CN104402985B - The antigen protein and its rabbit polyclonal antibody and purposes of anti-scaffold molecule 1-s antibody - Google Patents
The antigen protein and its rabbit polyclonal antibody and purposes of anti-scaffold molecule 1-s antibody Download PDFInfo
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- CN104402985B CN104402985B CN201410806542.8A CN201410806542A CN104402985B CN 104402985 B CN104402985 B CN 104402985B CN 201410806542 A CN201410806542 A CN 201410806542A CN 104402985 B CN104402985 B CN 104402985B
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Abstract
The invention discloses a kind of antigen protein of anti-scaffold molecule 1-s antibody and its rabbit polyclonal antibody and purposes, belong to the pharmacy preparation containing antigen or antibody.The sequence of the specific antigen protein of the anti-human scaffold molecule 1-s antibody of the present invention is qpvaissapafgmggias.The specific antigen immune rabbit of anti-human scaffold molecule 1-s antibody is obtained into antiserum, antigenic solution must be prepared after emulsification, the blood containing antibody is collected after injecting adult rabbits, its supernatant, that is, antiserum, the affinity column of TBS buffer solutions balance antigen polypeptide by precooling, addition sodium azide, centrifugal filtration degreasing wash column, and purifying obtains anti-human scaffold molecule 1-s rabbit polyclonal antibodies.The polyclonal antibody is significantly better than with the expression in tissue level on a cellular level utilizes commercialization antibody income effect on sale.It is poor to solve the current anti-human ITSN1-S antibody specificities of commodity on sale, the low problem of potency ratio.
Description
Technical field
The present invention relates to biomedicine technical field, the antigen protein of specifically a kind of scaffold molecule 1-s antibody and its rabbit
Polyclonal antibody and purposes.
Background technology
Scaffold molecule 1（Intersectin1 or ITSN1）It is the albumen being highly conserved during evolution, has in human body
Two kinds of spliced bodies of ITSN1-L and ITSN1-S.In central nervous system, ITSN1-L is mainly distributed in neuron, and
ITSN1-S is mainly expressed in spongiocyte.Two kinds of spliced bodies possess multiple common protein structure domains.ITSN1-L albumen with
A variety of nerve degenerative diseases are related, such as：Mongolism；Alzheimer disease（AD）And Huntington's chorea etc..First
The generation of its stupid type get muddled to the ITSN1-L vesicle transports regulated and controled it is related, and the generation of AD and Huntington's chorea with
ITSN1-L promotes the neurotoxicity of poly glumine related.ITSN1-S can participate in endocytosis, born of the same parents by its various structures domain
It spits, the physiological activities such as cell Proliferation.
Research report about ITSN1-S is less, and for ITSN1-S antibody on sale at present there are poor specificity, potency ratio is low
The problem of, it is difficult to it is applied to cellular level and organizes the detection of horizontal ITSN1-S protein.
Polyclonal antibody has the multiple epitopes that can identify same antigen, can be identified in immune detection more anti-
It is former, it is not easy to the advantages that influence by antigen conformation variation.Therefore under the same conditions, inspection can be improved using polyclonal antibody
The sensitivity of survey, the albumen relatively low to some abundance are easier to detect.
Invention content
The present invention is to solve ITSN1-S antibody on sale at present there are poor specificity, and potency ratio is low, it is difficult to be applied to
The problem of cellular level and tissue horizontal ITSN1-S protein detections, and a kind of antigen egg of anti-scaffold molecule 1-s antibody is provided
Its rabbit polyclonal antibody of bletilla and purposes.
The present invention is realized according to following technical scheme.
A kind of antigen protein of anti-scaffold molecule 1-s antibody, it is characterised in that：The specific antigen piece of people source ITSN1-S
For the amino acid sequence of section as shown in sequence table, the purity of synthesis polypeptide is greater than or equal to 95%.
A kind of antigen protein rabbit polyclonal antibody of anti-scaffold molecule 1-s antibody, it is characterised in that：The polyclonal antibody is
Protein immunization segment is carried out using people source ITSN1-S protein sequences, obtains the specific antigen segment for people source ITSN1-S
Qpvaissapafgmggias, artificial synthetic polypeptide, and immune adult rabbits, obtain antiserum, prepare antigenic solution, collect supernatant
Liquid is antiserum；It is finally purified and is obtained using antigen polypeptide antagonistic Serum；The antibody is in a degree of space structure
Upper identification people scaffold molecule 1-s, has the function of the specific recognition to people's scaffold molecule 1-s.
A kind of antigen protein rabbit polyclonal antibody of anti-scaffold molecule 1-s antibody detects anti-human scaffold molecule 1-S preparing
Application in protein expression preparation, for expression of the specific detection people scaffold molecule 1-S protein in cell and tissue.
The advantage of the present invention designed in this way is, using anti-ITSN1-S rabbit polyclonals antibody purification obtained by the present invention,
Have the function of to fight the specific recognition of scaffold molecule 1-S, i.e., identifies people's scaffold molecule 1-on a degree of space structure
S, therefore specific can accurately detect expression of the anti-human scaffold molecule 1-S protein in cell and tissue.On a cellular level
It is significantly better than commercial antibody income effect on sale with the expression in tissue level, it is horizontal for more accurate reacting cells
With the expression for organizing horizontal ITSN1-S albumen.
Description of the drawings
Fig. 1 be ELISA method of the present invention carry out antibody titer than measurement result compares figure；
Fig. 2 is the glioblastoma cells strain LN-229 cellular levels ITSN1- for expressing exogenous human ITSN1-S albumen
The detection figure of S；
Fig. 3 is the glioblastoma cells strain LN- of commercial antibody detection expression exogenous human ITSN1-S albumen on sale
The expression figure of ITSN1-S albumen in 229；
Fig. 4 is 200 times of figures of expression of ITSN1-S albumen in present invention detection infiltration ductal carcinoma of breast sample；
Fig. 5 is 400 times of figures of expression of ITSN1-S albumen in present invention detection infiltration ductal carcinoma of breast sample；
Fig. 6 is 200 times of figures of expression of ITSN1-S albumen in present invention detection people's Gene in Infiltrating Lobular Carcinoma of Breast sample；
Fig. 7 is 400 times of figures of expression of ITSN1-S albumen in present invention detection people's Gene in Infiltrating Lobular Carcinoma of Breast sample；
Fig. 8 is 200 times of figures of expression of ITSN1-S albumen in present invention detection people's fibroadenoma of breast sample；
Fig. 9 is 400 times of figures of expression of ITSN1-S albumen in present invention detection people's fibroadenoma of breast sample.
Specific implementation mode
The present invention will be described in detail with reference to the accompanying drawings and embodiments.
One, experiment materials：
People source malignant glioblastoma cell strain LN-229 cell strains are purchased from U.S.'s ATCC cell banks；
Human breast carcinoma tumor clinical samples are tissue-derived in the disease for implementing tumor resection patient in our unit's Breast Surgery
Reason stone wax stripping and slicing；
Antigen:Using people source ITSN1-S protein sequences（NM_001001132）The selection for carrying out protein immunization segment, obtains
For the specific antigen segment of people source ITSN1-S, qpvaissapafgmggias, artificial synthetic polypeptide, synthesis polypeptide it is pure
Degree is greater than or equal to 95%.
Phosphate buffer（PBS）：8 grams of sodium chloride, 0.2 gram of potassium chloride, phosphoric acid hydrogen two receive 3.63 grams, potassium dihydrogen phosphate
0.24 gram, it is dissolved in 1 liter of pure water, pH value 7.4.
TBS buffer solutions：The Tris of 10mmol/L contains 0.9%NaCl, with HC1 tune pH to 7.4.
Elution buffer solution：1.5% glycine solution is adjusted to PH1.9 with hydrochloric acid.
Neutralization buffer solution：The Tris-HCl solution of 1M adjusts pH value to 9.0 with concentrated hydrochloric acid.
Two, methods
1. anti-scaffold molecule 1-s（ITSN1-S）The design of the antigen protein sequence of antibody：
Using people source ITSN1-S protein sequences（NM_001001132）Carry out the selection of protein immunization segment.It utilizes
The online comparison search tool of Protein Blast protein sequences.It chooses and ITSN1-S protein sequences（NM_001001132）It is similar
Strong several variants are compared.Search sequence and its each variant are compared using " multalin " software
It is right, find out distinguished sequence, i.e. immune fragment.The distinguished sequence found out is subjected to " protein blast " again, see the sequence with
Whether there is specificity between other similar sequences in database.According to the above operation, obtain for the special of people source ITSN1-S
Property antigen fragment, qpvaissapafgmggias.
2. according to the immune fragment polypeptide sequence of design, artificial synthetic polypeptide.It is synthesized by Shanghai Gill polypeptide Co., Ltd
Polypeptide.Utilize the FMOC strategies of artificial synthetic polypeptide：Using Wang Resin resins, protected amino acid is synthesized, then utilizes contracting
Close reagent（HOBT, HBTU, DIEA）, cutting reagent（TFA/TIS/H2O）Artificial synthesized desired polypeptides.Using anti-after synthesis
Phase HPLC C18 are purified, and the Purity of synthesis is detected using HPLC.The purity of synthesis polypeptide is greater than or equal to
95%。
3. using the polypeptide immune new zealand rabbit of synthesis, antiserum is obtained.With two adult New Zealand rabbits, 100 μ g are closed
At polypeptide dissolve in 1ml phosphate buffer solutions, prepare antigenic solution.Mycobacteria is added in freund 's incomplete adjuvant to be made
Freund's complete adjuvant, and 1ml antigenic solutions are added, it is fully emulsified.It is subcutaneously injected at 4 different positions when injection, is respectively
At back two, two at thigh at.About 500 μ l antigenic solutions of each injection respectively.Per 4-6 weeks injections of antigens, and after injection 7
Blood is collected from rabbit auricular vein within it -10 days.The blood collected before the blood of collection and injection is compared, is checked whether there is
Antibody generates.The blood of collection is placed 30 minutes in 37 °C of insulating boxs, then is stood overnight at 4 °C.By blood be transferred to from
In heart pipe, 4 °C, 10,000g centrifugations 10 minutes, it is antiserum to collect supernatant.
4. being purified using antigen polypeptide antagonistic Serum, antibody purification is obtained.Prepare the affinity column of antigen polypeptide, utilizes
10 times of affine column volumes and the balance pillar of the TBS buffer solutions by being pre-chilled.Antiserum is placed in 4 °C of refrigerators and is slowly thawed
To avoid the aggregation of protein.Solid sodium azide is added to a concentration of 0.05%, 4 °C, 15,000g centrifugations 5 minutes remove clear
Clear antiserum is filtered to remove extra fat using filter.By antiserum with TBS buffer solutions with 1:5 ratio carries out dilute
It releases, then is filtered with filter.With the speed of 0.5 ml per minute by antiserum on column, to ensure antiserum and filler
Combination, need continuous upper prop 2 times and retain efflux.After cleaning pillar with TBS buffer solutions plus the elution buffer of pH2.7 is molten
Liquid is eluted to all albumen with the speed of 0.5ml/min and flows down.With having been added to the 1.5 of 100 μ l neutralization buffer solution
Ml EP pipes are in charge of collection eluent, check the pH of eluent after mixing with pH test paper, if pH can utilize neutralization buffer less than 7
Liquid is adjusted to about pH7.4 to prevent the denaturation of antibody.10ml is added in column, pH1.9 elution buffer solution is collected as stated above
Eluent.Utilize the content of protein in each pipe of spectrophotometric determination.It is preserved at -20 °C after the antibody of purifying is dispensed.
Three, results
1. antibody titer than measurement, Fig. 1 be using ELISA method carry out antibody titer of the present invention than measurement result compare
Figure.It is coated with elisa plate with people's scaffold molecule 1-s antigen polypeptides of synthesis, 100 μ l are added per hole, 4 DEG C overnight.Use cleaning solution
It washes 3 times, BSA room temperatures is added and close 4 h.After drying, sequentially adding the antibody purification of doubling dilution, (37 DEG C of 30 min, washes 5
It is secondary), 1:10 000 HRP- goat anti-rabbit iggs.Finally plus the colour developing of tmb substrate liquid reads A450 values after termination reaction.Knot
Fruit is referring to Fig. 1.Illustrate, using the anti-ITSN1-S rabbit polyclonals antibody purification of present invention gained, to tie in a degree of space
People scaffold molecule 1-s is identified on structure, and with the increase of antibody purification concentration, this recognition reaction is stronger.
2. the specificity and sensitivity of application Western Blotting methods antibody purification obtained by cellular level verification.
It is the glioblastoma cells strain LN-229 for utilizing expression exogenous human ITSN1-S albumen referring to Fig. 2, Fig. 2,
The detection of ITSN1-S is carried out in cellular level（Western Blotting methods）；It is more using anti-human ITSN1-S rabbits obtained by the present invention
Clone purification antibody test obtains the molecular weight of albumen that molecular weight is accurately located at after exogenous ITSN1-S and GFP amalgamation and expressions
The position of 170KDa instructions, almost without the interference of non-specific band.Absolutely prove that the antibody can be in a degree of space
People scaffold molecule 1-s is identified in structure, and being capable of expression of the accurate detection scaffold molecule 1-S protein of specificity in cell.
The glioma cell line of culture, is cracked with cell pyrolysis liquid, and high-temperature denatured at 95 degree, prepares loading sample
This.6% polyacrylamide gel is prepared, the vertical gel electrophoresis of the full lysate of cell is carried out.Albumen transfer is carried out after electrophoresis,
On protein delivery to nitrocellulose filter.The closing of film is carried out to remove the interference of non-specific factors using 5% milk.Then
Present invention gained antibody purification is added（1:1000）It is incubated overnight, secondary antibody uses Goat anti rabbit IRDye
800cw（1:8000）Be incubated 1 hour, the acquisition of result is then carried out by infrared laser imaging device.
Confirm that the antibody has the specificity of confrontation scaffold molecule 1-S, it can be special on a degree of space structure
Property identification people's scaffold molecule 1-s.
3. being significantly better than the result of the antibody of commercialization on sale using antibody purification acquired results obtained by the present invention.
Utilize the antibody of commercialization on sale（Santa Cruz, CA, USA）Acquired results are to utilize referring to Fig. 3, Fig. 3
The commercial antibody sold（Antibody goods number sc-9948）The glioblastoma of detection expression exogenous human ITSN1-S albumen
The expression of ITSN1-S albumen in cell strain LN-229；（Antibody goods number sc-9948）.As a result it shows without specific item
Band shows that commercial antibody recognition capability on sale is poor, and poor specificity cannot detect the expression of ITSN1-S albumen.
Using Cell immunohistochemical staining method tissue level on verification the present invention obtained by antibody purification specificity and
Sensitivity.Paraffin section is routinely dewaxed using dimethylbenzene after being placed in roasting piece machine baking 2-3h, then carries out graded ethanol
Rehydration.Antigen retrieval is carried out using citrate antigen retrieval buffers.Slice is rinsed 3 times with cool clear water, is put into 3% H2O2In tank
10min closes peroxidase.PBS is used after pulling out（Phosphate buffer：8 grams of sodium chloride, 0.2 gram of potassium chloride, phosphoric acid hydrogen two
Receive 3.63 grams, 0.24 gram of potassium dihydrogen phosphate is dissolved in 1 liter of pure water, pH value 7.4）Cleaning.It is closed in next step with lowlenthal serum,
Then present invention gained antibody is added to be incubated（1:200）It is placed in 4 DEG C of refrigerator overnights.Second day, slice was rinsed 3 times with PBS,
Secondary antibody is added（The goat anti-rabbit antibody of HRP labels（1:500）, it is placed in room temperature 1h.It is rinsed 3 times with PBS, is directly added into horseradish peroxide
Compound enzyme, covered tissue, was put into 37 DEG C of incubators, was incubated 20min.It is rinsed 3 times with PBS, is developed the color using DAB color developing agents.
Light brown, developing time ﹤ 10min are showed quickly after DAB liquid is added.PBS is rinsed 3 times after colour developing.Haematoxylin is carried out after DAB colour developings
It redyes about 5-10 minutes, is then placed in 1% hydrochloride alcohol, finally returns indigo plant with ammonium hydroxide again.Finally use resin mounting and drying.
4. the protein expression situation that the micro- sem observation present invention is detected.In order to verify the use effect of antibody obtained by the present invention
Fruit, we have chosen the tissue samples of 3 mammary gland disease patients, and immunohistochemistry is carried out using antibody purification obtained by the present invention
Dyeing.The tissue samples of 3 mammary gland disease patients are invasive ductal carcinoma, invasive lobular carcinoma and mammary gland fibroma respectively.Leaching
For lubricant nature duct carcinoma from intraductal carcinoma progress, cancerous tumor cell breaks through conduit basilar memebrane, and interstitial, referred to as Infiltrating ductal are arrived in infiltration
Cancer is most common type in breast cancer.Invasive lobular carcinoma is usually developed by lobular carcinoma in situ, and cancerous tumor cell breaks through conduit
Interstitial is arrived in basilar memebrane, infiltration, is in that multiple leaflets occur more, is fused to each other to form big tumour with tumour growth, volume is often bright
It is aobvious to be more than invasive ductal carcinoma.Mammary gland fibroma, also known as adenofibroma are a kind of connective tissue and epithelial tissue hyperplasia simultaneously,
The clear benign tumour of boundary of formation.
The immunohistochemical staining result figure of infiltration ductal carcinoma of breast tissue as an example of Fig. 4 and Fig. 5 is to utilize
Gained antibody purification of the invention detects the expression of ITSN1-S albumen in an example infiltration ductal carcinoma of breast tissue, explanation
ITSN1-S albumen expliciting the positions are in cytoplasm.The result shows that using the antibody acquired results, without non-specific coloring, fill
Bright antibody of defending oneself can identify people scaffold molecule 1-s on a degree of space structure, and being capable of the accurate inspection of specificity
Survey the expression of scaffold molecule 1-S protein in the tissue.
Fig. 6 and Fig. 7 is the immunohistochemical staining of an example people's Gene in Infiltrating Lobular Carcinoma of Breast tissue as a result, it is to utilize this
Invention gained antibody purification detects the expression of ITSN1-S albumen in an example people's Gene in Infiltrating Lobular Carcinoma of Breast tissue, explanation
ITSN1-S albumen expliciting the positions are in cytoplasm.The result shows that using the antibody acquired results, without non-specific coloring, fill
Bright antibody of defending oneself can identify people scaffold molecule 1-s on a degree of space structure, and being capable of the accurate inspection of specificity
Survey the expression of scaffold molecule 1-S protein in the tissue.
Fig. 8 and Fig. 9 is the immunohistochemical staining result figure of an example human milk adenofibroma patient tissue（Immuning tissue
Dyeing SP methods are learned, blue-hematoxylin element is colored as nucleus, and brown DAB painted areas shows the expression of ITSN1-S albumen）,
It is the expression that ITSN1-S albumen in an example human milk adenofibroma tissue is detected using present invention gained antibody purification, is said
Bright ITSN1-S albumen expliciting the position is in cytoplasm.The result shows that using the antibody acquired results, coloured without non-specific,
Absolutely prove that the antibody can identify people scaffold molecule 1-s on a degree of space structure, and can be specific accurate
Detect the expression of scaffold molecule 1-S protein in the tissue.The above result shows that：In tissue level, resist using obtained by the present invention
Body can identify people scaffold molecule 1-s on a degree of space structure, be accurately detected ITSN1-S albumen in various mammary gland
Expliciting the position in diseased tissue and expression.
In conclusion the result of Fig. 1-9 absolutely proves the antigen protein using the anti-scaffold molecule 1-s antibody of present invention gained
Rabbit polyclonal antibody, it is horizontal on cellular level in tissue, people's bridge point can be identified on a degree of space structure
Sub- 1-s, for accurately detecting expliciting the position and expression of the ITSN1-S albumen in cell and tissue.
SEQUENCE LISTING
<110>Tumour hospital of Medical University Of Tianjin
<120>The antigen protein and its rabbit polyclonal antibody and purposes of anti-scaffold molecule 1-s antibody
<130> protein
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<220>
<221> protein
<222> (1)..(18)
<400> 1
Gln Pro Val Ala Ile Ser Ser Ala Pro Ala Phe Gly Met Gly Gly Ile
1 5 10 15
Ala Ser
Claims (3)
1. a kind of antigen protein preparing anti-scaffold molecule 1-s antibody, it is characterised in that：The specificity of people source ITSN1-S
The amino acid sequence of antigen fragment such as SEQ ID NO:Shown in 1, the purity of synthesis polypeptide is greater than or equal to 95%.
2. a kind of rabbit polyclonal prepared by the antigen protein for preparing anti-scaffold molecule 1-s antibody as described in claim 1
Antibody, it is characterised in that：Using the specific antigen segment artificial synthetic polypeptide of the people source ITSN1-S, and it is immunized into
Year rabbit obtains antiserum, prepares antibody-solutions, and it is antiserum to collect supernatant；Finally apply antigen polypeptide antagonistic Serum into
Row is purified and is obtained.
3. a kind of rabbit polyclonal prepared by the antigen protein for preparing anti-scaffold molecule 1-s antibody as claimed in claim 2 is anti-
Body is preparing the application in detecting anti-human scaffold molecule 1-S protein expression preparation, is used for specific detection people's scaffold molecule 1-S
Expression of the albumen in cell and tissue.
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| CN113121683B (en) * | 2021-04-14 | 2022-01-14 | 南通大学 | Preparation method of cotton GraiRGA transcription factor specific recognition antibody |
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Non-Patent Citations (3)
| Title |
|---|
| GenBank：NP_001001132.1;Russo，A等;《GenBank》;20130608;全文 * |
| ITSN1-S的SH3功能域对恶性胶质瘤细胞U87增殖能力的影响;王丽等;《中国肿瘤临床》;20130930;第40卷(第18期);第1089-1093页 * |
| 人ITSN2基因新剪接异构体的克隆和鉴定;刘倩;《湖南师范大学论文集》;20120315;第23页最后1段－第24页第1段，第25页最后1段至第26页第5段，摘要 * |
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