CN1542135A - Preparation and use for phospholipase A2 congener from Agkistrodon blomhoffii Ussurensis of Changbai Mountains and genes encoding same - Google Patents
Preparation and use for phospholipase A2 congener from Agkistrodon blomhoffii Ussurensis of Changbai Mountains and genes encoding same Download PDFInfo
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- CN1542135A CN1542135A CNA2003101050457A CN200310105045A CN1542135A CN 1542135 A CN1542135 A CN 1542135A CN A2003101050457 A CNA2003101050457 A CN A2003101050457A CN 200310105045 A CN200310105045 A CN 200310105045A CN 1542135 A CN1542135 A CN 1542135A
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Abstract
The present invention relates to the amino acid sequence of one kind of phosphatidase A2 homology originated from snake venom of Changbai mountain pallas pit viper, the polynucleotide sequence encoding the homology and their use, and belongs to the field of biological engineering. The technological process the present invention provides can separate and purify snake venom phosphatidase A2 homology directly from snake venom of Changbai mountain pallas pit viper, and recombinant snake venom phosphatidase A2 homology may be purified from cell culture by means of gene engineering technology. This kind of snake venom phosphatidase A2 homology has several biological activities and excellent application foreground in clinical treatment of thrombus, tumor and pain.
Description
Technical field the invention belongs to bioengineering field, relates to a kind of new phospholipase A that comes from Changbai Mountain agkistrodon halys ussuriensis snake venom
2The homologue and this phospholipase A of encoding
2The polynucleotide sequence of homologue also relates to described polynucleotide and encoded protein matter purposes thereof.
The background technology phospholipase A
2(phospholipaseA
2, EC3.1.1.4), be called for short PLA
2, the hydrolysis of second acyl key of energy catalyzing glycerol phosphatide generates lysophospholipid and lipid acid.It extensively is present in the organism, especially is rich in the venom of the juice of Mammals pancreas and snake and insect.
Snake venom PLA
2By a class formation similar (containing 7 pairs of disulfide linkage usually), the protein families of molecular weight about 14kD formed.Except enzymic activity, also has multiple physiologically active, as neural poison, heart is malicious, muscle is malicious, cell toxicant, hemolytic action, the effect of anti-freezing collection etc.These are active or relevant with enzymic activity, perhaps are totally independent of outside the enzymic activity.The snake venom PLA of different sources
2Institute has above-mentioned physiologically active and is not quite similar every kind of PLA
2Usually have only a kind of or a few physiological action.Wu Xiang just waits and be separated to 3 kinds of PLA from the venom of Jiangsu and Zhejiang Provinces pallas pit viper
2(Wu XF etc., Acta Biochem Biophys Sin, 1984,16 (6): 664-671), and according to the difference of iso-electric point, the acid PLA of difference called after
2(APLA
2, pI4.5), neutral PLA
2(NPLA
2, pI6.9) and alkaline PLA
2(BPLA
2, pI9.3).Their primary structure homology is higher, and space structure is also more conservative, and pharmacologically active has sizable difference.Wherein, APLA
2The energy anticoagulant, NPLA
2Belong to presynaptic neurotoxin, BPLA
2Then has hemolytic activity.In order to make PLA clear
2The relation of 26S Proteasome Structure and Function, people are to the snake venom PLA in various sources
2Carried out a large amount of research, comprised by the method for biochemistry extraction snake venom is carried out separation and purification, then to gained PLA
2Carry out amino acid sequencing, explore the PLA of different structure by further functional study
2The rule of structure-function relationship.
Summary of the invention the purpose of this invention is to provide a kind of snake venom phospholipase A from Changbai Mountain agkistrodon halys ussuriensis (subspecies in the Japanese pallas pit viper Usu, Agkistrodon blomhoffii ussurensis, popular name Manushi)
2Homologue (Gln-49-PLA
2) and the preparation and the purposes of encoding gene.
The technical solution used in the present invention is: (1) separation and purification goes out a kind of new snake venom phospholipase A
2Homologue, and measure its partial amino-acid series; (2) the described phospholipase A of clones coding
2The gene of homologue is also measured its base sequence; (3) gene provided by the invention is imported host cell, use Ben Jiyin.(4) provide described phospholipase A
2Homologue character and purposes.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2Homologue and encoding gene thereof, be to derive from Changbai Mountain agkistrodon halys ussuriensis (subspecies in the Japanese pallas pit viper Usu, Agkistrodon blomhoffii ussurensis, popular name Manushi), the polypeptide that comprises aminoacid sequence shown in the SEQ ID No.2, or its conservative property variation polypeptide, or its active fragments, or its reactive derivative, active fragments and reactive derivative are meant the polypeptide with all or part of SEQID No.2 aminoacid sequence.
Prepare a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The method of homologue and encoding gene thereof is that separation and purification goes out a kind of new snake venom phospholipase A
2Homologue is cloned its encoding gene, inserts recombinant expression vector, and imports host cell, to use this gene.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof is:
(a) be fit to express snake venom phospholipase A
2Condition under, cultivate the described host cell of claim 3;
(b) from cell culture, isolate and have Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The active polypeptide of homologue;
(c) separating phospholipids enzyme A from the agkistrodon halys ussuriensis snake venom of Changbai Mountain
2Homologue.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof, encoding gene is meant a kind of isolating polynucleotide, it is characterized in that comprising (a) or (b) in the nucleotide sequence of at least 90% homogeny:
(a) coding has the polynucleotide of SEQ ID No.2 aminoacid sequence polypeptide;
(b) with (a) in polynucleotide complementary polynucleotide.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof, recombinant vectors is to stablize any plasmid and the carrier that duplicates and comprise polynucleotide in host cell.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof, host cell is a kind of genetically engineered host cell, can be selected from down a kind of of group:
(a) host cell of carrier conversion or transduction;
(b) host cell of polynucleotide conversion or transduction.
A kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A of preparation
2The purposes of homologue and encoding gene thereof is, utilizes characteristics such as polypeptide cytotoxicity, musclar toxicity, neurotoxicity and anticoagulant active, and preparation is used for the treatment of disease and analgesic compositions such as thrombus, tumour, nerve.
Snake venom phospholipase A provided by the invention
2Homologue can obtain by the combined utilization of ion exchange chromatography, gel chromatography and high performance liquid chromatography (HPLC), SDA-PAGE shows that it is single component, mass spectrum determines that its accurate molecular weight is 13881.83 ± 0.33D, and it is 8.56 that isoelectric focusing electrophoresis is measured its iso-electric point.Phospholipase A provided by the invention
2Homologue has the aminoacid sequence shown in the SEQ ID No.2.Sequence shows, this phospholipase A
2Homologue is a kind of new Gln49 phospholipase A
2The Gln49 phospholipase A
2Be meant phospholipase A
2The 49th amino acids residue in the aminoacid sequence is Gln, and in accordance with international practices, the protein sequence numbering that relates among the present invention is with ox pancreas PLA
2Sequence numbering as reference.
Phospholipase A provided by the invention
2Homologue can obtain by protein separation method appropriate combination separation and purification commonly used, and as ion exchange chromatography, gel chromatography and high performance liquid chromatography (HPLC) etc., but the adoptable method of the present invention is not limited thereto.
The present invention is not limited to proteinic source, and production method etc. are as long as it has the feature in the specification sheets.Protein provided by the invention can be natural protein, utilizes the protein of genetic engineering technique by recombinant dna expression, or the protein of chemosynthesis.
Protein provided by the invention not only comprises the protein with aminoacid sequence shown in the SEQ ID No.2, also comprises this proteinic fragment, derivative and analogue.Term used herein " fragment ", " derivative " are meant with " analogue " and keep identical biological function of polypeptide of the present invention or active polypeptide basically.
Fragment, derivative or the analogue of protein s EQ ID No.2 provided by the invention can be: (1) such peptide species, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can yes or no by the genetic codon coding; Perhaps (2) such peptide species, wherein one or more amino-acid residues comprise substituting group; Perhaps (3) such peptide species, wherein mature polypeptide and another kind of compound merge; Perhaps (4) such peptide species, wherein additional amino acid is integrated into mature polypeptide, and it is used for the purifying mature polypeptide; Perhaps (5) such peptide species, one or more amino-acid residues are lacked, and perhaps insert or add one or more amino acid.
The invention provides isolating polynucleotide, it is the polynucleotide of coding SEQ ID No.2 aminoacid sequence polypeptide basically.Polynucleotide sequence provided by the invention is included in the SEQ ID No.1 nucleotide sequence.Polynucleotide of the present invention separate from the agkistrodon halys ussuriensis poison gland of Changbai Mountain and obtain.It comprises 366 Nucleotide, 122 amino acid of encoding.
Term used herein " separation " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide then are separation and purification as separating in other materials that exist together from native state.
Polynucleotide provided by the invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with the identical or degeneracy varient of coding region sequence shown in the SEQ ID No.1.Term used herein " degeneracy varient " is meant that coding has the nucleotide sequence of SEQ ID No.2 protein or polypeptide, but because the degeneracy of codon, coding may be different with the coding region sequence shown in the SEQ ID No.1.
The polynucleotide of coding SEQ ID No.2 mature polypeptide comprise: the encoding sequence that natural polypeptides is only arranged; The encoding sequence of mature polypeptide and various additional code sequence; The encoding sequence of mature polypeptide and non-coding sequence.Term " polynucleotide of coded polypeptide " be meant the polynucleotide that comprise coded polypeptide and coding and (or) polynucleotide of non-coding sequence.
The invention still further relates to the varient of foregoing description polynucleotide, it is encoded polypeptide or polypeptide fragment, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise replacement varient, disappearance or insertion, but can not change its encoded polypeptides function basically.
The invention still further relates to can and the polynucleotide of the above sequence hybridization (have at least 90% between two sequences, preferred have 95% homogeny).The present invention relates under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the higher levels of humidity and wash-out, as 60 ℃, and 0.2 * SSC, 0.1%SDS; Or (2) when hybridization with denaturing agent, as 42 ℃, 50% (v/v) methane amide, 0.1% calf serum or 0.1%Ficoll etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And interfertile polynucleotide encoding polypeptide has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO.2.
The invention still further relates to the nucleic acid fragment of hybridization described above.As used herein, the length of " nucleic acid fragment " contain 15 Nucleotide at least, better are 20-30 Nucleotide, are more preferably 50-60 Nucleotide, preferably 100 more than the Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid with determine and (or) separate encode as described in snake venom phospholipase A
2Polynucleotide.
The present invention also relates to comprise the carrier of polynucleotide sequence of the present invention and the host cell that produces through gene work post method with carrier of the present invention, and the method that produces polypeptide of the present invention through recombinant technology.
Dna sequence dna provided by the invention can obtain with several method, for example uses hybridization technique DNA isolation well known in the art.These technology including, but not limited to: (1) with the hybridization of probe and genome cDNA library to detect the homology nucleotide sequence; (2) antibody screening of expression library is to detect the cloned DNA fragment that it has the homologous structure feature.
The described phospholipase A of encoding
2Homologue specific DNA fragment sequence produces also and can obtain with following method: (1) separates double chain DNA sequence from genomic dna; (2) the chemical synthesising DNA sequence is required to obtain
The double-stranded DNA of peptide coding.
In the above-mentioned method of mentioning, isolation of genomic DNA is the most commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic also to be optional method.When if the whole sequence of required polypeptide amino acid is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for selecting for use is the separation of cDNA sequence.The standard method that separates purpose cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qingene) from commercial channels.And the construction cDNA library also is usual method (Sambrook etc., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination polymerase chain reaction technique, even few expressing gene also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) measure the described Changbai Mountain of coding agkistrodon halys ussuriensis phospholipase A
2The level of homologue transcript; (4) applied immunology technology or mensuration biologic activity detect the protein product of genetic expression.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homologous nucleotide sequence of polynucleotide of the present invention, and at least 15 Nucleotide of its length better are 20-30 Nucleotide, be more preferably 50-60 Nucleotide, preferably 100 more than the Nucleotide.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence information provided by the invention normally.Gene provided by the invention itself or fragment certainly are used as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of described new protein genetic expression and can use immunological technique such as Western trace, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Saiki etc., Science, 1985 of round pcr DNA amplification/RNA; 230; 1350-1354) gene provided by the invention can be used to preferentially obtain.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE:cDNA), can suitably select according to sequence information provided by the invention at primer used aspect the above-mentioned PCR, and available ordinary method be synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
Gene provided by the invention, nucleotide sequences such as various dna fragmentations can with ordinary method such as double deoxidating chain termination measuring (Sanger etc., PNAS, 1977,74:5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
Changbai Mountain provided by the invention agkistrodon halys ussuriensis phospholipase A
2The polynucleotide sequence of homologue can be inserted in the recombinant expression vector.Term " recombinant expression vector " is meant and relates to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus as adenovirus, retrovirus or other carriers.In the present invention the carrier of Shi Yonging including, but not limited to: the expression vector based on T7 of in bacterium, expressing (Maeda M. etc., J. Biochem, 109 (4): 632-637,1991) and the expression vector based on AOX1 of in yeast cell, expressing; The expression vector based on MT of in the epidermic cell of Mammals mouse, expressing (Geyer H. etc., Eur.J.Biochem., 237:113-127,1996) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, duplicate as long as can stablize in host cell, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used for structure and contain described Changbai Mountain agkistrodon halys ussuriensis phospholipase A
2The DNA sequences encoding of homologue and suitable transcribing/the translate expression vector of control information.These methods comprise (Sambroook etc., Molecular Cloning, a laboratory Manual, Cold Spring Harbor Laboratory, New York, 1998) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.In these promotors there be representational example: colibacillary Lac or Trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTR promotor and some other known may command gene expression promoter in protokaryon, eukaryotic cell or its virus of CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also includes ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cell, as be used for Tetrahydrofolate dehydrogenase, new mould malicious resistance and the green fluorescent protein (GFP) that eukaryotic cell is cultivated, or be used for colibacillary tsiklomitsin or ammonia benzyl penicillin resistance.
Comprise above-described suitable dna sequence dna and suitable promotor or the carrier of control sequence and can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representational example has: intestinal bacteria, streptomycete, the bacterial cell of Salmonella typhimurium; Such as the zymic fungal cell; Vegetable cell; The insect cell of fruit bat S2 or Sf9; CHO, COS or Bowes melanoma zooblast etc.
When polynucleotide provided by the invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting an enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in 100 to 270 base pair SV40 enhansers of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell, when the host was prokaryotic organism such as intestinal bacteria, preparation can absorb the competent cell of DNA generally from exponential growth after date harvested cell, uses CaCl
2Method is handled, and used step is well-known in this area.Alternative is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, available following DNA transfection method: coprecipitation of calcium phosphate, microinjection, electroporation, methods such as liposome packing.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, culturing cell can be selected from various conventional substratum, cultivates under the condition that is suitable for the host cell growth.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (temperature transition or chemical induction), cell is cultivated for some time again.
Required in the above methods recombinant polypeptide can be expressed in the cell or express or be secreted into the extracellular on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well known to those skilled in the art more.More particularly, there is conventional renaturation to handle, handles (salt analysis method), centrifugal, infiltration smudge cells, uf processing, super centrifugal, sieve chromatography (gel-filtration), the combination of adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
The present invention also provides described phospholipase A
2The biologic activity of homologue.Experiment shows described snake venom phospholipase A
2Homologue does not have tangible phospholipase A
2Activity, but have anticoagulation, biologic activity such as antitumor, but also continuing described phospholipase A based on this research
2The biologic activity of homologue is not limited thereto.Described new phospholipase A
2Homologue is of use in many ways.These purposes include, but is not limited to: antitumor, anti-blood is fastened, pain relieving.
In sum, in view of snake venom PLA
2The complicacy of diversity structure and function, the phospholipase A that the present invention provides first
2Homologue aminoacid sequence and encoding gene thereof are to understanding fully the PLA of different sources
2Relation between structure and the function has important significance for theories.Phospholipase A provided by the invention
2Homologue has the various biological activity, makes natural or reorganization PLA
2Has the potential application prospect aspect clinical thrombus treatment, oncotherapy or the clinical pain relieving.
The present invention is further described in conjunction with example for embodiment below the embodiment.
Embodiment 1: Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The separation and purification of homologue
Changbai Mountain agkistrodon halys ussuriensis raw venin is available from the Huinan County, Jilin, and with 10mmol Tris-HCl (pH8.0) dissolving, the centrifugal precipitation of going is with twice of the ultra-filtration membrane ultrafiltration of 10kD before using.
Get above-mentioned raw venin sample and carry out cation-exchange chromatography, ion exchange column is Hitrap sp, and buffer A is 10mmol HAc-NaAc (pH 5.0), and buffer B is 10mmol HAc-NaAc+1molKCl (pH5.0), and flow velocity is 2ml/min.Each component on the collection of ions exchange column under the wash-out, carry out gel permeation chromatography behind the ultrafiltration and concentration: gel column is superdex75, and damping fluid C is 10mmol Tris-HCl+1molKCl, and flow velocity is 1ml/min.Collect the gel permeation chromatography component, after the ultrafiltration and concentration desalination, carry out anion-exchange chromatography: ion exchange column is source Q, and damping fluid D is 10mmol Tris-HCl (pH 8.0), damping fluid E is 10mmol Tris-HCl+1molKCl (pH 8.0), and flow velocity is 2ml/min.HPLC is further purified method: collect the anion-exchange chromatography component, carry out the HPLC preparation behind the ultrafiltration and concentration, chromatographic column is an anti-phase C18 preparative column (250 * 15); Moving phase F is acetonitrile+0.1% trifluoroacetic acid; G is water+0.1% trifluoroacetic acid; Flow velocity is 2ml/min.
Embodiment 2: Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The acquisition of homologue gene
Behind the agkistrodon halys ussuriensis toxin expelling of Changbai Mountain 4 days, take out poison gland, drop into rapidly among the TRIZOL, the operational guidance that provides according to test kit production firm carries out total RNA and extracts (TRIZOL test kit, U.S. GIBICOL company).According to known amino acid sequence, relatively design special primer P-F1 in conjunction with homologous sequence, adopt 3 '-FullRACE method to obtain Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2Homologue gene 3 ' terminal sequence.And then design primer P-F2, P-R2, adopt the RT-PCR method to obtain Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The encoding gene of homologue.3 '-Full RACE, RT-PCR all adopt test kit (TAKARA company, Japan), 3 '-FullRACE reverse transcription primer is Oligo dT-3 sites Adaptor Primer, is provided by TAKARA company, and all operations all carries out according to the operational guidance that test kit production firm provides.The condition of wherein relevant PCR reaction is such:
Primer is:
P-F1：5’-TATCGTCTGTGGAGGGGACGACCC-3’
3?sites?Adaptor?Primer：5’-CTGATCTAGAGGTACCGGATCC-3’
P-F2：5’-AGCCTGCTGCAATTCAGGAAGATGAT-3’
P-R2：5’-TCCCGGCCTGCAGAGACTTAGC-3’
The concrete reaction conditions of PCR is:
72℃ 7min
4℃ 5m
Gained PCR product cloning is in pMD18-T, and order-checking obtains coding Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The homologue gene order.This sequence and existing dna sequence data storehouse are compared, found that this polynucleotide sequence did not have report, we are with the protein called after Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A of this genes encoding
2Homologue, English by name: manushi phospholipaseA
2Homologue is abbreviated as Gln49-MPLA
2
Embodiment 3: Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The expression of homologue gene in intestinal bacteria
According to the corresponding gene order of this new protein, design a pair of specificity amplification primer, sequence is as follows:
F1?primer：5’-CCGAATTCATTGAGGGACGCAGCCTGCT?GCAATTCAG-3’
R1?primer：5’-CCGGATCCTCATTAGCATTTCTCTGACTT-3’
The F-primer sequence contains the recognition sequence of EcoRI restriction enzyme site and Factor Xa cutting.The R-primer sequence contains BamH I restriction enzyme site and translation termination subsequence, and its back is respectively the encoding sequence of target gene 5 ' end and 3 ' end, is template with the pMD118-T plasmid that contains goal gene, carries out the PCR reaction.
According to known coli expression carrier pET-32 (a)+complete sequence, design a pair of specificity amplification primer, with the reconstruction plasmid, obtain satisfied expression effect, primer sequence is as follows:
F2?primer?5’-CCGGATCCCAAAGCCCGAAAGGAAGC-3’
R2?primer?5’-GGGAATTCACCAGAAGAATGATGATGATG-3’
F primer contains BamH I restriction enzyme site and part T7 terminator sequence.R primer contains EcoRI restriction enzyme site, gentle sequence and section H is Tag sequence.With coli expression carrier pET-32 (a)+be template, carry out inverse PCR.
Respectively the target gene sequences of amplification and coli expression carrier pET-32 (a)+sequence being carried out enzyme with BmH I and EcoRI cuts and is connected.In recombinant plasmid electricimpulse transformed into escherichia coli BL21 (DE3), 37 ℃, IPTG induces after 4 hours and gathers in the crops thalline.The thalline ultrasonication, SDS-PAGE shows that recombinant protein has obtained expression.
Embodiment 4: Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The mensuration of homologue anticoagulant active
Get fresh Sodium Citrate anti-freezing pig blood, the centrifugal 10min of 1000g must be rich in thrombocyte blood plasma (PRP), gets PRP 0.5ml, contains the phospholipase A of different concns with 0.1ml
2The PBS solution of homologue (0.1,0.2,0.4 or 0.6g/L) is hatched 10min for 37 ℃, adds 0.1ml 0.25mol/L CaCl
2, the record clotting time.Experiment shows, Changbai Mountain agkistrodon halys ussuriensis PLA
2Homologue slightly reduces recalcification time when low concentration (0.1mg/ml), but when concentration is higher than 0.2mg/ml, with PLA
2The increase of concentration, recalcification time prolongs.
Embodiment 5: Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The cell numeration that the Cytotoxic mensuration of homologue is taken the logarithm frozen Hela, K562 cell vegetative period after recovering, going down to posterity is with 1 * 10
5/ ml cell adds 24 well culture plates, every hole 500 μ l, 5% CO
2Cultivate 24h in the incubator, add the PLA of different concns
2Homologue is cultivated 3h.Adopt mtt assay to measure the viable count of Hela, K562, calculate medium lethal dose (LD
50).Experiment shows, PLA
2Homologue has tangible toxic action to the Hela cell, but not obvious to the K562 cell, its LD
50Be respectively 1.3 μ mol/ml and 28.6 μ mol/l.
Claims (7)
1. Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2Homologue and encoding gene thereof, it is characterized in that, derive from the Changbai Mountain agkistrodon halys ussuriensis, the polypeptide that comprises aminoacid sequence shown in the SEQ ID No.2, or its conservative property variation polypeptide, or its active fragments, or its reactive derivative, active fragments and reactive derivative are meant the polypeptide with all or part of SEQ ID No.2 aminoacid sequence.
2. prepare a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A according to claim 1
2The method of homologue and encoding gene thereof is characterized in that, separation and purification goes out a kind of new snake venom phospholipase A
2Homologue is cloned its encoding gene, inserts recombinant expression vector, and imports host cell, to use this gene.
3. a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A according to claim 2
2The preparation method of homologue and encoding gene thereof is characterized in that,
(a) be fit to express snake venom phospholipase A
2Condition under, cultivate the described host cell of claim 2;
(b) from cell culture, isolate and have Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The active polypeptide of homologue;
(c) separating phospholipids enzyme A from the agkistrodon halys ussuriensis snake venom of Changbai Mountain
2Homologue.
4. a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A according to claim 2
2The preparation method of homologue and encoding gene thereof is characterized in that, encoding gene is meant a kind of isolating polynucleotide, it is characterized in that comprising (a) or (b) in the nucleotide sequence of at least 90% homogeny:
(a) coding has the polynucleotide of SEQ ID No.2 aminoacid sequence polypeptide;
(b) with (a) in polynucleotide complementary polynucleotide.
5. according to claim 2,4 described a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof is characterized in that, recombinant vectors is to stablize any plasmid and the carrier that duplicates and comprise polynucleotide in host cell.
6. according to claim 2,3 or 5 described a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A
2The preparation method of homologue and encoding gene thereof is characterized in that, host cell is a kind of genetically engineered host cell, can be selected from down a kind of of group:
(a) host cell of carrier conversion or transduction;
(b) host cell of polynucleotide conversion or transduction.
7. a kind of Changbai Mountain agkistrodon halys ussuriensis snake venom phospholipase A of claim 1,2 described preparations
2The purposes of homologue and encoding gene thereof is characterized in that, utilizes characteristics such as polypeptide cytotoxicity, musclar toxicity, neurotoxicity and anticoagulant active, and preparation is used for the treatment of disease and analgesic compositions such as thrombus, tumour, nerve.
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| CNA2003101050457A CN1542135A (en) | 2003-11-06 | 2003-11-06 | Preparation and use for phospholipase A2 congener from Agkistrodon blomhoffii Ussurensis of Changbai Mountains and genes encoding same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335496C (en) * | 2005-12-28 | 2007-09-05 | 南方医科大学 | Snake venom polypeptide and its preparation method |
| CN103804481A (en) * | 2014-01-28 | 2014-05-21 | 南宁培元基因科技有限公司 | Method for producing cobra CT and PLA2 in baculovirus-insect expression system |
| CN105969749A (en) * | 2016-03-28 | 2016-09-28 | 中鑫东泰(莱阳)纳米基因生物技术有限公司 | Method for purifying phosphodiesterase from deinagkistrodonacutus |
-
2003
- 2003-11-06 CN CNA2003101050457A patent/CN1542135A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335496C (en) * | 2005-12-28 | 2007-09-05 | 南方医科大学 | Snake venom polypeptide and its preparation method |
| CN103804481A (en) * | 2014-01-28 | 2014-05-21 | 南宁培元基因科技有限公司 | Method for producing cobra CT and PLA2 in baculovirus-insect expression system |
| CN105969749A (en) * | 2016-03-28 | 2016-09-28 | 中鑫东泰(莱阳)纳米基因生物技术有限公司 | Method for purifying phosphodiesterase from deinagkistrodonacutus |
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