CN1301772A - Antibody of specific identifying development regulating protein qBrn-2 and its preparing method and use - Google Patents
Antibody of specific identifying development regulating protein qBrn-2 and its preparing method and use Download PDFInfo
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Abstract
The present invlention belongs to the genetric engineering and especially relates to the antibody for specificity recognizing development regulatory protein qBrn-2 and its preparation method and application. Recombinatino polypeptide or chemically synthesized polypeptide antigen is used to immunize the mammal to obtain polyclonal antibody and the nonconservative amion acid sequence of the antiboby to N-terminal with recognition site qBrn-2 of development regulatory protein qBrn-2. The antibody does not recognize other POU structure domain protein, and recognizes specifically qunique protein in Western imprinting experiment. The qBrn-2 expression mode obtained immunohistochemistry process is identical with the resulted height of in-situ molecular hydridization. The antibody may be used in research of structure, function and embryonic development regulatory effect of development regulatory protein qBrn-2 and its homologous protein.
Description
The invention belongs to genetic engineering, particularly antibody of specific identifying development regulating protein qBrn-2-2 and its production and use.
Neural system is one of system that forms the earliest during animal embryo is grown, it the growing of animal, regenerate, play crucial effect in aging and the many biological phenomenas such as behavior and study.Biology of nerve growth is an important branch of developmental biology, is the main battle ground of present system animal development gene studies.Understanding to the Regulation Mechanism of the gene level in form generation, cytodifferentiation and the functional area position fixing process of central nervous system is one of key issue in the molecule biology of nerve growth.The research of development gene is a cross-centennial new comprehensive frontier branch of science.The molecule biology of nerve growth not only relates to the fundamental research of biology and medical science, has a wide range of applications aspect China's population quality, prenatal and postnatal care, the minimizing genetic diseases relevant with neural system with control improving simultaneously.Though the research of developmental biology is ten or twenty year only so far, has obtained suitable alarming development.Prove that now development gene in the animal embryo growth course form is taken place and organ forms the direct regulation and control effect that plays.The achievement in research in this field is used for preventing and treating the technology of genetic diseases and new high-tech pharmaceutical market with genetic engineering means probably.
Have been found that now some modulin molecules play keying action in nervous system development.As, the Hox gene is very identical in expression pattern and its merogenesis structure of mouse hindbrain, has determined the characteristic (Krumlauf, 1993. genetics trend (Trends Genet.) 9:106-112.) at this position in the combination of the Hox of each merogenesis of hindbrain gene.
POU family transcription factor also plays important effect to neural growth.Mainly express in the precursor of few dendron neurogliocyte and developmental schwann's cell as the Tst-1/Oct-6/SCIP of this family, these two kinds of cells are responsible for the central nervous system peripheral nervous system of unifying respectively and are covered the process of myelin.After this gene was knocked out, though the myelin of central nervous system is not subjected to obviously to influence, the myelin of peripheral nervous system was subjected to major injury (Bermingham et al 1996. genes and growth (Genes Dev) 10:1751-1762; Jaegle et al 1996. science (Science) 273:507-510).And for example, the neurocele wide expression of Brn-2 in the early stage generation of embryo, its normal physiological function is necessary (Nakai et al 1995. genes and growth (Genes Dev) 9:3109-3121 for hypothalamus and postpituitary some neuronic growth; Schonemann et al 1995. genes and growth (Genes Dev) 9:3122-3135).In the mankind, some POU transcription factor is undergone mutation and can be caused neural disease, is exactly (deKok et al 1995, science (Science) 267:685-688) due to POU3F4 suddenlys change as the deafness relevant with stapes.
Up to the present, though in a lot of species, found that the POU transcription factor is (about summary can be consulted Ryan et al 1997. genes and growth (Genes Dev) 11:1207-1225; Veenstra et al 1997. molecular biology reports (Molecular Biology Reports) 24:139-155), but the antibody of these transcription factors of specific recognition but for number seldom, the understanding to the function of these transcription factors is greatly limited.For example, expression pattern (the mechanism (Mech Dev) 58 that Baltzinger et al. grow of XLPOU 3 during the Xenopus laevis embryo is taken place in early days, 103-114), (Alvarez-Boladoet al. comparative neurology magazine (The Journal of Comparative Neurology) 355 237-295) all is the result who is obtained by antisense oligonucleotide probe to the expression pattern of Brn-2 in rat.Though these results make people to these expression of gene certain understanding arranged, but it is powerless to the other important information, as locating in the distribution of the proteins encoded of these genes each tissue in animal embryo is grown and the cell, information nucleic acid (mRNA) and its proteins encoded in time with the space on whether consistent, whether proteins encoded exists post transcriptional modificaiton.On method, use antisense oligonucleotide probe (anti-sense RNA probe) complicated operation in addition, and all will prepare corresponding probe for every kind of animal.
The objective of the invention is to obtain can specific identifying development regulating protein qBrn-2-2 antibody, for the research proteic structure of qBrn-2 and function relationship, in fetal development expression pattern and provide antibody of a kind of specific identifying development regulating protein qBrn-2-2 and its production and use to the regulating and controlling effect of growth course.
The antibody of specific identifying development regulating protein qBrn-2 of the present invention-2 is polyclonal antibodies that obtain for the antigen immune Mammals with the recombinant polypeptide, and this polypeptide is the terminal non-conservation peptide of the N-section of the qBrn-2 of dna recombinant expression and purifying; This antibody is the aminoacid sequence of non-conservation of the N-end of qBrn-2 to the recognition site of development regulating protein qBrn-2-2, other POU domain protein of this antibody nonrecognition; In the experiment of the Western marking, this antibody is discerned single albumen specifically; The qBrn-2 expression pattern that utilizes immunohistochemistry to connect to show is consistent with the height as a result of hybridization histochemistry.For example: in the experiment of the Western marking, this antibody is discerned single albumen among three days the quail embryo of hatching specifically, and its apparent molecular weight is 56,000.Its two, the qBrn-2 expression pattern that utilizes this antibody to connect to show is in the unanimity of height as a result of hybridization histochemistry.
Described recombinant polypeptide is to choose the POU box upstream of cDNA of qBrn-2 or the cDNA of chemosynthesis, through dna recombinant expression and purifying, and polypeptide that obtains or fusion polypeptide; Or according to the POU box upstream of the cDNA of qBrn-2 coded aminoacid sequence chemistry synthetic polypeptide or fusion polypeptide.The POU box upstream of the cDNA of the described qBrn-2 of choosing or the cDNA of chemosynthesis are meant the cDNA of qBrn-2 or-4～621 fragments of chemosynthesis.qBrn-2cDNA-4～621： AGTCATGGCGACCGCAGCCTCCAACCACTACAGC M A T A A S N H Y S 1053 CTGCTCGCCTCCGGCTCGCCCATGGTGCACGCCGAGCCGCCCGGCGGCATGCAGCCCGGC L L A S G S P M V H A E P P G G M Q P G 30113 GGAGGCTACCGCGACGCGGGCGCGCTGGTGCAGGCGGACTACGCGCTGCAGAGCAACGGG G G Y R D A G A L V Q A D Y A L Q S N G 50173 CACCCGCTGAGCCACGCTCACCAGTGGATCGCCGCGCTGTCCCACGGCGGCCCCGGCGGC H P L S H A H Q W I A A L S H G G P G G 70233 GGCGGCGGAGGAGGGGGCGGCGGCGGCGGAGGGGGCGGCGGAGGCGGCGAGGCTCCCTGG G G G G G G G G G G G G G G G G E A P W 90293 GCGGCGGCGGCGGCGGCGGCGGCGGGCGCGCTGGGCCCGCCCGACATCAAGCCGGCCGCG A A A A A A A A G A L G P P D I K P A A 110353 GTGCAGGCGGCCCCGCGCGGCGACGAGCTGCCGCCGCCTCCGCAGCACCCGCCGCCGCCG V Q A A P R G D E L P P P P Q H P P P P 130413 GGCCGAGCCCCGCACCTGGTGCACCACGGCGGCGGAGGCGGCGGGCACCACGCGGCGTGG G R A P H L V H H G G G G G G H H A A W 150473 CGGGCGGGCGGCGCGGCGCACCTGCCGCCGGGCATGGCCGCGGCCAACGGAGCGGCGCAG R A G G A A H L P P G M A A A N G A A Q 170533 GCGGGGCTGCTGTACCCGCAGCCGCCCGGCTTCACCGTGAACGGCATGTTGGGCGCCGCG A G L L Y P Q P P G F T V N G M L G A A 190593 CAGCCCGCCCTGCACCACCACGGCCTCCGCGACGCCCACGACGAGGCTCCC596 Q P A L H H H G L R D A H D E A P 207qBrn-2cDNA：
Dash area is the POU box.
The preparation method of specific identifying development regulating protein qBrn-2 of the present invention-2 antibody comprises:
(1). choose the POU box upstream of qBrn-2 or the cDNA of chemosynthesis, the construction expression plasmid;
(2). expression plasmid is imported host system express, the pure recombinant polypeptide of purified acquisition electrophoresis;
(3). with the pure recombinant polypeptide of this electrophoresis is antigen, and immune Mammals obtains the antibody of specific identifying development regulating protein qBrn-2-2, called after anti-qBrn-2.
The POU box upstream of described qBrn-2 is meant 1～207 amino acid whose encoding sequence of development regulating protein qBrn-2-2.Describedly expression plasmid is imported host system express and be meant pET is imported BL21 (DE3); The purifying of the pure recombinant polypeptide of described electrophoresis is to utilize the terminal His-tag of interpolation of its N-, uses Ni
++-chelating Sepharose 4B separation and purification.Described pET is meant pET 3b 207 or phis 207.The structure of described expression plasmid pET 3b 207 is to cut qBrn-2 cDNA with Hinf I and Sma I enzyme, gets its position and is positioned at Hinf I-Sma I fragment of-4～621, with end-filling, connects the BamH I linker of 12 mer with the Klenow archaeal dna polymerase; After BamH I enzyme is cut,, form expression plasmid pET 3b 207 with the BamH I site of this fragment cloning to pET 3b; The structure of described expression plasmid pHis 207, it is the Xba I-Nde I fragment of the Xba I-Nde I fragment displacement pET 3b 207 with pET 28a, form expression plasmid phis 207, so just added the His-tag coding region at the N-of expression plasmid pHis 207 end.The expression of described recombinant polypeptide is that pET 3b 207 or phis 207 are used CaCl
2Method transforms BL21 (DE3) bacterial strain, with 37 ℃ of liquid culture of LB; When OD600=0.6～0.8, adding final concentration is the IPTG of 0.05～0.4mM, continues to cultivate centrifugal collection thalline 2～3 hours; PHis 207 expressed proteic purifying are with the phosphoric acid buffer suspension thalline that adds proteinase inhibitor, the pH 7.3 of phosphoric acid buffer wherein, aprotinin 1 μ g/ml, PMSF100 μ g/ml; Per 1 liter of nutrient solution suspends ultrasonic degradation thalline in ice bath with the 50ml damping fluid; Subsequently, the adding final concentration is 1% Triton X-100, stirs gently 30 minutes; The centrifuging and taking supernatant, wherein the centrifugal condition temperature is 4 ℃, rotating speed is 10000rpm, uses Ni
++The affine absorption of-chelating Sepharose 4B post removes foreigh protein removing with 40mM imidazoles wash-out; Collect 200mM imidazoles elution fraction then, dialysis, frost drying obtains the pure recombinant polypeptide of electrophoresis.Described Mammals is New Zealand white rabbit, mouse, rat, guinea pig, goat, sheep, horse or ox.Described immunologic process is for making antigen with the pure recombinant polypeptide of electrophoresis that obtains, immunity New Zealand white rabbit in 8 age in week, multiple spot subcutaneous injection; During first immunisation, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 1.0mg electrophoresis, adds 1.0ml Fu Shi and does agent fully, fully the mixture of mixing; After 28 days, booster immunization, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 0.5mg electrophoresis, adds 1.0ml Fu Shi and not exclusively does agent, fully the mixture of mixing; The method of two weeks back use booster immunization is booster immunization once more; For the second time booster immunization begins to get the auricular vein blood examination and surveys and tire after 10 days, collects the antiserum(antisera) that is fit to experiment of the Western marking and immunohistochemical assay, and packing is frozen in-80 ℃.
The purposes of specific identifying development regulating protein qBrn-2 of the present invention-2 antibody, it is characterized in that this antibodies specific identification neurodevelopment modulin qBrn-2 and homologous protein thereof, so this antibody can be used for studying expression pattern and the developmental regulating and controlling effect of qBrn-2 in fetal development; Study the interaction of qBrn-2 and its target DNA and work in coordination with proteic interaction with qBrn-2.
The mode of described specific recognition is the variation by enzyme, vitamin H, fluorescein, the chemical group that adds lustre to, radio isotope or electrophoretic mobility, detects neurodevelopment modulin qBrn-2 albumen and homologous protein thereof.Described research qBrn-2 in fetal development expression pattern and the approach of developmental regulating and controlling effect be the immunohistochemistry of paraffin section, frozen section, full embryo or cell cultures; Studying the interaction of qBrn-2 and its target DNA and working in coordination with proteic interactional approach with qBrn-2 is EMSA (electrophoresismobility shift assay).
The antibody of specific identifying development regulating protein qBrn-2 of the present invention-2 can connect expression pattern and the regulating and controlling effect of homologous protein in fetal development that shows in qBrn-2 and the vertebrates thereof on cell levels; The homologous protein in research development regulating protein qBrn-2-2 and the vertebrates thereof and the interaction of its target DNA, and and its collaborative proteic interaction.On method, operate very easy.
Below in conjunction with drawings and Examples technical scheme of the present invention is further described.
The structure of Fig. 1, qBrn-2 cDNA;
The structure iron of Fig. 2, pET 3b 207 and pHis 207 and the purifying of recombinant polypeptide His-P207;
The specificity of Fig. 3, anti-qBrn-2;
Fig. 3 A, Western blot result, anti-qBrn-2 discern single albumen (56,000);
The hybridization histochemistry result of Fig. 3 B, E3 quail, and scale (B, C)=1mm;
The immunohistochemistry result of Fig. 3 C, adjacent sheet, and scale (B, C)=1mm;
The immunohistochemistry result of Fig. 4, the full embryo of 14 body segment quail embryos; QBrn-2 has obvious expression at whole neurocele, optic vesicle, ear nest;
The expression pattern of qBrn-2 in E4 that Fig. 5, anti-qBrn-2 disclose; Section is the transverse section that is positioned at the E4 of hind leg portion, and qBrn-2 expresses in neurocele, myotome, notochord; NT, neurocele; NC, notochord; Myo, myotome; Scale=53 μ m;
Fig. 6, EMSA connect the interaction of the development regulating protein qBrn-2-2 that shows and its target DNA;
Indicate among the figure: 1. 7. ultrasonic degradation supernatants, 8. ultrasonic degradations precipitation 9. was crossed the sample 4T. akrencephalon D. diencephalon M. midbrain R. hindbrain F. of sample 3 22. embodiment 7 of sample 2 21. embodiment 7 that posts penetrate sample 120. embodiment 7 of 15.E5.5 16.E5.5 trunk of liquid 10.20mM imidazoles eluent 11.40mM imidazoles eluent 12.200mM 13.E3.5 14.E3.5 trunk of imidazoles eluent 17. adult brains 18. adult kidneys 19. embodiment 7 and is represented unconjugated probe after 4. molecular weight markers 5. induced front 6. to induce after molecular weight marker 2. induced front 3. to induce
The cDNA of the qBrn-2 that following examples are used independently clones (Genbank AF091043) for this laboratory, expression vector is pET 3b (Novagen), Ni
++-chelating Sepharose 4B (Pharmacia), New Zealand white rabbit (301 Hospital) is planted quail (Beijing livestock corporation), HRP-goat anti rabbit IgG (Vector).
The molecular cloning of the cDNA of embodiment 1:qBrn-2.
Step 1: the preparation of sieve storehouse probe:
Synthetic following oligonucleotide sequence:
Primer 1:5 ' CGACCTGGAGCAGTTCGCCAA 3 '
Primer 2: 5 ' AACCAGACACGCACCACCT 3 '
Get five days quail embryo of hatching, adopt mRNA purification kit and cDNA synthesis kit (Pharmacia Biotech), Inc), according to product description provide method, preparation cDNA.Utilize primer 1 and primer 2, carry out the PCR experiment, use the Taq archaeal dna polymerase.97 ℃ of insulations are after 10 minutes, enter following circulation, annealed 1 minute for 55 ℃, 72 ℃ were extended 2 minutes, 94 ℃ of sex change 1 minute, circulate 35 times, obtain sieving the storehouse dna fragmentation, sequence is: 1 CGACCTGGAGCAGTTCGCCAAGCAGTTCAAGCAACGACGCATCAAGCTGGGCTTCA CCCA, 61 GGCCGACGTGGGACTGGCGCTGGGCACCCTCTACGGTAACGTGTTCTCGCAGACCA CCAT121 CTGCCGTTTCGAGGCCCTGCAGCTGAGCTTCAAGAACATGTGCAAGCTCAAGCCGC TGCT181 CAACAAGTGGCTGGAGGAGACCGACTCGTCCAGCGGCAGCCCCACCAACCTGGACA AGAT241 CGCGGCGCAGGGCCGCAAGCGCAAGAAGCGCACGTCCATCGAGGTGGGTGTCAAAG GCGC301 GCTCGGCCGTCTGCAGAGCCACTTTCTCAAGTGTCCCAAGCACGAGATCACCGGCC TGGC361 CGACAGCCTGCAACTGGAGAAGGAGGTGGTGCGTGTCTGGTT
Adopt random primering, this dna fragmentation is carried out
32P mark, preparation become sieve storehouse probe.Step 2: the structure in full embryo cDNA library of hatching five days quail.
Get five days quail embryo of hatching, 1 method prepares cDNA set by step, and the terminal EcoR I adaptor that connects is cloned in the pBluescript SK carrier then, transforms DH5 α bacterial strain competent cell, is built into the full embryo cDNA library of five days quail of hatching.This library is 3 * 10 before the amplification
5Individual clone.
Step 3: the probe with step 1 preparation screens the library of step 2 preparation, adopt ordinary method (molecular cloning, Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989), obtain the cDNA clone of development regulating protein qBrn-2-2, its sequence is as shown in specification sheets.
Embodiment 2: the structure of expression plasmid.Cut qBrn-2 cDNA (Fig. 1) with Hinf I and Sma I enzyme, get Hinf I-Sma I (4～621) fragment, with end-filling, connect BamH I linker (12mer) with the Klenow archaeal dna polymerase.After BamH I enzyme is cut,, form pET 3b 207 with the BamH I site of this fragment cloning to pET 3b.For ease of this recombinant polypeptide of later stage purifying, use Xba I-Nde I fragment of Xba I-Nde I (His-tag coding region) fragment displacement pET 3b 207 of pET 28a, form phis 207 (Fig. 2).Restriction enzyme mapping and dna sequencing result show that the structure of expression plasmid is accurate.
Embodiment 3: recombinant polypeptide expression and purifying.PHis 207 is transformed BL21 (DE3) bacterial strain, with 37 ℃ of liquid culture of LB.When OD600=0.6, add IPTG (final concentration 0.4mM), continue to cultivate centrifugal collection thalline 3 hours.With phosphoric acid buffer (pH7.3, aprotinin 1 μ g/ml, PMSF 100 μ g/ml) the suspension thalline that adds proteinase inhibitor, per 1 liter of nutrient solution suspends ultrasonic degradation thalline in ice bath with the 50ml damping fluid.Subsequently, add Triton X-100 (final concentration 1%), stirred gently 30 minutes.Centrifuging and taking supernatant (4 ℃ 10000rpm), are used Ni
++The affine absorption of-chelating Sepharose 4B post removes foreigh protein removing with 40mM imidazoles wash-out.Collect 200mM imidazoles elution fraction then, dialysis, frost drying obtains the pure recombinant polypeptide of electrophoresis (Fig. 2).
Embodiment 4: the preparation of antiserum(antisera) anti-qBrn-2.Get the pure recombinant polypeptide of electrophoresis that obtains among the embodiment 2 and make antigen, immunity New Zealand white rabbit in 8 age in week, multiple spot subcutaneous injection; During first immunisation, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 1.0mg electrophoresis, adds 1.0ml Fu Shi and does agent fully, fully the mixture of mixing; After 28 days, booster immunization, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 0.5mg electrophoresis, adds 1.0ml Fu Shi and not exclusively does agent, fully the mixture of mixing; The method of two weeks back use booster immunization is booster immunization once more; For the second time booster immunization begins to get the auricular vein blood examination and surveys and tire after 10 days, collects the antiserum(antisera) that is fit to experiment of the Western marking and immunohistochemical assay, and packing is frozen in-80 ℃
Embodiment 5: sero-fast specificity.Anti-qBrn-2 has good specificity, and this is proved by following result.One, the experiment of the Western marking.Get hatching 3.5 days, 5.5 it quail embryo's head and trunk, adult quail brain, nephridial tissue, add buffer suspension liquid (the 0.1 M NaCl that is equivalent to 1～2 times of tissue volume, 0.01 M Tris-Cl (pH7.6), 0.001 M EDTA (pH8.0), 1 μ g/mL aprotinin, 100 μ g/mLPMSF), tissue is shredded, 3 times SDS-PAGE sample-loading buffer (the 150mM Tris-Cl (pH6.8) that adds 0.5 volume then rapidly, 300mM DTT, 6%SDS, 0.3% tetrabromophenol sulfonphthalein, 30% glycerine), water-bath was boiled 10 minutes.Centrifugal (14000rpm, 4 ℃) get supernatant, determine the protein concentration of each supernatant liquor, and method is as follows.Respectively get 1 μ l supernatant liquor, add in the 200 μ l distilled waters.Get 5,10,15,20,25 μ g bovine serum albumins simultaneously respectively, add distilled water to 200 μ l.Add 20 μ l, 0.15% Sodium desoxycholate and 20 μ l, 72% trichoroacetic acid(TCA) in every pipe successively, centrifugal (3000g) gets precipitation, surveys protein concentration with the Lorry method then.When the immune marking is tested, respectively get 70 μ g albumen, carry out SDS-PAGE (12%).Electrophoresis, electrotransfer is to nitrocellulose filter then.Subsequently, incubate this film of bath to seal non-specific site with solution (PBS+0.2%Tween 20+5%skim milk+5% lowlenthal serum), use an anti-solution (PBS+0.2%Tween 20+5%skimmilk+5% lowlenthal serum+1: 800anti-qBrn-2) incubate this film of bath then, use the abundant rinsing of solution (PBS+0.2%Tween20+5%skim milk+5% lowlenthal serum) to resist again to remove one of surplus, next use two anti-solution (PBS+0.2%Tween 20+5%skim milk+5% lowlenthal serum+HRP-goat anti rabbit IgG, 1: 20,000, Vector) specific recognition one is anti-, use ECL reagent (Dupont) to soak at last, the exposure of X-sheet, mark position.The result shows that anti-qBrn-2 can discern single albumen (Fig. 3 A) specifically, illustrates that it has good specificity.Its two, immunohistochemistry is consistent with hybridization histochemistry result's height.Get the longitudinal section of 3 days quails of hatching, do immunohistochemistry and hybridization histochemistry respectively, method is with reference to reported (Xue 1993. developmental mechanisms (Mech Dev) 43:149-158) in the past.Found that the expression pattern of the two announcement is identical, qBrn-2 in the neurocele of brain and spinal cord wide expression (Fig. 3 B C), illustrates albumen that this antibody discerns qBrn-2 just.Immunohistochemical method in the experiment is specially, get paraffin section, Tuo Lajia water, remove nonspecific binding site with confining liquid (PBS+0.15%Tween 20+3% skimmed milk+5% lowlenthal serum), then anti-qBrn-2 (1: 200) is dissolved in the confining liquid 4 ℃ and hatches section and spend the night.Next anti-with confining liquid flush away unconjugated, (1: 2000, goat anti rabbitIgG Vector) is dissolved in and hatched the section room temperature in the confining liquid 2 hours, and was anti-with PBS flush away unnecessary two at last, DAB colour developing, microscopy with two anti-.
Embodiment 6:anti-qBrn-2 is applicable to the immunohistochemistry of full embryo.Get one to two day quail embryo of hatching (the concrete growth period presses the body segment number and judges), use PBS buffer solution for cleaning three times, fix 2 hours with 4% Paraformaldehyde 96 then.Remove remaining Paraformaldehyde 96 with PBS buffer solution for cleaning embryo subsequently, incubate on shaking table with 3%H2O2 and bathed 2 hours.Next use solution PBS-A (PBS+0.2% TritonX-100+2%skim milk+5% lowlenthal serum) on shaking table, to incubate and bathe 1.5 hours to remove nonspecific binding site.Use 4 ℃ of anti-qBrn-2 solution (PBS-A+anti-qBrn-2,1: 200) to incubate subsequently and bathe and spend the night.Next day, with the abundant rinsing of PBS-A to remove unconjugated antibody.Using two anti-solution (PBS-A+ goat anti-rabbit igg, 1: 2000) to incubate subsequently bathed 2 hours.At last with the abundant rinsing of PBS, and the DAB colour developing (300 μ l/ml DAB, 0.03%H2O2).Fig. 4 is the full embryo immunohistochemistry of the embryo of one 14 body segment result, can see that qBrn-2 has obvious expression (Fig. 4) at neurocele, optic vesicle, ear nest.
Embodiment 7:anti-qBrn-2 is applicable to upward immunohistochemistry of section.Get 4 days quail embryo of hatching, fix with 4% Paraformaldehyde 96, preparation paraffin section (7 μ m), press the immunohistochemical methods method operation among the embodiment 4, the result shows that qBrn-2 has obvious expression (Fig. 5) at neurocele, myotome, notochord, can see that under high power lens immune labeled position is positioned at the karyon of neuroepithelial cell, very identical with its function as transcription factor, these results suggest qBrn-2 plays an important role in the growth at these positions.
Embodiment 8:anti-qBrn-2 is applicable to that research qBrn-2 DNA interacts.Step 1: the extraction of cell whole protein.
Get 3 of 3.5 days quail embryos of hatching, use the PBS buffer solution for cleaning, add the homogenate of 0.5ml damping fluid (5mMHepes (pH7.9), 26%glycerol (v/v), 1.5mM MgCl
2, 0.2mM EDTA, 0.5mM DTT, 0.5mM PMSF).Measure the suspensions of tissues volume this moment, adds NaCl to 300mM, mixing, ice bath 30 minutes.Centrifuging and taking supernatant liquor (24000g, minute, 4 ℃) subsequently, packing after the quick-frozen, is deposited for-70 ℃ in dry ice/ethanol.The protein concentration of above-mentioned full cell extract is measured by the Lorry method.Step 2: the preparation of probe.Synthetic following dna sequence dna:
a:5’AGCT?TGCAT?AAATA?ATAGG?C?3’
b:5’TCGA?GCCTA?TTATT?TATGC?A?3’
Above-mentioned sequence a and sequence b are dissolved with distilled water, and by waiting mole mixing, final concentration is 2pmol/ μ l.This solution in 80 ℃ of water-baths 5 minutes, is slowly cooled to room temperature subsequently, get 2pmol and carry out the 32-P mark, adopt Klenow polysaccharase end-filling method, remove free dNTP with Sephadex G-50.Transfer to 200 μ ls with volume this moment.Step 3: albumen combines with DNA's.
The according to the form below preparation:
Table 1: albumen combines with DNA's
Damping fluid *: 20mM Hepes (pH7.9), 1mM MgCl
2, 4%Ficoll, 0.5mM DTT.
| Damping fluid * | The KCl final concentration | ??poly ??(dIdC ??) | Protein extract | ??anti-qBrn- ??2 ??1∶10 | Preimmune serum 1: 10 | |
| Sample 1 sample 2 samples 3 samples 4 | To 20 μ l to 20 μ l to 20 μ l to 20 μ l | ??50mM ??50mM ??50mM ??50mM | ??2μg ??2μg ??2μg ??2μg | ??15μg ??15μg ??15μg ??0 | ??0 ??0 ??1μl ??0 | ??0 ??0 ??0 ??1μl |
Next, ice bath is 30 minutes.Subsequently, at sample 1,2, respectively add probe 1 μ l in 3,4.Then, ice bath 40 minutes again.At last, electrophoresis 4%PAGE, 0.25xTBE, radioautograph.
Found that, add anti-qBrn-2 after, wherein a band and only have a band to disappear (Fig. 6, tip indication position) illustrates that this band is that qBrn-2 combines generation with probe.
Claims (16)
1. the antibody of a specific identifying development regulating protein qBrn-2-2, it is characterized in that: being one is the polyclonal antibody that the antigen immune Mammals obtains with recombinant polypeptide or chemically synthesized polypeptide, this antibody is the aminoacid sequence of non-conservation of the N-end of qBrn-2 to the recognition site of development regulating protein qBrn-2-2, other POU domain protein of this antibody nonrecognition; In the experiment of the Western marking, this antibody is discerned single albumen specifically; The qBrn-2 expression pattern that utilizes immunohistochemistry to connect to show is consistent with the height as a result of hybridization histochemistry.
2. the antibody of specific identifying development regulating protein qBrn-2-2 as claimed in claim 1, it is characterized in that described recombinant polypeptide is to choose the POU box upstream of cDNA of qBrn-2 or the cDNA of chemosynthesis, through dna recombinant expression and purifying, polypeptide that obtains or fusion polypeptide; Or according to the POU box upstream of the cDNA of qBrn-2 coded aminoacid sequence chemistry synthetic polypeptide or fusion polypeptide.
3. the antibody of specific identifying development regulating protein qBrn-2-2 as claimed in claim 2 is characterized in that the POU box upstream of cDNA of the described qBrn-2 of choosing or the cDNA of chemosynthesis are meant the cDNA of qBrn-2 or-4～621 fragments of chemosynthesis.
4．3qBrn-2,qBrn-2cDNA-4～621： AGTCATGGCGACCGCAGCCTCCAACCACTACAGC M A T A A S N H Y S 1053 CTGCTCGCCTCCGGCTCGCCCATGGTGCACGCCGAGCCGCCCGGCGGCATGCAGCCCGGC L L A S G S P M V H A E P P G G M Q P G 30113 GGAGGCTACCGCGACGCGGGCGCGCTGGTGCAGGCGGACTACGCGCTGCAGAGCAACGGG G G Y R D A G A L V Q A D Y A L Q S N G 50173 CACCCGCTGAGCCACGCTCACCAGTGGATCGCCGCGCTGTCCCACGGCGGCCCCGGCGGC H P L S H A H Q W I A A L S H G G P G G 70233 GGCGGCGGAGGAGGGGGCGGCGGCGGCGGAGGGGGCGGCGGAGGCGGCGAGGCTCCCTGG G G G G G G G G G G G G G G G G E A P W 90293 GCGGCGGCGGCGGCGGCGGCGGCGGGCGCGCTGGGCCCGCCCGACATCAAGCCGGCCGCG A A A A A A A A G A L G P P D I K P A A 110353 GTGCAGGCGGCCCCGCGCGGCGACGAGCTGCCGCCGCCTCCGCAGCACCCGCCGCCGCCG V Q A A P R G D E L P P P P Q H P P P P 130413 GGCCGAGCCCCGCACCTGGTGCACCACGGCGGCGGAGGCGGCGGGCACCACGCGGCGTGG G R A P H L V H H G G G G G G H H A A W 150473 CGGGCGGGCGGCGCGGCGCACCTGCCGCCGGGCATGGCCGCGGCCAACGGAGCGGCGCAG R A G G A A H L P P G M A A A N G A A Q 170533 GCGGGGCTGCTGTACCCGCAGCCGCCCGGCTTCACCGTGAACGGCATGTTGGGCGCCGCG A G L L Y P Q P P G F T V N G M L G A A 190593 CAGCCCGCCCTGCACCACCACGGCCTCCGCGACGCCCACGACGAGGCTCCC596 Q P A L H H H G L R D A H D E A P 207
5. the antibody of specific identifying development regulating protein qBrn-2-2 as claimed in claim 2 is characterized in that the cDNA sequence of described qBrn-2 and amino acid sequence coded thereof are:
6. as the preparation method of the described a kind of specific identifying development regulating protein qBrn-2-2 of claim 1-5 antibody, it is characterized in that this method comprises:
(1). choose the POU box upstream of qBrn-2 or the cDNA of chemosynthesis, the construction expression plasmid;
(2). expression plasmid is imported host system express, the pure recombinant polypeptide of purified acquisition electrophoresis;
(3). with the pure recombinant polypeptide of this electrophoresis is antigen, and immune Mammals obtains the antibody of specific identifying development regulating protein qBrn-2-2, called after anti-qBrn-2.
7. the preparation method of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 6, the POU box upstream that it is characterized in that described qBrn-2 is meant 1～207 amino acid whose encoding sequence of development regulating protein qBrn-2-2.
8. the preparation method of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 6 is characterized in that describedly expression plasmid is imported host system expressing and being meant pET is imported BL21 (DE3); The purifying of the pure recombinant polypeptide of described electrophoresis is to utilize the terminal His-tag of interpolation of its N-, uses Ni
++-chelating Sepharose 4B separation and purification.
9. the preparation method of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 8 is characterized in that described pET is meant pET 3b 207 or pHis 207.
10. the preparation method of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 9, the structure that it is characterized in that described expression plasmid pET 3b 207 is to cut qBrn-2cDNA with Hinf I and Sma I enzyme, get its position and be positioned at Hinf I-Sma I fragment of-4～621, with end-filling, connect the BamH I linker of 12mer with the Klenow archaeal dna polymerase; After BamH I enzyme is cut,, form expression plasmid pET 3b 207 with the BamH I site of this fragment cloning to pET 3b; The structure of described expression plasmid phis 207, it is the Xba I-Nde I fragment of the Xba I-Nde I fragment displacement pET 3b 207 with pET 28a, form expression plasmid pHis 207, so just added the His-tag coding region at the N-of expression plasmid pHis 207 end.
11. the preparation method of specific identifying development regulating protein qBrn-2 as claimed in claim 8-2 antibody, the expression that it is characterized in that described recombinant polypeptide is that pET 3b 207 or pHis 207 are used CaCl
2Method transforms BL21 (DE3) bacterial strain, with LB37 ℃ of liquid culture; When OD600=0.6～0.8, adding final concentration is the IPTG of 0.05～0.4mM, continues to cultivate centrifugal collection thalline 2～3 hours; Phis 207 expressed proteic purifying are with the phosphoric acid buffer suspension thalline that adds proteinase inhibitor, the pH7.3 of phosphoric acid buffer wherein, aprotinin 1 μ g/ml, PMSF 100 μ g/ml; Per 1 liter of nutrient solution suspends ultrasonic degradation thalline in ice bath with the 50ml damping fluid; Subsequently, the adding final concentration is 1% Triton X-100, stirs gently 30 minutes; The centrifuging and taking supernatant, wherein the centrifugal condition temperature is 4 ℃, rotating speed is 10000rpm, uses Ni
++The affine absorption of-chelating Sepharose 4B post removes foreigh protein removing with 40mM imidazoles wash-out; Collect 200mM imidazoles elution fraction then, dialysis, frost drying obtains the pure recombinant polypeptide of electrophoresis.
12. the preparation method of specific identifying development regulating protein qBrn-2 as claimed in claim 6～2 antibody is characterized in that described Mammals is New Zealand white rabbit, mouse, rat, guinea pig, goat, sheep, horse or ox.
13. the preparation method of specific identifying development regulating protein qBrn-2 as claimed in claim 6-2 antibody is characterized in that described immunologic process is for making antigen with the pure recombinant polypeptide of electrophoresis that obtains, immunity New Zealand white rabbit in 8 age in week, multiple spot subcutaneous injection; During first immunisation, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 1.0mg electrophoresis, adds 1.0ml Fu Shi and does agent fully, fully the mixture of mixing; After 28 days, booster immunization, the dosage of every rabbit use is dissolved in the 1.0ml phosphoric acid buffer for the pure recombinant polypeptide of 0.5mg electrophoresis, adds 1.0ml Fu Shi and not exclusively does agent, fully the mixture of mixing; The method of two weeks back use booster immunization is booster immunization once more; For the second time booster immunization begins to get the auricular vein blood examination and surveys and tire after 10 days, collects the antiserum(antisera) that is fit to experiment of the Western marking and immunohistochemical assay, and packing is frozen in-80 ℃.
14. purposes as the described a kind of specific identifying development regulating protein qBrn-2-2 of claim 1-5 antibody, it is characterized in that this antibodies specific identification neurodevelopment modulin qBrn-2 and homologous protein thereof, be used for studying expression pattern and the developmental regulating and controlling effect of qBrn-2 in fetal development; Study the interaction of qBrn-2 and its target DNA and work in coordination with proteic interaction with qBrn-2.
15. the purposes of a kind of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 14, the mode that it is characterized in that described specific recognition is the variation by enzyme, vitamin H, fluorescein, the chemical group that adds lustre to, radio isotope or electrophoretic mobility, detects neurodevelopment modulin qBrn-2 albumen and homologous protein thereof.
16. the purposes of a kind of specific identifying development regulating protein qBrn-2-2 antibody as claimed in claim 14, it is characterized in that described research qBrn-2 in fetal development expression pattern and the approach of developmental regulating and controlling effect be the immunohistochemistry of paraffin section, frozen section, full embryo or cell cultures; Studying the interaction of qBrn-2 and its target DNA and working in coordination with proteic interactional approach with qBrn-2 is EMSA.
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| CN 99127339 CN1301772A (en) | 1999-12-30 | 1999-12-30 | Antibody of specific identifying development regulating protein qBrn-2 and its preparing method and use |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99127339 CN1301772A (en) | 1999-12-30 | 1999-12-30 | Antibody of specific identifying development regulating protein qBrn-2 and its preparing method and use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306272C (en) * | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
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1999
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306272C (en) * | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
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