CN112724236B - Antigen peptide of amphoterin 1, antibody and application thereof - Google Patents
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Abstract
The invention discloses an antigenic peptide of amphoterin 1, an antibody and application thereof, belonging to the technical field of biology. The antigenic peptide of the invention is the antigenic peptide of the amphoterin 1(1-278) fragment, and the amino acid sequence of the antigenic peptide is CLPSPTASPN. The antibody prepared by using the antigen peptide as an antigen can specifically recognize the amphoterin 1(1-278) fragment but not recognize the full length of the amphoterin 1, can be used for detecting the shearing degree of the amphoterin 1 in a human brain specimen and an animal specimen, is used for researching the pathogenesis of the Alzheimer's disease, is used for early diagnosis of the Alzheimer's disease and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antigenic peptide of amphoterin 1 (Amphihyssin I), an antibody thereof and application thereof.
Background
Alzheimer Disease (AD) is the most common neurodegenerative disease in the clinic and also the most common cause of dementia in elderly people. The prevalence rate of old people over the age of 60 is as high as 5%, and the old people bring heavy burden to society and families [1,2 ]. However, the cause of AD is not clear so far, and therefore, there is a lack of therapeutic measures for delaying the course of disease by addressing the pathogenesis.
Amphoterin 1 (amphiusin I) is widely expressed in nerve endings and is involved in clathrin-mediated endocytosis [3,4 ]. Amphihysin I mediates internalisation and division of synaptic vesicles, senses and promotes membrane bending [5], and stimulates GTPase activity on dynamin re-membranes [6 ]. Reduced levels of ampiphysin I protein were found in the mouse model of tauopathy JNPL3 [7], but the mechanism is not yet clear. And the applicant researches and discovers that the Amphipsin I in the brain tissue of the AD patient is cut into fragments, and the protease mediating the cutting of the Amphipsin I is Asparagine Endopeptidase (AEP). During the attack of AD, AEP in brain is abnormally activated [8,9], and N278 site of Ampliphysin I is cut to form Ampliphysin I (1-278) segment, which further mediates the attack of nerve injury and AD.
In view of the fact that the Amphiphysin I (1-278) segment formed by cutting the Amphiphysin I by AEP plays an important role in AD pathogenesis, the detection of the Amphiphysin I (1-278) segment in experimental animal specimens and human body fluid plays an important role in understanding the AD pathogenesis. However, no antibody for detecting the ampiphysin I (1-278) fragment generated by the shearing of AEP in the brain has been found so far, which brings great inconvenience to related researches.
Reference documents:
1.Chan KY,Wang W,Wu JJ,Liu L,Theodoratou E,Car J,Middleton L,Russ TC,Deary IJ,Campbell H,Wang W,Rudan I.Epidemiology of Alzheimer's disease and other forms of dementia in China,1990-2010:a systematic review and analysis.Lancet.2013,381:2016-2023.
2.Jia J,Wang F,Wei C,Zhou A,Jia X,Li F,Tang M,Chu L,Zhou Y,Zhou C,Cui Y,Wang Q,Wang W,Yin P,Hu N,Zuo X,Song H,Qin W,Wu L,Li D,Jia L,Song J,Han Y,Xing Y,Yang P,Li Y,Qiao Y,Tang Y,Lv J,Dong X.The prevalence of dementia in urban and rural areas of China.Alzheimers Dement.2014,10:1-9.
3.Wu Y,Matsui H,Tomizawa K.Amphiphysin I and regulation of synaptic vesicle endocytosis.Acta Med Okayama.2009；63:305–323.
4.Takei K,Slepnev VI,Haucke V,De Camilli P.Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis.Nat Cell Biol.1999；1:33–39.
5.Zhang B,Zelhof AC(2002)Amphiphysins:raising the BAR for synaptic vesicle recycling and membrane dynamics.Bin-amphiphysin-Rvsp.Traffic 3:452–460.
6.Yoshida Y,Kinuta M,Abe T,Liang S,Araki K,Cremona O,Di Paolo G,Moriyama Y,Yasuda T,De Camilli P,Takei K(2004)The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature.EMBO J 23:3483–3491.
7.De Jesus-Cortes,H.J.,C.J.Nogueras-Ortiz,M.Gearing,S.E.Arnold and I.E.Vega(2012)."Amphiphysin-1protein level changes associated with tau-mediated neurodegeneration."Neuroreport 23(16):942-946.
8.Zhang Z,Song M,Liu X,Kang SS,Kwon IS,Duong DM,Seyfried NT,Hu WT,Liu Z,Wang JZ,Cheng L,Sun YE,Yu SP,Levey AI,Ye K.Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease.Nat Med.2014,20:1254-1262.
9.Zhang Z,Song M,Liu X,Su Kang S,Duong DM,Seyfried NT,Cao X,Cheng L,Sun YE,Ping Yu S,Jia J,Levey AI,Ye K.Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer's disease.Nat Commun.2015,6:8762.
disclosure of Invention
The invention aims to develop an antibody aiming at the Amphihysin I (1-278) fragment, provides an antigenic peptide of the Amphihysin I (1-278) fragment, an antibody and application thereof, and is used for pathogenesis research and early diagnosis of AD and aging-related diseases.
The purpose of the invention is realized by the following technical scheme:
an antigenic peptide of an Amphiphysin I (1-278) fragment, the amino acid sequence of which is as follows: CLPSPTASPN are provided.
An antibody prepared from the above antigen peptide as antigen. Furthermore, the antibody is prepared by using a cross-linked polypeptide obtained by cross-linking Ac-CLPSPTASPN with a carrier protein as an antigen, wherein the carrier protein is preferably KLH. The antibody can be a monoclonal antibody or a polyclonal antibody.
Further, the polyclonal antibody is a rabbit polyclonal antibody.
Further, the preparation method of the rabbit polyclonal antibody comprises the following steps: the cross-linked polypeptide is used as immunogen to immunize rabbits, rabbit blood is collected to prepare antiserum, and the antiserum is separated and purified to obtain the rabbit polyclonal antibody. The separation and purification of the antiserum are carried out by adopting a conventional technical means, namely, the antiserum is purified by adopting an antigen affinity chromatography method to obtain the purified rabbit polyclonal antibody.
The antigen peptide of the Amphiphysin I (1-278) fragment has good immunogenicity, and the rabbit polyclonal antibody obtained by taking the antigen peptide as immunogen has good specificity and high titer, only recognizes the Amphiphysin I (1-278) fragment formed by cutting AEP, but does not recognize the full-length Amphiphysin I; the titer can reach 1: 512000. Based on the specificity of the antibody, the method can be used for detecting the shearing degree of the Amphipsin I protein in a human brain specimen and an animal specimen, researching the pathogenesis of AD, early diagnosing AD and the like.
An agent for detecting the shearing degree of the Amphihyssin I protein comprises the antibody.
An AD early diagnosis reagent comprises the antibody.
Compared with the prior art, the invention has the following advantages and effects:
(1) the existing Amphihysin I antibody can identify the full-length Amphihysin I protein without difference, and researches find that the Amphihysin I (1-278) fragment formed by cutting the Amphihysin I is the part with the neurotoxicity effect, so that the detection of the fragment has important significance. At present, no antibody for specifically recognizing the Amphipsin I (1-278) fragment exists, and the problem is solved by the antibody for specifically recognizing the Amphipsin I (1-278) fragment obtained on the basis of the antigen peptide.
(2) The antibody of the invention for resisting the Amphiphysin I (1-278) fragment can be used for early diagnosis of AD.
Drawings
FIG. 1 is a diagram showing the result of detecting the titer of anti-Amphihysin I N278 antibody by ELISA.
FIG. 2 is a graph showing the results of specific detection of the anti-Amphihysin I-N278 antibody (anti-Amph 1N 278). The Amphipsin I is cut by AEP to form a (1-278) fragment, and the anti-Amphipsin I N278 antibody can specifically recognize the Amphipsin I (1-278) fragment formed by cutting by AEP and not recognize full-length Amphipsin I.
FIG. 3 is a graph showing the results of immunohistochemistry using an anti-Amphihysin I N278 antibody (anti-Amph 1N 278). The anti-Amphipsin I N278 antibody can detect the Amphipsin I (1-278) fragment in the brain of AD patients.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 preparation of Rabbit polyclonal antibody (anti-Amphipsin I N278 antibody)
The antigenic peptide (Ac-CLPSPTASPN) used in this example was obtained by artificial chemical synthesis (Nanjing Kingsrei Biotech Co., Ltd.). And carrying out mass spectrum identification on the synthesized antigen peptide, and purifying and identifying the purity of the antigen peptide by using HPLC after the identification is correct.
The antigen peptide is coupled to carrier protein KLH to obtain antigen peptide-KLH conjugate which is used as immunogen to immunize New Zealand rabbits to prepare polyclonal antibodies. The method comprises the following specific steps:
(1) animal blood was collected before the experiment and preimmune serum was prepared. Centrifuging the collected blood, and collecting the supernatant to obtain the serum.
(2) Primary immunization: each rabbit was injected subcutaneously in multiple doses with 1mL of an emulsified mixture of the antigenic peptide-KLH conjugate and Freund's complete adjuvant (volume ratio 1:1), wherein the antigenic peptide content was 0.5 mg.
(3) One week after the primary immunization, a second booster immunization was performed according to the method of the primary immunization.
(4) One week after the second boost, the third boost was performed according to the method of the primary immunization.
(5) One week after the third booster immunization, blood was collected to prepare antisera.
The antiserum was purified by antigen affinity chromatography to obtain a purified polyclonal antibody, an ampiphysin I N278 antibody (1mg/mL), for use in the following experiments.
Example 2 detection of antibody Titers
(1) The uncrosslinked antigenic peptide was dissolved in 4. mu.g/mL in a coating solution (0.05M carbonate buffer pH 9.6).
(2) To a 96-well plate, 100. mu.L of a coating solution prepared by dissolving the antigen peptide in (1) was added overnight at 4 ℃.
(3) The next day, the liquid was emptied and the residual liquid was patted dry, and the wash was added every 5 minutes, rinsing three times.
(4) mu.L of blocking solution was added to each well and incubated at 37 ℃ for 1 h.
(5) The liquid was emptied and the residual liquid was patted dry and washed three times with washing solution.
(6) mu.L of anti-Amphihysin I N278 antibody diluted in a gradient was added to each well and incubated at 37 ℃ for 1 h.
(7) The liquid was emptied and the residual liquid was patted dry and washed three times with washing solution.
(8) Horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1mg/mL) diluted 1:10000 was added to each well and incubated at 37 ℃ for 1 h.
(9) The liquid is emptied and the residual liquid is patted dry, and the wash is rinsed three to four times.
(10) The residual liquid in the wells was patted dry, 100. mu.L of mixed developing solution A, B was added to each well, and development was carried out for 30min at 37 ℃ in the dark.
(11) Adding 50-100 mu L of 2M H into each hole2SO4The color development is stopped, the OD value of 450nm is immediately read, the detection result is shown in figure 1, and the titer of the anti-Amphihysin I N278 antibody reaches 1: 512000.
Example 3 cytological detection of specificity of anti-Amphiphysin I-N278 antibodies
(1) Construction of a plasmid expressing GST-tagged Amphihyssin I (GST-Amph 1): taking a HEK293 cell cDNA library as a template, amplifying by using primers FP and RP to obtain an Amphipsin I gene sequence, connecting and transforming DH5 alpha to connect the Amphipsin I gene to a vector with pcDNA3.1-N-GST, and constructing to obtain a plasmid for expressing the Amphipsin I with a GST tag. Wherein the sequences of the primers FP and RP are as follows:
FP：GCGCGTCGACTATGGCAGAGATGGGCAGTAAAGGG；
RP：GCCGGCGGCCGCTCATGGGACCCTCTCAGTGAAGT。
(2) cell transfection: a plasmid expressing GST-tagged Amphilysin I (GST-Amph1) was transfected into HEK293 cells.
(3) Preparing a sample: after 2d of cell transfection, cells were harvested, washed once with PBS, lysed with lysis buffer (50mM sodium citrate, 5mM DTT, 0.1% CHAPS, 0.5% TX-100, pH 6.0) on ice for 30min, the supernatant was transferred to a new EP tube, and 20. mu.L of glutathione sepharose 4B GST-tagged protein purification resin (GE Healthcare) was added and incubated overnight at 4 ℃. The cells were washed 4 times with the lysate, centrifuged at 1000rpm for 5min and the supernatant discarded. mu.L of the lysate was added, mixed and divided into three tubes, 30. mu.L/tube, one of which was added with 5. mu.L of recombinant AEP enzyme (rAEP, Beijing Yiqianshengzhou), and the other two tubes were used as controls, and one was added with 5. mu.L of buffer (50mM sodium citrate, 5mM DTT, 0.1% CHAPS, 0.5% TX-100, pH 6.0) of pH 6.0, and the other was added with 5. mu.L of buffer (50mM sodium citrate, 5mM DTT, 0.1% CHAPS, 0.5% TX-100, pH 7.4) of pH 7.4, incubated at 37 ℃ for 30min, added with 5 XSDS loading buffer, mixed and boiled at 100 ℃ for 10 min.
(4) Glue running: the prepared samples are spotted according to the sequence of figure 2, after 20min of 80V constant pressure glue running, 120V constant pressure glue running to a Marker is separated to a proper position, and the same samples are spotted on two different glues for simultaneously detecting two different antibodies.
(5) Film transfer: preparing a membrane transferring solution in advance, precooling at 4 ℃, fixing the glue and the membrane by a membrane transferring clamp according to the sequence of a negative electrode, sponge, filter paper, glue, NC membrane, filter paper, sponge and a positive electrode, and transferring the membrane for 75min at a constant current of 220 mA.
(6) And (3) sealing: after the film transfer is completed, the film is washed once by TTBS solution, placed in 5% milk powder, and placed on a shaking bed to be sealed for 1h at room temperature.
(7) Incubating the primary antibody: after blocking, primary antibody was incubated with 3% milk powder (GST antibody (Proteitech, 1 mg/mL): 1:10000, anti-Amphihysin I N278 antibody (anti-Amph 1N 278): 1:2000) overnight at 4 ℃ with shaking.
(8) Washing the membrane: washing the membrane with TTBS solution at room temperature for 40min, and changing the membrane washing solution every 10 min.
(9) Incubation of secondary antibody: and (3) diluting a goat anti-mouse IgG secondary antibody (used for resisting a GST antibody) marked by horseradish peroxidase or a goat anti-rabbit IgG secondary antibody (used for resisting an Amphihysin I N278 antibody) marked by horseradish peroxidase according to the proportion of 1:10000 by adopting 3% milk powder, and incubating for 1h at room temperature.
(10) Washing the membrane: washing the membrane for 1h by adopting TTBS solution at room temperature, and replacing the membrane washing solution every 10 min.
(11) And (3) developing: the film was exposed and developed using ECL developer.
The results are shown in fig. 2, and the anti-ampiphysisin I N278 antibody (anti-Amph 1N 278) was able to recognize the ampiphysisin I (1-278) fragment formed by AEP cleavage, but not the full-length ampiphysisin I.
Example 4 specific detection of anti-Amphiphysin I N278 antibody in human brain tissue sections
(1) Brain slice: AD patients and non-AD brain tissue paraffin sections were from the university of Emory dementia research center, USA.
(2) Dewaxing, hydrating brain tablets: placing brain tissue paraffin section into xylene, dewaxing, removing wax, sequentially placing brain section into 95%, 85%, and 75% anhydrous ethanol, standing for 5min, and then placing into ddH2And (4) in O.
(3) Antigen retrieval: the brain slices are put into 100mM sodium citrate (pH 6.0) antigen retrieval solution, retrieved at 92 ℃ for 20min, and after natural cooling, washed with PBS for three times, each time for 5 min.
(4) Incubating with 3% hydrogen peroxide for 10min, blocking endogenous peroxidase to reduce nonspecific background staining, and washing with PBS for 5min for three times.
(5) And (3) sealing: blocking with 3% BSA for 30 min.
(6) Incubating primary antibody: the anti-Amphiphysin I N278 antibody was incubated, diluted with 3% BSA solution at a ratio of 1:500, and incubated overnight at 4 ℃.
(7) Washing primary antibody: PBS was washed three times for 5min each.
The following steps were performed using an immunohistochemical kit from abrin corporation:
(8) add 100. mu.L primary anti-amplifier (solution C in kit), incubate for 10min, wash three times with PBS, 5min each time.
(9) Add 100. mu.L of secondary antibody (D solution in kit), avoid light, incubate for 10min, wash three times with PBS, 5min each time.
(10) Preparing a fresh color developing solution: and (3) preparing the solution E and the solution F in the kit according to the ratio of 3:100, and uniformly mixing for later use.
(11) Color development: adding 100 mu L of fresh color developing solution to the brain slice for incubation, observing under a microscope, washing with deionized water after the brain slice is dyed, and stopping the color developing reaction.
(12) And (5) carrying out hematoxylin nucleus staining.
(13) And (4) transparent, sequentially placing the tissue slices into 75% absolute ethyl alcohol-85% absolute ethyl alcohol-95% absolute ethyl alcohol-xylene, and standing for 5min respectively.
(14) Sealing: and (5) sealing the neutral gum.
(15) And (5) observing through a microscope and taking a picture.
The results are shown in FIG. 3, and Amphiphosin I (1-278) fragments in the brains of AD patients were detectable using the anti-Amphiphosin I N278 antibody.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (7)
1. An antigenic peptide of a fragment of amphoterin 11-278, characterized by: the amino acid sequence is: CLPSPTASPN are provided.
2. An antibody, characterized by: the antigen is prepared by using cross-linked polypeptide obtained by cross-linking Ac-CLPSPTASPN and carrier protein KLH as an antigen; the antibody is a polyclonal antibody.
3. The antibody of claim 2, wherein: the polyclonal antibody is a rabbit polyclonal antibody.
4. The antibody of claim 3, wherein: the preparation method of the rabbit polyclonal antibody comprises the following steps: immunizing rabbit with the cross-linked polypeptide of claim 2 as immunogen, collecting rabbit blood to prepare antiserum, and separating and purifying the antiserum to obtain rabbit polyclonal antibody.
5. Use of the antibody of any one of claims 2-4 for the manufacture of a product for the study of the pathogenesis of alzheimer's disease.
6. A reagent for detecting the degree of cleavage of amphoterin 1, comprising: comprising the antibody of any one of claims 2-4.
7. An early diagnosis reagent for Alzheimer's disease, which is characterized in that: comprising the antibody of any one of claims 2-4.
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