CN101508723A - Polypeptide for preparing polyclonal antibody and uses thereof - Google Patents
Polypeptide for preparing polyclonal antibody and uses thereof Download PDFInfo
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- CN101508723A CN101508723A CNA2009100801600A CN200910080160A CN101508723A CN 101508723 A CN101508723 A CN 101508723A CN A2009100801600 A CNA2009100801600 A CN A2009100801600A CN 200910080160 A CN200910080160 A CN 200910080160A CN 101508723 A CN101508723 A CN 101508723A
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Abstract
The invention discloses a polypeptide for preparing a polyclonal antibody and application thereof. The polypeptide for preparing the polyclonal antibody comprises three polypeptides that are polypeptide 1, polypeptide 2 and polypeptide 3, wherein, the amino acid sequence of the polypeptide 1 is sequence 5 in a sequence table, the amino acid sequence of the polypeptide 2 is sequence 6 in the sequence table and the amino acid sequence of the polypeptide 3 is sequence 7 in the sequence table. A polypeptide immune animal for preparing the polyclonal antibody successfully obtains an antibody which can identify exogenous KIAA0649 protein and endogenous KIAA0649 protein, and the KIAA0649 polyclonal antibody has higher specificity. The polyclonal antibody can be used for analyzing Western blot and immunofluorescence, and provides an excellent antibody for functional studies on the KIAA0649.
Description
Technical field
The present invention relates to be used to prepare the polypeptide and the application thereof of polyclonal antibody.
Background technology
KIAA0649 is in research during 1A6/DRIM, a Unknown Function gene that utilizes the triage techniques of yeast two-hybrid to find first.Discover that in early days the expression of KIAA0649 in l cell NIH3T3 makes cell generation vicious transformation (Yang L, Zhao J, Lu W, Li Y, Du X, Ning T, Lu G, Ke Y, KIAA0649, a 1A6/DRIM-interacting protein with the oncogenic potential.
Biochem Bioph Res Co2005,334:884-890).Document Manabu Nakayama is seen in sequential analysis about KIAA0649, Reiko Kikuno and Osamu Ohara.Protein-Protein Interactions Between LargeProteins:Two-Hybrid Screening Using a Functionally Classified LibraryComposed of Long cDNAs.Genome Res., 2002,12:1773-1784.Fr é d é ric Colland, Xavier Jacq, etc.Functional Proteomics Mapping of a Human Signaling Pathway.Genome Res., 2004,14:1324-1332. and Ken-ichi Ishikawa, TakahiroNagase, etc.Prediction of the Coding sequence of Unidentified HumanGenes.X.The Complete Sequence of 100 New cDNA Clones from Brain Which CanCode for Large Protein in vivo.DNA Res., 1998,5:169-176.
New and gene disease-related are being separated in a lot of laboratories, for studying the function of these genes, at first to prepare antibody, the preparation monoclonal antibody need be expressed and purifying protein fragment or full-length proteins, there is very big problem in a lot of proteic expression, and can not prepare monoclonal antibody.Preparing polyclonal antibody by synthetic polypeptide becomes unique selection, and after synthetic polypeptide prepared polyclonal antibody, many times Zhi Bei antibody can only be discerned the albumen of heterogenous expression, and is the problem of a very difficult solution to the identification of intrinsic protein.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide and application thereof that is used to prepare polyclonal antibody.
The polypeptide that is used to prepare polyclonal antibody provided by the present invention, comprise 3 polypeptide, polypeptide 1, polypeptide 2 and polypeptide 3, the aminoacid sequence of described polypeptide 1 is the sequence 5 in the sequence table, the aminoacid sequence of described polypeptide 2 is the sequence 6 in the sequence table, and the aminoacid sequence of described polypeptide 3 is the sequence 7 in the sequence table.
Certainly the described polypeptide that is used to prepare polyclonal antibody can only be made up of described polypeptide 1, polypeptide 2 and polypeptide 3.
But described polypeptide 1, polypeptide 2 and polypeptide 3 independent packagings when the preparation polyclonal antibody, mix them as required.Described polypeptide 1, polypeptide 2 and polypeptide 3 also can be hybrid packed, and the quality of polypeptide described in the mixture 1, polypeptide 2 and polypeptide 3 is comparable to be (1-3): (1-3): (1-3).The mass ratio of polypeptide 1, polypeptide 2 and polypeptide 3 is 1:1:1 as described.
Polypeptide of the present invention can be used as the antigen prepd polyclonal antibody.
With described polypeptide is that the polyclonal antibody that antigen-immunized animal obtains also belongs to protection scope of the present invention.
Described polyclonal antibody is the proteic antibody of anti-KIAA0649.
The polypeptide immune animal that is used to prepare polyclonal antibody of the present invention has successfully obtained to discern external source and the proteic antibody of endogenous KIAA0649, and the KIAA0649 polyclonal antibody has higher specificity.This antibody can be used for the analysis of Western blot and immunofluorescence, and KIAA0649 is carried out functional study provides good antibody.
Description of drawings
Behave KIAA0649 wetting ability, antigenicity and surface of Fig. 1 exposes the property analysis.
Fig. 2 is the specific evaluation of KIAA0649 polyclonal antibody Ab1.
Fig. 3 is the specific evaluation of KIAA0649 polyclonal antibody Ab2.
Fig. 4 is the specific evaluation of KIAA0649 polyclonal antibody Ab3.
Fig. 5 is used for the evaluation of immunofluorescence at intracellular location and KIAA0649 polyclonal antibody Ab3 for KIAA0649.
Embodiment
Among the following embodiment if no special instructions method therefor be ordinary method.
The used experiment material of following embodiment:
New zealand rabbit is available from Department Of Medicine, Peking University's animal center.
Cell culture medium DMEM is available from Gibco company.
Foetal calf serum is available from Hyclone company.
Transfection uses liposome LipofectAMINE2000 available from Invitrogen company.
Pvdf membrane is available from Amersham Pharmacia Biotech company.
ECL Western blot detection luminescence reagent is available from GE Healthcare company.
Two anti-IgG of the anti-rabbit igg of fluorescein isothiocyanate (FITC) labelled goat, the anti-mouse IgG of TRITC red (TRITC) labelled goat, horseradish peroxidase-labeled are all available from biotech company of China fir Golden Bridge in Beijing.
Flag monoclonal antibody M2 is available from Sigma company.
Other reagent are homemade analytical pure or import divides installed reagents.
1) is used to prepare the acquisition of the polypeptide of polyclonal antibody
Utilize the proteic sequence of TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) software analysis KIAA0649; Utilize DNAstar software package (DNASTAR Inc., Madison, WI, USA) antigenicity, hydrophilic and hydrophobic and the surface of carrying out multisequencing comparison and KIAA0649 exposes property prediction (Zang Linquan, Qiu Pengxin, Xiao Ru, Deng proteic bioinformatic analysis of .YZ22 and Polyclonal Antibody Preparation thereof. cell and molecular biology magazine, 2006,22 (2): 205-207. Li Ting, Guo Xiaohuan, etc. anti-CMTM4 Polyclonal Antibody Preparation, purifying and evaluation. cell and molecular immunology magazine, 2008,24 (1): 41-44).
Utilize DNAstar software, KIAA0649 full length protein primary structure has been carried out analyzing (Fig. 1).Select the principle of polypeptide to be: to have higher wetting ability and antigenicity, to expose property stronger on the surface simultaneously.Choose different polypeptide (table 1) immune animals and prepare KIAA0649 antibody.
The polypeptide of table 1. preparation KIAA0649 antibody
| The title of polypeptide | |
| Polypeptide | |
| 4 | |
| Polypeptide 5 | |
| Polypeptide 6 | |
| Polypeptide 7 | |
| Polypeptide | |
| 1 | Sequence 5 in the |
| Polypeptide | |
| 2 | Sequence 6 in the |
| Polypeptide | |
| 3 | Sequence 7 in the sequence table |
Above-mentioned 7 peptide species are synthetic by peptide company in the Hangzhou, and with above-mentioned 7 peptide species respectively with mouse keyhole-limpet hemocyanin (KLH) coupling.
2) preparation antibody 1
The synthetic sequence dna fragment:
pCI-Flag-pl3-F：5’-CTAGACCACCATGGACTACAAGGACGACGATGACAAGGAATTCAAGCTTGGGCCCGGTACCGCTAGCGC-3’；
pCI-Flag-pl3-R：5’-GGCCGCGCTAGCGGTACCGGGCCCAAGCTTGAATTCCTTGTCATCGTCGTCCTTGTAGTCCATGGTGGT-3’。
PCI-Flag-pl3-F and pCI-Flag-pl3-R dna fragmentation are formed double-stranded DNA by renaturation.
Cut the double-stranded DNA that annealing forms with restriction enzyme NheI and NotI (available from NEB company) enzyme, be connected, obtain the pCI-neo-Flag carrier with the pCI-neo (Promega company) that same enzyme is cut.
With pcDNA3.1-KIAA0649 (Yang L, Zhao J, Lu W, Li Y, Du X, Ning T, Lu G, Ke Y, KIAA0649, a 1A6/DRIM-interacting protein with the oncogenic potential.
Biochem Bioph Res Co2005,334:884-890) (Peking University) is template, pcr amplification KIAA0649 full length DNA, and the primer sequence is as follows:
Forward?primer：
5’ATCG
AAGCTTGAATTCAGCGGCAGCGGCtttctcatgaacgcttctcca3’；
Reverse?primer：5’ATCG
GCTAGCTTActtagaccttcactactga3’。
The PCR product is connected with the pCI-neo-Flag carrier of cutting with same enzyme after NheI (available from NEB company) enzyme is cut through restriction enzyme EcoRI, obtains the pCI-neo-Flag-KIAA0649 plasmid.
The HeLa cell in containing the DMEM substratum of 10% foetal calf serum, 37 ℃, 5%CO
2, cell culture incubator is cultivated.Transfection the day before yesterday is with 1 * 10
6Individual transfectional cell kind is to culturing bottle, and by LipofectAMINE2000 transfection pCI-neo-Flag-KIAA0649 plasmid, transfection is undertaken by the LipofectAMINE2000 specification sheets, obtains to express the proteic HeLa cell of Flag-KIAA0649.With the HeLa cell that changes the pCI-neo-Flag carrier in contrast.
Send Nanjing Jin Site company to prepare the polyclonal antibody of anti-KIAA0649 KLH link coupled polypeptide 4.With this antibody called after antibody 1 (Ab1).
Western blot identifies antibody purified 1.
Western blot method is as follows:
With above-mentioned transfection the HeLa cell of Flag-KIAA0649 with EBC250 cracking (0.5%NP-40,250mMNaCl, 50mM Tris-HCl (PH7.4), 1mM EDTA, 1mM PMSF, 1mM NaF, 1mM Na
3VO4,2 μ g/ml leupeptins, 2 μ g/ml aprotinin proteinase inhibitor) 12000rpm after 10 minutes of cracking on ice, 4 ℃ centrifugal 30 minutes, supernatant liquor is expresses the proteic HeLa total protein of cell of Flag-KIAA0649.Total protein is separated by SDS-PAGE, be transferred on the pvdf membrane, film, was reacted 1 hour with antibody 1 after 1 hour with 5% skimmed milk-PBST (PBS that contains 0.5%Tween20) sealing; Washing the two anti-IgG (biotech company of China fir Golden Bridge in Beijing) that add horseradish peroxidase-labeled after 3 times with PBST reacted 1 hour.Luminous by ECL-Western blot Detection Reagent to specifications after the PBST washing, expose by the X-ray sheet in the darkroom then.
Western blot result as shown in Figure 2, Ab1 can only discern the Flag-KIAA0649 albumen of external source transfection, can not the endogenous KIAA0649 albumen of specific recognition cell, and have very high non-specific.
Among Fig. 2,1 and 3 is Flag-KIAA0649 albumen, and 2 and 4 is endogenous KIAA0649 albumen.
3) preparation antibody 2
With KLH link coupled polypeptide 5,6 and 7 according to 100ug, 100ug and 100ug mix the immune new zealand rabbit in back, initial immunity is diluted to behind the 1ml freund's adjuvant thorough mixing with equivalent with the polypeptide mixture of 300 μ g with PBS, carry out subcutaneous injection in two sufficient Plantar each 0.25ml of portion, all the other back part multiple spot subcutaneous injections, per afterwards 2 all booster immunizations 1 time, use the polypeptide of Isodose to mix all subcutaneous multi-point injection of back (Manabu Nakayama of back with isopyknic incomplete Freund's adjuvant, Reiko Kikuno and Osamu Ohara.Protein-ProteinInteractions Between Large Proteins:Two-Hybrid Screening Using aFunctionally Classified Library Composed of Long cDNAs.Genome Res., 2002,12:1773-1784).Get rabbit ear source venous samples can behind the 2nd booster immunization in 14 days and detect by ELISA and tire, reach 1:10 to tiring
5After above immunize rabbit is put to death, heart extracting blood obtains serum.Serum is hatched with polypeptide link coupled Sepharose 4B affinity column with PBS dilution back, and 4 ℃ are spent the night.Tris-HCl (pH9.0) with the 0.1mol/L of pH2.4 behind the PBS flushing post neutralizes and dialyses to PBS, acquisition antibody 2 (Ab2).Detect antibody concentration with BCA protein quantification test kit (U.S. Pierce company), the SDS-PAGE electrophoresis is identified antibody 2 purity.
Specificity by Western blot antagonist 2 is identified.
Western blot method is with reference to step 2).
Western blot result as shown in Figure 3, Ab2 can discern the Flag-KIAA0649 albumen of external source transfection, and the endogenous protein band of recognizing cells is greater than Flag-KIAA0649 albumen, because Flag-KIAA0649 albumen has Flag, bigger than endogenous KIAA0649 albumen, obviously antibody 2 can not the endogenous KIAA0649 albumen of specific recognition.
4) preparation antibody 3
KLH link coupled polypeptide 1,2 and 3 is mixed the immune new zealand rabbit in back, the same step 3) of method according to 100ug, 100ug and 100ug.Detect the antiserum(antisera) titre at ELISA and reach 1:10
5Back heart extracting blood obtains serum.Serum is hatched with polypeptide link coupled Sepharose4B affinity column with PBS dilution back, and 4 ℃ are spent the night.Tris-HCl (pH9.0) with the 0.1mol/L of pH2.4 behind the PBS flushing post neutralizes and dialyses to PBS, acquisition antibody 3 (Ab3).Detect antibody concentration with BCA protein quantification test kit (U.S. Pierce company), the SDS-PAGE electrophoresis is identified antibody 3 purity.
The U20S cell (available from ATCC, article No.: HTB-96) in containing the DMEM substratum of 10% foetal calf serum, 37 ℃, 5%CO
2, cell culture incubator is cultivated.Transfection the day before yesterday is with 1 * 10
6Individual transfectional cell kind is to culturing bottle, and by LipofectAMINE2000 transfection pCI-neo-Flag-KIAA0649 plasmid, transfection is undertaken by the LipofectAMINE2000 specification sheets, obtains to express the proteic U20S cell of Flag-KIAA0649.With the U20S cell that changes the pCI-neo-Flag carrier in contrast.
Express proteic U20S cell of Flag-KIAA0649 and control cells and use EBC250 cracking (0.5%NP-40,250mM NaCl, 50mM Tris-HCl (PH7.4), 1mM EDTA, 1mM PMSF, 1mMNaF, 1mM Na respectively
3VO4,2 μ g/ml leupeptins, 2 μ g/ml aprotinin proteinase inhibitor) 12000rpm after 10 minutes of cracking on ice, 4 ℃ centrifugal 30 minutes, supernatant liquor is respectively expresses proteic U20S total protein of cell of Flag-KIAA0649 and control cells total protein.To express the proteic U20S total protein of cell of Flag-KIAA0649 separates by SDS-PAGE respectively with the control cells total protein, be transferred on the pvdf membrane, film, was reacted 1 hour with antibody 3 after 1 hour with 5% skimmed milk-PBST (PBS that contains 0.5%Tween20) sealing; Washing the two anti-IgG (available from biotech firm of China fir Golden Bridge in Beijing) that add horseradish peroxidase-labeled after 3 times with PBST reacted 1 hour.Luminous by ECL-Western blot Detection Reagent to specifications after the PBST washing, expose by the X-ray sheet in the darkroom then.
The result is shown in Fig. 4 A, it is higher that AB3 can discern the specificity of the Flag-KIAA0649 albumen of external source and endogenous KIAA0649 albumen (Flag-KIAA0649 albumen is because the existence of Flag-tag is more bigger than endogenous KIAA0649 albumen) AB3 simultaneously, do not have non-specific band.
Among Fig. 4 A, " 1 " for expressing the proteic U20S cell of Flag-KIAA0649, " 2 " are control cells.
KIAA0649 albumen contains 1209 amino acid, and the theoretical calculate size is about 140KD, but the specific band that above-mentioned KIAA0649 polyclonal antibody is discerned all is positioned at about 175KD.For confirming that further this specific band is a KIAA0649 albumen, designed siRNA at KIAA0649, its nucleotide sequence of called after siKIAA is as follows:
5′-CGCUUCUCCAGUGGUUGCUUU-3′。Synthetic one section non-specific nucleotide sequence siNC (5 '-ACUACCGUUGUUAUAGGUG-3 ') in contrast 2.
The transfection of the siKIAA of synthetic and contrast 2 (Shanghai JiMa pharmacy Technology Co., Ltd is synthetic) difference is to the HeLa cell, obtain siKIAA0649 cell and contrast 2 cells, extract siKIAA0649 cell and contrast 2 total protein of cell after 72 hours respectively, method is with reference to the extraction of expressing the proteic U20S total protein of cell of Flag-KIAA0649.
The siKIAA0649 cell is separated by SDS-PAGE respectively with contrast 2 total protein of cell, be transferred on the pvdf membrane, film, was reacted 1 hour with antibody 3 after 1 hour with 5% skimmed milk-PBST (PBS that contains 0.5%Tween20) sealing; Washing the two anti-IgG that add horseradish peroxidase-labeled after 3 times with PBST reacted 1 hour.Luminous by ECL-Western blot Detection Reagent to specifications after the PBST washing, expose by the X-ray sheet in the darkroom then.
The result shown in Fig. 4 B, transfection the HeLa cell of siKIAA, the KIAA0649 specific band obviously weakens, and has confirmed that further this specific band is a KIAA0649 albumen, antibody 3 can specific recognition KIAA0649 albumen.
Among Fig. 4 B, " 2 " for transfection the HeLa cell of siKIAA, " 1 " for the contrast 2 cells.
5) KIAA0649 is in intracellular location
Express the proteic HeLa cell of Flag-KIAA0649 kind to cover glass, pass through formaldehyde: acetone (1:1) pair cell is fixed, the sheep blood serum sealing adds Flag monoclonal antibody M2 and 37 ℃ of overnight incubation of antibody 3 (Ab3) after 30 minutes, add anti-rabbit igg of FITC labelled goat and the anti-mouse IgG of TRITC labelled goat mixed solution behind the thorough washing, 1 hour after scouring of incubated at room, DAPI transfect cell nuclear, 90% glycerine mounting is observed at the fluorescence co-focusing microscopically.
Anti-Flag antibody M2 is by the crosslinked fluorescence two anti-identifications of TRITC, and is shown in red.Antibody 3 (Ab3) is by two crosslinked anti-identifications of FITC, and is shown in green; Dye nuclear with DAPI simultaneously, be blueness.
The fluorescence co-focusing microscopically carries out observations as shown in Figure 5, and KIAA0649 antibody specific recognition Flag-KIAA0649 locatees altogether with the Flag-KIAA0649 existence of M2 antibody recognition, is yellow in composite diagram.Simultaneously visible Flag-KIAA0649 shows as a kind of special positioning form in nucleus.In all cells, it is green that endogenous KIAA0649 is, and particulate state is distributed in the nucleus, and is similar with the Flag-KIAA0649 distribution form of external source transfection.Illustrate that anti-KIAA0649 polyclonal antibody can be used for the specificity of immunofluorescence analysis, analyzes in intracellular location KIAA0649 simultaneously.
Among Fig. 5, " 1 " nucleus for dying with DAPI, " 2 " immunofluorescence dyeing for carrying out with Ab3, the immunofluorescence dyeing that carry out for the antibody M2 with anti-Flag " 3 ", " 4 " are 1,2 and 3 integration map.
Sequence table
＜110〉Peking University
＜120〉be used to prepare the polypeptide and the application thereof of polyclonal antibody
<160>7
<210>1
<211>15
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>1
<210>2
<211>31
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>2
<210>3
<211>31
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>3
<210>4
<211>29
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>4
<210>5
<211>22
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>5
<210>6
<211>19
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>6
<210>7
<211>17
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>7
Claims (8)
1, is used to prepare the polypeptide of polyclonal antibody, comprise 3 polypeptide, polypeptide 1, polypeptide 2 and polypeptide 3, the aminoacid sequence of described polypeptide 1 are the sequence 5 in the sequence table, the aminoacid sequence of described polypeptide 2 is the sequence 6 in the sequence table, and the aminoacid sequence of described polypeptide 3 is the sequence 7 in the sequence table.
2, polypeptide according to claim 1 is characterized in that: described polypeptide 1, polypeptide 2 and polypeptide 3 independent packagings.
3, polypeptide according to claim 1 is characterized in that: described polypeptide 1, polypeptide 2 and polypeptide 3 are hybrid packed.
4, polypeptide according to claim 3 is characterized in that: the mass ratio of described polypeptide 1, polypeptide 2 and polypeptide 3 is (1-3): (1-3): (1-3).
5, polypeptide according to claim 4 is characterized in that: the mass ratio of described polypeptide 1, polypeptide 2 and polypeptide 3 is 1:1:1.
6, the application of arbitrary described polypeptide in the preparation polyclonal antibody in the claim 1 to 5.
7, a kind of polyclonal antibody is to be the polyclonal antibody of antigen-immunized animal acquisition with arbitrary described polypeptide in the claim 1 to 5.
8, polyclonal antibody according to claim 7 is characterized in that: described polyclonal antibody is the proteic antibody of anti-KIAA0649.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110204606A (en) * | 2019-05-31 | 2019-09-06 | 西安交通大学 | A kind of polypeptide being used to prepare rabbit polyclonal antibody and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110204606A (en) * | 2019-05-31 | 2019-09-06 | 西安交通大学 | A kind of polypeptide being used to prepare rabbit polyclonal antibody and its application |
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