CN101525602B - Polypeptide for preparing anti-PINK1 polyclonal antiserum and application thereof - Google Patents
Polypeptide for preparing anti-PINK1 polyclonal antiserum and application thereof Download PDFInfo
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Abstract
The present invention discloses a polypeptide for preparing anti-PINK1 polyclonal antiserum and application thereof. The polypeptide of the invention is one polypeptide selected from a) and b): a) the polypeptide which is composed of amino acid sequence shown by the sequence 2 in a sequence table; and b) the polypeptide which is derived from the amino acid sequence of sequence 2 in the sequence table through substitution and/or absence and/or adding one or a plurality of amino acids. The invention also discloses the DNA molecule encoding the polypeptide. The anti-PINK1 polyclonal antiserum prepared according to the invention not only can be used for detecting Western blot, but also can be applied to immunohistochemical analysis. The anti-PINK1 polyclonal antiserum prepared according to the invention is of great significance in diagnosing and treating the disease related with PD and PINK1.
Description
Technical field
The present invention relates to prepare sero-fast polypeptide of anti-PINK 1 polyclonal and application thereof.
Background technology
Parkinson's disease (Parkinson ' s Desease PD) are the second largest common nervous system degenerative diseases of senile dementia of continuing.Its clinical characters is static tremor, bradykinesia, and muscular tension strengthens.Typical neuropathology characteristics are that substantia nigra of midbrain dense area dopamine neuron selectivity sex change disappearance and striatum DOPAMINE CONTENT IN RABBIT obviously reduce.In addition, remain and occur the fiber-like eosinophilic inclusion in the neurone endochylema---Louis body (Lewy body), the composition of Lewy body has ubiquitin (ubiquitin), alpha-synapse nucleoprotein (α-Synuclein) and PETEN inductive kinases 1 (Miratul M.K.Muqit such as (PINK1), Patrick M.Abou-Sleiman, AdrianT.Saurin, et al.Altered cleavage and localization of PINK1 to aggresomesin the presence of proteasomal stress Journal of Neurochemistry 2006,98,156-169).So far the gene relevant with PD of Fa Xianing comprises α-Synuclein, LRRK2, UCH-L1, parkin, DJ-1, (Agnes Petit such as PINK1, Toshitaka Kawarai, Erwan Paitel, et al.Wild-type PINK1 Prevents Basal and Induced Neuronal Apoptosis, a ProtectiveEffect Abrogated by Parkinson Disease-related Mutations THE JOURNAL OFBIOLOGICAL CHEMISTRY 2005,280 (40): 34025-34032).Can cause protein denaturation after these transgenations, oxidative stress and mitochondria dysfunction, the change of kinase activity (
B.SchulzUpdate on the pathogenesis of Parkinson ' s disease J Neurol (2008) 255[Suppl5]: 3-7).A lot of laboratories all are devoted to these protein are summed up in the point that same signal path, to illustrate the PD pathogenesis, to find new treatment target spot.
PINK1 (PTEN-induced kinase 1) gene is to find in the research of oncogene PTEN, and therefore names.Found relevant (the Valente E.M. of PINK1 sudden change in 2004 with autosomal recessive inheritance PD, Abou-Sleiman P.M., Caputo V., et al.Hereditary early-onset Parkinson ' sdisease caused by mutations in PINK1 Science 2004,304 (5674): 1158-1160).It is positioned karyomit(e) 1p35-36, in detecting, part PD patient's pathology find that PINK1 albumen is the moiety of Louis body, gene comprises 8 exons, length 1.8Kb, 581 amino acid whose protein (Julia W.Pridgeon PINK1 Protects against Oxidative Stress byPhosphorylating Mitochondrial Chaperone PLoS BIOLOGY 2007,5 (7): 172) encodes.The PINK1 gene can be by the PTEN transcriptional activation, and its transcript is generally expressed, but being rich in the mitochondrial camber expression of organizing, as cardiac muscle, skeletal muscle and testis tissue etc.The directed protein kinase structural domain (156～509 amino-acid residue) that shifts a motif (cleavage site that a plastosome processing peptidase is arranged) and a high conservative between 34 and 35 residues of plastosome that 34 amino acid of predictive PI NK1 coding is formed is with Ca
2+The serine/threonine kinases of/calmodulin family has high homology (Petit, A., Kawaeai, T., Paitel, E., et al.Wild-type PINK1 prevents basal and induced neuronal apoptosis, a protectiveeffect abrogated by Parkinson disease-related mutations.J Biol Chem.2005280 (40): 34025-32.Silvestri, L., Caputo, V., Bellacchio, E., et al.Mitochondrial import and enzymatic activity of PINK1 mutants associated torecessive parkinsonism.Hum.Mol.Genet.2005,14 (22), 3477-3492).After PINK1 is transcribed in nucleus, in cytoplasm, be translated, be imported in the plastosome then, it imports only needs the plastosome of N end to shift motif (Chun Zhou, Yong Huang, Yufang Shao, et al.The kinasedomain of mitochondrial PINK1 faces the cytoplasm PNAS 2008,105 (33): 12022-12027).The position report of PINK1 in cell differs, and thinks that at present it both had been present in plastosome, also may reside in the endochylema.It can be positioned at mitochondrial inner membrance, across mitochondrial outer membrane, makes its kinase domain be exposed to cytoplasm, and this structure has the substrate interaction that is beneficial in itself and the endochylema.The formation of this structure mainly depends on the N end and shifts the membrane spaning domain (94～110 amino-acid residue) of motif back, and this structure of PINK1 mutant of common kinase domain does not change.The C of protein kinase end contains functional motif mostly, can regulate kinase whose catalytic activity, bonded substrate and adjusting albumen, the corresponding construction territory of PINK1 is positioned at the outside (512～581 amino-acid residue) (the Mills RD of C end kinase domain, Sim CH, Mok SS, etal.Biochemical aspects of the neuroprotective mechanism of PTEN-inducedkinase-1 (PINK1) J Neurochem 2008,105 (1): 18-33).PINK1 can be become different forms by proteolytic cleavage in cell, comprise total length (～66kDa) and the segment of different sizes (55kDa, 46kDa) etc., concrete mechanism is unclear.PINK1 can degrade by ubiquitin protein enzyme system system.DJ-1 and PINK1 can interact, and stability (the Beisha Tang of enhancing total length PINK1 protein level, Hui Xiong, PingSun, et al.Association of PINK1 and DJ-1 confers digenicinheritance ofearly-onset Parkinson ' s disease Human Molecular Genetics 2006,15 (11): 1816-1825), and Hsp90 and PINK1 interaction energy strengthen protein stability (the William Lin1 and Un Jung Kang1.Characterization of PINK1 processing of the various spliced bodies of PINK1, stability, and subcellular localization J Neurochem 2008,106 (1): 464-474).
Plastosome is eukaryotic important organoid, and it is the important place that produces ATP, regulating cell existence and dead aspect brought into play vital role.The caused PD symptom of MPTP is because the MPP that self metabolism forms
+Suppressed due to the respiratory chain complex body I, effect (Orth M, the Schapira AH Mitochondria and Degenerative DisordersAmerican Journal ofMedical Genetics Am J Med Gene of plastosome in PD so people begin one's study
PINK1 is the PD Disease-causing gene directly related with plastosome.PINK1 endogenic and the reorganization mark can be present in (Beilina A. in the plastosome, Van Der Brug M., Ahmad R., et al.Mutationsin PTEN-induced putative kinase 1 associated with recessive parkinsonismhave differential effects on protein stability.Proc Natl Acad Sci USA2005,102 (15): 5703-5708).But the fruit bat experiment shows PINK1 sudden change coup injury plastosome, reduce the amount of ATP, make dopamine neuron disappearance (Park J, Lee SB, Lee S, et al.Mitochondrialdysfunction in Drosophila PINK1 mutants is complemented by parkin 2006,441 (7097): 1157-61); Mitochondria dysfunctions such as the mitochondrial membrane potential difference that occurs after the PINK1 silence (Δ Ψ) decline, the decline of ATP output and apoptosis; The RNAi of PINK1 recovers experiment confirm, and PINK1 can alleviate mitochondria dysfunction.In the SH-SY5Y cell, PINK1 can cause unusually because the cell viability due to the plastosome apoptosis reduces; PINK1 lacks the mitochondria dysfunction widely then caused due to the oxidative stress and unusual plastosome form (Alison Wood-Kaczmar, Sonia Gandhi, Zhi Yao, et al.PINK1 IsNecessary for Long Term Survival and Mitochondrial Function in HumanDopaminergic Neurons PLoS BIOLOGY 2008,3 (6): e2455).These results show that the PINK1 defencive function is likely finishing unusually by plastosome form that anti-oxidation stress and neurotoxicity are caused and function.Because the PINK1 sudden change causes the plastosome obstacle to be crossed expression Bcl-2 to improve in the fruit bat, may be the release of apoptotic proteins cytochrome C etc. before suppressing by enhance Bcl-2 so infer the defencive function of PINK1.The PINK1 catastrophe point relevant with PD (as G309D, L347P, W437X etc.) infers thus that mostly all at the protein kinase structural domain performance of PINK1 defencive function mainly depends on the protein kinase structural domain.
More and more evidences shows that PINK1 can interact with other signal protein that participates in PD pathology and plastosome obstacle.So far three substrates of Fa Xianing are respectively E3 ubiquitin ligase Parkin, plastosome kinases HtrA2 and plastosome chaperone Tumor Necrosis Factor Receptors associated protein TRAP1 (claiming heat shock protein(HSP) 75 again).How PINK1 regulates these albumen to keep the survival of cell, still is not very clear so far.
The mutant of finding PINK1 and Parkin in the fruit bat has a lot of similar phenotypes, the myofiber of the disorder of expanding as plastosome, and myocyte's accent is died, the disappearance of the dopamine neuron of specific nerve bunch etc.Parkin crosses expression can improve the symptom that the PINK1 sudden change causes, otherwise then can not, show that PINK1 and Parkin act on same approach and PINK1 regulate cell in the upstream apoptosis (Angela C.Poole, Ruth E.Thomas, LaurieA.Andrews, et al.The PINK1/Parkin pathway regulates mitochondrialmorphology PNAS 2008,105 (5): 1638-1643).And there is document to show that the PINK1/Parkin path can promote chondriokinesis, the plastosome that its mutant causes and the disorder of weave construction cause (Pridgeon J.W. owing to chondriokinesis reduces, Olzmann J.A., Chin L.-S., et al.PINK1Protects against Oxidative Stress by Phosphorylating MitochondrialChaperone TRAP1 PLoS Biol.2007,5 (7): 1494-1503).The PINK1/Parkin path may participate in many signal paths, as IKK/NF κ B, and JNK and PI3-kinase/Akt, this awaits further to studies have shown that.
The concrete function of TRAP1 is still unclear so far.Reported in literature disturbs the release that can increase the cytochrome C that oxidative stress causes behind the TRAP1 and the apoptosis of cell, cross generation (Hua G., Zhang Q.and Fan Z.Heat shock protein 75 (TRAP1) the antagonizes reactive oxygen species generation and protects cells fromgranzyme M-mediated apoptosis J Biol Chem. 282 (28): 20553-20560) that then can suppress the ROS that cytotoxin causes after expressing.TRAP1 is considered to one of substrate of PINK1, external it can be directly by the PINK1 phosphorylation.Both can be positioned mitochondrial membrane space and mitochondrial inner membrane altogether.After experiment showed, the oxidated stress stimulation of cell, the phosphorylation level of TRAP1 improves, and can improve its phosphorylation level and cross expression PINK1.This has pointed out TRAP1 may participate in the cytoprotective function of PINK1.Mechanism such as its phosphorylation site still need further to inquire into.
HtrA2 is a kind of serine protease (claiming Omi again), and under apoptotic stimulus, it can be discharged into from plastosome in the endochylema, combines (IAPs) with the apoptotic proteins supressor, thereby suppresses the generation of apoptosis.HtrA2 and PINK1 all can be positioned at mitochondrial membrane space (Strauss K.M., Martins L.M., Plun-Favreau H., et al.Loss of function mutations in the gene encoding Omi/HtrA2 in Parkinson ' sdisease Hum Mol Genet 2005,14 (15): 2099-2111).Discover that both can mutually combine and form mixture in plastosome, and this combination has promoted the Ser-142 site of HtrA2 stress be activated kinase p 38 γ phosphorylation (Plun-Favreau H., Klupsch K., Moisoi N., et al.Themitochondrial protease HtrA2 is regulated by Parkinson ' s disease-associatedkinase PINK1 Nat Cell Biol.9 (11): 1243-1252), and this phosphorylation form can suppress the apoptosis that neurotoxicity causes.Its concrete mechanism is still among research.
The research report is arranged in addition, and DJ-1 and PINK1 can interact, and strengthen the stability of total length PINK1 protein level, resist the apoptosis of cytotoxicity mediation jointly.
In sum, in PD PINK1 bring into play its defencive function and plastosome closely related.The adjusting of PINK1, substrate, how each signal paths that action site and PINK1 participate in interacts, and these problems still remain further to be discovered.
Summary of the invention
The purpose of this invention is to provide sero-fast polypeptide of a kind of preparation anti-PINK 1 polyclonal and application thereof.
The sero-fast polypeptide of preparation anti-PINK 1 polyclonal provided by the present invention, the kinase domain of called after PINK1, be following a) or b) polypeptide:
A) polypeptide that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;
B) in sequence table the aminoacid sequence of sequence 2 through replacing and/or disappearance and/or add one or several amino acid by a) polypeptides derived.
In order to make polypeptide a) be convenient to purifying, can in by sequence table, connect label as shown in table 1 by the N-terminal or the C-terminal of the polypeptide shown in the sequence 2.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep- |
8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned b) but in the polypeptide synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of the polypeptide above-mentioned b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The encoding gene of described polypeptide also belongs to protection scope of the present invention.
The encoding gene of described polypeptide is following 1) or 2) or 3):
1) its nucleotide sequence is a sequence 1 in the sequence table;
2) under stringent condition with 1) the dna fragmentation hybridization that limits and the dna molecular of coding said polypeptide;
3) with 1) or 2) dna molecular have the homology more than 90% and the dna molecular of coding said polypeptide.
Dna molecular in the described step 3) is with 1) dna molecular homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 68 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The described dna molecular total length that increases or arbitrary segmental primer are to also belonging to protection scope of the present invention.
The recombinant vectors, transgenic cell line and the reorganization bacterium that contain above-mentioned described dna molecular also belong to protection scope of the present invention.
Described polypeptide can be used to prepare the anti-PINK 1 polyclonal antiserum(antisera).
This anti-PINK 1 polyclonal antiserum(antisera) is to be the polyclonal antiserum of antigen-immunized animal acquisition with described polypeptide.Described polypeptide forms inclusion body.
Described anti-PINK 1 polyclonal antiserum(antisera) also belongs to protection scope of the present invention.
Experimental results show that the kinase domain immunity new zealand white rabbit with PINK1, successfully obtained the anti-PINK 1 polyclonal antiserum(antisera), Dot blot demonstration is tired and can be reached 1: 3000.The Western blot that tentatively saltouts behind the purifying shows that this anti-PINK 1 polyclonal antiserum(antisera) can discern recombinant human PINK1, also can discern the PINK1 in rat cerebral even slurry, mouse brain homogenate, normal brain homogenate and the people's meningioma brain homogenate.This shows that the anti-PINK 1 polyclonal antiserum(antisera) that obtains is specific to people PINK1, and can cross reaction take place with rat, mouse.People PINK1 and rat, mouse PINK1 aminoacid sequence have high homology, and the part of this antibody recognition is people, rat, the common fragment of mouse PINK1.Illustrate that with the cross reaction of rat, mouse PINK1 this anti-PINK 1 polyclonal antiserum(antisera) not only can be applied to animal PD Study of model, but also can be applicable to the early diagnosis of human nerve's degenerative disease such as Parkinson's disease and Louis body relative disease, significant to further illustrating the proteinic physiological function of PINK1 and its effect in the PD pathogenic process.Carried out the immunohistochemistry research of rat brain slice with the anti-PINK 1 polyclonal antiserum(antisera), the result shows that the PINK1 immunity substance is distributed in the extensive position of full brain, as olfactory bulb, cortex, hippocampus, striatum, black substance, aqueduct of midbrain and rubrum etc., for its Subcellular Localization of different cells also difference to some extent.Therefore described, the anti-PINK 1 polyclonal antiserum(antisera) of the present invention's preparation can be used for the detection of Western blot, also can be applied to the analysis of immunohistochemical methods, this PD and with the diagnosis of PINK1 relative disease and treatment in significant.
Description of drawings
Fig. 1 identifies with antigen for immunity.
Fig. 2 detects the anti-PINK 1 polyclonal antiserum(antisera) of tiring for Dot blot.
Fig. 3 detects sero-fast susceptibility of anti-PINK 1 polyclonal and specificity for Western blot.
PINK1 is proteic in anti-PINK 1 polyclonal antiserum(antisera) and the homogenate of people's meningioma combines Fig. 4 for western blot detects.
Fig. 5 is the distribution of PINK1 immunologic active material at olfactory bulb.
Fig. 6 is the distribution of PINK1 immunologic active material at cortex.
Fig. 7 is the distribution of PINK1 immunologic active material hippocampus.
Fig. 8 is the distribution of PINK1 immunologic active material at black substance.
Fig. 9 is the distribution of PINK1 immunologic active material at cerebellum.
Figure 10 is the distribution of PINK1 immunologic active material at cerebral aqueduct.
Figure 11 is the distribution of PINK1 immunologic active material rubrum.
Figure 12 is the distribution of PINK1 immunologic active material at cortex.
Figure 13 is the distribution of PINK1 immunologic active material at cerebral peduncle.
Embodiment
Among the following embodiment if no special instructions method therefor be ordinary method, agents useful for same all can obtain from commercial channels.
One, animal immune expression and the purifying of recombinant human PINK1
The kinase domain of people PINK1 (kinase domain) is made up of 425 amino acid, and its aminoacid sequence is shown in sequence in the sequence table 1.From normal people's embryo and brain, extract total RNA, reverse transcription becomes cDNA, DNA with the kinase domain of primer 1 (5`CCC AAG TTT GTT GTG ACC G3`) and primer 2 (3`TGT AAT TTC CCA CTC CCGTA5`) pcr amplification coding PINK1, the DNA of the kinase domain of the coding people PINK1 that pcr amplification is obtained inserts the pET-28a carrier, constitutes recombinant plasmid pET-28a-PINK1.PET-28a-PINK1 is converted into BL21 (DE3) competent cell, (every liter contains the 16g peptone to get single bacterium colony implantation 10mL 2 * YTA-amp liquid nutrient medium, the 10g yeast extract, 5g NaCl and 0.1g Ampicillin Trihydrate), be transferred in the fresh 2 * YTA-amp substratum of 1L after 37 ℃ of pre-cultivations of overnight shaking, 37 ℃ of shaking culture 3h, add IPTG (final concentration 0.1mmol/L), collect thalline after continuing to cultivate 4h, with ice-cold 0.01mol/L PBS suspension bacterium, ultrasonic abundant fragmentation, add Triton X-100 (final concentration is 1%) in the bacterium liquid of fragmentation, room temperature is placed 30min, centrifugal (4 ℃, 15000g) 1h abandons clean supernatant.The aqueous solution suspension that contains 4%triton X-100 with 30ml precipitates 37 ℃ of concussions (230 rev/mins) 30 minutes.It is centrifugal that (4 ℃, 15000g) 1h abandons supernatant.Repeat the solution washing process of 1 4%Triton X-100 again.With deionized water washing precipitation 2 times (each 30 minutes), abandon clean supernatant.5ml is added into precipitation with the 6mol/L Guanidinium hydrochloride, fully suspends.It is centrifugal that (4 ℃, 15000g) 1h is transferred to supernatant in the dialysis tubing, with PBS damping fluid dialysed overnight (2 PBS damping fluids of middle replacing).To there be cotton-shaped dialysis tubing content of separating out to be transferred in the 15ml centrifuge tube, use ultrasonication protein liquid 20 minutes, obtain recombinant human PINK1 protein.Proteinic quantitative employing BCA method is a standard with bovine serum albumin (BSA).Purity of protein is identified and is adopted SDS-PAGE.
SDS-PAGE as shown in Figure 1, recombinant human PINK1 efficiently expresses the formation inclusion body in intestinal bacteria.Handle inclusion body with the 6M Guanidinium hydrochloride, the recombinant human PINK-1 of acquisition after dialysis and ultrasonic wave dissolving again, relative molecular mass is about the single band of 40kd.
Among Figure 1A, swimming lane 1: the supernatant liquor after bacteria breaking liquid is centrifugal; Swimming lane 2: the precipitation resuspending liquid after bacteria breaking liquid is centrifugal.
Among Figure 1B, swimming lane 1: common standard molecular weight Marker; Swimming lane 2: PINK1 100ng behind the purifying; Swimming lane 3: PINK1 140ng behind the purifying; Swimming lane 4: PINK1 180ng behind the purifying; Swimming lane 5: PINK1 220ng behind the purifying; Swimming lane 6: PINK1 260ng behind the purifying; Swimming lane 7: PINK1 300ng behind the purifying; Swimming lane 8: PINK1 340ng behind the purifying; Swimming lane 9: color standard molecular weight Marker.
Two, immune animal
250 μ g (250 μ l) recombinant human PINK1 is mixed in the complete freund adjuvant of 250 μ l (Sigma company), ultrasonicly in the water reach complete emulsification degree.By 125 μ g/ antigen dose only, respectively immune 2 New Zealand's large ear rabbits (back subcutaneous injection, per injection one site).Week about reinforced immunological once, totally 3 times.Use freund 's incomplete adjuvant (Sigma company) mixing and emulsifying during reinforced immunological, method, antigen amount are with for the first time.Blood sampling in three days behind the reinforced immunological for the third time, Dot blot detects and tires.
Dot blot detection method is as follows:
With 0.1% gelatin bag by nitrocellulose membrane (NC film).After the seasoning, on the NC film, drip recombinant human PINK1 3000,1500,300 and 150ng respectively, 80 ℃ of crosslinked 30m of Paraformaldehyde 96,4 ℃ down with preliminary purification after antiserum(antisera) (being diluted to volume fraction 1: 3000) overnight incubation jointly.Two anti-(available from companies of China fir Golden Bridge in Beijing) are biotinylation goat anti-rabbit igg (being diluted to volume fraction 1: 1200), under the room temperature with NC film reaction 3h after again with horseradish peroxidase (company of China fir Golden Bridge in Beijing, be diluted to volume fraction 1: 1200) hatch 3h jointly, the DAB method develops the color.
Dot blot analytical results such as Fig. 2 show, with the new zealand white rabbit that the kinase domain immunity of PINK1 is crossed, after the antiserum(antisera) that is obtained was diluted to 1: 3000, still can discern the PINK1 in the MN9D cell.
Tiring reaches 1: 3000, blood sampling, separation of serum ,-20 ℃ of preservations.
Each batch adopted homogeneity serum mix twice of 38-40% ammonium sulfate precipitation; For the second time centrifugal back gained precipitation is resuspended with 0.01M PBS, puts into the semi-permeable membranes bag, with the semi-permeable membranes bag at the 50mMTris-Hcl that contains 0.15mol/L NaCl (PH7.4)) in 4 ℃ of 3h, change liquid once, spend the night; Collect and obtain the anti-PINK 1 polyclonal antiserum(antisera), add 0.01% sodium azide, packing ,-20 ℃ of preservations.
Three, Western blot analyzes
Get normal rat cerebral tissue, mouse brain tissue, normal brain tissue and people's meningioma are made homogenate respectively, method is as follows: 6000g, 4 ℃ of centrifugal 30min obtain supernatant, add 1: 4 (Beijing Puli comes company) back 95 ℃ of sex change 5min of 5 * loading, obtain rat cerebral tissue, mouse brain tissue, normal brain tissue and the homogenate of people's meningioma respectively; Separate the back rat cerebral even slurry with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein transduction in the gel is moved to PVDF membrane (pvdf membrane) go up (half-dried shifting method); With 5% (quality percentage composition) skimmed milk sealing pvdf membrane 1h, again the anti-PINK 1 polyclonal antiserum(antisera) behind film and the preliminary purification (be diluted to volume fraction 1: 5000, and wherein contained 1% milk) is spent the night for 4 ℃; TBST washes 3 times, 10m/ time; The goat anti-rabbit igg of horseradish peroxidase-labeled (be diluted to volume fraction 1: 10000, and wherein contained 1% milk) is hatched 1h jointly; TBST washes 3 times, 10m/ time; With ECL method colour developing (ECL reagent is available from Beijing Zhong Shan company).
Western blot analytical results is shown in Fig. 3 and 4, show that this anti-PINK 1 polyclonal antiserum(antisera) was diluted to volume fraction at 1: 7500 o'clock, both can discern recombinant human PINK1, also can discern rat cerebral even slurry, mouse brain homogenate, PINK1 in normal brain tissue and the people's meningioma brain homogenate, the relative molecular mass of institute's identification of protein is about 40kd, but has the kind difference.
Among Fig. 3, A is the sero-fast susceptibility of anti-PINK 1 polyclonal, the kinase domain of 1-12:PINK1 (130ng-20ng);
B detects anti-PINK 1 polyclonal sero-fast specificity-1 for Western blot, a left side is detected for Western blot, it is right that (absorption experiment is to spend the night for 4 ℃ with q.s antigen and gained antiserum(antisera) thorough mixing for absorption experiment, afterwards mixed solution is served as one and resists western blot test sample) 1: normal people's embryo and brain homogenate 10ug:2, mouse brain homogenate 10ug 3: rat cerebral even slurry 10ug, 4: reorganization PINK1 100ng.
C detects anti-PINK 1 polyclonal sero-fast specificity-2 for Western blot; A left side is detected for Western blot, and the right side is an absorption experiment, and 1: people's meningioma homogenate 10ug, 2: mouse brain homogenate 10ug, 3: rat cerebral even slurry 10ug, 4: reorganization PINK1 100ng.
Figure B and C illustrated together, PINK1 reaches in the meningioma molecular weight difference to some extent in normal brain.
Among Fig. 4,1-10: anti-PINK 1 polyclonal antiserum(antisera) (dilution in 1: 1000, dilution in 1: 2000, dilution in 1: 3000, dilution in 1: 4000, dilution in 1: 5000, dilution in 1: 6000, dilution in 1: 7000, dilution in 1: 8000, dilution in 1: 9000, dilution in 1: 10000); 1-10 swimming lane applied sample amount is people's meningioma homogenate 25ug/ swimming lane.
Four, immunohistochemical analysis
Normal rat is got brain after 4% (quality percentage composition) Paraformaldehyde 96 (PFA) perfusion fixation, fix 48 hours after 30% sucrose (disposing with 4%PFA), freezing microtome section (40 μ m/ sheet).Section is through PBST (wherein containing 0.03% (volumn concentration) TritonX-100), 1N hydrochloric acid, 3% (volumn concentration) H
2O
2After the processing of 5% (volumn concentration) milk, spend the night with the anti-PINK 1 polyclonal antiserum(antisera) (being diluted to volume fraction 1: 5000) behind the preliminary purification, two anti-(biotinylated goat anti-rabbit iggs, be diluted to volume fraction 1: 1000) after room temperature 3h and horseradish peroxidase (being diluted to volume fraction 1: 1000) the room temperature 3h reaction, the colour developing of DAB method.
DAB Faxian look result such as Fig. 5 be to shown in Figure 13, carries out the immunohistochemical study of rat brain slice with the anti-PINK 1 polyclonal antiserum(antisera) behind the preliminary purification, in visible stronger PINK1 immunocompetence such as olfactory bulb, cortex, hippocampus, striatum, black substance and rubrum.
Sequence table
＜110〉Capital University of Medical Sciences
＜120〉preparation sero-fast polypeptide of anti-PINK 1 polyclonal and application thereof
<160>2
<210>1
<211>1278
<212>DNA
<213>
<400>1
ctgatagggc?agtccattgg?taagggctgc?agtgctgctg?tgtatgaagc?caccatgcct 60
acattgcccc?agaacctgga?ggtgacaaag?agcaccgggt?tgcttccagg?gagaggccca 120
ggtaccagtg?caccaggaga?agggcaggag?cgagctccgg?gggcccctgc?cttccccttg 180
gccatcaaga?tgatgtggaa?catctcggca?ggttcctcca?gcgaagccat?cttgaacaca 240
atgagccagg?agctggtccc?agcgagccga?gtggccttgg?ctggggagta?tggagcagtc 300
acttacagaa?aatccaagag?aggtcccaag?caactagccc?ctcaccccaa?catcatccgg 360
gttctccgcg?ccttcacctc?ttccgtgccg?ctgctgccag?gggccctggt?cgactaccct 420
gatgtgctgc?cctcacgcct?ccaccctgaa?ggcctgggcc?atggccggac?gctgttcctc 480
gttatgaaga?actatccctg?taccctgcgc?cagtaccttt?gtgtgaacac?acccagcccc 540
cgcctcgccg?ccatgatgct?gctgcagctg?ctggaaggcg?tggaccatct?ggttcaacag 600
ggcatcgcgc?acagagacct?gaaatccgac?aacatccttg?tggagctgga?cccagacggc 660
tgcccctggc?tggtgatcgc?agattttggc?tgctgcctgg?ctgatgagag?catcggcctg 720
cagttgccct?tcagcagctg?gtacgtggat?cggggcggaa?acggctgtct?gatggcccca 780
gaggtgtcca?cggcccgtcc?tggccccagg?gcagtgattg?actacagcaa?ggctgatgcc 840
tgggcagtgg?gagccatcgc?ctatgaaatc?ttcgggcttg?tcaatccctt?ctacggccag 900
ggcaaggccc?accttgaaag?ccgcagctac?caagaggctc?agctacctgc?actgcccgag 960
tcagtgcctc?cagacgtgag?acagttggtg?agggcactgc?tccagcgaga?ggccagcaag 1020
agaccatctg?cccgagtagc?cgcaaatgtg?cttcatctaa?gcctctgggg?tgaacatatt 1080
ctagccctga?agaatctgaa?gttagacaag?atggttggct?ggctcctcca?acaatcggcc 1140
gccactttgt?tggccaacag?gctcacagag?aagtgttgtg?tggaaacaaa?aatgaagatg 1200
ctctttctgg?ctaacctgga?gtgtgaaacg?ctctgccagg?cagccctcct?cctctgctca 1260
tggagggcag?ccctgtga 1278
<210>2
<211>425
<212>PRT
<213>
<400>2
Leu?Ile?Gly?Gln?Ser?Ile?Gly?Lys?Gly?Cys?Ser?Ala?Ala?Val?Tyr?Glu
1 5 10 15
Ala?Thr?Met?Pro?Thr?Leu?Pro?Gln?Asn?Leu?Glu?Val?Thr?Lys?Ser?Thr
20 25 30
Gly?Leu?Leu?Pro?Gly?Arg?Gly?Pro?Gly?Thr?Ser?Ala?Pro?Gly?Glu?Gly
35 40 45
Gln?Glu?Arg?Ala?Pro?Gly?Ala?Pro?Ala?Phe?Pro?Leu?Ala?Ile?Lys?Met
50 55 60
Met?Trp?Asn?Ile?Ser?Ala?Gly?Ser?Ser?Ser?Glu?Ala?Ile?Leu?Asn?Thr
65 70 75 80
Met?Ser?Gln?Glu?Leu?Val?Pro?Ala?Ser?Arg?Val?Ala?Leu?Ala?Gly?Glu
85 90 95
Tyr?Gly?Ala?Val?Thr?Tyr?Arg?Lys?Ser?Lys?Arg?Gly?Pro?Lys?Gln?Leu
100 105 110
Ala?Pro?His?Pro?Asn?Ile?Ile?Arg?Val?Leu?Arg?Ala?Phe?Thr?Ser?Ser
115 120 125
Val?Pro?Leu?Leu?Pro?Gly?Ala?Leu?Val?Asp?Tyr?Pro?Asp?Val?Leu?Pro
130 135 140
Ser?Arg?Leu?His?Pro?Glu?Gly?Leu?Gly?His?Gly?Arg?Thr?Leu?Phe?Leu
145 150 155 160
Val?Met?Lys?Asn?Tyr?Pro?Cys?Thr?Leu?Arg?Gln?Tyr?Leu?Cys?Val?Asn
165 170 175
Thr?Pro?Ser?Pro?Arg?Leu?Ala?Ala?Met?Met?Leu?Leu?Gln?Leu?Leu?Glu
180 185 190
Gly?Val?Asp?His?Leu?Val?Gln?Gln?Gly?Ile?Ala?His?Arg?Asp?Leu?Lys
195 200 205
Ser?Asp?Asn?Ile?Leu?Val?Glu?Leu?Asp?Pro?Asp?Gly?Cys?Pro?Trp?Leu
210 215 220
Val?Ile?Ala?Asp?Phe?Gly?Cys?Cys?Leu?Ala?Asp?Glu?Ser?Ile?Gly?Leu
225 230 235 240
Gln?Leu?Pro?Phe?Ser?Ser?Trp?Tyr?Val?Asp?Arg?Gly?Gly?Asn?Gly?Cys
245 250 255
Leu?Met?Ala?Pro?Glu?Val?Ser?Thr?Ala?Arg?Pro?Gly?Pro?Arg?Ala?Val
260 265 270
Ile?Asp?Tyr?Ser?Lys?Ala?Asp?Ala?Trp?Ala?Val?Gly?Ala?Ile?Ala?Tyr
275 280 285
Glu?Ile?Phe?Gly?Leu?Val?Asn?Pro?Phe?Tyr?Gly?Gln?Gly?Lys?Ala?His
290 295 300
Leu?Glu?Ser?Arg?Ser?Tyr?Gln?Glu?Ala?Gln?Leu?Pro?Ala?Leu?Pro?Glu
305 310 315 320
Ser?Val?Pro?Pro?Asp?Val?Arg?Gln?Leu?Val?Arg?Ala?Leu?Leu?Gln?Arg
325 330 335
Glu?Ala?Ser?Lys?Arg?Pro?Ser?Ala?Arg?Val?Ala?Ala?Asn?Val?Leu?His
340 345 350
Leu?Ser?Leu?Trp?Gly?Glu?His?Ile?Leu?Ala?Leu?Lys?Asn?Leu?Lys?Leu
355 360 365
Asp?Lys?Met?Val?Gly?Trp?Leu?Leu?Gln?Gln?Ser?Ala?Ala?Thr?Leu?Leu
370 375 380
Ala?Asn?Arg?Leu?Thr?Glu?Lys?Cys?Cys?Val?Glu?Thr?Lys?Met?Lys?Met
385 390 395 400
Leu?Phe?Leu?Ala?Asn?Leu?Glu?Cys?Glu?Thr?Leu?Cys?Gln?Ala?Ala?Leu
405 410 415
Leu?Leu?Cys?Ser?Trp?Arg?Ala?Ala?Leu
420 425
Claims (9)
1. a peptide species, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 2.
2. the dna molecular of coding claim 1 described polypeptide.
3. dna molecular according to claim 2 is characterized in that: the nucleotide sequence of described dna molecular is a sequence 1 in the sequence table.
4. the recombinant expression vector that contains claim 2 or 3 described dna moleculars.
5. the transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described dna moleculars.
6. the application of the described polypeptide of claim 1 in preparation anti-PINK 1 polyclonal antiserum(antisera).
7. claim 2 or the 3 described dna moleculars application in preparation anti-PINK 1 polyclonal antiserum(antisera).
8. an anti-PINK 1 polyclonal antiserum(antisera) is to be the antiserum(antisera) of antigen-immunized animal acquisition with the described polypeptide of claim 1.
9. antiserum(antisera) according to claim 8 is characterized in that: the described polypeptide of claim 1 is an inclusion body.
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Families Citing this family (3)
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| CN108290930A (en) * | 2015-05-01 | 2018-07-17 | 胡文聪 | PINK1 C-terminals Domain Polypeptide and its method use for cancer treatment |
| WO2020000472A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Recombinant vector for promoting overexpression of pink1 protein and construction method thereof |
| CN116284416A (en) * | 2023-01-09 | 2023-06-23 | 暨南大学 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Gandhi,S.,et al..Homo sapiens PTEN induced putative kinase 1 (PINK1), nuclear gene encoding mitochondrial protein, mRNA.《Genbank：NM_032409》.2009,1-5. * |
| Scheele S,et al..The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function.《BMC Genomics》.2007,第8卷(第74期),1-13. * |
| 范春香等.帕金森病相关蛋白PINK1和αO突触核蛋白相互作用研究.《中国生物工程杂志》.2008,第28卷(第12期),7-11. * |
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| CN101525602A (en) | 2009-09-09 |
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