CN101935338A - Reticuloprotein epitope, anti-reticuloprotein antibody and application thereof - Google Patents

Abstract

The invention belongs to the fields of molecular biology and immunology and relates to a reticuloprotein epitope, an anti-reticuloprotein antibody and application thereof. Particularly, the reticuloprotein epitope has a sequence shown as SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The invention also relates to an anti-reticuloprotein polyclonal antibody, wherein the polyclonal antibody is specifically combined with the epitope and has high specificity and valence. The invention also relates to a preparation method and application of the polyclonal antibody, a composition containing the polyclonal antibody and a method for detecting the reticuloprotein.

Description

A kind of reticuloprotein epitope, antireticulin antibody and uses thereof

Technical field

The invention belongs to molecular biology and field of immunology.Particularly, relate to a kind of reticuloprotein epitope, antireticulin antibody.The invention still further relates to the composition and the purposes of described antibody in detecting reticuloprotein that contain described epitope or antibody respectively.

Background technology

Reticuloprotein (Reticulon protein) family is a class formation albumen, also is the constituent of endoplasmic reticulum.Eukaryotic reticuloprotein family all is closely related with the composition of endoplasmic reticulum.The amino terminal region of reticuloprotein does not link to each other mutually, at C-terminal a similar sequences (SEQ ID NO:1) is arranged but, and this zone comprises two big water repellent region, is separated by 66 hydrophilic residue fragments.Experiment showed, that the conservative hydrophobicity fragment of C end plays a significant role in the contact of reticuloprotein and endoplasmic reticulum.Prediction hydrophobicity fragment is embedded in the film kernel and 66 hydrophilic residues are positioned at film outer surface.Most of reticuloproteins all have a Methionin maintenance motif at the C end.Because the inner link that reticuloprotein and rough surfaced endoplasmic reticulum and smooth endoplasmic reticulum may exist, they may and keep in the cell transportation, and the Ca ion concentration is stable in the cell plays a significant role.Reticuloprotein is considered to protein (the Thereticulons:a family of proteins with diverse functions YvonneS Yang and Stephen MStrittmatter Genome Biol.2007 with the closely-related channel-like of endoplasmic reticulum; 8 (12): 234.Published online 2007 December 28. doi:10.1186/gb-2007-8-12-234.PMCID:PMC2246256).

In paddy rice, reticuloprotein is by being arranged in gene Os04t0670000 (ID of GenBank number the is NM_001060744) coding on the karyomit(e) No. 4, this gene has 2915 bases, in 2004 first by Sasaki, T. wait the people to find, (Submitted (25-OCT-2004) Contact:Takuji Sasaki National Institute of AgrobiologicalSciences, Rice Genome Research Program; Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan).The long 600bp of this genes encoding frame (SEQID NO:2), the reticuloprotein of coding (SEQ ID NO:3) has 199 amino acid, the about 22kda of molecular weight.

Yet up to now, the paddy rice reticuloprotein still belongs to highly albumen to be identified, and the activity of this albumen on transcriptional level is the evidence of its existence.And in the prior art, the polyclonal antibody specificity of preparation is not high usually, is used to detect target substance and can has the insufficient problem of accuracy.On the other hand, if prepare antigenic words with reticuloprotein, no matter be from recombinant expressed or chemosynthesis, the aminoacid sequence of total length has 1996 amino acid, the reconstruction of protein steric structure is a very difficult job, reproduce with organism in identical space conformation remain the present stage difficult point studied of proteinology.In general epitope is synthetic simple and easy, the accuracy height, with low cost, space conformation reproduces easily, therefore be necessary to develop a kind of epitope (antigenic determinant that represents reticuloprotein itself that has, AD) (claim antigenic determinant again, be meant the special chemical group of decision antigen-specific in the antigen molecule) to replace full-length proteins.

Although be useful on the software of prediction epitope at present, BEPITOPE software for example, but have the non-effective epitope about 30% in the epitope that prediction obtains, promptly can not represent antigenic epitope specifically, perhaps can not cause immunne response (Chang HT, LiuCH, Pai TW.Estimation and extraction of B-cell linear epitopespredicted by mathematical morphology approaches.J MolRecognit.2008 Nov-Dec; 21 (6): 431-41).

Summary of the invention

In order to address the above problem, the inventor is obtaining by the BEPITOPE software prediction on the basis of a plurality of epitope sequences, a large amount of experiments and unremitting effort have been carried out, found that it is effective antigens epi-position that an epitope sequence D TIISKTGESTKQK (SEQID NO:4) is arranged in 12 epitopes of prediction, and it has good antigen-specific, and prepared can with reticuloprotein specific immune bonded antibody, this antibody has enough susceptibilitys and accuracy.Following invention is provided thus:

One aspect of the present invention relates to a kind of reticuloprotein epitope, and its aminoacid sequence is shown in SEQ ID NO:4.

In one embodiment of the invention, in order to increase the bridging property with carrier, added a halfcystine (halfcystine provide disulfide linkage and carrier crosslinked) at the C-terminal of above-mentioned epitope, obtaining sequence is the epitope of DTIISKTGESTKQKC (SEQ ID NO:5).

Halfcystine also can be added in the N-terminal of polypeptide, and obtaining sequence is the epitope of CDTIISKTGESTKQK (SEQ ID NO:6).

It is just passable that end adds 1 halfcystine.If add a plurality of halfcystines, cause cost to rise, and disulfide linkage density is too big, and does not in fact also need so many disulfide linkage at same end.Should not all add halfcystine at N-terminal and C-terminal in addition, because an epitope and a carrier combination, if add 1 halfcystine respectively at two ends, halfcystine is with after carrier combines, and epitope has lacked free end, is unfavorable for producing antibody on the contrary.

Epitope of the present invention can obtain by the peptide synthetic technology chemosynthesis of routine, also can express obtaining in suitable host; Preferably chemosynthesis.

Of the present inventionly also relate in one aspect to a kind of composition, it comprises the reticuloprotein epitope shown in SEQ ID NO:4 or SEQID NO:5 or the SEQ ID NO:6, randomly, can contain immunological adjuvant, for example aluminium hydroxide, Freund's complete adjuvant or Freund's incomplete adjuvant etc.

Of the present inventionly also relate in one aspect to a kind of reticuloprotein epitope-carrier complexes; Wherein, described reticuloprotein epitope is a reticuloprotein epitope of the present invention, and described carrier can be keyhole limpet hemocyanin (KLH), BSA or casein etc.

Of the present inventionly also relate in one aspect to a kind of antireticulin antibody, described antireticulin antibody can be specifically in conjunction with the reticuloprotein epitope shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQ ID NO:6.Described antireticulin antibody can be monoclonal antibody, also can be polyclonal antibody.

Antireticulin polyclonal antibody of the present invention can derive from the animals of various conventional preparation polyclonal antibodies, goat for example, rabbit, rat, mouse etc.

To those skilled in the art, can prepare monoclonal antibody according to the reticuloprotein epitope shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQ ID NO:6, concrete operations can be referring to the technical manual of this area, also can reference Nature 1975Kohler﹠amp for example; Milstein Vol256, p495.

Of the present inventionly also relate in one aspect to a kind of serum (abbreviation polyvalent antibody) that contains the antireticulin polyclonal antibody, it makes by using the epitope immune animal shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQID NO:6.

Of the present inventionly also relate in one aspect to a kind of antireticulin preparation method of polyclonal antibody, comprise the step of the reticuloprotein epitope shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQ ID NO:6 as antigen-immunized animal.Randomly, described immune step can add adjuvant, for example aluminium hydroxide, Freund's complete adjuvant or Freund's incomplete adjuvant, or the like.

In one embodiment of the invention, described antireticulin preparation method of polyclonal antibody comprises the steps:

1) with the reticuloprotein epitope shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQ ID NO:6 as antigen-immunized animal;

2) get blood, centrifugal collection polyvalent antibody; With

3) purification step 2) in polyvalent antibody, obtain the antireticulin polyclonal antibody.

Of the present inventionly also relate in one aspect to a kind of composition, it comprises antireticulin polyclonal antibody of the present invention.

Of the present inventionly also relate in one aspect to a kind of reticuloprotein detection agent, it comprises antireticulin polyclonal antibody of the present invention.

Wherein because reticuloprotein is (the SEQ ID NO:1 of face as follows) that guards in a plurality of species, and the SEQ ID NO:4 as epitope is present in (referring to the sequence that adds frame among the SEQ ID NO:1) in the conserved sequence among the present invention, therefore polyclonal antibody of the present invention not only can detect the reticuloprotein in the paddy rice, also can detect the reticuloprotein in other a plurality of species (for example the mankind, diphtheria sparrow, Caenorhabditis elegans, Arabidopis thaliana, or the like).

MATTSRRSXXXXXXXXXXXXXXXWRRRNXXXXXXXXXXXXWFLFERAGYSFPSVMANALLLLVAILFFWAKSASXXXXXXXXXXXXEVSDVVVEKAADRALVWINKVLAVGHDIAIKRDRSVFIKVILILWVVSYIGMLFNFLTLIYIGVMFSLLVPPLYEKYQDQVDEKIGMAHSVLSRHL

KTE(SEQ?ID?NO：1)

Wherein, X represents arbitrary amino acid, and the sequence that adds frame is SEQ ID NO:4.

The purposes of antireticulin polyclonal antibody of the present invention in the medicine of preparation detection reticuloprotein that also relate in one aspect to of the present invention.

Of the present inventionly also relate in one aspect to a kind of method that detects reticuloprotein, described method comprises the step of using antireticulin polyclonal antibody of the present invention.Particularly, comprise the steps:

1) testing sample and antireticulin polyclonal antibody of the present invention are hatched, described antireticulin polyclonal antibody is combined with reticuloprotein specificity in the testing sample, form immunocomplex thus; With

2) detect whether there is immunocomplex.

The method of above-mentioned detection reticuloprotein can detect the having or not of reticuloprotein, it is carried out sxemiquantitative (for example western blot method); Under the situation that the reticuloprotein standard substance are arranged, can also carry out detection by quantitative to reticuloprotein by the ELISA method.

In one embodiment of the invention, described reticuloprotein is the reticuloprotein of paddy rice.Concrete, described paddy rice is paddy rice 93-11.

The beneficial effect of the invention

1) polyclonal antibody provided by the invention, the specificity height.A specific band (see figure 1), specificity height only appear in Western blot when detecting;

2) reticuloprotein antigen provided by the invention is synthetic simple and easy, the accuracy height.Epitope provided by the invention is chemosynthesis after the epitope fragment that software prediction is selected, and fragment is short and small, and synthetic easily, space conformation reproduces easily.

3) polyclonal antibody provided by the invention can be used for the reagent whether all detection reticuloproteins exist.

4) polyclonal antibody provided by the invention can be analyzed and researched to the expression of reticuloprotein in the paddy rice under different space-times, different growth conditions, will provide important clue for the function and the mechanism of action of further explaining reticuloprotein.

Description of drawings

Fig. 1: use the immunoblotting detected result of 1 pair of reticuloprotein of antireticulin polyclonal antibody at the paddy rice different tissues.

Embodiment

Below in conjunction with embodiment embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples only are used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment, according to the described technology of the document in this area or condition (for example with reference to works such as J. Sa nurse Brookers, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

Embodiment 1: the prediction of candidate antigens epi-position 1

The gene I of gene in GenBank of paddy rice reticuloprotein correspondence is NM-001060744.The reading frame sequence is as follows:

ATGGCCACCACCTCCCGCCGATCCCTCCACGCCCTCCTCGGCGGCGGCGCAGTCGCCGACTTGGTGCTGTGGCGGCGGAGGAACGTGTCCGCGGCGGCCGTGGCGGGCGCCACGGCGGTGTGGTTCCTCTTCGAGCGCGCGGGGTACAGCTTCCCGTCAGTCATGGCCAACGCCCTGCTCCTCCTCGTCGCCATCCTCTTCTTCTGGGCCAAGTCCGCCTCGCTGCTCAACAGGCCTCTTCCACCACTCCCTAAACTAGAGGTCTCAGATGTGGTTGTTGAGAAAGCTGCAGATCGGGCTCTTGTATGGATCAACAAGGTGTTGGCTGTTGGCCATGATATTGCCATCAAGAGAGATAGGAGTGTTTTTATAAAGGTTATATTGATTTTATGGGTGGTTTCATACATTGGAATGCTCTTCAACTTCCTTACGCTCATTTACATTGGTGTAATGTTTTCTCTGTTAGTTCCACCATTGTATGAGAAGTACCAGGACCAAGTCGATGAAAAGATTGGTATGGCGCACAGTGTACTATCAAGGCACTTAGATACCATCATTAGCAAGACTGGAGAATCAACCAAGCAAAAGAAGACTGAGTAA(SEQ?ID?NO：2)

Coded paddy rice reticuloprotein full length sequence is as follows:

MATTSRRSLHALLGGGAVADLVLWRRRNVSAAAVAGATAVWFLFERAGYSFPSVMA NALLLLVAILFFWAKSASLLNRPLPPLPKLEVSDVVVEKAADRALVWINKVLAVGH DIAIKRDRSVFIKVILILWVVSYIGMLFNFLTLIYIGVMFSLLVPPLYEKYQDQVD EKIGMAHSVLSRHL

KTE (SEQ ID NO:3, the sequence that wherein adds frame is SEQ ID NO:4)

Then according to SEQ ID NO:3, the protein of paddy rice reticuloprotein genes encoding is carried out the prediction of epitope with BEPITOPE software.Five kinds of method Standard, the Karplus, Emini, Amphiphi, the Pellequer that have used BEPITOPE software to provide in the present embodiment, and the integrated approach cons_Sta_Kar_Emi_Amp_Pel of these five kinds of methods, all parameters are selected acquiescence.Concrete grammar can be with reference to Odorico M, Pellequer J L.BEPITOPE:predicting the location of continuous epitopes and patterns inproteins[J] .J Mol Recognit, 2003,16 (1): 20-22.

Prediction has obtained 12 candidates' epitope altogether, selects the higher fragment DTIISKTGESTKQK of epitope peak value (SEQ ID NO:4).

This fragment is carried out uniqueness retrieval (protein sequence comparison) at TIGR paddy rice database (http://rapdb.dna.affrc.go.jp/) then, has determined the uniqueness of this fragment in the rice protein storehouse.

Embodiment 2: the chemosynthesis of epitope 1

The two ends of the candidate antigens epi-position 1 that obtains among the embodiment 1 do not have halfcystine, and in order to realize crosslinked with carrier, the synthetic sequence need add halfcystine, therefore wants the synthetic peptide sequence to be: DTIISKTGESTKQKC (SEQ ID NO:5).

Peptide sequence shown in the SEQ ID NO:5 is carried out chemosynthesis (synthetic by the gill biochemical corp), obtain the epitope 1 of reticuloprotein.

Embodiment 3: the preparation of epitope 1-KLH mixture

Adopt the glutaraldehyde connection method, the C end of synthetic epitope 1 among the embodiment 2 is crosslinked with crosslinked carrier proteins-keyhole limpet hemocyanin (KLH), obtain epitope 1-KLH mixture.

Concrete implementation step is as follows:

The synthetic polypeptide of 5mg is added among the 7mg KLH, slowly add freshly prepared 3g/L glutaraldehyde solution 1ml, incubated at room 2h while shaking.Borate buffer dialysis 24h with pH8.5 obtains epitope 1-KLH mixture.

Embodiment 4: the preparation of polyvalent antibody 1

Get the epitope 1-KLH mixture of preparation among the 1-2mg embodiment 3, immune new zealand white rabbit, every 14 days booster immunizations once; Behind the 2nd booster immunization 7 days, ear vein was got blood, and separation of serum (the centrifugal 10min of 5000r pm) is collected supernatant, and survey is tired.Simultaneously according to identical step, in contrast with epitope 1 (SEQ ID NO:5).

Concrete steps are as follows:

The epitope 1-KLH mixture of getting among the 1-2mg embodiment 3 preparation is fully emulsified with the complete freund adjuvant of equivalent, formation profit bag, and in the subcutaneous multi-point injection of rabbit neck part and back, every about 100 μ g.Booster immunization after 2 weeks, dosage is the same, after the full freund adjuvant that toos many or too much for use is fully emulsified, in the subcutaneous multi-point injection of rabbit back; Later on every 2 all booster immunizations 1 time; Since the 2nd booster immunization, each immunity was got tiring of hematometry antibody through ear vein after 7 days.

Wherein, to get the tire step of detection (ELISA method) of blood as follows for ear vein:

On 96 orifice plates, every hole adds 50 μ g/ml Tyrosine O-phosphates, 100 μ l, 4 ℃ spend the night after, wrap quilt, the washing.To be 1: 100 according to the polyvalent antibody dilution of the ear vein blood of preceding method preparation, 1: 500,1: 2500,1: 3200,1: 12800,1: 25600, every hole adds 100 μ l, 37 ℃ of insulation 30min, washing.Each adds the goat-anti rabbit I g enzyme conjugates 100 μ l of dilution in 1: 100,37 ℃ of insulation 30min, washing.Add TMB (3,3,5, the 5-tetramethyl benzidine) 100 μ l, behind the 20min, add the H of 2mol/l ₂SO ₄Termination reaction.Use microplate reader to measure the A490nm value, be higher than the positive of 10 times of preimmune serums.

Through behind 2 booster immunizations, the result that detects of tiring is as follows:

Polyvalent antibody 1 (immunity of epitope 1-KLH mixture)＞102400 (it is 102400 that epitope 1-KLH mixture has immunogenic maximum dilution multiple);

Polyvalent antibody＞102400 that naked peptide immunity (epitope 1, SEQ ID NO:5) obtains (only the naked peptide of forming by epitope 1 have immunogenic maximum dilution multiple be 102400).

The above results meets the requirements, and satisfies in the rear neck artery bloodletting in 7 days of last booster immunization, collects blood sample.The blood sample of collecting was left standstill under 3-4 ℃ 3-4 hour, and 5000rpm is centrifugal 10 minutes then, collects serum, obtain polyvalent antibody 1 (ear vein detect tire meet the requirements after, the blood sample that carotid artery is got needn't detect again and tire).Aseptic subpackaged being stored in-80 is ℃ standby.

Embodiment 5: the preparation of antireticulin polyclonal antibody 1 (resisting 1) more

The polyvalent antibody 1 of preparation among the embodiment 4 is carried out purifying, make polyclonal antibody 1 (resisting 1) more.

With epitope DTIISKTGESTKQK CThe Sepharose 4B coupling of (SEQ ID NO:5) polypeptide and cyanogen bromide-activated, preparation polypeptide affinity column.

The polyvalent antibody 1 of preparation is joined in the chromatography column of above preparation, be positioned over 4 ℃ of overnight incubation after, wash-out antibody promptly obtains antireticulin polyclonal antibody (how anti-1).

Embodiment 6: resist the specificity check of 1 pair of reticuloprotein more

Choose fresh paddy rice 93-11 (available from national hybrid rice engineering center) overground part in seedling stage, tillering phase blade, boot stage sword-like leave, flowering period sword-like leave, ripening stage sword-like leave, seedling stage underground part, tillering phase stem, flowering period tassel, ripening stage seed (totally 9 positions), extract gross protein respectively.

The preparation of gross protein sample: extremely Powdered with the above-mentioned fresh rice tissue of liquid nitrogen grinding, divide to install in the precooling centrifuge tube, per 300 μ l powder add 800 μ l protein cleavage liquid (62.5mmol/L pH7.4 TrisHCl, 10% glycerine, 2%SDS, 20mmol/LNaF, 2mmol/L EDTA, 1mmol/L PMSF, 5% beta-mercaptoethanol), rapid mixing also places on ice, hatches in the mixture of ice and water 10 minutes, and the concussion mixing was 1 time in per approximately 2 minutes.4 ℃, centrifugal 15 minutes of 12000r/min.Get supernatant, transfer in the new centrifuge tube ,-70 ℃ of preservations.Obtain the paddy rice total protein.

The Western marking:

The above-mentioned rice protein that extracts is carried out SDS-PAGE (12%), during detection, the applied sample amount of the paddy rice total protein at Marker (Transgen Biotech, article No.: DM 201) and above-mentioned 9 positions is followed successively by from left to right and is 10 μ l, 7 μ l, 8 μ l, 7 μ l, 6 μ l, 6 μ l, 6 μ l, 10 μ l, 3 μ l, 5 μ l (Fig. 1).

Electrotransfer is to pvdf membrane behind the SDS-PAGE, and the skim-milk with 5% seals pvdf membrane.Use prepare among the embodiment 5 to resist 1, incubated at room is 3 hours after diluting with 1: 1000 more, and (2mmol/L TrisHCl pH7.6,13.6mmol/L NaCl 0.1%Tween-20) wash film 3 times to TTBS, each 5 minutes.Add afterwards with the rabbit source of dilution in 1: 15000 how anti-(producer's article No.: middle China fir ZB2301), incubated at room 1 hour, TTBS washes film 3 times, each 5 minutes.Add ECL Pluc chromogenic reagent, darkroom exposure 5 minutes.The result as shown in Figure 1, antibody has specificity, has only 1 tangible master tape, and at the different development stage of paddy rice, this albumen all has expresses and expression amount tends towards stability.

Need to prove, just to have only one of reticuloprotein with many anti-1 specificity bonded although have a lot of molecular weight albumen similar in the paddy rice whole protein to it.About specific prediction: SEQ ID NO:5 is (identical with the TIGR paddy rice database effect among the embodiment 1 through http://rice.plantbiology.msu.edu/blast.shtml, purpose is to check uniqueness once more) inspection, determine that this peptide sequence is special, this sequence is unique definite reticuloprotein in the paddy rice total protein.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Claims (10)

1. reticuloprotein epitope, it is made up of the aminoacid sequence shown in SEQ ID NO:4 or SEQ ID NO:5 or the SEQ ID NO:6.

2. a composition wherein contains the described reticuloprotein epitope of claim 1, randomly, also contains the adjuvant that is useful on immunity.

3. reticuloprotein epitope-carrier complexes, wherein, described reticuloprotein epitope is the described reticuloprotein epitope of claim 1, described carrier is selected from keyhole limpet hemocyanin, BSA and casein.

4. the preparation method of an antireticulin antibody comprises the step of the mixture of the composition of the reticuloprotein epitope that uses claim 1 or claim 2 or claim 3; Randomly, described antireticulin antibody is monoclonal antibody or polyclonal antibody; Particularly, described preparation method comprises the steps:

1) blood sample that the mixture immune animal of the composition of described reticuloprotein epitope of claim 1 or claim 2 or claim 3 is obtained carries out centrifugally, obtains polyvalent antibody; With

2) purification step 1) in polyvalent antibody, obtain the antireticulin polyclonal antibody.

5. polyvalent antibody, it is made by the reticuloprotein epitope of claim 1 or the composition of claim 2 or the mixture immune animal of claim 3.

6. antireticulin antibody, it can be specifically in conjunction with the described reticuloprotein epitope of claim 1, and randomly, it is polyclonal antibody or monoclonal antibody.

7. composition, it comprises the described antireticulin antibody of claim 6.

8. reticuloprotein detection agent, it comprises the described antireticulin antibody of claim 6.

9. the described antibody of claim 6 detects purposes in the medicine of reticuloprotein in preparation.

10. method that detects reticuloprotein, described method comprise the step of using the described antireticulin polyclonal antibody of claim 6; Particularly, described reticuloprotein is the reticuloprotein of paddy rice.

Images