CN106167793A - A kind of people's GTPBP9 polypeptide and preparation method for antibody thereof - Google Patents

Abstract

The invention discloses people's GTPBP9 polypeptide and the preparation method of antibody of a kind of N terminal specific, belong to the biological product of the experiment in vitro being characterized with antibody.The aminoacid sequence of people's GTPBP9 polypeptide of N terminal specific is: RAFEDDDITHVEGS.Anti-human GTPBP9 polypeptide antibody is prepared as follows: (1) people GTPBP9 Characterization of antigenic epitopes；(2) people GTPBP9N end Peptide systhesis；(3) synthesis polypeptide cross-links with carrier protein；(4) rabbit anti-human GTPBP9 polypeptide antibody is prepared；(5) collection, isolated contain the serum of antibody, antibody purification, i.e. obtain anti-human GTPBP9 polypeptide antibody.The anti-human GTPBP9 synthesis of polypeptide antibody titer of N terminal specific prepared by the present invention is high, affinity is strong, specificity good, with natural human GTPBP9, specific binding reaction can occur；Preparation cost is low；Antibody after purification may be used for immunoblotting and SABC.This antibody is the basic research of GTPBP9 albumen, and such as to the characteristic of GTPBP9, function, express spectra and containing quantitative analysis, and the research of relevant disease provides an important instrument.

Description

A kind of people's GTPBP9 polypeptide and preparation method for antibody thereof

1. technical field

The present invention relates to a peptide species and preparation method for antibody thereof, this antibody is mainly used in native protein and resists Former detection.

2. background technology

1) GTPBP9 function: GTPBP9 is a kind of new NTP hydrolysis in Obg family GTP hydrolytic enzyme Enzyme (Koller-Eichhon et al., J.Biol.Chem.2007 282:19928-19937).Research display GTPBP9 can more effectively combine and hydrolysising ATP rather than GTP molecule.This type of in different population Albumen has the function of two kinds of hydrolytic enzyme of ATP and GTP the most simultaneously.By X-ray crystal structure analysis, And with AMPPCP, the ATP molecular mimics of a kind of non-hydrolysable is as analyzing substrate, it has been found that make The reason becoming this kind of NTP hydrolytic enzyme nucleotide binding specificity to change is all relevant Obg families A hydrophobic amino acid groups in GTP hydrolytic enzyme is substituted by glutamine molecule, thus causes these Hydrolytic enzyme is higher than the combination with GTP molecule with the joint efficiency of ATP molecule.Same replacement can also Make Ras sample GTP inactivation of hydrolytic enzyme.The both sides in the G region, center of GTPBP9 molecule are respectively by a spiral shell The TGS region (ThrRS, GTPase, SpoT) of spin line coil structures and a Unknown Function is comprised. According to these architectural features, people predict that GTPBP9 is a kind of regulation albumen, it and the effect egg in downstream White interact, and be combined the conformational change of generation with ADP and ATP by it and exercise its regulatory function. 2) GTPBP9 antibody product information: through retrieval, in addition to my Products, market does not has other Anti-GTPBP9 polyclonal antibody.

3) application of GTPBP9 antibody:

It is thin that the data display GTPBP9 that a group is published on NAS's magazine has suppression human body The effect of born of the same parents' anti-oxidation function.GTPBP9 gene is pounded out can increase human body cell to oxidant, including uncle Butylhydroperoxide thing (tBH) and the resistivity of diamides, and do not affect the propagation of cell, base Plinth apoptosis and sensitivity (Zhang et al., the Proc Natl Acad Sci USA. to other cytotoxic factor 2009 Sep 8；106 (36): 15356-61).On the contrary, overexpression GTPBP9 can increase cell to tBH Sensitivity with oxides such as diamides.After peroxide treatment, the people of GTPBP9 gene knockout The activating oxide horizontal assembled in somatic cell is significantly lower than the level in blast cell, and its reproducibility The utilization rate of glutathion is significantly reduced.This research group also finds that GTPBP9 is in non-transcribed level The inhibitory action to anti-oxidation function of upper generation.GTPBP9 gene pounds out descendant's somatic cell antioxidation merit The result that can strengthen shows that this target protein will become the medicine of very promising Antioxidation Treatment in the near future Thing target spot.

3. summary of the invention

The invention provides a kind of GTPBP9 polypeptide, its sequence is: RAFEDDDITHVEGS.Many with this Anti-GTPBP9 antibody prepared by peptide can be in immunoblotting (Western blot) and SABC (IHC) Natural GTPBP9 albumen in specific recognition tissue or cell in analysis.

Anti-GTPBP9 antibody through the following steps that obtain:

Step one: the analysis of peptide sequence and design: utilize the DNAstar software amino to GTPBP9 albumen Acid sequence carries out Characterization of antigenic epitopes, mainly assesses hydrophilic, antigenicity, surface probability, flex region Deng index, prepare the practical experience of antibody in conjunction with the past, finally determine GTPBP9 protein 12 5-138 14, position aminoacid is as synthesis polypeptid acid sequence, and sequence is RAFEDDDITHVEGS.

Step 2: Peptide systhesis and crosslinking: use ACT396 fully-automatic multi-channel Peptide synthesizer synthesis purpose Polypeptide, and use mass spectrum to identify；For strengthening the antigenicity of polypeptide, by GTPBP9 polypeptide and carrier Albumen KLH uses Sulfo-SMCC cross-linking method to cross-link.

Step 3: prepared by polypeptide immune and antiserum: the GTPBP9-KLH after crosslinking is mixed with Freund adjuvant Close emulsifying, carry out intradermal injection immunity, and booster immunization repeatedly at new zealand rabbit back, survey to taking blood examination Antibody titer reaches to stop immunity during standard.

Step 4: antibody purification: after experimental rabbit antibody titer reaches standard, uses heart extracting blood, separates anti-blood Clearly, after using Protein A purification whole antibody, use peptide affinity purification further, obtain target antibody.

Anti-GTPBP9 antibody through the following steps that identify:

Step one: immunoblotting: resisted as one by the GTPBP9 antibody obtained, uses the Diagnosis of Sghistosomiasis of standard Mark method, confirms that this antibody can be used for exempting from the natural GTPBP9 protein-interacting after degeneration Epidemic disease blot analysis.

Step 2: SABC: resisted as one by the GTPBP9 antibody obtained, uses the immune group of standard Change method, is used for detecting organization chip (9 kinds of tissues), confirm this antibody can with there is natural knot The GTPBP9 protein-interacting of structure, can be used for immunohistochemical assay.

4. accompanying drawing explanation

Fig. 1 is western blot figure (Western blot)

Fig. 2 is SABC (IHC) figure

5. detailed description of the invention

1. the analysis of peptide sequence and design

Utilize DNAstar software that the aminoacid sequence of GTPBP9 albumen is carried out Characterization of antigenic epitopes, mainly comment Estimate the indexes such as hydrophilic, antigenicity, surface probability, flex region, prepare the reality of antibody in conjunction with the past Border experience, it is considered to amino acid structure complexity, oxidizable degree, synthesize difficulty, aminoacid classification and Distributions etc., finally determine that 14, position of GTPBP9 protein 12 5-138 aminoacid is as synthesis polypeptide amino Acid sequence, sequence is RAFEDDDITHVEGS.Meanwhile, for ensureing later stage polypeptide crosslinking carrier protein And peptide affinity purification, increasing a cysteine C at N-terminal, final peptide sequence to be synthesized is C-RAFEDDDITHVEGS。

2. Peptide systhesis and crosslinking

Use ACT396 fully-automatic multi-channel Peptide synthesizer, many according to the Automatic Program synthesis purpose woven Peptide, is dissolved in 50% acetonitrile by the polypeptide after synthesis, uses mass spectrograph to identify, confirmation is obtained many Polypeptide for the purpose of peptide.Sulfo-SMCC is used to be entered with synthesis polypeptide by carrier protein KLH as cross-linking agent Row crosslinking: take 10mg KLH and be dissolved in 0.5ml ultra-pure water；Take 3mg sulfo-SMCC and be dissolved in 0.5ml In ultra-pure water, adjust pH value about 7 with 3M NaOH.In the case of mixing, sulfo-SMCC solution Dropwise it is slowly added in KLH solution, under room temperature, rotates mixing reactant 30min.By completely reacted Sulfo-SMCC/KLH mixed liquor is loaded to equilibrated with level pad (0.05M PB, pH6.0) in advance In the Sephadex G25 post of 30min, collect light grey eluent, the sulfo-SMCC/KLH i.e. activated Solution.Dissolve with 200ul PBS (pH7.3) and treat 2mg cross linking polypeptides, 0.2 volume Sulfo-SMCC/KLH complex solution joins in polypeptide solution, adjust pH value to 7.3, rocked at room temperature 4 Hour, after-70 freezings, with standby after freeze dryer lyophilizing 24 hours.By the detection crosslinking of Ellman method Front and back polypeptide sulfydryl determines polypeptide cross-linking efficiency.

3. prepared by polypeptide immune and antiserum

The KLH-polypeptide 400 μ g cross-linked is dissolved in 400 μ l phosphate buffers (0.01M PBS), adds Equal-volume Freund's complete adjuvant fully emulsified (being as the criterion to indiffusion in water).Use 3 months ages of rabbit, The healthy new zealand rabbit of body weight 1.75-2.25Kg carries out immunity, carries out back intradermal injection immunity, at least Inject more than 20.After first immunisation 3 weeks, 300 μ g polypeptide are dissolved in 300 μ l phosphate buffers In (0.01M PBS), fully emulsified with the incomplete Freund's adjuvant of equivalent after carry out intradermal immunization, make For first time booster immunization, it is desirable to back intradermal injection immunity, at least to inject more than 15.Second After secondary immune 3 weeks, carry out second time booster immunization, method and requirement with booster immunization for the first time.1 week After, use auricular vein trace to take blood, with uncrosslinked synthesis polypeptide coated elisa plate, indirect ELISA Method detection immune serum titer.Repeat booster immunization and titration, until serum titer reaches 1: 60000 Above, using heart extracting blood, standard method obtains antiserum.

4. antibody affinity purification

(1), TIgG purification: the Protein-A Sepharose suspension 10ml of 50% is added to pipettor In 30ml chromatographic column, removing top lid and bottom cap, the bed volume after liquid flows out is 5ml, then uses 25ml deionized water rinsing 3 times.Take out corresponding serum 10ml, after mixing with 2ml PBS, add 30ml In chromatographic column, on impeller, room temperature (20-25 DEG C) DL 1 hour, allows blood serum sample flow out.Again Purify washing liquid with 15ml and wash chromatographic column 3 times, add 10ml eluent and carry out eluting.

(2), peptide affinity purification: in chromatographic column add 1ml Sulfo-link gel suspension (0.5ml gel), Treat dried liquid stream in post, rinse chromatographic column with 4ml coupling buffer.Dissolve with 1ml coupling buffer and close The RBPMS polypeptide become, and adds chromatographic column, adds 1ml coupling buffer in chromatographic column, room The reverse mixing of temperature 1 hour.Rinse chromatographic column with 6ml coupling buffer, be subsequently adding 3ml confining liquid, Room temperature mixes 1 hour.Rinse chromatographic column 3 times, in chromatographic column, then add 6ml IgG and 3ml PBS, The reverse mixing of room temperature 1 hour.Rinse chromatographic column 3 times with PBS, then use 2ml elution.Will The antibody purification obtained loads 4 DEG C of dialysis in bag filter.Dialysed overnight, then 4000rpm × 35min Centrifugal except precipitation, collect supernatant.Measure antibody titer with indirect elisa method and survey by Bradford method Determine protein concentration.

Anti-GTPBP9 antibody through the following steps that identify:

1. immunoblotting assay

Prepare PAGE gel according to standard method, by cell that 5 μ l protein concentrations are 5mg/ml or Tissue lysates is loaded successively, constant voltage 80V about 30 minutes, treats that sample ran concentration gum base originally in one During straight line, changing 160V voltage, electrophoresis to bromophenol blue indicator runs out of separation gel (about 60 minutes) completely Time terminate electrophoresis, use electricity transferring film method constant voltage 100V electricity turn 80 minutes transferring films to pvdf membrane.By institute The GTPBP9 antibody obtained resists as one, and using concentration is the antigen core that 1 μ l/ml obtains with above-mentioned transferring film Sheet at room temperature hybridizes 1 hour, then at room temperature hybridizes 1 with the goat anti-rabbit antibody of HRP labelling little Time, use ECL development process to develop the color, carry out developing the color and exposing with X sheet in darkroom, exempted from Epidemic disease Blot results.

2. immunohistochemical analysis

The tissue that 4% formalin is fixing is cut into the piece of tissue of 1.5mm × 1.5mm × 2.0-3.0mm, Transparent 30 minutes of dimethylbenzene after Gradient elution using ethanol, 62 DEG C of paraffin waxdips are after 2 hours, at organization embedding On machine, 9 kinds of tissues are embedded according to the arrangement mode paraffin of 3 × 3.Employing standard paraffin is cut into slices The organization chip wax disk(-sc) of the 4-5 μ m-thick cut is attached on the microscope slide that APES processed by method, The roasting sheet of 60 DEG C of roasting sheet machine 1 hour, 60 DEG C of roasting sheets 6 hours the most in an oven.Use standard immunoassay Groupization method, closes section 30 minutes in wet box with 100 μ l 10% sheep blood serums under room temperature, adds 100 μ l concentration are the GTPBP9 antibody of 2-4 μ g/ml, in wet box 4 DEG C overnight, add after PBS Enter biotin labeling goat anti-rabbit igg in wet box 37 DEG C hatch 30 minutes, then add after PBS The strepto-avidin concentrated solution of HRP labelling, hatches 30 minutes for 37 DEG C in wet box, 1%DAB after developing a film Colour developing, haematoxylin is redyed, multiple blue, and dehydration is transparent, mounting, and microscopy checks that result is also taken pictures.

Claims (6)

1. people's GTPBP9 polypeptide, it is characterised in that the aminoacid sequence of polypeptide is: RAFEDDDITHVEGS.

2. the preparation method for antibody of an anti-human GTPBP9 polypeptide, it is characterized in that the terminal modified peptide of sequent synthesis N as described in claim 1, the terminal modified peptide of N of synthesis is cross-linked with carrier protein, peptide immune animal with crosslinking, the blood taking immune animal prepares antiserum, from serum, isolated and purified IgG, wherein said N are terminal modified is N end one cysteine residues of increase at aminoacid sequence.

Preparation method for antibody the most according to claim 2, it is characterised in that carrier protein is keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

Preparation method for antibody the most according to claim 2, it is characterised in that its sulfydryl is cross-linked with carrier protein amino covalence by the terminal modified peptide of N by cross-linking agent.

Preparation method for antibody the most according to claim 2, it is characterised in that after crosslinking peptide and immunological adjuvant mixing and emulsifying, at rabbit back by multiple spot subcutaneous injection, and through the above booster immunization of secondary, the titer of serum is more than 1: 10000.

Preparation method for antibody the most according to claim 2, it is characterised in that highly purified IgG can be obtained from antiserum through ammonium sulfate precipitation, protein A affinity purification and peptide affinity purification.