Polyclonal antibody aiming at near-infrared fluorescent protein emiRFP as well as preparation and application of polyclonal antibody

Abstract

The invention relates to the technical field of biological medicine, in particular to a polyclonal antibody aiming at near-infrared fluorescent protein emiRFP as well as preparation and application of the polyclonal antibody. The preparation method comprises the following steps: taking the emiRFP polypeptide as shown in SEQ ID NO.1 as an antigen immune animal, collecting serum, and collecting the polyclonal antibody aiming at the emiRFP from the serum. Compared with the prior art, the polyclonal rabbit antibody has the beneficial effects that the polyclonal rabbit antibody of the near-infrared fluorescent protein emiRFP is successfully prepared for the first time, the technical blank is filled, and an effective supplement scheme is provided for detection and application of the near-infrared fluorescent protein.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a polyclonal antibody against the near-infrared fluorescent protein emiRFP, its preparation and application. Background Technology

[0002] Fluorescently labeled proteins are widely used for gene expression detection and cell lineage tracking. Commonly used fluorescent proteins include visible light-excited green fluorescent protein (GFP) and red fluorescent protein (RFP), as well as their variants. However, studies have reported that the immunogenicity and cytotoxicity of GFP can confound the interpretation of in vivo experimental data, leading to false positives or false negatives. Furthermore, GFP expression accumulates a metabolic burden over time, making GFP-labeled cells more susceptible to death under environmental stress. RFP's disadvantages include a slow and incomplete maturation process and oligomerization. The slow maturation process limits its application as a reporter gene, such as detecting short-term gene expression and conditions during rapid organism development. It is generally believed that long-wavelength photon excitation has low phototoxicity to cells and tissues, and exhibits minimal autofluorescence and light absorption in animal tissues. Therefore, the modification of fluorescent proteins has gradually shifted towards red. Among the many fluorescent proteins, near-infrared fluorescent proteins (NIIR) have advantages such as low background imaging and the ability to perform in vivo imaging, thus gaining favor among researchers.

[0003] The research on fluorescent proteins with emission wavelengths above 600 nm has two main objectives: First, fluorescent proteins that emit light within the near-infrared window (650-900 nm) are highly needed in bioimaging techniques because biomolecules such as melanin, hemoglobin, and water exhibit minimal absorption in the near-infrared window when light penetrates tissue. Second, such fluorescent proteins can provide more options for multi-wavelength imaging techniques, and their application in in vivo imaging techniques can produce less phototoxicity (Shcherbakova DM. Near-infrared and far-red genetically encoded indicators of neuronal activity[J]. Journal of Neuroscience Methods, 2021, 362:109314.). Although scientists have made numerous attempts to find far-infrared red fluorescent proteins, a very stable and widely used in vivo and in vitro reporter system is still lacking in the field. In addition to the stability of fluorescence spectroscopy, it is also necessary to systematically evaluate the stability, specificity and compatibility with existing genetic marker systems of in vivo cell fate lineage tracing (Hontani Y, Balban M, Escobar FV, et al. Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome[J]. Communications chemistry, 2021, 4(1): 3.).

[0004] Regarding near-infrared fluorescent proteins, CN117286183A describes the construction of emiRFP near-infrared fluorescent protein transgenic reporter mice, but antibodies against near-infrared fluorescent proteins are currently lacking. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a polyclonal antibody against the near-infrared fluorescent protein emiRFP, and its preparation and application.

[0006] One or more embodiments of this application provide a method for preparing a polyclonal antibody against emiRFP, the method comprising the following steps:

[0007] Animals were immunized with the emiRFP polypeptide shown in SEQ ID NO.1 as an antigen, and serum was collected. Polyclonal antibodies against emiRFP were collected from the serum.

[0008] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0009] (1) The animals mentioned include rabbits;

[0010] (2) Immunization methods include injection; and,

[0011] (3) The number of immunizations is multiple.

[0012] In some embodiments of this application, multiple immunizations include:

[0013] In the immunization composition 1 used for the first immunization, the immunization adjuvant is a complete adjuvant;

[0014] In the second, third, and fourth immunizations, the immunizing adjuvants used in immunizing compositions 2, 3, and 4, respectively, were incomplete adjuvants.

[0015] In some embodiments of this application, multiple immunizations satisfy one or more of the following conditions:

[0016] 1) The complete adjuvant comprises paraffin oil, mannitol monooleate, and inactivated Mycobacterium tuberculosis;

[0017] 2) The incomplete adjuvants in immune compositions 2, 3, and 4 all include paraffin oil and mannitol monooleate; and,

[0018] 3) The volume percentage of the immune adjuvant in immune composition 1, immune composition 2, immune composition 3 and immune composition 4 is 45%-55% respectively.

[0019] In some embodiments of this application, the concentration of the antigen in each of the immune compositions 1, 2, 3 and 4 is independently 0.5 g / mL to 1.5 g / mL.

[0020] In some embodiments of this application, multiple immunizations include:

[0021] The dosage of the antigen used for the initial immunization is 0.45g-0.55g per animal;

[0022] The dosage of the antigen used for the second, third, and fourth immunizations is 0.2g-0.3g per animal.

[0023] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0024] (A) The second immunization should be administered 13-15 days after the first immunization;

[0025] (B) The interval between the second and third immunizations is 6-8 days;

[0026] (C) The fourth immunization will be administered 6-8 days after the third immunization.

[0027] In some embodiments of this application, the pH of the collected product is maintained at 6.5-7.5 during the collection process;

[0028] Optionally, the preparation method further includes a step of purifying the polyclonal antibody against emiRFP in the collected product, the purification step including sequentially performing membrane filtration and column chromatography on the collected product;

[0029] Optionally, the membrane filtration uses a microporous membrane with a pore size of 0.4μm-0.5μm;

[0030] Optionally, column chromatography satisfies one or more of the following conditions:

[0031] A) Chromatographic columns include affinity chromatography columns;

[0032] B) The eluent consists of a glycine solution with a concentration of 0.1M-0.3M and a pH of 2.5-3; and,

[0033] C) The number of column chromatography steps is 1 to 3.

[0034] Alternatively, column chromatography may also satisfy one or more of the following conditions:

[0035] D) The sample loading rate is 35 mL / h - 45 mL / h;

[0036] E) The washing solution includes 1×PBS buffer with a pH of 7-8; and,

[0037] F) The flow rate of the cleaning solution is 65 mL / h-75 mL / h.

[0038] One or more embodiments of this application also provide polyclonal antibodies against emiRFP prepared by the preparation method described above.

[0039] One or more embodiments of this application also provide an emiRFP detection kit comprising the polyclonal antibody against emiRFP described above.

[0040] Compared to traditional technologies, the beneficial effects of this application include:

[0041] This application successfully prepared a polyclonal rabbit antibody against the near-infrared fluorescent protein emiRFP for the first time, filling a technological gap and providing an effective supplementary solution for the detection and application of near-infrared fluorescent proteins. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 The results of electrophoresis for the high-level expression and purification of the antibody in Example 1;

[0044] Figure 2 This is a diagram showing the expression results of emiRFP protein in 293T-emiRFP cells in Example 2;

[0045] Figure 3 The image shows the genotype identification results of hGFAP-Cre;H11-LSL-emiRFP mice in Example 3. Detailed Implementation

[0046] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0048] the term

[0049] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0050] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0051] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0052] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0053] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0054] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0055] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0056] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0057] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0058] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0059] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0060] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0061] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0062] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0063] Based on this, one or more embodiments of this application provide a polyclonal antibody against the near-infrared fluorescent protein emiRFP, and its preparation and application.

[0064] A first aspect of this application provides a method for preparing a polyclonal antibody against emiRFP, the method comprising the following steps:

[0065] Animals were immunized with the emiRFP polypeptide shown in SEQ ID NO.1 as an antigen, and serum was collected. Polyclonal antibodies against emiRFP were collected from the serum.

[0066] In one embodiment, the preparation method satisfies one or more of the following conditions:

[0067] (1) The animals mentioned include rabbits;

[0068] (2) Immunization methods include injection; and,

[0069] (3) The number of immunizations is multiple.

[0070] In one implementation, multiple immunizations include:

[0071] In the immunization composition 1 used for the first immunization, the immunization adjuvant is a complete adjuvant;

[0072] In the second, third, and fourth immunizations, the immunizing adjuvants used in immunizing compositions 2, 3, and 4, respectively, were incomplete adjuvants.

[0073] In one implementation, multiple immunizations satisfy one or more of the following conditions:

[0074] 1) The complete adjuvant comprises paraffin oil, mannitol monooleate and inactivated Mycobacterium tuberculosis; the complete adjuvant in the examples was purchased from Merk, catalog number F5881 (each 1 mL contains 1 mg of heat-killed and dried Mycobacterium tuberculosis (H37Ra, ATCC 25177), 0.85 mL of paraffin oil and 0.15 mL of mannitol monooleate);

[0075] 2) The incomplete adjuvants in immune compositions 2, 3, and 4 include paraffin oil and mannitol monooleate; the incomplete adjuvants in the examples were purchased from Merk, catalog number F5506 (containing 0.85 mL paraffin oil and 0.15 mL mannitol monooleate per milliliter); and,

[0076] 3) The volume percentage of the immune adjuvant in immune composition 1, immune composition 2, immune composition 3 and immune composition 4 is 45%-55% respectively.

[0077] In one embodiment, the concentration of the antigen in each of the immune compositions 1, 2, 3 and 4 is independently 0.5 g / mL to 1.5 g / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 g / mL).

[0078] In one implementation, multiple immunizations include:

[0079] The dosage of the antigen used for the initial immunization is 0.45g-0.55g per animal (e.g., 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55g).

[0080] The dosage of the antigen used for the second, third, and fourth immunizations is 0.2g-0.3g per animal.

[0081] In one embodiment, the preparation method satisfies one or more of the following conditions:

[0082] (A) The second immunization should be administered 13-15 days after the first immunization (e.g., 13, 14, 15 days);

[0083] (B) The interval between the second and third immunizations is 6-8 days (e.g., 6, 7, 8 days);

[0084] (C) The fourth immunization is administered 6-8 days after the third immunization (e.g., 6, 7, 8 days).

[0085] In one embodiment, the pH of the collected product is maintained at 6.5-7.5 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5) during the collection process;

[0086] Optionally, the preparation method further includes a step of purifying the polyclonal antibody against emiRFP in the collected product, the purification step including sequentially performing membrane filtration and column chromatography on the collected product;

[0087] Optionally, the membrane filtration uses a microporous membrane with a pore size of 0.4μm-0.5μm;

[0088] Optionally, column chromatography satisfies one or more of the following conditions:

[0089] A) Chromatographic columns include affinity chromatography columns;

[0090] B) The eluent comprises a glycine solution with a concentration of 0.1M-0.3M (e.g., 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3) and a pH of 2.5-3 (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3); and,

[0091] C) The number of column chromatography steps is 1 to 3 (e.g., 1, 2, 3).

[0092] Alternatively, column chromatography may also satisfy one or more of the following conditions:

[0093] D) The sample loading flow rate is 35 mL / h-45 mL / h (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 mL / h);

[0094] E) The washing solution comprises 1×PBS buffer at pH 7-8 (e.g., pH 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8); and,

[0095] F) The flow rate of the cleaning solution is 65 mL / h-75 mL / h (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 mL / h).

[0096] A second aspect of this application provides a polyclonal antibody against emiRFP prepared by the preparation method.

[0097] A third aspect of this application also provides an emiRFP detection kit, which includes the aforementioned polyclonal antibody against emiRFP.

[0098] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0099] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0100] Example 1: Preparation of emiRFP antibody

[0101] (1) Based on the amino acid sequence of emiRFP, protein fragments were designed, and expression plasmids were constructed by synthesizing the whole gene to express and purify the protein in the E. coli system, and the antigen shown in SEQ ID NO.1 was prepared.

[0102] SEQ ID NO.1:

[0103] MAEGSVARQPDLLTCEHEEIHLAGSIQPHGALLVVSEHDHRVIQASANAAEFLNLGSVLGVPLAEIDGDLL

[0104] IKILPHLDPTAEGMPVAVRCRIGNPSTEYCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRAL

[0105] CDDTVLLFQQCTGYDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLV

[0106] DVTYQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVVCHHYLPRFIRFELRAICKRLAERIATRITALES.

[0107] (2) Immunization, serum titer evaluation and serum collection: Two New Zealand white rabbits were selected for antigen immunization. Specifically, antigen was extracted (antigen was completely mixed), the antigen concentration for the first immunization was 1g / mL, and the amount of antigen for each rabbit was 0.5mL. The amount of antigen was halved for the second to fourth immunizations.

[0108] The adjuvant and antigen are drawn in a 1:1 volume ratio. For the first immunization, a complete adjuvant (commercially available, specifically liquid paraffin, lanolin, and BCG) is used; for the second to fourth immunizations, an incomplete adjuvant (commercially available, specifically liquid paraffin and lanolin) is used. The adjuvant must be thoroughly mixed before being drawn into the syringe. Two syringes are connected using a syringe plunger for complete emulsification. The emulsification standard is: the emulsified immunogen should not disperse when dropped into 37°C water.

[0109] Immunization: Rabbits were given multiple subcutaneous injections, 0.2 mL at each point, 0.5 mL per rabbit.

[0110] Immunization schedule: The second immunization is administered 14 days after the first immunization, with a 7-day interval between the second and third immunizations. A small serum sample is collected from the middle ear artery 7 days after the third immunization for testing. If the test is satisfactory, a booster immunization (fourth immunization) is administered 7 days later. Whole blood can be collected 7 days after the booster immunization. An ELISA titer of 1:64000 with an OD value greater than 0.6 indicates successful immunization.

[0111] (3) Antibody purification:

[0112] The affinity chromatography column (packing material purchased from Tiandi Renhe, protein A packing material, SA012025, 5 mL) was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0113] Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0114] The filtered serum sample was loaded onto the affinity chromatography column at a flow rate of 40 mL / h, and the process was repeated once.

[0115] Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) reading to 100; adjust the absorbance (1A) reading of the protein analyzer to 0. At this time, turn on the HDA computer acquisition device on the computer desktop and adjust the full-screen range to 5. Elute the antibody with glycine solution (pH 2.7, 0.2M) at a rate of 40 mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0116] During antibody collection, the pH of the antibody was adjusted to approximately 7 using 1M sodium bicarbonate, and the highest peak value of the elution peak was recorded.

[0117] After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tubing connected to the collector with purified water.

[0118] The affinity chromatography column was washed sequentially with 20 mL of 1×PBS and water at a rate of 70 mL. Then, an ethanol-water solution with a volume ratio of 20% was added to seal the column, and it was stored at 4°C.

[0119] The purified antibodies were sent for testing according to different requirements. After sufficient antibody quantity was purified for delivery and the titer of the semi-finished product was qualified, all antibodies were mixed and concentrated using an ultrafiltration concentrator to achieve a certain concentration and volume. The concentrated antibody was then dialyzed in 1L of 0.01M PBS (pH 7.4) at room temperature, with the medium changed every 3 hours for a total of 3 changes (overnight dialyzing was performed in a refrigerator at 2-8℃). The dialyzed antibody was then transferred to a clean centrifuge tube and filtered through a 0.22μm disposable low-adsorption filter in a clean bench. A small sample was sent for testing, and another 5μL was tested for concentration using a Protein A280 application on a micro-volume spectrophotometer (denovix DS-11). The purified antibody was considered qualified if the ELISA titer was greater than 1:64000. The electrophoresis results of antibody large-scale expression and purification are as follows. Figure 1 The ELISA titer test results are shown in Table 1.

[0120] Table 1. Detection of purified antibody titer

[0121]

[0122] Example 2: Constructing a cell line overexpressing emiRFP protein and performing antibody detection.

[0123] (1) The near-infrared fluorescent protein emiRFP (Matlashov ME, Shcherbakova DM, Alvelid J, et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales[J]. Nat Commun, 2020, 11(1):239.) was integrated into the lentiviral expression vector (rLV-EF1A-pemiRFP-N1-PGK-PURO-WPRE) and infected 293T (human embryonic kidney cells) cells to establish a 293T-emiRFP overexpression cell line.

[0124] (2) Proteins from the 293T-emiRFP cell line were collected, loaded with loading buffer, and subjected to protein electrophoresis to detect the expression of the near-infrared fluorescent protein emiRFP in the 293T-emiRFP cell line, verifying the application of anti-emiRFP antibody in cellular proteins. Western blotting results are shown below. Figure 2 .

[0125] Figure 2 The figures show the expression of emiRFP protein in 293T-emiRFP cells; Figure A shows 293T cells successfully transfected with emiRFP lentivirus, bar = 50 μm; Figure B shows the expression of emiRFP protein in 293T-emiRFP cells (protein size 35 kDa).

[0126] The specific operation steps in this embodiment are as follows:

[0127] 1) Viral infection

[0128] Day 1: Seed healthy 293T cells into 24-well plates at a cell concentration of 3 × 10⁻⁶ cells / well. 5 / mL, 500μL / well, incubate overnight in a 37℃, 5% CO2 incubator.

[0129] Day 2: Viral Infection (1 / 2 Volume Infection Method). For viral infection, add 1 / 2 volume of fresh culture medium. After 4 hours of lentivirus infection, replenish to the total culture volume. The culture medium volume for a 24-well plate is 500 μL, and the half-volume culture volume is 250 μL. Before infection, remove the virus from the freezer and thaw it slowly on ice. Aspirate the original culture medium from the cells, add 1 / 2 volume of fresh culture medium, and add an appropriate volume of virus based on the determined MOI (virus volume per well (μL) = MOI × cell number / virus titer (TU / mL) × 1000). For cells requiring polybrene, add an appropriate amount of polybrene simultaneously. After 4 hours of lentivirus infection, replenish to the total culture volume.

[0130] Day 3: Change the medium. On the second day after infection (about 24 hours), aspirate the culture medium containing the virus, replace it with fresh complete culture medium, and continue incubation at 37°C.

[0131] Days 4-5: Observe fluorescence. 48 hours post-infection, emiRFP expression efficiency can be initially observed using a fluorescence microscope, reaching its peak at 72 hours post-infection. In addition, the virus carries a Puromycin resistance gene; after replacing the medium with fresh complete culture medium containing an appropriate concentration of Puromycin, stable transduced cell lines are screened.

[0132] 2) Protein electrophoresis Western blotting

[0133] A standard curve was prepared using standard proteins. Based on the relationship between absorbance and concentration of the standard curve, loading buffer was added to the sample to prepare the sample protein. The sample was boiled in 100℃ boiling water for 6 min. The protein sample was added to an SDS-PAGE gel for electrophoresis for 2 h, then transferred to a PVDF membrane for 2 h, and then blocked with 5% skim milk at room temperature for 1 h. The primary antibody (anti-emiRFP) was incubated overnight (at least 8 h) at 4℃. The next day, the sample was washed 3 times with 1×TBST for 5 min each time. The secondary antibody was incubated at room temperature for 1 h, washed 3 times with 1×TBST for 5 min each time, and then exposed and photographed after adding ECL developer.

[0134] Example 3: Constructing transgenic mice overexpressing emiRFP protein and performing antibody detection.

[0135] hGFAP-cre;H11-LSL-emiRFP transgenic mice were established (following the method described in CN117286183A). Brain and intestinal tissue proteins were collected, and emiRFP fluorescent protein was detected by protein electrophoresis. Results are as follows: Figure 3 As shown.

[0136] Figure 3 Genotyping of hGFAP-Cre;H11-LSL-emiRFP mice; Figure A shows the small genotyping of hGFAP-Cre;H11-LSL-emiRFP mice (GFAP-Cre+IC:300 / 500kd; H11-LSL-emiRFP:335kd); Figure B shows immunofluorescence staining of brain tissue from hGFAP-Cre;H11-LSL-emiRFP mice.

[0137] Brain tissue sections were collected and prepared, and anti-emiRFP antibodies were used to detect whether they were co-labeled with emiRFP. The results are as follows: Figure 3 As shown in Figure B, bar = 25 μm.

[0138] The operation steps in this embodiment are as follows:

[0139] hGFAP-cre;H11-LSL-emiRFP transgenic mice were perfused with pre-cooled 4% PFA and PBS, respectively. Brain and intestinal tissues were collected, embedded in OCT, sectioned, and then subjected to subsequent immunofluorescence staining. Specifically: the mice were washed three times with 1×PBS for 5 min each time; a blocking agent (2% normal donkey serum containing 0.3% Triton X-100) was added and incubated at room temperature for 20 min; the blocking agent was removed, anti-emiRFP antibody was added, and the mice were placed in a 4°C refrigerator overnight; the next day, after returning to room temperature, the mice were washed three times with 1×PBST; fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 h, followed by washing three times with 1×PBST; finally, the mice were incubated with DAPI for 10 min, anti-fluorescence quencher was added, and the slides were mounted, observed under a microscope, and photographed.

[0140] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0141] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a polyclonal antibody against emiRFP, characterized in that, The preparation method includes the following steps: Animals were immunized with the emiRFP polypeptide shown in SEQ ID NO.1 as an antigen, and serum was collected. Polyclonal antibodies against emiRFP were collected from the serum.

2. The method for preparing a polyclonal antibody against emiRFP according to claim 1, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The animals mentioned include rabbits; (2) Immunization methods include injection; and, (3) The number of immunizations is multiple.

3. The method for preparing a polyclonal antibody against emiRFP according to claim 2, characterized in that, Multiple immunizations include: In the immunization composition 1 used for the first immunization, the immunization adjuvant is a complete adjuvant; In the second, third, and fourth immunizations, the immunizing adjuvants used in immunizing compositions 2, 3, and 4, respectively, were incomplete adjuvants.

4. The method for preparing a polyclonal antibody against emiRFP according to claim 3, characterized in that, Multiple immunizations meet one or more of the following conditions: 1) The complete adjuvant comprises paraffin oil, mannitol monooleate, and inactivated Mycobacterium tuberculosis; 2) The incomplete adjuvants in immune compositions 2, 3, and 4 all include paraffin oil and mannitol monooleate; and, 3) The volume percentage of the immune adjuvant in immune composition 1, immune composition 2, immune composition 3 and immune composition 4 is 45%-55% respectively.

5. The method for preparing a polyclonal antibody against emiRFP according to claim 3, characterized in that, The concentration of the antigen in each of the immune compositions 1, 2, 3 and 4 is independently 0.5 g / mL to 1.5 g / mL.

6. The method for preparing a polyclonal antibody against emiRFP according to claim 3, characterized in that, Multiple immunizations include: The dosage of the antigen used for the initial immunization is 0.45g-0.55g per animal; The dosage of the antigen used for the second, third, and fourth immunizations is 0.2g-0.3g per animal.

7. The method for preparing a polyclonal antibody against emiRFP according to claim 3, characterized in that, The preparation method satisfies one or more of the following conditions: (A) The second immunization should be administered 13-15 days after the first immunization; (B) The interval between the second and third immunizations is 6-8 days; (C) The fourth immunization will be administered 6-8 days after the third immunization.

8. The method for preparing a polyclonal antibody against emiRFP according to any one of claims 1 to 7, characterized in that, During the collection process, maintain the pH of the collected product at 6.5-7.5; Optionally, the preparation method further includes a step of purifying the polyclonal antibody against emiRFP in the collected product, the purification step including sequentially performing membrane filtration and column chromatography on the collected product; Optionally, the membrane filtration uses a microporous membrane with a pore size of 0.4μm-0.5μm; Optionally, column chromatography satisfies one or more of the following conditions: A) Chromatographic columns include affinity chromatography columns; B) The eluent consists of a glycine solution with a concentration of 0.1M-0.3M and a pH of 2.5-3; and, C) The number of column chromatography steps is 1 to 3; Alternatively, column chromatography may also satisfy one or more of the following conditions: D) The sample loading rate is 35 mL / h - 45 mL / h; E) The washing solution includes 1×PBS buffer with a pH of 7-8; and, F) The flow rate of the cleaning solution is 65 mL / h-75 mL / h.

9. A polyclonal antibody against emiRFP prepared by the preparation method according to any one of claims 1 to 8.

10. An emiRFP detection kit comprising the polyclonal antibody against emiRFP as described in claim 9.

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