Preparation method of beta 2-microglobulin monoclonal antibody
阅读说明：本技术 一种β2-微球蛋白单克隆抗体的制备方法 (Preparation method of beta 2-microglobulin monoclonal antibody ) 是由 高健 陈旭华 汪渊 谌敦华 于 2019-10-11 设计创作，主要内容包括：本发明公开了一种β2-微球蛋白单克隆抗体的制备方法。该方法首先筛选出免疫原性相对较强的两个多肽表位序列分别作为第一、第二抗原表位,再在第一、第二抗原表位的碳端氨基酸上分别连接1个半胱氨酸,通过SMCC偶联方法将半胱氨酸的巯基与蓝钥蛋白的伯胺基偶联,获得体外合成的β2-MG双表位多肽抗原；然后利用双表位多肽抗原对小鼠进行常规免疫、分离出脾淋巴细胞与骨髓瘤细胞进行细胞融合；最后筛选出阳性单克隆细胞,经腹腔注射及腹水回收提取出单克隆抗体。本发明通过对传统单表位抗原免疫方法进行改进,优化了抗原的免疫原性,缩短了免疫周期,提高了免疫阶段的血清效价,从而提升了抗β2-MG的阳性单克隆抗体获得效率。(The invention discloses a preparation method of a beta 2-microglobulin monoclonal antibody. Firstly, screening two polypeptide epitope sequences with relatively strong immunogenicity as a first antigen epitope and a second antigen epitope respectively, connecting 1 cysteine to the carbon-terminal amino acids of the first antigen epitope and the second antigen epitope respectively, and coupling the sulfydryl of the cysteine and the primary amino of a blue-key protein by an SMCC coupling method to obtain a beta 2-MG double-epitope polypeptide antigen synthesized in vitro; then, conventional immunization is carried out on the mouse by using the double-epitope polypeptide antigen, and spleen lymphocytes and myeloma cells are separated out for cell fusion; and finally, screening out positive monoclonal cells, and extracting the monoclonal antibody through intraperitoneal injection and ascites recovery. The invention optimizes the immunogenicity of the antigen, shortens the immune cycle and improves the serum titer in the immune stage by improving the traditional single epitope antigen immune method, thereby improving the obtaining efficiency of the anti-beta 2-MG positive monoclonal antibody.)
1. A preparation method of a beta 2-microglobulin monoclonal antibody is characterized by comprising the following specific steps:
(1) screening two polypeptide epitope sequences IQRTPKIQVYSRHPAEN and YLLYYTEFTPTEK with relatively strong immunogenicity based on an NCBI platform and an IEDB platform to serve as a first epitope and a second epitope respectively;
(2) respectively connecting 1 cysteine Cys to the asparagine N at the tail end of a first antigen epitope and the tyrosine Y at the tail end of a second antigen epitope, and then coupling the sulfydryl of the cysteine and the primary amino of the blue-key protein KLH by using an SMCC coupling method to simultaneously connect two polypeptide epitope sequences to a KLH molecule to serve as a beta 2-MG double-epitope polypeptide antigen synthesized in vitro;
(3) after 3 times of continuous immunization is carried out on 8 w-age BALB/C mice by adopting beta 2-MG bi-epitope polypeptide antigen, spleen is taken out 3 days after the last immunization, spleen lymphocytes are separated by using lymphocyte separation liquid, and the spleen lymphocytes are fused with myeloma cells SP2/0-Ag 14; after cell fusion, screening by adopting an indirect ELISA method, cloning a positive hybridoma cell strain by using a limiting dilution method, and extracting the monoclonal antibody of the anti-beta 2-MG from the obtained monoclonal cell of the anti-beta 2-MG antibody by intraperitoneal injection and ascites recovery.
2. The method of claim 1, wherein in step (1), two polypeptide epitope sequences with relatively strong immunogenicity are screened by using hydrophobic and surface accessibility indexes in a web.
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a beta 2-microglobulin monoclonal antibody.
Background
The current clinical detection methods of the beta 2-MG are mainly Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), and the methods all need monoclonal or polyclonal antibodies of the beta 2-MG. However, because β 2-MG participates in the formation of histocompatibility complex (MHC I and MHC II) or similar dimers, and as a result, there is some homology between human and mouse β 2-MG, it is difficult to obtain a large number of positive monoclonal antibodies in mice subjected to natural antigen immuno-fusion [ 2 ] and [ 3 ]. In order to solve the above problems, the conventional scheme is to express the amino acid sequence of a specific target epitope fragment in vitro by a eukaryotic expression system, and perform immunization and hybridoma fusion by using a single epitope polypeptide design and in vitro synthesis method, but it is difficult to obtain a high number of positive monoclonal antibodies, and the research has proved.
Reference to the literature
1.Calabrese M F,Miranker AD.Formation of a stable oligomer of beta-2microglobulin requires only transient encounter with Cu(II).Journal ofMolecular Biology,2007,367(1)：1-7.
2.Frey B F,Jiang J,Sui Y,Boyd L F,et al.Effects of cross-presentation,antigen procession,and peptide binding in HIV Evasion of T cellimmunity.Journal of Immunology,2018,200(5):1853-1864.
3.Joseph W,Becker and George N,Reeke JR.Three-dimensional structure ofβ2-microglobulin.Proc.Natl.Acad Sci,1985,82:4225-4229.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for preparing a beta 2-microglobulin (beta 2-MG) monoclonal antibody. Based on the traditional method for improving the immunogenicity by synthesizing the single epitope coupling carrier in vitro, the invention implements the connection of the same carrier with two different double epitopes, improves the immunogenicity and the number of effective groups, thereby improving the immune effect, and effectively improving the positive rate of the beta 2-microglobulin (beta 2-MG) monoclonal antibody while ensuring the immunogenicity of the synthetic peptide. The technical scheme of the invention is specifically introduced as follows.
The invention provides a preparation method of a beta 2-microglobulin monoclonal antibody, which comprises the following specific steps:
(1) screening two polypeptide epitope sequences IQRTPKIQVYSRHPAEN and YLLYYTEFTPTEK with relatively strong immunogenicity based on an NCBI platform and an IEDB platform to serve as a first epitope and a second epitope respectively;
(2) respectively connecting 1 cysteine Cys to the asparagine N at the tail end of a first antigen epitope and the tyrosine Y at the tail end of a second antigen epitope, and then coupling the sulfydryl of the cysteine and the primary amino of the blue-key protein KLH by using an SMCC coupling method to simultaneously connect two polypeptide epitope sequences to a KLH molecule to serve as a beta 2-MG double-epitope polypeptide antigen synthesized in vitro;
(3) after 3 times of continuous immunization is carried out on 8 w-age BALB/C mice by adopting beta 2-MG bi-epitope polypeptide antigen, spleen is taken out 3 days after the last immunization, spleen lymphocytes are separated by using lymphocyte separation liquid, and the spleen lymphocytes are fused with myeloma cells SP2/0-
In the invention, in the step (1), two polypeptide epitope sequences with relatively strong immunogenicity are screened out through hydrophobic and surface accessibility indexes in a web.
Compared with the prior art, the invention has the beneficial effects that:
the traditional single epitope immunization method is optimized into double epitope immunization, and the positive rate of the beta 2-microglobulin (beta 2-MG) monoclonal antibody is effectively improved by increasing the antigen epitope on the basis of the immunogenicity of the polypeptide epitope synthesized in vitro by the barrier.
The method realizes the preparation and increase of the number of the monoclonal antibodies for resisting the beta 2-MG by increasing the antigen epitope. Compared with the monoclonal antibody for resisting the beta 2-MG prepared by the single epitope antigen, the quantity of the monoclonal antibody for resisting the beta 2-MG prepared by the antigen containing two epitopes after immune fusion can be further improved. The monoclonal antibody produced by the invention has specific reaction with beta 2-MG natural antigen, and 3 different specific monoclonal antibodies are obtained: a monoclonal antibody of an anti-beta 2-
Drawings
FIG. 1 is a graph of the different epitope hydrophobicity scores (Hphop. Kyte & Doolittle) for β 2-MG; wherein: A. b, C, D, E, F are the hydrophobicity scores of the
FIG. 2 is the surface accessibility of the different epitope amino acids of β 2-MG.
FIG. 3 shows the detection results of specific indirect ELISA of beta 2-MG
FIG. 4 shows the detection results of specific indirect ELISA of beta 2-MG
FIG. 5 shows the detection results of specific indirect ELISA of beta 2-MG
FIG. 6 shows the result of specific indirect ELISA detection of beta 2-MG
Detailed Description
The technical solution of the present invention is described in detail below with reference to examples.
The invention adopts double polypeptide epitopes to connect the same carrier in vitro synthesis method, which is based on NCBI platform and IEDB platform, firstly, based on the amino acid sequence of beta 2-MG in NCBI platform www.ncbi.nlm.nih.gov/protein/3CIQ _ L and the theoretical epitope of beta 2-MG in IEDB platform http:// www.iedb.org, classifying the epitope information and finishing all the theoretical epitope amino acid sequences, screening by hydrophobic (Hphob.Kyte & Doolittle) and surface accessibility (Access) indexes in web.expay.org platform, theoretically screening out beta 2-MG antigen polypeptide epitope 1(IQRTPKIQVYSRHPAEN) and antigen polypeptide epitope 2(YLLYYTEFTPTEK) with stronger immunogenicity,
according to the preliminary comparison of the hydrophobicity (figure 1) and the surface accessibility (figure 2) of different epitope amino acids of the beta 2-MG, the first epitope and the second epitope with the best immunogenicity are screened according to the principle that the lower the hydrophobicity score is and the higher the surface accessibility ratio is, the better the immunogenicity is. In order to further improve the immunogenicity of the polypeptide epitope, firstly, connecting 1 cysteine to the carbon-terminal amino acid of the first and second epitopes respectively as a bond bridge, and connecting the sulfhydryl of the cysteine and the primary amino group of the cyankey protein (KLH) with 1 KLH molecule respectively by an SMCC coupling method to form polypeptide antigen 1(IQRTPKIQVYSRHPAEN-Cys-KLH) and polypeptide antigen 2 (YLLYYTEFTPTEK-Cys-KLH); secondly, the beta 2-MG double
On the basis of the same immunization program, 8 w-age BALB/C mice were immunized with beta 2-MG polypeptide antigen 1(IQRTPKIQVYSRHPAEN-Cys-KLH), polypeptide antigen 2(YLLYYTEFTPTEK-Cys-KLH), polypeptide antigen 3(IQRTPKIQVYSRHPAEN-CYS-KLH-CYS-YLLYYTEFTPTEK) and beta 2-MG native antigen, respectively, 3 of each antigen, for a total of 12. After 3 times of continuous immunization, 12 mice were subjected to ELISA for serum titer detection, spleen was collected 3 days after the final immunization, spleen lymphocytes were separated from the lymphocyte separation solution, and cell fusion was performed with the known myeloma cells SP2/0-
The fusion agent used in the present invention is polyethylene glycol (also referred to as PEG), and the fusion method is the same as that of the conventional method. Myeloma cells and splenic lymphocytes were mixed at 1:10, and PEG with a molecular weight of 5000 at a concentration of 45% was selected for fusion. The hybridoma fusion is cultured by using a complete medium feeder layer cell containing 20% FBS, a 5 XHAT selective medium is supplemented for 16 hours after fusion for culture, a limited dilution method is adopted for detecting and screening a target antibody generating cell strain on the 10 th to 14 th days after fusion, and the positive cloning rate (%) obtained by calculation is as follows:
positive cloning efficiency (%) (positive well/fused well) × 100%
The method for obtaining monoclonal cell strain capable of producing target antibody mainly adopts indirect ELISA method to select hybridoma cell capable of producing target antibody, and the produced antibody can produce specific reaction with beta 2-MG natural antigen and can produce reaction with said same epitope antigen labeled with other carrier.
Specifically, monoclonal antibodies in culture supernatants of hybridoma monoclonal cells were reacted with the purified β 2-MG antigen after solid-phase treatment, and then reacted with an anti-IgG-labeled antibody, and monoclonal hybridoma cells capable of producing an anti- β 2-MG antibody were selected by indirect ELISA. The obtained hybridoma cells are further cultured, monoclonal cells for generating the anti-beta 2-MG antibody are screened and purified, and a large amount of anti-beta 2-MG monoclonal antibodies are extracted from the obtained monoclonal cells of the positive beta 2-MG antibody through intraperitoneal injection and ascites recovery.
The following is a description of specific implementations and details of the invention, in which various processes and methods, which are conventional and well known in the art, have not been described in detail.
EXAMPLE 1 preparation of anti-beta 2-MG monoclonal antibody hybridoma cells
a was prepared by ion chromatography using the amino acid sequence described in the non-patent literature (Andrea Gloger, Danilo Ritz, Tim Fugman, Dario Neri. cancer Immunol Immunother,2016,65(11): 1377-1393; Heyder T, Kohler M, Tarasova N K, et al. Applich for Identifying Human Leucocyte Antigen (HLA) -DRbound peptides from score Samples.) and synthetically purified polypeptide Antigen 1(IQRTPKIQVYSRHPAEN-Cys-KLH), Antigen 2(YLLYYTEFTPTEK-Cys-KLH) and Antigen 3 (IQRTPKIQVYSRHPAEN-CYS-KLH-CYS-YLLYYTEFTPTEK). 1MG of synthetic beta 2-
b selecting 12 Balb/c mice with no significant difference in body weight (18-22g), week age (8w) and sex (female), randomly dividing into three groups, namely an
TABLE 1 comparison of serum titers and fusion rates for four different groups of beta 2-MG antigens
Example 2 ELISA detection and identification of monoclonal antibodies against four antigens of beta 2-MG
a, respectively adding a polypeptide detection antigen 1(IQRTPKIQVYSRHPAEN-Cys-OVA), a polypeptide detection antigen 2(YLLYYTEFTPTEK-Cys-OVA), a polypeptide detection antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) and a beta 2-MG natural antigen solution which are diluted by PBS and have a final concentration of 1 mu g/ml into a small hole of a 96-hole ELISA plate, standing overnight at 4 ℃ for one night, washing the 96-hole ELISA plate coated by the
b 10-14d after the fusion, 100 μ l of cell supernatant from each well of the hybridoma obtained in example 1 is added to a 96-well elisa plate, incubated at room temperature for 1 hour, the supernatant is poured off, washed on a plate washing machine with a washing solution (PBS buffer containing 0.05% Tween) for 3 cycles, then the 96-well elisa plate is taken down and dried on absorbent paper, then 100 μ l of goat anti-mouse IgG antibody labeled with HRP (horse radish peroxidase) is added to the small hole of the 96-well elisa plate, incubated at room temperature for 1 hour, washed 2 times with the washing solution, 100 μ l of TMB developing solution (3,3 ', 5, 5' -tetramethylbenzidine) is added for developing at room temperature for 3min, after 50 μ l of HCl of 1M stops the reaction, detected at the absorbance of 450nm, the monoclonal well with the highest absorbance value compared with the positive clone is picked, and further subcloned and screened, and the result is detected after the fusion, see table 2. Comparing the proportion of positive clone cells of anti-detection antigen 1(IQRTPKIQVYSRHPAEN-Cys-OVA), anti-detection antigen 2(YLLYYTEFTPTEK-Cys-OVA), anti-detection antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) and anti-beta 2-MG natural antigen generated after the four groups are fused, the fusion hole positive rate of the anti-corresponding antigen (+) and the anti-beta 2-MG natural antigen (+) obtained by the beta 2-
Table 2 fusion positive rate comparison units for four different groups of β 2-MG antigens: [ n ═ n/% ]
Remarking: after each mouse is fused, 20 pieces of 96-well cell culture plates are paved, and the total number of the fusion holes is 1920 mice; corresponding antigens are respectively polypeptide detection antigen 1(IQRTPKIQVYSRHPAEN-Cys-OVA), polypeptide detection antigen 2(YLLYYTEFTPTEK-Cys-OVA), polypeptide detection antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) and beta 2-MG natural antigen
EXAMPLE 3 cloning culture of anti-beta 2-MG Natural antigen (+) monoclonal antibody hybridoma cells
The positive hybridoma cells MC obtained in example 2 were diluted by limiting dilution and then positive monoclonals were selected and cloned. The specific method comprises the following steps: respectively counting the hybridoma cells resisting the natural antigen (+) obtained in the example 2, preparing 1/200 mul of single cell suspension through gradient dilution, adding 200 mul of single cell suspension into a small hole of a 96-hole cell culture plate, culturing in a 5% carbon dioxide incubator at 37 ℃, selecting the supernatant of a monoclonal hole for indirect ELISA detection after about 9d of culture, setting a negative control (PBS) and a positive control (mouse eye serum with high immune titer of beta 2-MG antigen), selecting the monoclonal hybridoma cells with the absorbance (OD) value higher than that of the positive control hole and being monoclonal for continuous 3-time dilution passage, finally selecting the monoclonal hybridoma cells with the absorbance (OD) value higher than that of the positive control hole, having complete cell shape, uniform size and normal growth state and having the live cell rate of more than 98% through 0.4% trypan blue staining, and carrying out expanded culture on the monoclonal hybridoma cells, Establishing a strain and freezing and storing.
EXAMPLE 4 ascites production of monoclonal antibodies against the beta 2-MG native antigen (+)
Firstly, determining a Pristane pre-stimulation time table; secondly, selecting healthy Balb/c female mice with the age of 10-12 weeks, pre-stimulating the mice with Pristane 10-14 days before planned cell injection, and injecting 0.5 ml/mouse on the right side of the midline of the lower abdomen of the mice; thirdly, 1-2 days before the abdominal cavity inoculation of the hybridoma monoclonal cells, the hybridoma cells are subcultured by using a fresh 1640 culture medium (containing 10% FBS) to ensure that the hybridoma cells are in a logarithmic phase, and meanwhile, the activity of the hybridoma cells on the day of injection is determined to be more than 95% by using 0.4% trypan blue staining; fourthly, injecting 7 th to 10 th d intraperitoneally at Pristane, adding the hybridoma cell suspension which is obtained by the example 4 and has the cell activity of more than 95 percent and is in logarithmic phase into a 15ml centrifuge tube, centrifuging for 5min at 200g,resuspending with D-PBS after discarding to adjust cell content to 0.6-1.2 × 106Each dose is injected into the abdominal cavity as soon as possible within 1h after the resuspension, and 0.5ml of injection is carried out into the abdominal cavity by using a sterile syringe with a 21-gauge needle head; fifthly, paying attention to health condition observation of mice injected with hybridoma cells in an abdominal cavity within 24h, observing and aseptically collecting ascites after 7-10 days of hybridoma cell injection, combining the ascites obtained each time, centrifuging for 10min by 1500g, removing upper-layer grease, transferring supernatant of the centrifuged ascites into a 50ml centrifuge tube, adding thimerosal in a ratio of 1:1000, mixing uniformly, and storing at 4 ℃.
Example 5 specific detection of monoclonal antibodies against specific epitope antigen (+) of beta 2-MG and against the native antigen (+) by Indirect ELISA
Firstly, the ascites obtained in example 4 is purified by an octanoic acid precipitation method, and different antibodies after dialysis purification are marked with names; secondly, all the purified antibodies are respectively added into an ELISA coated plate containing a beta 2-MG natural antigen to carry out indirect ELISA detection (see example 2), and antibodies which react with the beta 2-MG natural antigen are screened (the result is the same as the result in the table 2); thirdly, the screened antibody against the natural beta 2-MG antigen is diluted with PBS (pH 7.4) at a ratio of 1:10000, and is added to an ELISA plate coated with polypeptide detection antigen 1(IQRTPKIQVYSRHPAEN-Cys-OVA), polypeptide detection antigen 2(YLLYYTEFTPTEK-Cys-OVA), polypeptide detection antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) and the natural beta 2-MG antigen respectively to perform indirect ELISA detection, and the results are shown in Table 3 and FIG. 3, tables 4 and 4, Table 5 and FIG. 5, Table 6 and FIG. 6. As can be seen from Table 3 and FIG. 3, 8 monoclonal antibodies against specific epitope 1(IQRTPKIQVYSRHPAEN) and native β 2-MG antigen were obtained in total for the β 2-MG
The anti-beta 2-MG monoclonal antibody obtained by the invention is detected by enzyme linked immunosorbent assay (ELISA) with the antigen coated by the natural beta 2-MG, compared with the traditional single
The monoclonal antibody obtained by the invention reacts with a polypeptide detection antigen 1(IQRTPKIQVYSRHPAEN-Cys-OVA) of beta 2-MG (figure 3 and table 3), reacts with a polypeptide detection antigen 2(YLLYYTEFTPTEK-Cys-OVA) (figure 4 and table 4) or reacts specifically with a polypeptide detection antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) (figure 5 and table 5), and reacts specifically with a natural antigen of beta 2-MG.
TABLE 3 detection results of specificity indirect ELISA of beta 2-MG
TABLE 4 detection results of specificity indirect ELISA of beta 2-MG
TABLE 5 detection results of specific indirect ELISA of beta 2-MG
TABLE 6 result of specific indirect ELISA detection of beta 2-MG
The in vitro synthesized double epitope polypeptide antigen 3(IQRTPKIQVYSRHPAEN-CYS-OVA-CYS-YLLYYTEFTPTEK) is compared with single
Therefore, the beta 2-MG double epitope polypeptide antigen immune method synthesized in vitro effectively improves the positive rate of the beta 2-microglobulin (beta 2-MG) monoclonal antibody by increasing the antigen epitope, and meanwhile, the antibody of the beta 2-MG natural antigen obtained in the method is suitable for any one of common immunological determination methods, including a competitive ELISA method, a double antibody Elisa sandwich method, an immunochromatography method, a latex gel method and the like, and the antibody is suitable for the immunological determination of any beta 2-MG.