CN114622287A - Epitope screening method and device based on polypeptide microarray - Google Patents

Epitope screening method and device based on polypeptide microarray Download PDF

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Publication number
CN114622287A
CN114622287A CN202210265753.XA CN202210265753A CN114622287A CN 114622287 A CN114622287 A CN 114622287A CN 202210265753 A CN202210265753 A CN 202210265753A CN 114622287 A CN114622287 A CN 114622287A
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polypeptide
chip
microarray
amino acid
synthesizer
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梁洞泉
赵媛
梁柏堂
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Yaoming Jichuang Foshan Biotechnology Co ltd
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Yaoming Jichuang Foshan Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention discloses an epitope screening method and device based on a polypeptide microarray, wherein the method comprises the following steps: determining a primary protein sequence, setting the length of a polypeptide sequence and the distribution of a microarray by a polypeptide microarray synthesizer, preparing a polypeptide chip, activating the polypeptide chip and applying the polypeptide chip; the device includes polypeptide microarray synthesizer, and polypeptide microarray synthesizer includes high-efficient filtration safety guard, arm, sample application needle, amino acid solution place the platform, sample application needle cleaning module, carrier place the platform, sample application platform, micro-syringe pump in advance. According to the immune reaction principle, antigen epitopes of COVID-19Omicron and other viruses can be screened, and early scientific guidance is provided for research and development of vaccines and therapeutic antibodies, development of target drugs and the like. The invention has the advantages of trace amount, high precision, high flux, high sensitivity and the like, provides a high-efficiency method for screening antigen epitopes of COVID-19Omicron and other diseases, and provides a scientific data basis for drug development.

Description

Epitope screening method and device based on polypeptide microarray
[ technical field ] A
The invention belongs to the technical field of polypeptide array chips, and particularly relates to an epitope screening method and device based on a polypeptide microarray.
[ background of the invention ]
The polypeptide microarray is a novel technology for researching intermolecular interaction, and provides a powerful platform for biomedical research and drug development. The technology researches the interaction between molecules by combining polypeptide mimic protein molecules with antibodies or medicines and adopting a high-throughput analysis technology, and has remarkable advantages in the aspects of disease screening, early diagnosis, medicine screening and the like. The polypeptide chip technology is a microarray chip formed by decomposing protein molecules into continuous polypeptide fragments and fixing the polypeptide fragments on a specially treated carrier matrix by adopting a solid phase synthesis technology. The polypeptide fragments have corresponding biological molecular characteristics, can capture proteins or drugs specifically bound with the polypeptide fragments, and can obtain corresponding action relationships, such as epitope recognition, drug action target selection and the like, through corresponding biochemical analysis.
The polypeptide chip is a biochip, and is prepared by fixing amino acids on a specially treated carrier matrix by a polypeptide microarray synthesizer according to a set sequence through a contact dot printing technology and performing subsequent treatment. The polypeptide arrays have extremely high density characteristics, including hundreds to thousands of polypeptide microarrays. The microarrays react with antibodies, serum and proteins to be detected, can be used for high-throughput epitope screening, and is used for revealing the interrelation between amino acids and between proteins. Most of the existing polypeptide synthesis methods are artificially customized and single polypeptide, and the synthesis technology has low product efficiency, low product purity and high manufacturing cost and cannot meet the requirements of customers.
[ summary of the invention ]
The invention aims to provide a polypeptide synthesis instrument which can be used for simultaneously synthesizing polypeptides with different sequences by accurately and quantitatively transferring liquid to the same carrier matrix, has high synthesis efficiency, saves the waiting time of gradual synthesis reaction, effectively saves the labor time and meets the requirements of various polypeptides of customers; the instrument carries a polypeptide microarray chip manufactured by a chemical solid-phase synthesis method, has high product purity and high stability, and is applied to epitope screening and vaccine development assistance;
in order to realize the purpose of the invention, the following technical scheme is adopted:
designing the polypeptide microarray chip:
(1) according to the use requirements of customers, introducing the primary structure sequence of the antigen/protein to be researched into synthesizer software;
(2) 12 amino acids are used as a polypeptide, and the number of the amino acids of the polypeptide can be more than or less than 12 by designing a polypeptide microarray through the software;
(3) setting microarray arrangement on a polypeptide chip, wherein the microarray arrangement comprises array point sample loading volume, array arrangement and inter-point distance;
the preparation of the polypeptide microarray chip comprises the following steps:
(1) preparing amino acids and other reagents required by synthesis, wherein the amino acids can be natural amino acid derivatives or compounds containing at least one amino group and one carboxyl group;
(2) operating the instrument, and accurately and quantitatively transferring the specified solution from the amino acid solution placing platform by the sample application needle according to the preset value;
(3) the mechanical arm stably moves to drive the sample application needle to print the transferred amino acid solution to a specified position on the carrier substrate;
(4) the carrier matrix is stably fixed on a carrier placing platform, one or more carrier matrixes can be placed on the platform at the same time, and the matrix material used by the carrier is prepared according to the requirements of users;
(5) preparing 40 sample pore plates by using an amino acid solution placing platform, wherein the reagent is fully opened, wherein 20 sample pore plates fix amino acid positions, and 20 sample pore plates are used in an open mode;
(6) the spotting needle will automatically clean every time it has removed one amino acid to prevent sample cross-contamination. The point sampling needle cleaning tank comprises a waste liquid collecting tank and a cleaning tank which are respectively and independently arranged at two sides of the point sampling needle cleaning module;
(7) the sample application needle is connected with a micro-injection pump to realize accurate quantitative washing liquid and spraying liquid;
the polypeptide microarray chip activation treatment comprises the following steps:
(1) the instrument prints the amino acid solution on the appointed position of the carrier matrix after special treatment to obtain a semi-finished product of the polypeptide chip;
(2) and passivating and sealing the chip. Standing the synthesized chip in a sealing solution, sealing unreacted activated groups on the chip, washing with ethanol, and naturally drying for the next step;
(3) repeating the steps (1) and (2) until the set dot printing setting is completed, and obtaining a semi-finished product of the polypeptide chip;
(4) and activating the chip activity. Standing the chip in an activating solution to remove the protecting groups on the amino acid so as to release the activity of the amino groups on the chip, thereby obtaining an active polypeptide chip;
(5) air drying, and storing at-20 deg.C under sealed condition;
the polypeptide microarray chip is applied as follows:
(1) hydrating the active polypeptide chip prepared as above;
(2) hybridizing the hydrated chip with a corresponding antibody;
(3) and placing the reacted chip in an ECL chemiluminescent reagent for reaction, scanning the chip by using a corresponding instrument, and analyzing the result.
Compared with the background technology, the invention has the following beneficial effects:
by adopting the technical scheme, the polypeptide microarray synthesis device is used for solid-phase synthesis of the polypeptide chip for epitope screening. Polypeptide microarray instrument is adopted to design polypeptide sequence, and amino acid is printed on the specially treated carrier matrix layer by layer to synthesize polypeptide chip. The synthesized chip can be applied to the development of antigens of COVID-19Omicron and the development and research of corresponding vaccine-based drugs.
[ description of the drawings ]
FIG. 1 is a schematic view of a polypeptide microarray synthesizer;
FIG. 2 is a schematic diagram of a platform of a polypeptide microarray synthesizer;
FIG. 3 is a left side view of the micro-syringe;
fig. 4 is a front view of the micro-syringe.
[ detailed description ] embodiments
The technical scheme and the beneficial effects of the invention are clearer and clearer by further describing the specific embodiment of the invention with the accompanying drawings of the specification. The embodiments described below are exemplary and are intended to be illustrative of the invention, but are not to be construed as limiting the invention.
An epitope screening method based on a polypeptide microarray comprises the following steps:
1. a polypeptide array synthesis method for screening antigen epitopes of COVID-19Omicron spike protein (S protein), comprising the following steps:
1.1, introducing a primary structure sequence of COVID-19Omicro spike protein (S protein) into a polypeptide microarray instrument VERSA 110 SPOTTER;
1.2, setting the length of a peptide chain on the prepared polypeptide microarray chip, taking 12 amino acids as a polypeptide, designing a polypeptide array point through a VERSA 110SPOTTER software of an Aurora polypeptide microarray instrument, wherein the number of the amino acids of the polypeptide can be more than or less than 12 amino acids;
1.3, setting the microarray arrangement on the polypeptide chip, the sample loading volume of array points, the array arrangement and the distance between the points;
2. preparing a polypeptide microarray chip:
2.1, preparing an amino acid solution required by the experiment, subpackaging, refrigerating and storing, and balancing to room temperature before dotting;
2.2, operating an instrument, and accurately transferring the amino acid solution from the amino acid solution placing platform by using a sample application needle;
2.3, stably moving the mechanical arm to drive the sample application needle to print the transferred amino acid solution to a specified position on the carrier substrate;
2.4, preparing 40 sample pore plates by using an amino acid solution placing platform, wherein the reagent is fully opened, wherein 20 sample pore plates fix amino acid positions, and 20 sample pore plates are used in an open mode;
2.5, the sample application needle is automatically cleaned after taking one amino acid to prevent the cross contamination of samples. The sample application needle cleaning tank comprises a waste liquid collecting tank and a cleaning tank which are respectively and independently arranged at two sides of the sample application needle cleaning module;
2.6, printing a layer of amino acid solution on each spot, drying, placing the semi-finished product of the polypeptide chip in a chip passivation solution, sealing unreacted activated groups on the chip, washing with ethanol, and naturally drying for the next step;
2.7, repeating the operation until the set point printing setting is completed to obtain a semi-finished product of the polypeptide chip;
2.8, standing the prepared semi-finished polypeptide chip in activating solution to remove a protecting group on amino acid so as to release the activity of amino on the chip, and obtaining the polypeptide chip with activity;
3. the active polypeptide chip prepared by the method is dried and then is stored at minus 20 ℃ in a sealing way for later use, or is directly hydrated for antibody hybridization reaction.
As shown in fig. 1-4, a device comprises a polypeptide microarray synthesizer, the polypeptide microarray synthesizer comprises a high-efficiency filtering safety cover 1, a mechanical arm 2, a sample application needle 3, a workbench 4, an amino acid solution placing platform 6, a sample application needle cleaning module 7, a carrier placing platform 8, a pre-sample application platform 9 and a micro-injection pump 10, and the device comprises the following steps;
the high-efficiency filtering safety cover 1 comprises a closed UV outer cover, negative pressure exhaust, ultraviolet sterilization and LED lamps. The ultraviolet-resistant transparent resin glass sealed protective cover can effectively prevent toxic volatile gas from damaging human bodies, ensure the safety of operators and samples, and reduce the damage of ultraviolet lamps to users and other articles during operation;
the mechanical arm 2 can stably and precisely move in the X, Y, Z direction, and accurate positioning is realized;
the sample application needle 3 can be set according to a customer and accurately and quantitatively transfers and takes liquid;
preparing 40 sample pore plates by using an amino acid solution placing platform 6, wherein the reagent is fully opened, wherein 20 sample pore plates fix amino acid positions, and 20 sample pore plates are used in an open mode;
the point sampling needle cleaning module 7 comprises a cleaning tank for collecting waste liquid and preventing sample cross contamination, and the point sampling needle cleaning tanks are respectively and independently arranged at two sides of the point sampling needle cleaning module;
the carrier placing platform 8 can simultaneously place one or more carrier matrixes, and the matrix materials used by the carriers are prepared according to the requirements of users;
the micro-injection pump 10 is provided with a first interface 14 and a second interface 15, the first interface 14 is connected with the sample application needle 3, and the second interface 15 is connected with the solvent;
a control circuit board 11 and a stepping motor 12 are provided at one side of the micro syringe pump 10.
In the description of the specification, reference to the description of "one embodiment", "preferably", "an example", "a specific example" or "some examples", etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention, and schematic representations of the terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
With the above structure and principle in mind, those skilled in the art should understand that the present invention is not limited to the above embodiments, and modifications and substitutions based on the known technology in the field are within the scope of the present invention, which should be limited by the claims.

Claims (7)

1. An epitope screening method based on a polypeptide microarray is characterized by comprising the following steps:
1.1, opening a switch of a power supply of the polypeptide microarray synthesizer and connecting a computer;
1.2, opening synthesizer operating software, introducing a primary sequence of a protein to be researched into the software according to the use requirement of a customer, and setting the length of the polypeptide and the microarray arrangement of chips;
1.3, preparing amino acid and other reagents required by synthesis, operating an instrument, and dotting the amino acid solution to a set position by the instrument until the preparation of the polypeptide chip is completed.
2. The method of claim 1, wherein the synthesizer operating software comprises the steps of:
2.1, taking 12 amino acids as a polypeptide, and designing a polypeptide microarray through the software, wherein the number of the amino acids of the polypeptide can be more than or less than 12 amino acids;
2.2, the polypeptide microarray layout can be set by the software, including the array spot loading volume, array layout and inter-spot distance.
3. An apparatus comprising the polypeptide microarray synthesizer of claim 1, wherein the polypeptide microarray synthesizer comprises a high efficiency filtration safety shield, a mechanical arm, a spotting needle, a work table, an amino acid solution placement platform, a spotting needle cleaning module, a carrier placement platform, a pre-spotting platform, and a micro-syringe pump.
4. The apparatus of claim 3, wherein the high efficiency filtration safety enclosure comprises a closed UV enclosure, negative pressure exhaust, UV sterilization, LED light.
5. The device of claim 3, wherein the micro syringe pump has a second first port and a second port, the first port is connected to the sample application needle, and the second port is connected to the solvent.
6. The device of claim 3, wherein the micro syringe pump is provided with a control circuit board and a stepping motor on one side.
7. The method for screening epitopes based on polypeptide microarray of claim 1, wherein the preparation of the polypeptide chip comprises the steps of:
7.1, setting the length of the polypeptide on the chip and the distribution of the microarray according to software, and dotting an amino acid solution to a specified position of a carrier matrix by an instrument according to a software instruction to obtain a semi-finished product of the polypeptide chip;
7.2, preparing a chip passivation sealing liquid, standing the synthesized chip in the sealing liquid, sealing unreacted activated groups on the chip, washing with ethanol, and naturally drying for the next step;
7.3, repeating the steps 7.1 and 7.2 until the set spot printing setting is completed, and obtaining a semi-finished product of the polypeptide chip;
7.4, preparing a chip activating solution, standing the chip in the activating solution to remove a protecting group on the amino acid so as to release the activity of the amino on the chip, and obtaining a polypeptide chip with activity;
7.5, air-drying, and storing at-20 ℃ in a sealing way for later use;
7.6, adding corresponding antibody for hybridization reaction;
7.7, placing the chip in an ECL chemiluminescence reagent for reaction, scanning the chip by using a corresponding instrument, and analyzing the result.
CN202210265753.XA 2022-03-17 2022-03-17 Epitope screening method and device based on polypeptide microarray Pending CN114622287A (en)

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CN202210265753.XA CN114622287A (en) 2022-03-17 2022-03-17 Epitope screening method and device based on polypeptide microarray

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Application Number Priority Date Filing Date Title
CN202210265753.XA CN114622287A (en) 2022-03-17 2022-03-17 Epitope screening method and device based on polypeptide microarray

Publications (1)

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CN114622287A true CN114622287A (en) 2022-06-14

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