CN103087154A - Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide - Google Patents
Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide Download PDFInfo
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Abstract
The invention provides a polypeptide for preparing a monoclonal antibody for detecting acetylcholin esterase. The polypeptide comprises an ammonia acid sequence represented by either SEQ ID No:1 or SEQ ID No:2, wherein the ammonia acid sequence represented by the SEQ ID No:1 is NH2-GDPNEPRDPKAPQWPPYTAGGC-COOH, and the ammonia acid sequence represented by the SEQ ID No:2 is NH2-CGGPRRFLPPEPKQPWSGVVD-COOH. The polypeptide is specifically combined with an acetylcholin esterase antibody. The polypeptide for preparing the monoclonal antibody for detecting the acetylcholin esterase is high in affinity and titer; and the monoclonal antibody prepared from the polypeptide is applied to a piezoelectric immunosensor and is high in flexibility and accuracy of detection on the acetylcholin esterase.
Description
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to a polypeptide for preparing a monoclonal antibody for detecting acetylcholinesterase, and application of the polypeptide in preparing the monoclonal antibody for detecting acetylcholinesterase.
Background
Acetylcholinesterase (Acetylcholinesterase, AChE, EC3.1.1.7) is a key enzyme in biological nerve conduction, is a main signal transmission molecule, is mainly present on chemical synapses among neurons and between neurons and muscle cells of multicellular animals, is a membrane-bound protein, is positioned on cell membranes and presynaptic and postsynaptic membranes, can rapidly hydrolyze neurotransmitter Acetylcholine (ACh) so as to stop the excitation of ACh on cholinergic receptors and maintain the sensitivity of nerve impulse conduction. Acetylcholinesterase is one of the fastest reacting enzymes in the organism, degrading a molecule for about 80 microseconds.
Because the content of acetylcholinesterase of motor nerve tracts and sensory nerve tracts in peripheral nerves is different by tens of times, the quantitative analysis of the content of acetylcholinesterase of the surgical severed nerve tracts can quickly and accurately distinguish the sensory nerves and the motor nerves clinically, and has great application potential clinically.
The content of the existing acetylcholinesterase is generally determined by adopting a piezoelectric immunosensor with mature and stable technology, and the method is quick and accurate and has the function of on-line analysis. The detection interval of the piezoelectric immunosensor mainly depends on a chip for detecting acetylcholinesterase, and the detection interval of the chip mainly depends on the affinity and titer of the monoclonal antibody used in the chip. The linear interval of the detection concentration value of the antibody chip prepared by the existing commercial antibody is 0.5-2 mug/ml, and is similar to the detection interval of sandwich ELISA. However, due to the smaller cross-section of the surgically excised specimens, the amount of protein solubilized in the cell lysate is low, with acetylcholinesterase outside the linear range of the sandwich ELISA assay (less than 1 ng/ml). Therefore, the method for detecting the content of acetylcholinesterase by using the immune piezoelectric sensor is applied to clinic, and the preparation of monoclonal antibodies with higher affinity and high titer is urgently needed.
The existing monoclonal antibody used for piezoelectric immunosensor mainly selects polypeptide with higher immunogenicity in acetylcholinesterase antigen protein, couples large molecules (such as BSA and BTG) as antigen, and after immunization, the monoclonal antibody is screened out by using the antigen with the same polypeptide amino acid sequence and different coupled macromolecules; however, polypeptides with higher immunogenicity are often at the hydrophobic end of the antigenic protein, resulting in lower affinity and potency of existing acetylcholinesterase antibodies.
Disclosure of Invention
The invention aims to provide a polypeptide with high affinity and high titer, namely a preparation method thereof.
In a first aspect of the present invention, there is provided a polypeptide for use in the preparation of a monoclonal antibody for detecting acetylcholinesterase, comprising the amino acid sequence of SEQ ID No: 1 or 2, and the amino acid sequence shown in SEQ ID No: 1 is NH2-GDPNEPRDPKAPQWPPYTAGGC-COOH, and the amino acid sequence of SEQ ID No: 2 is NH 2-CGGPRRFLPPEPKQPWSGVVD-COOH. The polypeptide can be specifically combined with acetylcholinesterase antibody.
In a second aspect of the invention, there is provided a polypeptide conjugate consisting of a polypeptide, a cross-linking agent and a macromolecular substance, wherein the polypeptide comprises the amino acid sequence of SEQ ID No: 1 or 2, the cross-linking agent is a mixture of formaldehyde and sodium acetate, and the macromolecule is Bovine Serum Albumin (BSA), Ovalbumin (OVA), keyhole limpet hemocyanin (LKH) or thyroglobulin (BTG).
In a third aspect of the invention, there is provided an antibody for use in the preparation of an antibody for detecting acetylcholinesterase, said antibody specifically binding to the polypeptide of claim 1.
Preferably, the antibody of the third aspect of the present invention is a monoclonal antibody.
In the fourth aspect of the invention, the application of the polypeptide in preparing the monoclonal antibody for detecting acetylcholinesterase is provided.
The application comprises the following steps:
(1) a polypeptide comprising SEQ ID No: 1 or 2 to obtain a whole antigen;
(2) immunizing the whole antigen to prepare a monoclonal antibody for detecting acetylcholinesterase;
(3) the monoclonal antibody is applied to a piezoelectric immunosensor to detect acetylcholinesterase.
Has the advantages that: the monoclonal antibody for detecting acetylcholinesterase provided by the invention has high polypeptide affinity and high titer, and the monoclonal antibody prepared by the polypeptide is applied to a piezoelectric immunosensor, so that the detection sensitivity and accuracy on acetylcholinesterase are high.
According to the invention, through the hydrophilic and antigenic analysis of each amino acid in the protein structure of acetylcholinesterase, 2 amino acid sequences with higher hydrophilicity and antigenicity are found and designed; then the two amino acid sequences with higher hydrophilicity and antigenicity are coupled with macromolecules to obtain complete antigens, and the monoclonal antibody for detecting acetylcholinesterase is prepared by immunization, and the prepared monoclonal antibody has high affinity and high titer. The monoclonal antibody is applied to a piezoelectric immunosensor for detecting acetylcholinesterase, and the detection sensitivity and accuracy can be obviously improved.
Drawings
FIG. 1 is a graph of co-immunoprecipitation of AChE in human leukemia cells by murine mAb prepared in example 3.
FIG. 2 is a graph of co-immunoprecipitation of AChE in murine brain tissue with murine mAb prepared in example 3.
FIG. 3 is a diagram of immunohistochemistry of AChE mab in murine brain tissue.
FIG. 4 is a diagram of immunohistochemistry of human spinal cord foreroot AChE mab.
FIG. 5 is a graph showing the results of the application of the murine mAb prepared in example 3 to a piezoelectric immunosensor.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
EXAMPLE 1 polypeptide Synthesis
The polypeptides were synthesized by solid-phase synthesis using a MultiPep RS polypeptide synthesizer according to the amino acid sequences of the polypeptides shown in Table 1.
TABLE 1 amino acid sequence of the polypeptide used for preparing monoclonal antibody for detecting acetylcholinesterase
| SEQ ID No： | Sequence of |
| 1 | NH2-GDPNEPRDPKAPQWPPYTAGGC- |
| 2 | NH2-CGGPRRFLPPEPKQPWSGVVD-COOH |
EXAMPLE 2 preparation of conjugates
(1) Preparation of KLH polypeptide conjugates
15mg of KLH was dissolved in 0.5ml of deionized water, 1mg of SEQ ID No: 1 or 2 in 0.5ml of a mixed solution of deionized water and ethanol (wherein the volume ratio of the deionized water to the ethanol is 2: 1); mixing the KLH solution with the polypeptide solution, adding 0.3ml of 3mol/L sodium acetate and 0.5ml of 7.5% formaldehyde, and stirring and reacting for 24 hours in a dark place; dialyzing with 0.05mol/L pH7.4PBS at 4 deg.C for more than 24 hr, lyophilizing, and packaging to obtain KLH polypeptide conjugate, and storing at-20 deg.C.
(2) The BSA, BTG and OVA polypeptide conjugates were prepared as in (1).
EXAMPLE 3 immunization of animals with antigenic Polypeptides to prepare monoclonal antibodies
The preparation of monoclonal antibody by immunizing animal with antigen polypeptide includes the following steps:
1. preparation of eukaryotic expressed AChE protein:
(1) extracting and purifying RNA from brain tissue of aborted fetus, and performing reverse transcription to obtain cDNA;
(2) design of primer with EcoRI cleavage site (P15 '-tagaattcggccggctgcggggcattcgc-3', AChE full-length sequence 292-312 comprising sequence encoding AChE polypeptide precursor; P2
5 '-gtcgaattctcacaggtctgag cagcgatcctgcttgct-3' AChE CDS 1971-2001), the product after PCR amplification is proved to be consistent with the mRNA sequence of acetylcholinesterase (E4-E6, NM-000665) through sequencing, and is cloned into a pGEM-T vector and is subcloned into a eukaryotic expression vector pcDNA3.1;
(3) the eukaryotic expression vector is transfected into 293T cells, stable recombinant hAChE expression cell strains are screened out by G418, and the AChE protein expression quantity can reach 25 micrograms/ml.
2. Immunization:
mice were immunized conventionally with the KLH polypeptide conjugate obtained in example 2 as an antigen once every two weeks; injecting 0.8ml of KLH polypeptide conjugate antigen into each mouse, and injecting the KLH polypeptide conjugate antigen into a two-foot pad, a neck and a back of the animal at multiple points respectively; and after three times of immunization, collecting antiserum, measuring the titer of the antiserum in the serum of the mouse by a thyroglobulin-coupled polypeptide ELASA method, and immunizing twice again when the titer reaches 1: 10000.
3. Preparing a monoclonal antibody:
taking the spleen of the immunized mouse in the step 2, preparing a suspension, and fusing the suspension with hybridoma cells; culturing the fusion cell, and screening positive clone cells by using the AChE protein expressed by the eukaryon. Measuring the titer of the culture supernatant of the positive clone cells, selecting 3-4 positive clone cell strains (more than 1: 10000) with the highest titer, injecting the positive clone cell strains into the abdominal cavity of a mouse, extracting ascites, refining and purifying to obtain the AChE mouse monoclonal antibody, wherein the clone number is as follows: AP 0691.
The method can be used for preparing the monoclonal antibody of chicken and the monoclonal antibody of rabbit.
The AChE mouse monoclonal antibody is prepared by the method, and the clone number is as follows: AP 0692; wherein the conjugate antigen comprises SEQ id no: 3, and the amino acid sequence shown in SEQ ID No: 3 is the amino acid sequence as follows:
NH2-CKLLSATDTLDEAERQWKH-COOH。
example 4 affinity assay
1. Co-immunoprecipitation experiment of AChE in human leukemia cells (Hela cells) with the AChE murine mAb prepared in example 3
The co-immunoprecipitation experiments of AChE were carried out on the AChE murine monoclonal antibody (clone No. AP 0691), the AChE murine monoclonal antibody (clone No. AP 0692), and the AChE antibody ab78228 from Abcam in human leukemia cells (Hela cells), respectively.
The co-immunoprecipitation experiment was performed by a method conventional in the art, and the affinity of the antibody to AChE was examined by developing with western blot (western blot), and the results are shown in fig. 1.
Wherein,
tape No. 1: the lysate of Hela cells was developed with western blot of AChE murine monoclonal antibody (clone No: AP 0691) to show a specific band at a molecular weight of 68 kd;
band No. 2: after the human IgG and the Hela cell lysate are subjected to co-immunoprecipitation, a western blot color development result of an AChE mouse monoclonal antibody (clone number: AP 0691) is used, no specific strip exists, and the fact that the human IgG and the AChE have no affinity is shown;
band No. 3: after the AChE mouse monoclonal antibody (clone number: AP 0691) and the Hela cell lysate are subjected to immune coprecipitation, a western blot color development result of the AChE mouse monoclonal antibody (clone number: AP 0691) is used, and a specific band is formed at the position with the molecular weight of 68kd, which shows that the AChE mouse monoclonal antibody (clone number: AP 0691) and the AChE have affinity;
band No. 4: after the AChE mouse monoclonal antibody (clone number: AP 0691) and the Hela cell lysate are subjected to immune coprecipitation, a western blot color development result of the AChE mouse monoclonal antibody (clone number: AP 0692) is used, and no specific band exists, which indicates that the AChE mouse monoclonal antibody (clone number: AP 0692) can not identify AChE;
band No. 5: after the AChE antibody ab78228 and Hela cell lysate of Abcam company are subjected to co-immunoprecipitation, a western blot color development result of an AChE mouse monoclonal antibody (clone number: AP 0691) is used, no specific band exists, and the antibody ab78228 and AChE have no affinity;
band No. 6: after the antibody BS3694 of the Baaode company and the Hela cell lysate are subjected to immune coprecipitation, a western blot result of an AChE mouse monoclonal antibody (clone number: AP 0691) is used for developing a color, no specific band exists, and the antibody BS3694 and the AChE have no affinity;
band No. 7: after the antibody BS3694 of the Baaode company and Hela cell lysate are subjected to co-immunoprecipitation, a western blot result of an AChE antibody ab78228 of the Abcam company has no specific band, which indicates that the antibody BS3694 has no affinity with AChE;
tape No. 8: after the AChE mouse monoclonal antibody (clone number: AP 0691) and Hela cell lysate are subjected to co-immunoprecipitation, a western blot color development result of an AChE antibody ab78228 of Abcam company is used, and no specific band exists, which indicates that the AChE antibody ab78228 can not identify AChE;
tape No. 9: after the AChE mouse monoclonal antibody (clone number: AP 0692) and Hela cell lysate are subjected to co-immunoprecipitation, a western blot color development result of an AChE antibody ab78228 of Abcam company is used, and no specific band exists, which indicates that the AChE antibody ab78228 can not identify AChE;
tape No. 10: after the AChE antibody ab78228 and Hela cell lysate of Abcam company are subjected to co-immunoprecipitation, a western blot result of the AChE antibody ab78228 of Abcam company is used for developing a color, and no specific band exists, which indicates that the AChE antibody ab78228 has no affinity with AChE;
band No. 11: hela cell lysates were subjected to western blot analysis using Abcam's AChE antibody ab78228, and showed no specific bands, indicating that Abcam's AChE antibody ab78228 could not recognize human AChE.
The results show that the AChE murine monoclonal antibody (clone number: AP 0691) prepared in example 3 can specifically bind to human AChE, and the affinity thereof is significantly better than that of the commercial product.
2. Co-immunoprecipitation experiment of mouse monoclonal antibody prepared in example 3 on AChE in mouse brain tissue
AChE co-immunoprecipitation experiments were performed on the mouse brain tissue with AChE murine monoclonal antibody (clone No.: AP 0691), AChE murine monoclonal antibody (clone No.: AP 0692), and AChE antibody ab78228 from Abcam, respectively.
The co-immunoprecipitation experiment was performed by a method conventional in the art, and the affinity of the antibody to AChE was examined by developing with western blot (western blot), and the results are shown in fig. 2.
Wherein,
band No. 1': the result of western blot coloration of mouse brain tissue lysate with AChE mouse monoclonal antibody (clone number: AP 0691) shows that there is a specific band at a molecular weight of 68 kd;
band No. 2': after the mouse IgG and the mouse brain tissue lysate are used for immune coprecipitation, the result of western blot color development of the AChE mouse monoclonal antibody (clone number: AP 0691) is used, no specific band exists, and the result shows that the mouse IgG and the AChE have no affinity.
3' band: after the AChE mouse monoclonal antibody (clone number: AP 0691) and the mouse brain tissue lysate are subjected to immune coprecipitation, a western blot color development result of the AChE mouse monoclonal antibody (clone number: AP 0691) is used, and a specific band is formed at the position of a molecular weight of 68kd, which shows that the AChE mouse monoclonal antibody (clone number: AP 0691) and the AChE have affinity.
Band No. 4': after the AChE mouse monoclonal antibody (clone number: AP 0691) and the mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot color development result of the AChE mouse monoclonal antibody (clone number: AP 0692) is used, and no specific band exists, which indicates that the AChE mouse monoclonal antibody (clone number: AP 0692) can not identify AChE.
Band No. 5': after the AChE antibody ab78228 of Abcam company and the mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot result of the AChE mouse monoclonal antibody (clone number: AP 0691) is used for developing a color, and no specific band exists, which shows that the antibody ab78228 has no affinity with AChE.
Band No. 6': after the antibody BS3694 of Baaode corporation and the mouse brain tissue lysate are subjected to immune coprecipitation, a western blot result of an AChE mouse monoclonal antibody (clone number: AP 0691) is used for developing a color, and no specific band exists, which indicates that the antibody BS3694 has no affinity with AChE.
Band No. 7': after the antibody BS3694 of the Baaode company and the mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot result of an AChE antibody ab78228 of the Abcam company is used for developing a color, and no specific band exists, so that the antibody BS3694 and the AChE have no affinity.
Band No. 8': after the AChE mouse monoclonal antibody (clone number: AP 0691) and the mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot result of an AChE antibody ab78228 of Abcam company is used for developing, and no specific band exists, which indicates that the AChE antibody ab78228 can not recognize AChE.
Band No. 9': after the AChE mouse monoclonal antibody (clone number: AP 0692) and the mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot result of an AChE antibody ab78228 of Abcam company is used for developing a color, and no specific band exists, which indicates that the AChE antibody ab78228 can not recognize AChE.
Band No. 10': after the AChE antibody ab78228 of Abcam and mouse brain tissue lysate are subjected to co-immunoprecipitation, a western blot result of the AChE antibody ab78228 of Abcam is used for developing a color, and no specific band exists, which indicates that the AChE antibody ab78228 and AChE have no affinity.
Band No. 11': the result of western blot of mouse brain tissue lysate using AChE antibody ab78228 from Abcam shows that there is no specific band, indicating that AChE antibody ab78228 from Abcam cannot recognize AChE.
The results show that the AChE mouse monoclonal antibody (clone number: AP 0691) prepared in example 3 can be specifically combined with AChE in mouse brain tissue lysate, and the affinity is remarkably superior to that of the commercial product.
3. Immunohistochemical results of AChE monoclonal antibody of mouse brain tissue
The AChE content in brain tissue is high, so the mouse cerebral cortex is taken to be cut after being fixed and embedded conventionally, the conventional immunohistochemical staining in the field is carried out, and the DAB is photographed after color development, which is shown in figure 3.
As can be seen from FIG. 3, the mouse cerebral cortex was immunohistochemically stained with the AChE murine monoclonal antibody (clone No. AP 0691) prepared in example 3 to form a large number of tan particles, which were positive cells.
It can be seen that the AChE murine mAb (clone No.: AP 0691) prepared in example 3 specifically recognized AChE in murine brain tissue.
4. Immunohistochemical results of human spinal cord anterior root AChE monoclonal antibody
The AChE content in the human spinal nerve anterior root is higher, so the human spinal nerve anterior root is taken to be cut after being fixed and embedded conventionally, and is subjected to the conventional immunohistochemical staining in the field, and the DAB is photographed after color development, which is shown in figure 4.
As can be seen from FIG. 4, the human spinal nerve anterior root section was immunohistochemically stained with the AChE murine monoclonal antibody (clone No. AP 0691) prepared in example 3 to form a large number of brown-yellow particles, which were positive cells.
It can be seen that the AChE murine mAb (clone No.: AP 0691) prepared in example 3 specifically recognizes AChE in human spinal nerve foreroots.
5. Mouse monoclonal antibody (clone number: AP 0691) prepared in example 3 was used in piezoelectric immunosensor assay
The content of AChE was determined on-line using the murine mAb (clone No. AP 0691) prepared in example 3, and the content of AChE was determined on-line using the AChE antibody ab78228 of Abcam as a comparative example, and the results are shown in FIG. 5.
Wherein:
FIG. 5a is the primary detection result of the murine mAb (clone No. AP 0691) prepared in example 3 on the eluate of the anterior spinal root, showing that the vibration frequency is reduced by 9.8 Hz;
FIG. 5b shows the primary detection result of AChE antibody ab78228 of Abcam corporation on the spinal cord anterior root eluate, showing that the vibration frequency is reduced by 4.8 Hz;
FIG. 5c shows the primary detection result of the murine monoclonal antibody (clone No. AP 0691) prepared in example 3 on the erythrocyte suspension, showing that the vibration frequency is reduced by 1.2 Hz;
FIG. 5d shows the result of a single measurement of the erythrocyte suspension by the AChE antibody ab78228 from Abcam, which shows that the vibration frequency is reduced by 0.2 Hz.
FIG. 5e is a statistical graph of the frequency change of the anterior and posterior roots of the spinal cord measured in 3 different replicates; wherein,
the English meaning in the figure is:
frequency of Frequency;
injecting a sample by Injection;
reaction;
time;
a rate of change of Δ F frequency;
ventiral root anterior root;
dornal root.
The results show that the murine monoclonal antibody (clone No. AP 0691) prepared in example 3 is more accurate and sensitive in detecting the spinal cord anterior root eluate and erythrocyte suspension.
Claims (5)
1. A polypeptide for preparing a monoclonal antibody for detecting acetylcholinesterase, comprising the amino acid sequence of SEQ ID No: 1 or 2, and the amino acid sequence shown in SEQ ID No: 1 has the amino acid sequence of
NH2-GDPNEPRDPKAPQWPPYTAGGC-COOH, SEQ ID No: 2 is NH 2-CGGPRRFLPPEPKQPWSGVVD-COOH.
2. A polypeptide conjugate, comprising: the polypeptide conjugate is composed of a polypeptide, a cross-linking agent and a macromolecular substance, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID No: 1 or 2, the cross-linking agent is a mixture of formaldehyde and sodium acetate, and the macromolecules are bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or thyroglobulin.
3. An antibody for preparing a reagent for detecting acetylcholinesterase, which is characterized in that: the antibody specifically binds to the polypeptide of claim 1.
4. The method for preparing an antibody for detecting acetylcholinesterase according to claim 3, wherein: the antibody is a monoclonal antibody.
5. Use of the polypeptide of claim 1 for the preparation of a monoclonal antibody for the detection of acetylcholinesterase.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6258780B1 (en) * | 1996-11-20 | 2001-07-10 | Yissum Research Development Company | Method and composition for enabling passage through the blood-brain-barrier |
| CN1376798A (en) * | 2001-03-23 | 2002-10-30 | 中国科学院上海细胞生物学研究所 | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence |
| CN1772766A (en) * | 2004-11-12 | 2006-05-17 | 中国科学院上海生命科学研究院 | Anti-apoptosis-related acetylcholinesterase monoclonal antibody and use thereof |
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2012
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6258780B1 (en) * | 1996-11-20 | 2001-07-10 | Yissum Research Development Company | Method and composition for enabling passage through the blood-brain-barrier |
| CN1376798A (en) * | 2001-03-23 | 2002-10-30 | 中国科学院上海细胞生物学研究所 | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence |
| CN1772766A (en) * | 2004-11-12 | 2006-05-17 | 中国科学院上海生命科学研究院 | Anti-apoptosis-related acetylcholinesterase monoclonal antibody and use thereof |
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