CN107266573A - The polyclonal antibody and preparation method of a kind of ox cross-film epididymal proteins 1 - Google Patents
The polyclonal antibody and preparation method of a kind of ox cross-film epididymal proteins 1 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- Biomedical Technology (AREA)
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Abstract
The invention belongs to gene engineering technology field, the polyclonal antibody and preparation method of a kind of ox cross-film epididymal proteins 1 are specifically disclosed, the polyclonal antibody is prepared from using artificial synthetic polypeptide as antigen using animal immune method；The amino acid sequence of the artificial synthetic polypeptide is as shown in SEQ ID NO.1.Polyclonal antibody prepared by the present invention can have an effect with the antigen of ox cross-film epididymal proteins 1, be that the expression study work of ox cross-film epididymal proteins 1 lays the foundation.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of polyclonal antibody of ox cross-film epididymal proteins 1 and
Preparation method.
Background technology
Up to the present only it has been reported that cross-film epididymal proteins 1 (Transmembrane epididymal protein 1,
TEDDM1) expressed in epididymis, most study is people's epididymal proteins 4 (HE4), and HE4 is a kind of new tumor markers, it is sent out
Sick rate occupies the 3rd of three big gynecologic malignant tumors, studies its biological function, has weight for the treatment of gynecologic malignant tumor
Want clinical meaning.
We study discovery TEDDM1 first also has expression in ox ovary, we have discovered that TEDDM1 is that G-protein is even
Join acceptor, obtain TEDDM1 total length CDS sequences by being expanded in the granular cell of ox ovary, analyzing its topology discovery has G
The structure of G-protein linked receptor, there is 7 transmembrane helix structures, 3 extracellular rings, 3 intracellular rings, 1 C-terminal, 1 N-terminal.In order to enter
One step studies the effect that it is played during Bovine follicular development during signal transduction and hormone control, need to carry out TEDDM1
Expression study.But domestic market has no TEDDM1 polyclonal antibody sale, limits TEDDM1 expression study work
Make.
The content of the invention
The polyclonal antibody and preparation method for a kind of ox cross-film epididymal proteins 1 that the present invention is provided, the Anti-TNF-α physical efficiency
The enough antigen with TEDDM1 is had an effect, and the expression study for being TEDDM1 work lays the foundation.
The purpose that the present invention is provided is a kind of preparation method of the polyclonal antibody of ox cross-film epididymal proteins 1, described many grams
Grand antibody is prepared from using artificial synthetic polypeptide as antigen using animal immune method；The amino acid of the artificial synthetic polypeptide
Sequence is as shown in SEQ ID NO.1.
It is preferred that, the preparation method of the polyclonal antibody of above-mentioned ox cross-film epididymal proteins 1, detailed process is as follows：
Using the new zealand white rabbit of 2.5kg body weight as object, head exempts from every and adds 250 μ g artificial synthetic polypeptides and 250 μ g
The mixture of Freund's complete adjuvant, the subcutaneous multi-point injection of neck；Secondary immunity after three weeks, every 250 μ g of addition are artificial synthesized more
The mixture of peptide and 250 μ g Freund's complete adjuvants, the subcutaneous multi-point injection of neck；Three immune, 250 μ g people of same addition after two weeks
Work synthesis polypeptide and the mixture of 250 μ g Freund's complete adjuvants, the subcutaneous multi-point injection of neck；14d after third time is immune, arteria carotis
Blood sampling, prepares serum, obtains rabbit polyvalent antibody, the rabbit polyvalent antibody is the polyclonal antibody of ox cross-film epididymal proteins 1.
Present invention also offers a kind of polyclonal antibody prepared according to the above method.
Compared with prior art, the polyclonal antibody and preparation method for a kind of ox cross-film epididymal proteins 1 that the present invention is provided,
Have the advantages that：
We study discovery ox cross-film epididymal proteins 1 (TEDDM1) first also expression in ox ovary, and TEDDM1 is G eggs
White coupled receptor, TEDDM1 total length CDS sequences are obtained by being expanded in the granular cell of ox ovary, analyze its topology discovery tool
There is the structure of g protein coupled receptor, there is 7 transmembrane helix structures, 3 extracellular rings, 3 intracellular rings, 1 C-terminal, 1 N-terminal.This
Polyclonal antibody prepared by invention can have an effect with the antigen of ox cross-film epididymal proteins 1, be the table of ox cross-film epididymal proteins 1
Laid the foundation up to research work.
Brief description of the drawings
Fig. 1 is TEDDM1 and β-actin albumen Western in the large follicle of ox second and maximum follicular cell
Blotting result figures；
Wherein, ODF1 swimming lanes are maximum ovarian follicles, and ODF2 swimming lanes are the second large follicles；
Fig. 2 is that the second large follicle and TEDDM1 expressing quantities in maximum follicular cell are quantified in Niu Tongyi Follicular waves
Analysis；
Wherein, ODF1 is maximum ovarian follicle, and ODF2 is the second large follicle；
Fig. 3 is immunohistochemical location analysis results (amplification 400 times) of the TEDDM1 in Niu Tongyi Follicular waves；
Wherein, Fig. 3 A, Fig. 3 B are maximum ovarian follicles；Fig. 3 C, Fig. 3 D are the second large follicles；GC is ovarian follicle layer in Fig. 3；TC is layer.
Embodiment
The present invention is described in detail with reference to the accompanying drawings and detailed description, it is to be understood that the protection of the present invention
Scope is not limited by embodiment.The test method of unreceipted actual conditions in the following example, generally according to normal
Conditional operation is advised, the reagent such as goat-anti rabbit HRP is commercially available.Unless otherwise defined, all technologies and science used in the present invention
Term is identical with the meaning that those skilled in the art of the present technique are generally understood that.
The invention provides a kind of preparation method of the polyclonal antibody of ox cross-film epididymal proteins 1, the polyclonal antibody
Using artificial synthetic polypeptide as antigen, it is prepared from using animal immune method；The amino acid sequence of the artificial synthetic polypeptide is such as
Shown in SEQ ID NO.1, the nucleotide sequence of the artificial synthetic polypeptide is the specific antigen nucleosides of ox cross-film epididymal proteins 1
Acid sequence is modified, i.e., " TGC is added before first nucleotides of 859-900 bit sequences shown in SEQ ID NO.2
(corresponding is cysteine) " synthesizes afterwards, and its antigenic site is not on the transmembrane domain of ox cross-film epididymal proteins 1.Addition
Cysteine is that it is to combine on the one hand to be easy to purification column filler because cysteine carries sulfydryl；Another aspect because
When preparing polyclonal antibody for early stage immune rabbit, by cysteine by artificial synthetic polypeptide and protein carrier KLH
((Keyhole Limpet Hemocyanin) is coupled, and immunogenicity can be increased after protein carrier KLH in coupling.
First, the preparation method of the polyclonal antibody is as follows：
Step 1, animal immune
The new zealand white rabbit of two 2.5kg body weight, immunization method is as follows:It is artificial synthesized many that head exempts from every 250 μ g of addition
Peptide and the mixture (500 μ g dosage) with 250 μ g Freund's complete adjuvant, the subcutaneous multi-point injection of neck；It is secondary after three weeks to exempt from
The mixture (500 μ g dosage) of epidemic disease, every 250 μ g artificial synthetic polypeptides of addition and 250 μ g Freund's complete adjuvants, neck is subcutaneous
Multi-point injection；Three immune, same mixtures for adding 250 μ g artificial synthetic polypeptides and 250 μ g Freund's complete adjuvants after two weeks
(500 μ g dosage), the subcutaneous multi-point injection of neck；14d after third time is immune, arteria carotis blood sampling, prepares serum, obtains rabbit and resists more
Serum, the rabbit polyvalent antibody is the polyclonal antibody of ox cross-film epididymal proteins 1.
Wherein, Freund's complete adjuvant plays emulsification to artificial synthetic polypeptide.
Step 2, ELISA detections (rabbit polyvalent antibody prepared by serum antibody titration detection)
Step 2.1, antigen title：Artificial synthetic polypeptide, the nucleotide sequence such as SEQ ID of the artificial synthetic polypeptide
Shown in NO.3, amino acid sequence is as shown in SEQ ID NO.1；
Step 2.2, it is coated with：With coating buffer solution (coating buffer：Na2CO3And NaHCO3Buffer solution) by step
2.1 antigen diluent adds 50 μ l dilutions to 1 μ g/ml in the reacting hole of each XPS, and 4 DEG C overnight, next day, are abandoned
Solution in hole is removed, is washed 1 time with every μ l of hole 180 with 1 × TBST buffer solutions.
Step 2.3, close：Every hole incubates 1h under the conditions of adding 1%BSA, 37 DEG C.
Step 2.4, antibody dilutes：The rabbit polyvalent antibody that step 1 is obtained is with 1:200,1:1000,1：5000,1:10000,
1:20000,1:60000,1:240000 dilution, respectively obtains the antibody diluent of various concentrations.
Step 2.5, it is loaded：Antibody diluent is loaded with 50 μ L/ holes.
Step 2.6, incubate：1h is incubated under the conditions of 37 DEG C, is washed twice with 200 μ l/ holes with TBST buffer solutions.
Plus ELIAS secondary antibody step 2.7,：Goat-anti rabbit HRP is with 1:5000 are diluted, and 45min is incubated under the conditions of 37 DEG C.
Step 2.8, wash：Washed three times with 200 μ l/ holes with TBST buffer solutions.
Step 2.9, develop the color：TMB nitrite ions 5min is added with 100 μ l/ holes.
Step 2.10, reading (OD)：ELIASA determines OD450nm readings, as a result as shown in table 1.
The antibody ELISA of table 1 detects data
As shown in Table 1, sample dilution 1:When 20000, testing result is 1.207 (average values of two groups of data), explanation
The polyclonal antibody potency of ox cross-film epididymal proteins 1 is high in the rabbit polyvalent antibody of preparation.
Step 3, polyclonal antibody purification
Step 3.1, chromatographic stuffing：Sulfo-link-gel (west treasured biotechnology (Shanghai) limited company).
Step 3.2, column production is chromatographed：Naked peptide crosslinked fillers, cysteine closing, the naked peptide is A albumen or G eggs
In vain.
Step 3.3, post is pre-processed：50mM PBS, pH7.4,20ml, flow velocity 60ml/h.
Step 3.4, serum loading：Polyclonal antibody prepared by 15ml steps 1,50mM PBS, pH7.4 are diluted to 20ml,
Repeat loading once.
Step 3.5, clean：50mM PBS, pH7.4,30ml, flow velocity 60ml/h.
Step 3.6, antibody elution：0.1M glycine-HCl, pH3.0, collect eluent, the Anti-TNF-α purified
Body.
Step 3.7, chromatographic column is washed：Merthiolate containing 0.02g/100ml in 50mM PBS, pH7.4, the PBS.
Step 3.8, chromatographic column is stored：4℃.
Step 3.9, pH value is adjusted：PH7.4 is adjusted with 1M sodium acid carbonate.
Without preservative during polyclonal antibody purification, not glycerol adding, BSA.
Step 4, the polyclonal antibody of ELISA detections purifying
Step 4.1, antigen coat：Antigen (artificial synthetic polypeptide) is coated with 1 μ g/ml concentration, and 4 DEG C of coatings are stayed overnight.
Step 4.2, close：1%BSA, incubates 1h under the conditions of 37 DEG C.
Step 4.3, the polyclonal antibody dilution of purifying：With 1:200,1:1000,1:5000,1:10000,1:20000,1:
60000,1:240000 dilution, respectively obtains the antibody purification dilution of various concentrations.
Step 4.4, it is loaded：Antibody purification dilution is loaded with 50 μ l/ holes, and 1h is incubated under the conditions of 37 DEG C.
Step 4.5, wash：Washed 2 times with 200 μ l/ holes with TBST buffer solutions.
Plus ELIAS secondary antibody step 4.6,：Goat-anti rabbit HRP is with 1:5000 dilutions are used, and 45min is incubated under the conditions of 37 DEG C.
Step 4.7, wash：Washed 3 times with 200 μ l/ holes with TBST buffer solutions.
Step 4.8, develop the color：TMB nitrite ions, 5min. are added with 100 μ l/ holes
Step 4.9, reading (OD)：ELIASA determines OD450nm readings, as a result as shown in table 2.
The ELISA detection data for the polyclonal antibody that table 2 is purified
The polyclonal antibody of purifying is needed again by ELISA detections, as shown in Table 2, the polyclonal antibody 1 of purifying:5000>
0.6, potency is not reaching to 1 after detection finds antibody purification:10000>0.6, antibody titer is too low to also need to concentration step
5, if potency reaches 1 after antibody purification:10000>0.6 without concentration, it is necessary to explanation, with two groups of data in table 2
Average value calculates potency as detection OD values.
Step 5 Antibody Concentration
The polyclonal antibody that pipe is purified is concentrated by ultrafiltration with millipore, obtains antibody concentrated solution, then carries out antibody inspection
Survey.
(1) consumption of antibody concentrated solution：5ml
(2) IgG OD280 concentration is determined using nucleic acid-protein analyzer, IgG concentration is obtained for 0.92mg/ml.
(3) ELISA method is with step 4, as a result as shown in table 3.
The ELISA detection data of the antibody concentrated solution of table 3
Conclusion：ELISA detections 1:10000>0.6, antibody test is qualified, it is necessary to explanation, with two groups of data in table 2
Average value calculates potency as detection OD values.
2nd, antibody Western bloting are detected
All experiments are all provided with 3 repetitions (3 biology are repeated, and 3 technologies are repeated).
(1) method：Every group of ox ovarian follicle total protein (including maximum is extracted using Protein Extraction Reagent kit (the green skies, China)
Ovarian follicle ODF1 and the second large follicle ODF2).Using bovine serum albumin (BSA) as standard, protein concentration is determined using BCA methods.
Albuminous degeneration, 97 DEG C, 5min.12% sds polyacrylamide gel electrophoresis protein isolate, per equal (25 μ of histone applied sample amount
G/ swimming lanes), then transferring film (nitrocellulose NC films, 0.22 μm).Skimmed milk power closes 1.2h.It is anti-with the dilution of TBST buffer solutions
Body：anti-TEDDM1(1:500, the polyclonal antibody of foregoing preparation), anti-β-actin (1:1000, CWBIO, China),
The antibody incubation NC films of dilution, 4 DEG C overnight.Cleaned with the TBS added with 0.1%Tween-20 after NC films, goat-anti rabbit HRP secondary antibodies
(1:10000, CWBIO, China) 2h is incubated at room temperature.Clean NC films after, with eECL western Blot kits (CWBIO,
China) detect and expose.With Image-Pro Plus6.0 (Media Cybernetics, USA) software to every histone
Optical density signal carries out quantitative analysis, and is used as internal reference with β-actin values.All experiments are all provided with 3 repetitions.
(2) result：
The total of the middle extraction of ox Granulosa cells (granulosa cells, GCs) is determined by Western bloting
TEDDM1 expression in albumen, its molecular weight is 34kDa (Fig. 1).Fig. 1 is TEDDM1 and β-actin albumen in ox the
Western blotting result figures in two large follicles and maximum follicular cell；Wherein ODF1 swimming lanes are maximum ovarian follicles,
ODF2 swimming lanes are the second large follicles.Fig. 2 is the second large follicle and TEDDM1 in maximum follicular cell in same Follicular wave
Expressing quantity quantitative analysis；Wherein, ODF1 swimming lanes are maximum ovarian follicles, and ODF2 swimming lanes are the second large follicles, and * represents difference
Significantly, P<0.05, n=3, error line representative ± S.E..Second large follicle (The second in the Follicular wave of ox oestrous cycle first
Largest follicle at onset of deviation, ODF2) in granular cell TEDDM1 expressing quantities than maximum
It is high 1.73 times in ovarian follicle (The largest follicle at onset of deviation, ODF1), significant difference (figure
2)。
Illustrate that the polyclonal antibody that the inventive method is prepared from has good effect.
3rd, the immunohistochemical localization of antibody
All experiments are all provided with 3 repetitions (3 biology are repeated, and 3 technologies are repeated).
(1) method：Ovarian follicle is placed in magazine, fixed with 4% paraformaldehyde solution, FFPE.TEDDM1's is immune
Histochemistry's detection is completed with the method (Western bloting methods) described before.It is (preceding with TEDDM1 polyclonal antibody
State the polyclonal antibody of preparation) detection.Parallel control, including PBS or pre- overnight with 4 DEG C of 10 μ g/ml oxen TEDDM1 are set
The section for the rabbit-anti TEDDM1 antibody incubations being incubated.Three serial section that every group of sample is obtained are verified.
(2) result：Positioning of the ox TEDDM1 in ovarian follicle is determined with SABC.Fig. 3 is ox TEDDM1 in same ovum
Steep the immunohistochemical location analysis result (400 times of amplification) in ripple；Wherein, the primary antibody that Fig. 3 A, Fig. 3 C are used is PBS；Fig. 3 B, figure
The primary antibody that 3D is used is TEDDM1 polyclonal antibodies；Fig. 3 A, Fig. 3 B are maximum ovarian follicles；Fig. 3 C, Fig. 3 D are the second large follicles；Fig. 3
Middle GC is ovarian follicle layer；TC is layer, 20 μm of scale.As can be seen from Figure 3 TEDDM1 ODF1 and ODF2 granular cell layer and
There is expression (Fig. 3 B, Fig. 3 D) in theca cell layer.When negative control incubation is made in adjacent section of PBS, do not examined in GCs
Measure significant immunoreactivity (Fig. 3 A, Fig. 3 C).
Further illustrate that the polyclonal antibody that the inventive method is prepared from has good effect.
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, appended claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without departing from the present invention to the present invention
God and scope.So, if these modifications and variations of the present invention belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these changes and modification.
Sequence table
<120>The polyclonal antibody and preparation method of a kind of ox cross-film epididymal proteins 1
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213>Artificial sequence
<400> 1
Cys Ser Glu Lys Asp Asp Gln Ala Leu Leu Ser Lys Ser Ser Pro
1 5 10 15
<210> 2
<211> 903
<212> DNA
<213>Artificial sequence
<400> 2
atgggaacct ttatcggtca cgtgtatcca gggttgttcc tactcgcata cggactgtat 60
cgagcggtag tggtctccaa agctgtgata ctcagcgact ctttcccgga tccttctttg 120
cctcccaaga ataaggggag atgggccagg ctgtggaaaa tgtcccacag aggtttgctg 180
aagatgctgg ccggctttgg cttgatggcg tatgagatca gctgtgttga gagaggattg 240
acgctgatga acagggacct gccaccaaga ttcatgtacg ccaaacagtg gcagcacctc 300
accatgttca tcctcctcac cctcaatggc tgtatagaca tcatgagcaa aaacttgctg 360
tcccaacgct gtgtgcccct agagaaaggg atcttggtgc tgacctttta tgtgctcctg 420
ctgctattgg tgtcgcatgt ccaggactct gtgggggtgg agatgcatgt tcactctctg 480
ctcatcttgg tggtgttgct gctgatgctg gtgttcaccg ccaagctgtg ggctcccgac 540
acgtttcaac tcgctgtgat cgagaccttt ctgtttcaga tgatgggttc ctggctggta 600
caggcaggct tcattctgta caaaccagtc tctggctacc cgtgggagga tgataacatt 660
agtgacatca tgtttgttac caccttcttc tgttggcatg tgatgatcaa tgctttgtgc 720
ctgctgggta tctatggagt gtcttccttt tggcatcgtt gctaccatcc tagctggaag 780
ctgacggggt ccagagaagc tccaggttac atatactcca ggagacccct ctaccaattg 840
ctacaggaag tggagcagtc agagaaagat gaccaggctc tgctttcaaa gagttcaccc 900
tga 903
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence
<400> 3
tgctcagaga aagatgacca ggctctgctt tcaaagagtt caccc 45
Claims (3)
1. a kind of preparation method of the polyclonal antibody of ox cross-film epididymal proteins 1, it is characterised in that the polyclonal antibody is with people
Work synthesis polypeptide is antigen, is prepared from using animal immune method；The amino acid sequence of the artificial synthetic polypeptide such as SEQ
Shown in ID NO.1.
2. the preparation method of the polyclonal antibody of ox cross-film epididymal proteins 1 according to claim 1, it is characterised in that tool
Body process is as follows：
Using the new zealand white rabbit of 2.5kg body weight as object, head exempts from every and adds 250 μ g artificial synthetic polypeptides and 250 μ g Freunds
The mixture of Freund's complete adjuvant, the subcutaneous multi-point injection of neck；Secondary immunity after three weeks, every add 250 μ g artificial synthetic polypeptides with
The mixture of 250 μ g Freund's complete adjuvants, the subcutaneous multi-point injection of neck；It is immunized for three times after two weeks, the same 250 μ g that add manually are closed
Into polypeptide and the mixture of 250 μ g Freund's complete adjuvants, the subcutaneous multi-point injection of neck；14d after third time is immune, arteria carotis is adopted
Blood, prepares serum, obtains rabbit polyvalent antibody, and the rabbit polyvalent antibody is the polyclonal antibody of ox cross-film epididymal proteins 1.
3. the polyclonal antibody that a kind of method described in utilization claim 1 or 2 is prepared from.
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|---|---|---|---|---|
| CN103509107A (en) * | 2013-08-07 | 2014-01-15 | 浙江大学 | Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic polypeptide |
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|---|---|---|---|---|
| CN103509107A (en) * | 2013-08-07 | 2014-01-15 | 浙江大学 | Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic polypeptide |
Non-Patent Citations (1)
| Title |
|---|
| LIJUN ZHOU等: "Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody‐Based Sandwich Enzyme‐Linked Immunosorbent Assay (ELISA)", 《JOURNAL OF CLINICAL LABORATORY ANALYSIS》 * |
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