CN1332030C - Polypeptide, its coding sequence and preparation method and application of fungus galactose agglutinin protein activity - Google Patents

Abstract

The present invention discloses a polypeptide with the fungus galactose agglutinin protein activity, a code sequence thereof, a preparation method thereof and an application thereof. The code sequence of the isolated protein has a sequence with at least 70% of autoploidy to a nucleotide sequence disclosed by SEQ ID NO. 1. The present invention relates to an isolated polypeptide which has a sequence with at least 80% of autoploidy to an amino acid sequence disclosed in SEQ ID NO. 2, the polypeptide coded by the nucleotide sequence, the preparation method of the polypeptide, and the application of the polynucleotide and the polypeptide in medicine and agriculture. The protein of the present invention has anti-tumor activity and anti-plant virus activity, and has great application value in the aspects of anti-tumor drug development, plant protection, etc.

Description

A kind of polypeptide of fungus galactose agglutinin protein activity, its encoding sequence and preparation method and application

Technical field

The present invention relates to the genetically engineered field, specifically, the present invention relates to fungi Agrocybe aegerita (Brig) Sing (Agrocybeaegerita) galactose agglutinin (Agrocybe aegerita Galectin) gene order.The invention still further relates to by this nucleotide sequence coded polypeptide and preparation method thereof, also relate to the application of polypeptide in medical, antitumor, agriculture, Antiphytoviral simultaneously.

Background technology

Agrocybe aegerita (Brig) Sing (Agrocybe aegerita) is under the jurisdiction of Mycophyta, Basidiomycetes, Agaricales, excrement rust umbrella section, field mushroom genus.Have another name called Agroeybe cylindracea mycelia, Agrocybe aegerita (Brig) Sing, chaxingu mushroom, Liu Songrong etc.Be rich in eight seed amino acids of needed by human, wherein Methionin is up to 1.75%, its nutritive value surpasses other edible mushroomss such as mushroom, needle mushroom, has simultaneously unique pharmaceutical use again, among the peoplely be usually used in anticancer, step-down, the treatment stomach is cold, ephritis, oedema etc. have unique curative effect, and the good reputation of " Chinese refreshing mushroom " is arranged, but pharmaceutical component wherein is also indeterminate, and the Agrocybe aegerita Galectin that relates among the present invention is the valuable pharmacological composition that we find first.

Galactose agglutinin (Galectin) is the protein families of a class to galactoside (galactoside) specific combination, has all found their existence from the fungi to the Mammals etc. in the various organisms.Galectin has two features: 1) with the galactoside specific combination; 2) have conservative sugared calmodulin binding domain CaM (carbohydraterecongnition domain, CRD).

In Mammals, found 14 kinds of Galectin at present, and unified to number called after Galectin 1-14 (hereinafter to be referred as Gal 1-14) according to its sequencing that enters Genome Data Base.Existing many pieces of documents are with regard to the structure of Gal, and problems such as function and expression regulation are summarized.They are distributed widely in the multiple tissue and organ of animal, comprise normal tissue (thymus gland, lymphoglandula, prostate gland, spleen, developmental brain, intestines, lung, placenta, endotheliocyte, skin, heart, the olfactory nerve cell, liver, kidney, skeletal muscle, unstriated muscle, testis etc.) and pathological tissue (thyroid carcinoma, the rectum cancer, ovarian cancer, melanoma, fibrosarcoma, lymphoma etc.) in expression is all arranged.They are at the eukaryote body development, and immune stable state is kept in differentiation, cell-cell adhesion, and cell-medium is done mutually, growth regulating, apoptosis plays a significant role in the various physiological processes such as RNA shearing and metastases.

The research of galectin in other species is less relatively.From birds, also separated several Galectins in succession in other species such as batrachians, fish, worm and sponge, from fungi, only separated 3 kinds of galectin, i.e. CCL-I, CCL-II and ACG.CCL-I, CCL-II utilize semi-lactosi affinity column protein isolate, and the albumen that the clone gene analytical separation obtains belongs to galectin family.And ACG is found and has coagulation function, and through the amino acid complete sequence determination, analysis is the galectin family member.3 kinds of fungi galectin on function other pharmacology, physiological all without any research.

Summary of the invention

One object of the present invention is to provide a kind of polypeptide nucleotide sequence, this sequence above-mentioned Agrocybe aegerita Galectin peptide sequence of encoding.Show at least 70% homology from the nucleotides sequence of Nucleotide 1-474 position among this sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ IDNO.1 in from the nucleotide sequence hybridization of Nucleotide 1-474 position.70% homologous gene can suddenly change by the sudden change test kit, and perhaps by inserting, genetic manipulations such as disappearance obtain.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.Preferably at least 80%, at least 90% nucleotide sequence more preferably, best, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 1-474 position.This sequence is to clone first, can design primer according to this sequence, and this nucleotide sequence that increases in a large number of the method by PCR or RT-PCR is connected in expression vector, produces Agrocybe aegerita Galectin.

Another object of the present invention is to provide a kind of polypeptide of fungus galactose agglutinin protein activity, has various active: anti-tumor activity, anti-phytoviral activity is with a wide range of applications on medicine, agricultural.This galactose agglutinin be the report first have galactose agglutinin antitumor, antiviral activity.

The invention still further relates to a kind of preparation method of Agrocybe aegerita Galectin polypeptide, this method is to make up prokaryotic expression carrier, utilizes affinity chromatography method purifying Agrocybe aegerita Galectin, and this method is easy, and is with low cost.

The invention still further relates to a kind of isolated polypeptide, its sequence is the application of polypeptide in conduct preparation or prophylaxis of tumours disease medicine of SEQ ID NO.2.It can suppress kinds of tumor cells (rectum cancer, cervical cancer, cancer of the stomach, the leukemia cell) growth, the kill tumor cell is better than the chemotherapeutics Zorubicin effect of clinical application to the restraining effect of tumour cell, and this galactose agglutinin has the potentiality that are developed to antitumor drug.

The invention still further relates to a kind of isolated polypeptide, its sequence is that the polypeptide of SEQ ID NO.2 is in Antiphytoviral (tobacco mosaic disease, TMV) application in.(tobacco mosaic disease TMV) has good resistance to this galactose agglutinin, can be used as the agricultural chemicals of resisting tobacco mosaic virus, and the gene of this galactose agglutinin can be used as a kind of antiviral genetic resources, is used for seed selection antivirus plant kind to plant virus.

In order to realize above-mentioned task, the present invention adopts following technical measures:

An object of the present invention is to provide a kind of new nucleotide sequence, a kind of new Agrocybe aegerita Galectin of this sequence encoding.In the present invention, the cDNA nucleotide sequence of fungi Agrocybe aegerita Galectin is to be template with total RNA of Agrocybe aegerita (Brig) Sing or mRNA, obtains by the RT-PCR method.Concrete grammar is as follows: No. 3, the cultivar that the Agrocybe aegerita (Brig) Sing bacterial classification provides for Sanming City, Fujian Province institute, get the fresh mycelia or the sporophore of Agrocybe aegerita (Brig) Sing, extracting total RNA or mRNA is template (commercially available extraction RNA and mRNA test kit, as Clontech, Promega, company's products such as Stratagene, operation to specifications), synthetic reverse transcriptase primer 5 ' gac cac gcg tat cga tgt cga ct16 (a/c/g) 3 ' carries out reverse transcription and obtains cDNA first chain.N terminal sequence according to Agrocybe aegerita Galectin mensuration, design degenerated primer 5 ' ccc aag ctt ca (a/g) ggcgtc aac at (t/c) ta (t/c) aa (t/c) at3 ' is a forward primer, with 5 ' gac cac gcg tat cga tgt cga c3 ' is reverse primer, with above-mentioned cDNA first chain is template, carry out PCR, obtain the purpose fragment of 635bp,, obtain the full length cDNA sequence of SEQ ID NO.1 this fragment cloning order-checking.

Required for protection and the above-mentioned cDNA sequence of the present invention has at least the gene of 70% homology can lead to insertion, and genetic manipulations such as disappearance, sudden change obtain.As can on the sequence basis that SEQ ID NO.1 provides, inserting arbitrarily when designing the PCR primer, the primer of disappearance and sudden change is synthetic, carries out PCR with this primer, then can obtain having at least the gene of 70% homology.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.Preferably at least 80%, at least 90% nucleotide sequence more preferably, best, this sequence has the nucleotide sequence of 1-474 position among the SEQ ID NO.1.According to the nucleotide sequence of SEQ ID NO.1 1-474 position, design primer, primer be generally comprise 1-20 position nucleotide sequence and with 457-474 position complementary nucleotide sequence, this nucleotide sequence that increases in a large number of the method by PCR or RT-PCR.

The claimed active polypeptide of Agrocybe aegerita Galectin of the present invention can be produced with the following method.This method comprises: following concrete operation is according to the normal experiment condition, and the condition described in " molecular cloning laboratory manual " (third edition) [J. Sa nurse Brooker etc. is write, 2003, Beijing: Science Press] is carried out.

1) the total RNA or the mRNA of extraction Agrocybe aegerita (Brig) Sing are template, obtain cDNA first chain by RT-PCR,

With this chain is pcr template, and it is the full length sequence shown in the SEQ ID NO.1 that amplification obtains sequence;

2) acquisition and cDNA have the gene order of 70% homology at least: on the 1-474 sequence basis of SEQ ID NO.1, as required, (be generally 20-40bp with wherein any one section sequence, also can reach 100bp) be the PCR primer, the autotelic insertion of when primer is synthetic, introducing base, disappearance and sudden change, two terminal sequences (454-474 sequence with design restriction enzyme site among this primer and the SEQ ID NO.1, or 454-474 complementary sequence, or 1-20 sequence, or 1-20 complementary sequence) be a pair of primer, carry out pcr amplification can be in former sequence the insertion of autotelic introducing base, lack and sudden change, obtain having at least the gene order of 70% homology with cDNA.

3) enzyme is cut the sequence that step 2 obtains, enzyme is cut prokaryotic expression carrier pET28 (commercially available, Novagen company also can be with the various expression vectors of commercially available other), cDNA sequence after ligase enzyme is cut and expression vector pET28b make up recombinant expression vector pET28-Agrocybe aegerita Galectin;

4) with the recombinant expression vector transformed into escherichia coli host cell BL21 that obtains in the step 3 (commercially available, Novagen company can select the host cell of coupling according to used expression vector), form and express the proteic reconstitution cell of Agrocybe aegerita Galectin;

5) reconstitution cell that obtains in the step 4 is obtained saturated culture 37 ℃ of overnight incubation; 50 μ L saturated culture are inoculated in 5mL contain in the substratum of penbritin 100 μ g/mL, cultivated 2 hours in 37 ℃.Getting the 1mL culture is transferred in the Eppendorf tube.Add isopropylthiogalactoside IPTG to final concentration be 1m mol/L, culture is induced, induced 10 hours.

6) utilize Ni2+ affinity chromatography purifying protein, separate, in the purification step 5 in reconstitution cell the polypeptide of abduction delivering.

Active and sugar suppresses activity experiment according to the haemagglutination that provides in " lectin " [Sun Ce etc. write, 1986, Beijing: Science Press], can check the activity of the claimed polypeptide of the present invention.Purity detecting can detect with the SDS-PAGE method.

The claimed active polypeptide of Agrocybe aegerita Galectin of the present invention can suppress kinds of tumor cells (rectum cancer, cervical cancer, cancer of the stomach, leukemia cell's) growth, kill tumor cell.Substantially pure this galactose agglutinin polypeptide or its variant form can be developed to antitumor drug.This galactose agglutinin is to plant virus (tobacco mosaic disease, TMV) good resistance is arranged, substantially the agricultural chemicals that this pure galactose agglutinin polypeptide or its variant form can be used as resisting tobacco mosaic virus, the encoding sequence of this galactose agglutinin can be used as a kind of antiviral genetic resources, is used for seed selection antivirus plant kind.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA refer to, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and with cell in the protein followed separate.

In the present invention, term " Agrocybe aegerita Galectin albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with Agrocybe aegerita Galectin protein-active is as 1-474 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 1-474 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 1-474 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the rigorous condition of moderate, more preferably under highly rigorous condition can with the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 1-474 position among the SEQ ID NO.1.In addition, this term also comprise with SEQ IDNO.1 in from Nucleotide 1-474 position homology of nucleotide sequence at least 70%, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises the variant form of open reading frame sequence among the SEQ ID NO.2 with Agrocybe aegerita Galectin functional protein that can encode.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and add several (in usually with 120 at 5 ' and/or 3 ' end, preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.In embodiment 2, the nucleotides sequence that the expression vector of structure is transcribed is listed in SEQ ID NO.1 Nucleotide 5 ' end and inserts 120 Nucleotide, and expression product keeps former proteic anti-tumor activity.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.

In the present invention, term " Agrocybe aegerita Galectin protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of Agrocybe aegerita Galectin protein-active, and this term also comprises having and the variant form Agrocybe aegerita Galectin identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 40, preferably being in 20, more preferably is in 10) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.In embodiment 2, the recombinant protein that the expression vector of structure is expressed has increased by 40 amino acid at SEQ ID NO.2 sequence of N end, and expression product keeps former proteic anti-tumor activity.This term also comprises proteic active fragments of Agrocybe aegerita Galectin and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of Agrocybe aegerita Galectin DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-Agrocybe aegerita Galectin polypeptide to obtain.The present invention also provides other polypeptide, as comprises the soluble fragments of Agrocybe aegerita Galectin polypeptide.Usually, this fragment have the Agrocybe aegerita Galectin peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

The present invention also provides the analogue of Agrocybe aegerita Galectin albumen or polypeptide.The difference of these analogues and natural Agrocybe aegerita Galectin polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, can also pass through site-directed mutagenesis method or other molecular biological technology.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external, in the synthetic of polypeptide and the further procedure of processing of processing neutralization, carry out glycosylation modified and polypeptide that produce as those.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises the sequence of phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

The present invention also comprises the antisense sequences of Agrocybe aegerita Galectin polypeptid coding sequence.This antisense sequences can be used to suppress the expression of Agrocybe aegerita Galectin in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of Agrocybe aegerita Galectin polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the Agrocybe aegerita Galectin of encoding.

The present invention also comprises the method that detects the Agrocybe aegerita Galectin nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing, whether detection probes combination has taken place then, preferably, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to the encoding sequence of Agrocybe aegerita Galectin polypeptide, and can be positioned at the both sides or the centre of this encoding sequence, and primer length is generally 20-50 Nucleotide.

On the other hand, the present invention also comprises Agrocybe aegerita Galectin or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into Agrocybe aegerita Galectin gene product or fragment.Preferably, refer to those can combine with Agrocybe aegerita Galectin gene product or fragment but nonrecognition and in conjunction with the antibody of other irrelevant antigen molecules.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of Agrocybe aegerita Galectin, comprise that also those do not influence the proteic antibody of Agrocybe aegerita Galectin.The present invention also comprise those can with modify or without the Agrocybe aegerita Galectin gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; The strand Fv molecule that genetic process is transformed; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Agrocybe aegerita Galectin gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Agrocybe aegerita Galectin or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block the Agrocybe aegerita Galectin function and the antibody that does not influence the Agrocybe aegerita Galectin function.Each antibody-like of the present invention can utilize the fragment or the functional zone of Agrocybe aegerita Galectin gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (as E.coli) with the unmodified form bonded antibody of Agrocybe aegerita Galectin gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

Advantage of the present invention and effect:

Agrocybe aegerita Galectin nucleotide sequence that provides among the present invention and protein sequence are a kind of gene and protein sequences of new galactose agglutinin, do not appear in the newspapers.In the galactose agglutinin of having reported, that nucleotide sequence and Agrocybe aegerita Galectin dna homolog are the highest is CCL-I, CCL-II (Coprinuscinereus lectin), homology only is 30%, with Agrocybe aegerita Galectin protein sequence homology the highest be ACG (Agrocybe cylindracea galectin), homology is 87%, but ACG does not obtain nucleotide sequence.Up to the present, the galactose agglutinin of having found is not all reported antitumor, antiviral activity.Therefore, advantage of the present invention is " newly "---the present invention is a kind of new, and first report has the galactose agglutinin of anti-tumor activity, anti-phytoviral activity, can be applied in field of medicaments and agriculture field.

The protein that relates among the present invention has very strong anti-tumor activity and very strong anti-phytoviral activity.Under identical concentration, SGC-7901 and HeLa cell inhibiting effect are better than the chemotherapeutics Zorubicin of clinical use; To SW480, MGC80-3, BGC-823, kinds of tumor cells such as S-180 have similar inhibiting rate to the chemotherapeutics Zorubicin.Therefore, this albumen and gene thereof will have very big using value aspect developing anti-tumor medicaments.

This albumen also has very strong resistance to tobacco mosaic virus (TMV), when concentration reaches 25 μ g/mL, is 47.3% to the inhibiting rate of tobacco mosaic virus (TMV).This albumen can be used as the resisting tobacco mosaic virus agricultural chemicals and uses; This proteic gene can be used as a kind of genetic resources of Antiphytoviral, and very big using value is arranged in the resisting tobacco mosaic virus plant breeding.

Description of drawings

Fig. 1 is a kind of structure synoptic diagram of pET28-Agrocybe aegerita Galectin.Aal is the gene of Agrocybe aegerita Galectin among the figure, and pGEM-aal is the preservation plasmid of aal, with this plasmid of EcoR I single endonuclease digestion, the aal gene fragment is discharged; With EcoR I single endonuclease digestion expression vector pET28b, make it become wire simultaneously, and (CalfIntestinalAlkaline Phosphatase CIAP) handle, and makes the carrier dephosphorylation with the calf alkaline phosphatase by ring-type.Connect aal gene fragment and wire expression vector pET28b by ligase enzyme again, make the multiple clone site of aal insertion pET28b, finish the structure of pET28-Agrocybe aegerita Galectin expression vector.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to the normal experiment condition, " molecular cloning laboratory manual " (third edition) [J. Sa nurse Brooker etc. writes 2003, Beijing: Science Press] described in condition carry out or the condition of advising according to manufacturer.

The preparation method of coding Agrocybe aegerita Galectin nucleotide sequence, Agrocybe aegerita Galectin polypeptide preparation method, Application Example, Antibody Preparation embodiment, antitumor embodiment, Antiphytoviral embodiment.

Embodiment 1

The cDNA clone and the order-checking of Agrocybe aegerita Galectin: obtaining a new cDNA sequence in the present embodiment, is that forefathers did not report.

1. primer amplification

With the total RNA of Agrocybe aegerita (Brig) Sing is template, use oligonucleotide A:5 ' gac cac gcg tat cga tgt cga ct16 (a/c/g) 3 ' to carry out reverse transcription for reverse transcriptase primer, obtain first chain of cDNA, with oligonucleotide B (according to the degenerated primer of N terminal amino acid design): 5 ' ccc aag ctt ca (a/g) ggc gtc aac at (t/c) ta (t/c) aa (t/c) at3 ' is a forward primer, oligonucleotide C:5 ' gac cac gcg tat cga tgt cga c3 ' is a reverse primer, carry out PCR, the PCR condition is 95 ℃ of initial 10min, enter three steps circulation (95 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 2min totally 30 circulations), last 72 ℃ are extended 10min.20 μ L reaction systems comprise: 1 μ g cDNA, 0.2mMdNTP, 20pM forward primer and reverse primer, 2 μ L, 10 * buffer, 1 μ L Taq enzyme and 1.5mM MgCl ₂The PCR product is-635bp purpose fragment.

2. cloning and sequencing

According to the operational manual of this pGEMT carrier, this amplified fragments is cloned on the pGEM carrier, make up plasmid pGEM-Agrocybe aegerita Galectin, check order, be total to 635bp, detailed sequence is seen SEQ ID NO.1, and wherein 1-474 is the open reading frame sequence of encoding amino acid sequence.

Translate the aminoacid sequence of Agrocybe aegerita Galectin according to the cDNA sequence that obtains, totally 158 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

Expression, the purifying of Agrocybe aegerita Galectin in intestinal bacteria: the protein example that can obtain great expression, purifying in the present embodiment.

EcoRI single endonuclease digestion pGEM-Agrocybe aegerita Galectin carrier at first, discharge the Agrocybe aegerita Galectin fragment, simultaneously with EcoRI digestion pET28b plasmid, with the CIAP enzyme handle linearizing plasmid pET28b half hour, reclaim Agrocybe aegerita Galectin fragment and purpose plasmid, carry out ligation according to the amount that reclaims, make up prokaryotic expression carrier pET28b-Agrocybe aegerita Galectin, as shown in Figure 1.

According to aforesaid prokaryotic expression method, the result according to different time abduction delivering reorganization Agrocybe aegerita Galectin selects the optimization time, great expression.Utilize Ni column purification reorganization Agrocybe aegerita Galectin, expression amount is about 7mg/L.

Embodiment 3

The preparation of Agrocybe aegerita Galectin antibody: this embodiment can obtain the polyclonal antibody of Agrocybe aegerita Galectin, is used to detect Agrocybe aegerita Galectin.

The recombinant protein that obtains among native protein and the embodiment 2 is used for immune animal to produce antibody, and concrete grammar is as follows.It is standby that recombinant protein separates the back with chromatography, and also available SDS-PAGE gel electrophoresis separates, electrophoretic band is downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.With the albumen of 50-100ug/0.2mL emulsification, mouse is carried out peritoneal injection.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100ug/0.2mL with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least 3 times.The sero-fast specific reaction that obtains is active is assessed (Western method) with it in external ability in conjunction with Agrocybe aegerita Galectin gene translation product.

Embodiment 4

Agrocybe aegerita Galectin is to the restraining effect of tumour cell and tumor-bearing mice tumor tissues: Agrocybe aegerita Galectin suppresses 7 kinds of tumor cell lines detecting in the present embodiment, confirms that Agrocybe aegerita Galectin has the broad-spectrum antitumor action; The tumor tissue growth that suppresses lotus S-180 sarcoma mouse, the survival time that prolongs tumor-bearing mice confirms that Agrocybe aegerita Galectin is woven with the growth effect that suppresses to the tumor group of tumor animal.

1. Agrocybe aegerita Galectin is in external restraining effect to tumor cell line

Present embodiment has detected the inhibition activity of Agrocybe aegerita Galectin to 7 kinds of tumor cell lines (SW480, HeLa, SGC-7901, MGC80-3, BGC-823, HL-60 and S-180) growth.All tumor cell lines are cultivated in RPMI1640 substratum (wherein comprising 10% foetal calf serum, the penicillin of 100mg/mL Streptomycin sulphate and 100U/mL).(be mixed with 5,10,50 with substratum, 100ug/mL) add in the tumour cell, incubation detected the restraining effect of Agrocybe aegerita Galectin to growth of tumour cell with the MTT method after 24 hours with the Agrocybe aegerita Galectin of different concns.Detected result is as shown in table 1.Agrocybe aegerita Galectin has the good restraining effect to the tumor cell line that is detected, SGC-7901 and HeLa cell inhibiting effect are better than the chemotherapeutics Zorubicin, at SW480, MGC80-3, BGC-823 shows in the kinds of tumor cells such as S-180 with the chemotherapeutics Zorubicin have similar inhibiting rate under identical concentration.

Table 1 Agrocybe aegerita Galectin is in external restraining effect to tumour cell

Cell line	[AAL](μg/ml)	Inhibition ratio(％)
							5	10	50	100	Adriamyoin (1000μg/ml)
		SW480 SGC-7901 HeLa MGC80-3 BGC-823 HL-00 S-180		44.75 69.06 34.14 35.00 35.10 10.60 19.01	51.10 71.21 50.00 48.40 48.10 22.10 22.17	68.78 72.23 64.52 61.80 60.80 37.64 46.33						82.60 73.98 67.74 67.70 67.40 42.79 68.50	80.66 65.88 40.96 64.40 64.00 69.12 69.44

2. Agrocybe aegerita Galectin is to the growth-inhibiting effect of tumor-bearing mice tumor tissues

In the present embodiment, adopt BALB/c mouse (7 weeks, 20 grams), at armpit subcutaneous vaccination 0.1mL murine osteosarcoma oncocyte S-180 (1.18 * 10 ⁸Cells/mL), inoculate after 3 days intratumor injection Agrocybe aegerita Galectin (with physiological saline solution 1.67mg/mL, an injection 0.06mL) 0.1mg/ mouse, contrast injecting normal saline.After this, injection every other day.Put to death mouse at inoculated tumour after 20 days.The cutting-out tumour is weighed, and calculates inhibiting rate.Inhibiting rate (%)=[(C-T)/C] * 100% (C is a control group average tumor weight, and T is the weight in average of treatment group tumour).The result is as shown in table 2.Agrocybe aegerita Galectin can reach 36.36% to the inhibiting rate of the tumour of the mouse of processing, and has prolonged the life-span of tumor-bearing mice.

Table 2 Agrocybe aegerita Galectin is to the restraining effect of lotus S-180 knurl mouse tumor

Treatment	Death ratio	Inhibition ratio
			0.06ml of 1.67mg/ml AAL 0.06ml of 0.9％NaCl(control)	10％ 50％	36.36％ -

Embodiment 5

The Antiphytoviral effect of Agrocybe aegerita Galectin: Agrocybe aegerita Galectin has the good restraining effect to tobacco mosaic virus (TMV) in the present embodiment, can be used as a kind of antiviral agricultural chemicals and be developed, its encoding sequence can be used as the seed selection that the Antiphytoviral genetic resources is applied to the Antiphytoviral crop varieties.

Detect the inhibition activity of Agrocybe aegerita Galectin to plant virus (TMV) with half leaf method: 0.01M PBS (PH7.2) is a reaction buffer, common strain 10 μ g/mL of tobacco mosaic virus (TMV) and Agrocybe aegerita Galectin (25,50,100,200 μ g/mL) equal-volume mixes, with 0.01M PBS (PH7.2) serve as the contrast be inoculated in respectively Nicotiana glutinosa about half leaf, the withered spot number order of counting left and right sides half vane after 48 hours, calculate inhibition ratio: inhibiting rate %=(withered spot number/contrast withered spot number that the 1-Agrocybe aegerita Galectin is handled) * 100%, experimental result is as shown in table 3.Agrocybe aegerita Galectin has the good restraining effect to TMV.

Table 3 Agrocybe aegerita Galectin is in the last restraining effect to TMV of Nicotiana glutinosa (Nicotiana glutinosa)

Concentration of AAVP (μg·ml -1)	Inhibition ratio (％)
		25 50 100 200	47.3 62.5 78.7 84.3

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition and reading after the present invention divides above-mentioned teachings that those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

SEQUENCE LISTING

＜110〉Wuhan University

＜120〉a kind of polypeptide of fungus galactose agglutinin protein activity, its encoding sequence and preparation method and application

＜130〉a kind of polypeptide of fungus galactose agglutinin protein activity, its encoding sequence and preparation method and application

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<170>PatentIn version 3.1

<210>1

<211>635

<212>DNA

＜213〉Agrocybe aegerita (Brig) Sing

<400>1

cagggcgtca acatctataa catcagtgcc gggaccagcg tcgacctcgc tgctcctgtt 60

accactggcg acattgtcac tttcttctcc tcagcgctca acctcaacgc gggcgcaggg 120

aacccaaaca acaccacgct gaacctattc gccgaaaatg gcgcatacct cctccacatc 180

gccttccgcc tccaagagaa tgtaattatc ttcaacagtc gccagcccga tgggccgtgg 240

ctcgtcgagc agcgtgtatc tgacgtcgca aatcaattcg ccgggattga tgggaaggcc 300

atggtgacgg tgttcgacca cggcgacaag tatcaggtag tgataaatga gaagacggtg 360

attcagtaca cgaagcaaat ctcgggtctg acattgagcc tctcctataa tgcaacggaa 420

gagacttcca tattctccac cgtggtcgaa gcggtgacat acacgggttt ggcgtagatt 480

ccgcgacatc ggaaggctag ggactgccag gccaagctag tagtaatcaa agaaaatgtc 540

gttcattccc catcaggtca gaaacaaagt cgaatgtaca gtagtctttt cagcaatgca 600

gaagtgtgta gttatgtgct aaaaaaaaaa aaaaa 635

<210>2

<211>158

<212>PRT

＜213〉Agrocybe aegerita (Brig) Sing

<400>2

Gln Gly Val Asn Ile Tyr Asn Ile Ser Ala Gly Thr Ser Val Asp Leu

1 5 10 15

Ala Ala Pro Val Thr Thr Gly Asp Ile Val Thr Phe Phe Ser Ser Ala

20 25 30

Leu Asn Leu Asn Ala Gly Ala Gly Asn Pro Asn Asn Thr Thr Leu Asn

35 40 45

Leu Phe Ala Glu Asn Gly Ala Tyr Leu Leu His Ile Ala Phe Arg Leu

50 55 60

Gln Glu Asn Val Ile Ile Phe Asn Ser Arg Gln Pro Asp Gly Pro Trp

65 70 75 80

Leu Val Glu Gln Arg Val Ser Asp Val Ala Asn Gln Phe Ala Gly Ile

85 90 95

Asp Gly Lys Ala Met Val Thr Val Phe Asp His Gly Asp Lys Tyr Gln

100 105 110

Val Val Ile Asn Glu Lys Thr Val Ile Gln Tyr Thr Lys Gln Ile Ser

115 120 125

Gly Leu Thr Leu Ser Leu Ser Tyr Asn Ala Thr Glu Glu Thr Ser Ile

130 135 140

Phe Ser Thr Val Val Glu Ala Val Thr Tyr Thr Gly Leu Ala

145 150 155

Claims (9)

1, a kind of nucleotide sequence of separated coding fungus galactose agglutinin protein, its sequence are nucleotide sequence shown in the SEQ IDNO.1.

2, a kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID NO.2.

3, a kind of polypeptide preparation method of fungus galactose agglutinin protein activity, it comprises the following steps:

The total RNA or the mRNA of A, extraction Agrocybe aegerita (Brig) Sing are template, obtain cDNA first chain by RT-PCR, are pcr template with this chain, and it is the full length sequence shown in the SEQ ID NO.1 that amplification obtains sequence;

B. enzyme is cut sequence and the prokaryotic expression carrier that steps A obtains, and makes up recombinant expression vector;

C, with the recombinant expression vector transformed into escherichia coli host cell that obtains among the step B, form to express the proteic reconstitution cell of Agrocybe aegerita Galectin;

D, the reconstitution cell that obtains among the step C is obtained saturated culture 37 ℃ of overnight incubation, 50 μ L saturated culture are inoculated in 5mL to be contained in the substratum of penbritin 100 μ g/mL, cultivated 2 hours in 37 ℃, getting the 1mL culture is transferred in the Eppendorf tube, add isopropylthiogalactoside IPTG to final concentration be 1m mol/L, culture is induced, induced 10 hours;

E, utilize Ni2+ affinity chromatography purifying protein, separate, among the purification step D in reconstitution cell polypeptide expressed.

4, the application of a kind of isolated polypeptide according to claim 2 in preparation prevention rectum cancer medicine.

5, the application of a kind of isolated polypeptide according to claim 2 in preparation prevention cervical cancer medicine.

6, the application of a kind of isolated polypeptide according to claim 2 in preparation prevention cancer of the stomach medicine.

7, the application of a kind of isolated polypeptide according to claim 2 in preparation prevention leukemia medicament.

8, the application of a kind of isolated polypeptide according to claim 2 in preparation prevention tobacco mosaic disease poison.

9, the application of a kind of isolated polypeptide according to claim 2 in the resisting tobacco mosaic virus plant breeding.

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