thr-Ala-Glu-Asp (SEQ ID NO.30), or sequence or their conservative variations aminoacid sequence or their analogue of one of Val-Lys-Ala-Glu-Asp (SEQ ID NO.31), preferably this sequence is that 5 amino-acid residues form; 10. A10 is one section sequence or its conservative variations aminoacid sequence or its analogue that includes aminoacid sequence Asn-Met-Glu-Phe-Ile-Tyr-Pro (SEQ ID NO.32).
In a fifth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP9 gene, it is to be undertaken with 3 '-RACE (cDNA end rapid amplifying) and RT-PCR amplification, obtaining reverse transcription by the total RNA from dog hookworm adult.The polynucleotide sequence total length that it comprises is 443 bases (13 adenylic acid (AMP) A that comprise 3 ' tail end), its open reading frame (26-310) 94 amino acid of having encoded.According to nucleotide homology relatively, (GenBank typing number: AY253915) have maximum homology, their consistence is 86% for this polynucleotide sequence and Ancylostoma caninum anticoagulant peptide AcAPc4 gene.With (the GenBank typing number: U30793) there is 80% consistence of AcAPc2 gene.
In a sixth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP10 gene, it is to be undertaken with the RT-PCR amplification, obtaining reverse transcription by the total RNA from dog hookworm adult.The polynucleotide sequence that it comprises is 243 bases (containing terminator codon), 80 amino acid of encoding.According to nucleotide homology relatively, this polynucleotide sequence and (the GenBank typing number: AY253915) there is maximum homology of Ancylostoma caninum anticoagulant peptide AcAPc4 gene, their consistence is 93%, with (the GenBank typing number: AY232998), (GenBank typing number: DQ435781) consistence is respectively 84%, 80% to the AcaNAP7 gene of AcAPc3 gene.
In a seventh aspect of the present invention, the present invention relates to a kind of polynucleotide of separation-AcaNAP11 gene, it is to be undertaken with the RT-PCR amplification, obtaining reverse transcription by the total RNA from dog hookworm adult.The polynucleotide sequence that it comprises is 372 bases (containing 11 adenylic acid (AMP) A of terminator codon, 3-UTR and 3 ' tail end), polynucleotide sequence 1-240 80 amino acid of encoding.According to nucleotide homology relatively, this polynucleotide sequence and (the GenBank typing number: AY253915) there is maximum homology of Ancylostoma caninum anticoagulant peptide AcAPc4 gene, their consistence is 95%, with (the GenBank typing number: AY232998), (the GenBank typing number: DQ435781), (GenBank typing number: U30793) gene identity is respectively 84%, 81%, 80% to AcAPc2 of AcaNAP7 gene of AcAPc3 gene.
In a eighth aspect of the present invention, the present invention relates to a kind of polynucleotide of separation, the conservative property variation polypeptide that it comprises encoding sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 or polynucleotide or its degenerate sequence of their active fragments or their reactive derivative or their analogue.Those skilled in the art know, and identical amino acid can have 1-6 codon.In the present invention, " degenerate sequence " refer in the Nucleotide of code book invention anticoagulant peptide, the nucleotide sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.
Polynucleotide full length sequence or its fragment of coding anticoagulant peptide of the present invention can obtain by several different methods, these methods comprise with hybridization technique from dog hookworm eDNA library, screen, RT-PCR TRAP, recombination method or the synthetic method acquisition of artificial chemistry, but be not limited only to the above-mentioned method listed.The method of more often selecting is to extract RNA from the dog hookworm, mRNA more preferably, carry out reverse transcription after amplification obtain; Or extract mRNA, build in the eDNA library and screening by hybridization or amplification separation.Extract RNA or mRNA, the existing multiple proven technique in construction cDNA library.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and with this carrier or the host cell that directly produces through genetically engineered with the hookworm coagulate peptide resistant encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.
In the present invention, coding anticoagulant peptide polynucleotide sequence (DNA) is inserted in carrier, the recombinant vectors that contains polynucleotide of the present invention with formation.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, mammalian cell virus are as adenovirus, retrovirus or other carrier.Also be not limited in the present invention the above-mentioned carrier of enumerating.
In the present invention, the polynucleotide of coding anticoagulant peptide or the recombinant vectors that contains these polynucleotide can be transformed or transduced into host cell, the genetically engineered host cell that contains these polynucleotide or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as intestinal bacteria; Or eukaryotic cell, as yeast cell and CHO etc.These host cells are that those skilled in the art understand.
With DNA sequence dna of the present invention or the recombinant vectors transformed host cell that contains described DNA sequence dna, can carry out with routine techniques well known to those skilled in the art.By conventional recombinant DNA technology, utilize polynucleotide sequence of the present invention to can be used to the anticoagulant peptide of expression or Restruction.In general step is once arranged:
(1). with the polynucleotide of coded polypeptide of the present invention, or transform or transduction appropriate host cell with the recombinant expression vector that contains these polynucleotide;
(2). cultivate host cell in suitable medium;
(3). separation, protein purification from substratum or host cell.
Except recombination method produces, the polypeptide basis in the present invention is available solid phase technique also, is produced by direct peptide synthesis, or synthesizes each fragment respectively, then by chemical process, is connected to produce the polypeptide in the present invention.
The recombinant protein energy significant prolongation human plasma prothrombin time (PT) that comprises polypeptide of the present invention and activated partial thrombozyme time (aPTT), have remarkable anticoagulating active, can be used for as anticoagulant or anti-freezing preparation.
The recombinant protein that comprises polypeptide of the present invention has anti-animal thrombosis formation effect, can be used for conduct or develops into the medicine for treating thrombus thing.
The accompanying drawing explanation
Fig. 1: the aminoacid sequence of Ancylostoma caninum anticoagulant peptide AcaNAP9 nucleotide sequence and coding thereof.
Fig. 2: the aminoacid sequence of Ancylostoma caninum anticoagulant peptide AcaNAP10 nucleotide sequence and coding thereof.
Fig. 3: the aminoacid sequence of Ancylostoma caninum anticoagulant peptide AcaNAP11 nucleotide sequence and coding thereof.
The SDS-PAGE figure that Fig. 4: AcaNAP9/pET-32a expresses at Host Strains BL21 (DE3).Wherein: swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is without the pET-32a/AcaNAP9 induced; Swimming lane 3 is the pET-32a/AcaNAP9 through inducing; Swimming lane 4,5 is without the empty plasmid pET-32a induced; Swimming lane 6 is the soluble protein expression; Swimming lane 7 is soluble protein expression situation; Swimming lane 8 is the Trx-AcaNAP9 fusion rotein through the solidifying FF affinity purification of nickel agarose.
The SDS-PAGE figure that Fig. 5: AcaNAP10/pET-32a expresses at Host Strains BL21 (DE3).Wherein: swimming lane 1 is without the pET-32a/AcaNAP10 induced; Swimming lane 2 is without the empty plasmid pET-32a induced; Swimming lane 3 is the pET-32a/AcaNAP10 through inducing; Swimming lane 4 is the Trx-AcaNAP10 fusion rotein through the solidifying FF affinity purification of nickel agarose; Swimming lane 5 is the molecular weight of albumen standard.
The SDS-PAGE figure that Fig. 6: AcaNAP11/pET-32a expresses at Host Strains BL21 (DE3).Wherein: swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is the empty plasmid pET-32a through inducing; Swimming lane 3 is the pET-32a/AcaNAP11 through inducing; Swimming lane 4 is the Trx-AcaNAP11 fusion rotein through the solidifying FF affinity purification of nickel agarose; Swimming lane 5 is the pET-32a/AcaNAP11cd (AcaNAP11cd represents 3 amino acid of AcaNAP11 mature peptide carboxy terminal deletion, i.e. SEQ ID NO.5 sequence) through inducing; Swimming lane 6 is the Trx-AcaNAP11cd fusion rotein through the solidifying FF affinity purification of nickel agarose; Swimming lane 7 is the pET-32a/AcaNAP11cnd (AcaNAP11cnd represents that AcaNAP11 mature peptide aminoterminal and carboxyl terminal lack respectively 3 amino acid, i.e. SEQ ID NO.6 sequence) through inducing; Swimming lane 6 is the Trx-AcaNAP11cnd fusion rotein through the solidifying FF affinity purification of nickel agarose.
Embodiment
Below in conjunction with specific examples, further set forth the present invention.Should be understood that these enforcements only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of hookworm coagulate peptide resistant gene
1. design of primers
According to AcaNAP7 (GenBank typing number: DQ435781), AcAPc4 (GenBank typing number: AY253915), AcAPc3 (GenBank typing number: AY232998) and AcAPc2 (GenBank typing number: U30793) nucleotide sequence design primer downstream primer N2; According to hookworm Spliced leader sequence design upstream primer NSL1, sequence is as follows:
NSL1：5’-GGTTTAATTACCCAAGTTTGAG-3’
N2：5’-TTTGGTCATTTTCTGTTAGG-3’
2.RNA separation and reverse transcription
Get 20 of dog hookworm adults (the host dog is from Zhanjiang area and Guangxi), with TRIzol Reagent (Invitrogen), by operation instructions, extract total RNA.With 3 '-Full RACE Core Set (Dalian Takara), the total RNA of dog hookworm extracted is carried out to reverse transcription according to operation instructions and obtain cDNA.
3. the amplification of anticoagulant peptide gene fragment, Clone and sequence
The 3 μ l dog hookworm adult cDNA that the preceding method of take obtains are template, and NSL1 and N2 are primer, and amplification obtains about 300bp nucleic acid fragment.The PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 50 ℃, 30s; Extend, 72 ℃, 1min, 30 circulations; Finally extend 72 ℃, 10min.Amplified production, with after DNA purification kit (TIANGEN company) purifying, is connected to pUCm-T Vector with pUCm-T Vector PCR product cloning test kit (Shanghai Sangon), Transformed E .coli DH5 α competent cell.Random 10 positive colonies of picking, deliver Shanghai Sangon and checked order, and obtained the gene of 2 kinds of coding different aminoacids sequences.
According to sequencing result, design primer upstream primer N9-1, N10-1, anchor primer (SAP) sequence that downstream primer provides with 3 '-Full RACE Core Set test kit, each primer sequence is as follows:
N9-1：5’-GCAGTACTAACATTGTTAAACCAAA-3’
N10-1：5’-GCAGTGTAATGCAAATCCAAG-3’
SAP：5’-CTGATCTAGAGGTACCGGATCC-3’
(1) clone of AcaNAP9 full length gene sequence
The 2 μ l dog hookworm cDNA of take are template, and N9-1 and SAP are primer, and the PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 50 ℃, 30s; Extend, 72 ℃, 1min, 30 circulations; Finally extend 72 ℃, 10min, amplification obtains about 400bp nucleic acid fragment.Amplified production is purified, TA delivers Shanghai Sangon order-checking after cloning.Sequencing result, after splicing, obtains AcaNAP9 full length gene sequence, i.e. SEQ ID NO.11.
(2) screening of AcaNAP11 sequence and clone
The 2 μ l dog hookworm cDNA of take are template, and N10-1 and SAP are primer, and the PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 50 ℃, 30s; Extend, 72 ℃, 1min, 30 circulations; Finally extend 72 ℃, 10min, amplification obtains about 400bp nucleic acid fragment.Amplified production is purified, TA delivers Shanghai Sangon order-checking after cloning, and obtains anticoagulant peptide AcaNAP11 encoding sequence and 3-UTR thereof, i.e. SEQ ID NO.13.
(3) screening of AcaNAP10 sequence and clone
For obtaining the AcaNAP11 encoded peptide, according to AcaNAP11 sequence results design primer, design primer NAP11-2, sequence is as follows:
N11-2：5’-CGAAGCTTGGTCATTTTCTATTAGGG-3’。
The 2 μ l dog hookworm eDNA of take are template, and N10-1 and N11-2 are primer, and the PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 50 ℃, 30s; Extend, 72 ℃, 1min, 30 circulations; Finally extend 72 ℃, 10min, amplification obtains about 250bp nucleic acid fragment.Amplified production is purified, after TA clone, select positive colony and deliver Shanghai Sangon order-checking.Except obtaining coding AcaNAP11 sequence, also obtain coding other one anticoagulant peptide-AcaNAP10 gene order, i.e. SEQ ID NO.12.
Embodiment 2: Ancylostoma caninum anticoagulant peptide AcaNAP9 sequence information and homology analysis
The Ancylostoma caninum anticoagulant peptide AcaNAP9 full length cDNA sequence total length of separating is 443 bases (13 adenylic acid (AMP) A that comprise 3 ' tail end) (SEQ ID NO.11), its open reading frame (26-310) peptide classes (SEQ ID NO.1) that 94 amino acid form of having encoded, wherein mature peptide forms (SEQ ID NO.4) by 81 amino-acid residues, the signal peptide that also has 13 amino-acid residues to form in addition.(Fig. 1).
Ancylostoma caninum anticoagulant peptide AcaNAP9 full length cDNA sequence and coded protein thereof are carried out to nucleic acid and protein homology search in the Genbank+EMBL+DDBJ+PD database with blast program, show in database with AcaNAP9 amino acid sequence homology maximum be that (GenBank typing number: AAP82926), their consistence is 65% (62/94) to Ancylostoma caninum anticoagulant peptide AcAPc4; With AcaNAP9 nucleotide sequence homology maximum be (the GenBank typing number: AY253915) of Ancylostoma caninum anticoagulant peptide AcAPc4 gene, their consistence is that (GenBank typing number: U30793) gene also has 80%-causing property for 86%, AcaNAP9 nucleotide sequence and AcAPc2.
Embodiment 3: Ancylostoma caninum anticoagulant peptide AcaNAP10 sequence information and homology analysis
The Ancylostoma caninum anticoagulant peptide AcaNAP10cDNA sequence of separating is 243 bases (containing terminator codon) (SEQ ID NO.12), polynucleotide sequence 1-240 80 amino acid (SEQ ID NO.2) of encoding.(Fig. 2).
Carry out nucleic acid and protein homology search in the Genbank+EMBL+DDBJ+PD database with blast program, show in database with AcaNAP10 amino acid sequence homology maximum be Ancylostoma caninum anticoagulant peptide AcAPc4 (GenBank typing number: AAP82926), their consistence is 87%, other is followed successively by AcAPc3 (GenBank typing number: AAP57305) (consistence is 76%), AcaNAP7 (consistence is 70%), AcAPc2 (consistence is 66%).; With AcaNAP10 nucleotide sequence homology maximum be (the GenBank typing number: AY253915) of Ancylostoma caninum anticoagulant peptide AcAPc4 gene, their consistence is 93%, AcaNAP10 nucleotide sequence and AcAPc3 (GenBank typing number: AY232998), (GenBank typing number: DQ435781) gene identity is respectively 84%, 80% to AcaNAP7.
Embodiment 4: Ancylostoma caninum anticoagulant peptide AcaNAP11 sequence information and homology analysis
The Ancylostoma caninum anticoagulant peptide AcaNAP11 cDNA sequence of separating is 372 bases (containing 11 adenylic acid (AMP) A of terminator codon, 3 '-UTR and 3 ' tail end) (SEQ ID NO.13), polynucleotide sequence 1-240 80 amino acid (SEQ ID NO.3) of encoding.(Fig. 3).
Carry out nucleic acid and protein homology search in the Genbank+EMBL+DDBJ+PD database with blast program, show in database with AcaNAP11 amino acid sequence homology maximum be Ancylostoma caninum anticoagulant peptide AcAPc4 (GenBank typing number: AAP82926), their its consistence is 92%, other and Ancylostoma caninum anticoagulant peptide AcAPc3 (GenBank typing number: AAP57305), AcaNAP7 (GenBank typing number: ABD98795), (GenBank typing number: consistence AAC47080) is followed successively by 76%, 73%, 69% to AcAPc2.; With AcaNAP11 nucleotide sequence homology maximum be (the GenBank typing number: AAP82926) of Ancylostoma caninum anticoagulant peptide AcAPc4 gene, their consistence be 95%, AcaNAP11 nucleotide sequence with (the GenBank typing number: AY232998), (the GenBank typing number: DQ435781), (GenBank typing number: U30793) consistence is respectively 84%, 81%, 80% to the AcAPc2 gene of AcaNAP7 gene of AcAPc3 gene.
Embodiment 5: the vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP9, separation and purifying
(1) vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP9 mature peptide, separation and purifying
According to coding region sequence shown in sequence SEQ ID NO.11 and Fig. 1, design a pair of Auele Specific Primer, sequence is as follows:
N9-3:5 '-GCGGATCCAAACCAAACTGTGGTGAGA-3 ' (containing restriction enzyme site BamH I and protection bases G C)
N9-4:5 '-CGAAGCTTGGTCATTTTCTTTTAGGG-3 ' (containing restriction enzyme site Hind III and protection base CG)
BamH I and Hind III restriction enzyme site are corresponding to the selectivity restriction enzyme site on expression vector PET-32a.Take recombinant plasmid AcaNAP9/pUCm-T as template, the nucleotide sequence of amplification coding AcaNAP9 albumen (SEQ ID NO.1).The PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 54 ℃, 30s; Extend, 72 ℃, 30s, 30 circulations.Finally extend 72 ℃, 10min.The PCR product carries out double digestion with BamH I and Hind III after reclaiming test kit recovery purifying with sepharose; Expression plasmid PET-32a also uses BamH I and Hind III double digestion.Reclaim respectively aforementioned two enzymes and cut product, both are used to T
4dNA ligase connects and will connect product and is converted in E.coli DH5a competent cell, in containing the substratum of penbritin, is cultivating.With PCR method screening positive clone, and the evaluation of checking order, the recombinant plasmid called after AcaNAP9/pET-32a that gained is correct.Through identifying that correct recombinant expression plasmid is converted into e. coli bl21 (DE3) competent cell.Containing recombinant expression plasmid AcaNAP9/pET-32a Host Strains BL21 (DE3) containing cultivation in the LB nutrient solution of penbritin, adding IPTG is that 1.0mmol/L is induced to final concentration.Centrifugal collection thalline is collected supernatant after ultrasonication, gets supernatant liquor and carries out purifying with the solidifying FF (Beijing Zhuo Guan Science and Technology Ltd.) of nickel agarose.Fusion protein molecule amount size and purity that row SDS-PAGE electrophoresis detection is expressed.Result has shown to obtain aminoterminal and has merged the Trx-AcaNAP9 fusion rotein that Trx (Trx) is arranged, and fusion protein molecule amount size is about 29Kda.Because the molecular size range of the N-terminal fusion rotein of AcaNAP9 is about 20Kda, calculate that AcaNAP9 molecular weight of albumen size is about 9Kda, conforms to theoretical prediction.This electrophoresis result is shown in Fig. 4.
(2) Ancylostoma caninum anticoagulant peptide AcaNAP9 mature peptide does not contain vivoexpression, separation and the purifying of 3 amino acid whose peptide sections of carboxyl terminal (AcaNAP9cd)
According to 3 amino acid peptide sections of coding disappearance carboxyl terminal in coding region sequence shown in Fig. 1, be SEQ ID NO.5 encoding sequence design upstream primer N9-3 (5 '-GCGGATCCAAACCAAACTGTGGTGAGA-3 ', containing restriction enzyme site BamH I and protection bases G C) and downstream primer N9-6 (5 '-CGAAGCTTAGGGTAGATGAACTCCATATT-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP9/pUCm-T as template, amplification coding AcaNAP9 mature peptide does not contain the nucleotide sequence of 3 amino acid whose peptide sections of carboxyl terminal (SEQ ID NO.5).Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP9cd, mature peptide does not contain the fusion rotein of 3 amino acid whose peptide section AcaNAP9cd of carboxyl terminal and Trx (Trx).
(3) Ancylostoma caninum anticoagulant peptide AcaNAP9 mature peptide containing the vivoexpression of carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal (AcaNAP9cnd), separate and purifying
According to coding in coding region sequence shown in Fig. 1 containing 3 amino acid whose encoding sequences design upstream primer N9-5 of carboxyl terminal and aminoterminal (5 '-GCGGATCCTGTGGTGAGAATGAAGAGTA-3 ', containing restriction enzyme site BamH I and protection bases G C) and downstream primer N9-6 (5 '-CGAAGCTTAGGGTAGATGAACTCCATATT-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP9/pUCm-T as template, amplification coding AcaNAP9 mature peptide is not containing carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal, it is the nucleotide sequence of SEQ ID NO.6.Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP9cnd, mature peptide does not contain the fusion rotein of carboxyl terminal and 3 amino acid whose peptide section AcaNAP9cnd of aminoterminal and Trx (Trx).
Embodiment 6: the vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP10, separation and purifying
(1) vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP10 mature peptide, separation and purifying
According to coding region sequence shown in sequence SEQIDNO.12 and Fig. 2, design a pair of Auele Specific Primer, sequence is as follows:
N10-3:5 '-GAGGATCCAATCCAAGCTGTGGTGAG-3 ' (containing restriction enzyme site BamH I and protection bases G A)
N11-2:5 '-CGAAGCTTGGTCATTTTCTATTAGGG-3 ' (containing restriction enzyme site Hind III and protection base CG)
BamH I and Hind III restriction enzyme site are corresponding to the selectivity restriction enzyme site on expression vector PET-32a.Take recombinant plasmid AcaNAP10/pUCm-T as template, the nucleotide sequence of amplification coding AcaNAP10 albumen (SEQ ID NO.2).The PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 54 ℃, 30s; Extend, 72 ℃, 30s, 30 circulations.Finally extend 72 ℃, 10min.The PCR product carries out double digestion with BamH I and Hind III after reclaiming test kit recovery purifying with sepharose; Expression plasmid PET-32a also uses BamH I and Hind III double digestion.Reclaim respectively aforementioned two enzymes and cut product, both are used to T
4dNA ligase connects and will connect product and is converted in E.coli DH5a competent cell, in containing the substratum of penbritin, is cultivating.With PCR method screening positive clone, and the evaluation of checking order, the recombinant plasmid called after AcaNAP10/pET-32a that gained is correct.Through identifying that correct recombinant expression plasmid is converted into e. coli bl21 (DE3) competent cell.Containing recombinant expression plasmid AcaNAP10/pET-32a Host Strains BL21 (DE3) containing cultivation in the LB nutrient solution of penbritin, adding IPTG is that 1.0mmol/L is induced to final concentration.Centrifugal collection thalline is collected supernatant after ultrasonication, gets supernatant liquor and carries out purifying with the solidifying FF (Beijing Zhuo Guan Science and Technology Ltd.) of nickel agarose.Fusion protein molecule amount size and purity that row SDS-PAGE electrophoresis detection is expressed.Result has shown to obtain aminoterminal and has merged the Trx-AcaNAP10 fusion rotein that Trx (Trx) is arranged, and fusion protein molecule amount size is about 29Kda.Because the molecular size range of the N-terminal fusion rotein of AcaNAP10 is about 20Kda, calculate that AcaNAP10 molecular weight of albumen size is about 9Kda, conforms to the theoretical prediction result.Electrophoresis result is shown in Fig. 5.
(2) Ancylostoma caninum anticoagulant peptide AcaNAP10 mature peptide does not contain vivoexpression, separation and the purifying of 3 amino acid whose peptide sections of carboxyl terminal (AcaNAP10cd)
According to 3 amino acid peptide sections of coding disappearance carboxyl terminal in coding region sequence shown in Fig. 2, be SEQ ID NO.7 encoding sequence design upstream primer N10-3 (5 '-GAGGATCCAATCCAAGCTGTGGTGAG-3 ', containing restriction enzyme site BamH I and protection bases G A) and downstream primer N10-6 (5 '-CGAAGCTTAGGGTAGATAAACTCCATGTTGTC-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP10/pUCm-T as template, amplification coding AcaNAP10 mature peptide does not contain the nucleotide sequence of 3 amino acid whose peptide sections of carboxyl terminal (SEQ ID NO.7).Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP10cd, mature peptide does not contain the fusion rotein of 3 amino acid whose peptide section AcaNAP10cd of carboxyl terminal and Trx (Trx).
(3) Ancylostoma caninum anticoagulant peptide AcaNAP10 mature peptide containing the vivoexpression of carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal (AcaNAP10cnd), separate and purifying
Do not contain carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal according to coding in coding region sequence shown in Fig. 2, be SEQ ID NO.8 encoding sequence design upstream primer N10-5 (5 '-GCGGATCCTGTGGTGAGAATGAAAGGC-3 ', containing restriction enzyme site BamH I and protection bases G C) and downstream primer N10-6 (5 '-CGAAGCTTAGGGTAGATAAACTCCATGTTGTC-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP10/pUCm-T as template, amplification coding AcaNAP10 mature peptide is not containing carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal, it is the nucleotide sequence of SEQ ID NO.8.Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP10cnd, mature peptide does not contain the fusion rotein of carboxyl terminal and 3 amino acid whose peptide section AcaNAP10cnd of aminoterminal and Trx (Trx).
Embodiment 7: the vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP11, separation and purifying
(1) vivoexpression of Ancylostoma caninum anticoagulant peptide AcaNAP11 mature peptide, separation and purifying
According to coding region sequence shown in sequence SEQ ID NO.13 and Fig. 3, design a pair of Auele Specific Primer, sequence is as follows:
N10-3:5 '-GAGGATCCAATCCAAGCTGTGGTGAG-3 ' (containing restriction enzyme site BamH I and protection bases G A)
N11-2:5 '-CGAAGCTTGGTCATTTTCTATTAGGG-3 ' (containing restriction enzyme site Hind III and protection base CG)
BamH I and Hind III restriction enzyme site are corresponding to the selectivity restriction enzyme site on expression vector PET-32a.Take recombinant plasmid AcaNAP11/pUCm-T as template, the nucleotide sequence of amplification coding AcaNAP11 albumen (SEQ ID NO.3).The PCR reaction conditions is: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30s; Annealing, 54 ℃, 30s; Extend, 72 ℃, 30s, 30 circulations.Finally extend 72 ℃, 10min.The PCR product carries out double digestion with BamH I and Hind III after reclaiming test kit recovery purifying with sepharose; Expression plasmid PET-32a also uses BamH I and Hind III double digestion.Reclaim respectively aforementioned two enzymes and cut product, both are used to T
4dNA ligase connects and will connect product and is converted in E.coli DH5 α competent cell, in containing the substratum of penbritin, is cultivating.With PCR method screening positive clone, and the evaluation of checking order, the recombinant plasmid called after AcaNAP11/pET-32a that gained is correct.Through identifying that correct recombinant expression plasmid is converted into e. coli bl21 (DE3) competent cell.Containing recombinant expression plasmid AcaNAP11/pET-32a Host Strains BL21 (DE3) containing cultivation in the LB nutrient solution of penbritin, adding IPTG is that 1.0mmol/L is induced to final concentration.Centrifugal collection thalline is collected supernatant after ultrasonication, gets supernatant liquor and carries out purifying with the solidifying FF (Beijing Zhuo Guan Science and Technology Ltd.) of nickel agarose.Fusion protein molecule amount size and purity that row SDS-PAGE electrophoresis detection is expressed.Result has shown to obtain aminoterminal and has merged the Trx-AcaNAP11 fusion rotein that Trx (Trx) is arranged, and fusion protein molecule amount size is about 29Kda.Because the molecular size range of the N-terminal fusion rotein of AcaNAP11 is about 20Kda, calculate that AcaNAP11 molecular weight of albumen size is about 9Kda, conforms to the theoretical prediction result.Electrophoresis result is shown in Fig. 6.
(2) Ancylostoma caninum anticoagulant peptide AcaNAP11 mature peptide does not contain vivoexpression, separation and the purifying of 3 amino acid whose peptide sections of carboxyl terminal (AcaNAP11cd)
According to 3 amino acid peptide sections of coding disappearance carboxyl terminal in coding region sequence shown in Fig. 3, be SEQ ID NO.9 encoding sequence design upstream primer N10-3 (5 '-GAGGATCCAATCCAAGCTGTGGTGAG-3 ', containing restriction enzyme site BamH I and protection bases G A) and downstream primer N11-6 (5 '-CGAAGCTTAGGGTAGATAAACTCCATATTGTC-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP11/pUCm-T as template, amplification coding AcaNAP11 mature peptide does not contain the nucleotide sequence of 3 amino acid whose peptide sections of carboxyl terminal (SEQ ID NO.9).Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP11cd, mature peptide does not contain the fusion rotein of 3 amino acid whose peptide section AcaNAP11cd of carboxyl terminal and Trx (Trx).The SDS-PAGE electrophoresis result is shown in Fig. 6.
(3) Ancylostoma caninum anticoagulant peptide AcaNAP11 mature peptide containing the vivoexpression of carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal (AcaNAP11cnd), separate and purifying
Do not contain carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal according to coding in coding region sequence shown in Fig. 3, be SEQ ID NO.10 encoding sequence design upstream primer N11-5 (5 '-GCGGATCCTGTGGTGAGAATGAAAGGTAT-3 ', containing restriction enzyme site BamH I and protection bases G C) and N11-6 (5 '-CGAAGCTTAGGGTAGATAAACTCCATATTGTC-3 ', containing restriction enzyme site Hind III and protection base CG), take recombinant plasmid AcaNAP11/pUCm-T as template, amplification coding AcaNAP11 mature peptide is not containing carboxyl terminal and 3 amino acid whose peptide sections of aminoterminal, it is the nucleotide sequence of SEQ ID NO.10.Amplified production is cut and is connected into expression vector PET-32a through enzyme, be converted into Host Strains BL21 (DE3) and carry out abduction delivering and purifying, obtain Trx-AcaNAP11cnd, mature peptide does not contain the fusion rotein of carboxyl terminal and 3 amino acid whose peptide section AcaNAP11cnd of aminoterminal and Trx (Trx).The SDS-PAGE electrophoresis result is shown in Fig. 6.
Embodiment 8: the anticoagulating active experiment of Ancylostoma caninum anticoagulant peptide fusion protein Trx-AcaNAP9, Trx-AcaNAP10, Trx-AcaNAP11, Trx-AcaNAP9cd, Trx-AcaNAP10cd, Trx-AcaNAP11cd, Trx-AcaNAP9cnd, Trx-AcaNAP10cnd and Trx-AcaNAP11cnd
(use disposable plastic tube, specification: 12 * 75mm) the manual PT of mensuration and aPTT with test tube method.The service requirements and the step measurements data that after the suitable proportion dilution, according to test kit (Shanghai sun biotech company product), provide are provided for fusion rotein Trx-AcaNAP9, Trx-AcaNAP10, Trx-AcaNAP11, Trx-AcaNAP9cd, Trx-AcaNAP10cd, Trx-AcaNAP11cd, Trx-AcaNAP9cnd, Trx-AcaNAP10cnd and Trx-AcaNAP11cnd to purifying, and each concentration all repeats 3 times.The Trx-AcAPc2 simultaneously prepared with this use for laboratory same method (Trx-NAPc2) and Trx-AcAP5 (Trx-NAP5) fusion rotein are made positive control, and physiological saline is made negative control.
Prothrombin time (PT) is measured: get certain density protein purification product 10 μ l and 90 μ l human normal plasmas (taking from the healthy human body of Subsidiary Hospital of Guangdong Medical College's health check-up) and mix latter 37 ℃ and hatch 3min, the PT reagent 0.2ml that adds 37 ℃ of pre-temperature, record setting time, be the PT value.Each concentration all repeats 3 times, gets each concentration PT value mean and blank group PT value and relatively is this concentration prolongation PT multiple.
Activated partial thromboplastin time (aPTT) is measured: get after certain density protein purification product 10 μ l and 90 μ l human normal plasmas (taking from the healthy human body of Subsidiary Hospital of Guangdong Medical College's health check-up) mix, add 37 ℃ of 37 ℃ of pre-warm aPTT reagent 0.1ml to hatch 5min, add the CaCl of the 0.025mol/L of 37 ℃ of pre-temperature
2solution 0.1ml, record setting time, is the aPTT value.Each concentration all repeats 3 times, gets each concentration aPTT value mean and blank group aPTT value and relatively is this concentration prolongation aPTT multiple.
Each recombinant dog hookworm coagulate peptide resistant fusion rotein on the impact of human normal plasma PT, aPTT value in Table 1, table 2.
It is as shown in the table, the peptide fusion protein of Ancylostoma caninum anticoagulant described in the present invention Trx-AcaNAP9, Trx-AcaNAP10, Trx-AcaNAP11, Trx-AcaNAP9cd, Trx-AcaNAP10cd, Trx-AcaNAP11cd, Trx-AcaNAP9cnd, Trx-AcaNAP10cnd and Trx-AcaNAP11cnd be equal significant prolongation human plasma PT, aPTT in experiment, has remarkable anticoagulating active.Prolonged human blood plasma PT be take Trx-AcaNAP9, Trx-AcaNAP9cd and Trx-AcaNAP9cnd as active best; Prolonged human blood plasma aPTT be take Trx-AcaNAP11, Trx-AcaNAP11cd and Trx-AcaNAP11cnd as active best.During 3 amino acid of each hookworm coagulate peptide resistant carboxyl terminal or aminoterminal disappearance, biological activity does not have considerable change, because the little polypeptide of molecular weight forms the antibody to it may can avoid or reduce the body medication time, to the benefit of body innerlich anwenden, is apparent.The polypeptide that molecular weight is little also is easier to produce, especially when polypeptide is produced in chemosynthesis.
With NAPc2, compare, although the fusion rotein of hookworm coagulate peptide resistant described in the present invention has different (table 1) aspect prolongation PT activity, they are being (tables 2) that significantly is better than NAPc2 aspect prolongation aPTT activity; With NAP5, compare, although the fusion rotein of hookworm coagulate peptide resistant described in the present invention has different (table 2) aspect active extending aPTT, they are being significantly to be better than NAP5 extending PT aspect active, as at 60.0nmolL
-1during concentration, fusion rotein Trx-NAP5 is substantially without extending the PT activity, but the fusion rotein of hookworm coagulate peptide resistant described in the present invention all has the activity (table 1) of significant prolongation PT.These results show that hookworm coagulate peptide resistant described in the present invention has unique effect characteristics.
The table 1 hookworm coagulate peptide resistant fusion rotein prolonged human blood plasma PT multiple table (in table, numerical value is for extending the PT multiple) of respectively recombinating
Remarks:
1. " ※ " each letter represents that anticoagulant peptide fusion rotein kind is:
A：Trx-NAPc2；B：Trx-NAP5；C：Trx-AcaNAP9；D：Trx-AcaNAP10；
E：Trx-AcaNAP11；F：Trx-AcaNAP9cd；G：Trx-AcaNAP10cd；H：Trx-AcaNAP11cd；
I：Trx-AcaNAP9cnd；J：Trx-AcaNAP10cnd；K：Trx-AcaNAP11cnd
2.
*p<0.05vs physiological saline group
The table 2 hookworm coagulate peptide resistant fusion rotein prolonged human blood plasma aPTT multiple table (in table, numerical value is for extending the aPTT multiple) of respectively recombinating
Remarks:
1. " ※ " each letter represents that anticoagulant peptide fusion rotein kind is:
A：Trx-NAPc2；B：Trx-NAP5；C：Trx-AcaNAP9；D：Trx-AcaNAP10；
E：Trx-AcaNAP11；F：Trx-AcaNAP9cd；G：Trx-AcaNAP10cd；H：Trx-AcaNAP11cd；
I：Trx-AcaNAP9cnd；J：Trx-AcaNAP10cnd；K：Trx-AcaNAP11cnd
2.
*p<0.05vs physiological saline group
Embodiment 9: Ancylostoma caninum anticoagulant peptide fusion protein Trx-AcaNAP9, the mouse venous thrombosis experiment of the Trx-AcaNAP11 Chinese People's Anti-Japanese Military and Political College.
70 of male SD rats (Guangdong Medical College's Experimental Animal Center provides), 200～250g/ is only; By the inventor laboratory, prepared by Ancylostoma caninum anticoagulant peptide fusion protein Trx-AcaNAP9, Trx-AcaNAP11.
Male rat is divided into 7 groups at random, i.e. Normal group, the large, medium and small dosage group of Trx-AcaNAP9, the large, medium and small dosage group of Trx-AcaNAP11 (in Table 3), every group of 10 rats.By whole rats by intraperitoneal injection 3% vetanarcol 1ml/kg anesthesia, the separation postcava of cutting open the belly, put the standby ligation blood vessel of a silk thread uses in the left renal vein below, from the tested medicine of tongue intravenous injection different concns, after 5min, the ligation postcava causes extravasated blood, then closes abdominal cavity, again open abdominal cavity after 4h, in 2.0cm place, ligation below, folder closes blood vessel, and stringer is cut inspection open and had or not thrombosis, and the Normal group of take is respectively organized the thrombosis ratio as benchmark.The results are shown in Table 3, show that hookworm coagulate peptide resistant Trx-AcaNAP9, Trx-AcaNAP11 have the mouse venous thrombosis effect of the significant Chinese People's Anti-Japanese Military and Political College.
The impact that table 3 Ancylostoma caninum anticoagulant peptide fusion protein Trx-AcaNAP9, Trx-AcaNAP11 form the rat inferior vena cava thrombosis
*p<0.05vs Normal group.
Claims (7)
1. the Ancylostoma caninum anticoagulant peptide of a separation, is characterized in that, this anticoagulant peptide is selected from lower group:
(a) polypeptide formed by SEQ ID NO.3 aminoacid sequence;
(b) polypeptide formed by SEQ ID NO.9 aminoacid sequence;
(c) polypeptide formed by SEQ ID NO.10 aminoacid sequence.
2. the polynucleotide of a separation, is characterized in that its Ancylostoma caninum anticoagulant peptide claimed in claim 1 of encoding.
3. a carrier, is characterized in that it comprises polynucleotide claimed in claim 2.
4. a host cell, is characterized in that it comprises polynucleotide claimed in claim 2 or carrier claimed in claim 3.
5. the preparation method of the described Ancylostoma caninum anticoagulant peptide of claim 1, its feature, the method comprises:
(a) cultivate host cell claimed in claim 4 in suitable medium;
(b) separation, purifying Ancylostoma caninum anticoagulant peptide from substratum or host cell.
6. a pharmaceutical composition, is characterized in that, it includes Ancylostoma caninum anticoagulant peptide claimed in claim 1 and pharmaceutically acceptable carrier.
7. the application of Ancylostoma caninum anticoagulant peptide claimed in claim 1 in preparing anticoagulant, antithrombotic reagent.
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Non-Patent Citations (7)
| Title |
|---|
| AcAPc2自剪切融合蛋白表达载体pTWIN1-AcAPc2的构建和表达;杨博 等;《生物医学工程学杂志》;20061231;第23卷(第3期);630-634 * |
| Biochemical Parasitology》.2004,第133卷319-323. * |
| Juliusz et al.Anticoagulant peptides from Ancylostoma caninum are immunologically distint and localize to separate structures within the adult hookworm.《Molecular & Biochemical Parasitology》.2004,第133卷319-323. |
| Juliusz et al.Anticoagulant peptides from Ancylostoma caninum are immunologically distint and localize to separate structures within the adult hookworm.《Molecular & * |
| 杨博 等.AcAPc2自剪切融合蛋白表达载体pTWIN1-AcAPc2的构建和表达.《生物医学工程学杂志》.2006,第23卷(第3期),630-634. |
| 犬钩虫抗凝血肽;郑玉强 等;《重庆医科大学学报》;20031231;第28卷(第1期);119-121 * |
| 郑玉强 等.犬钩虫抗凝血肽.《重庆医科大学学报》.2003,第28卷(第1期),119-121. |
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