CN108424448B - anti-Ebola virus VP40 protein monoclonal antibody F1B4 and application thereof - Google Patents
anti-Ebola virus VP40 protein monoclonal antibody F1B4 and application thereof Download PDFInfo
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- CN108424448B CN108424448B CN201810143429.4A CN201810143429A CN108424448B CN 108424448 B CN108424448 B CN 108424448B CN 201810143429 A CN201810143429 A CN 201810143429A CN 108424448 B CN108424448 B CN 108424448B
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Abstract
The invention discloses an anti-Ebola virus VP40 protein monoclonal antibody F1B4 and application thereof, wherein the monoclonal antibody subtype is IgG1 and kappa type, and can be specifically combined with a C-terminal antigen peptide of ZEBOV-VP40 protein to play an antiviral effect, hybridoma cells for generating the monoclonal antibody are hybridoma cells obtained by fusing, screening, cloning and passaging immune BA L B/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, can stably secrete F1B4, the heavy chain amino acid sequence of the antibody is shown as SEQ ID No.1, the light chain amino acid sequence is shown as SEQ ID No.2, F1B4 antagonizes the antiviral effect of Zaire type Ebola virus-like particles, and can be combined with other monoclonal antibodies for application.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to preparation and application of a monoclonal antibody against Zaire type Ebola virus VP40 protein, wherein a hybridoma cell line secreting the monoclonal antibody against VP40 protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, a monoclonal antibody F1B4 against VP40 protein is prepared and identified as IgG1 and kappa type, the sequence of the antibody is determined by gene sequencing, and the application of the antibody is realized by technologies such as affinity purification, electrophoresis, immunity, genetic engineering and the like.
Background
Ebola virus disease is a fatal hemorrhagic fever caused by ebola virus infection. In 1976, ebola virus was first discovered in southern sudan and congo (gold) (formerly zaire) and then subsequently exploded in small scale in the middle and non-districts, causing 2387 patients to suffer from diseases and 1310 cases of death with 54.9% mortality by 2013. The ebola virus outbreak again in 2014, with zaire-type ebola virus as the major strain, schinfei, and spanning continents, ebola cases occurred sequentially in the united states, uk, spain. By 2016, 12 and 31 days, 28712 cases (including confirmed and suspicious cases) were reported, 11372 deaths were reported, and the mortality rate was 39.6%. Although great progress has been made in research on rapid diagnosis, antiviral drugs, vaccines and the like of ebola viruses since western non-epidemic situations, no specific treatment means and no vaccine for clinical popularization and application exist internationally at present. The Ebola virus becomes an international serious infectious disease, and the research on the anti-Ebola virus treatment is very important for the safety of human beings.
Research shows that the proteins GP and VP40 of Ebola virus play an important role in the pathogenesis of Ebola virus, the GP protein is closely related to virus invasion and cell membrane fusion, and the VP40 protein plays an important role in promoting membrane fusion, maintaining the integrity of virus particles, assisting virus budding and the like, so the GP and VP40 protein is an important target point for researching medicines, vaccines and the likeTM. Currently, only ZMAPTMPhase I/II clinical trials are being conducted. However, the VP40 monoclonal antibody with anti-Ebola virus effect is not reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an anti-Ebola virus VP40 protein monoclonal antibody F1B4 and application thereof.
A monoclonal antibody F1B4 for resisting Ebola virus VP40 protein is disclosed, which has the subtypes IgG1 and kappa, and can be specifically combined with the C-terminal antigen peptide of ZEBOV-VP40 protein to play the role of antivirus, wherein the hybridoma cell for generating the monoclonal antibody is the hybridoma cell obtained by fusing, screening, cloning and passaging immune BA L B/C mouse spleen lymphocyte and mouse myeloma cell SP2/0, and can stably secrete F1B4, the heavy chain amino acid sequence of the antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
The preparation method of the monoclonal antibody F1B4 comprises the following steps:
(1) immunizing mice by selecting male mice BA L B/C of 6 weeks old, immunizing the mice by specific antigenic peptide 13 peptide CHSPAS L PAMVEK and SEQ ID No.3 aiming at the C end of ZEBOV-VP40 protein, immunizing the mice for 3 times in total, immunizing the mice for intramuscular injection of the inner thigh of the mice after 100 mu g of antigenic peptide is added with an adjuvant and mixed uniformly for the 1 st time, immunizing the mice for 2 times by using the same dosage of antigenic peptide and adjuvant mixture on the 21 st day, immunizing the mice for 3 times by using the same dosage of antigenic peptide and adjuvant mixture on the 35 th day, and treating the mice for fusion experiment on the 38 th day;
(2) culture of mouse myeloma cells: culturing mouse myeloma cell SP2/0 and keeping it in good growth state for hybridoma cell fusion;
(3) using BA L B/C mouse abdominal cavity macrophages as feeder cells, inoculating BA L B/C mouse abdominal cavity macrophages on a 96-hole culture plate one day before fusion, culturing the mouse with HAT culture medium containing 20% FCS for one day, killing the mouse prepared in the step (1) by cervical dislocation, taking out spleen, obtaining spleen lymphocytes, collecting mouse myeloma cells in the step (2), mixing and centrifuging the two cells, mediating cell fusion by polyethylene glycol (PEG 6000), properly diluting the fused cells, inoculating the cells to a cell culture plate, and culturing under proper conditions;
(4) screening of hybridoma cells: culturing the culture in selective medium containing HAT, when the cell colony grows to proper size, absorbing culture supernatant, performing antibody identification by enzyme-linked immunosorbent assay, and screening positive clone;
(5) cloning and culturing hybridoma cells: the hybridoma cells of the positive clone are diluted by a limiting dilution method, and the cells diluted to a certain density are inoculated to a 96-well cell culture plate, so that only one hybridoma cell per well grows. Taking supernatant from the hole where the cell colony is formed for enzyme-linked immunosorbent assay, screening and identifying positive clones until the positive rate of the hybridoma cell planting hole reaches 100%, carrying out expanded culture on the cloned hybridoma cells, and carrying out antibody physicochemical property analysis and identification;
(6) inducing ascites in mice by injecting 0.5 ml/mouse with pristane into BA L B/C mouse abdominal cavity 1 week before inoculating hybridoma cells, and then inoculating 5 × 10 with good growth6Collecting ascites after 10 days, centrifuging, measuring the antibody titer, and purifying the monoclonal antibody in the ascites;
(7) purification of monoclonal antibodies: monoclonal antibodies in ascites were purified by Protein G affinity purification.
The application of the monoclonal antibody F1B4 is used for detecting the expression of the ZEBOV-VP40 protein or neutralizing Zaire type Ebola virus.
The Zaire type Ebola virus is constructed Zaire type Ebola virus-like particles ZEBOV-trV L Ps.
The monoclonal antibody F1B4 is combined with one or more of an anti-GP monoclonal antibody ZJEB8-01, an anti-VP 40 monoclonal antibody G7A6 and an anti-VP 40 monoclonal antibody A2G7 for application.
The invention has the beneficial effects that: F1B4 has an antiviral effect, and the combined application of F1B4, an anti-GP monoclonal antibody (ZJEB 8-01) and other anti-VP 40 monoclonal antibodies (A2G7 and G7A6) has a more remarkable antiviral effect, so that a new reference scheme is provided for the treatment of anti-Zaire type Ebola virus.
Drawings
FIG. 1 immunoglobulin subtype analysis of monoclonal antibody F1B 4;
FIG. 2 detection of the titer of ascites and culture supernatant of monoclonal antibody F1B 4;
FIG. 3 construction of in vitro replication model of ZEBOV-trV L Ps infection;
FIG. 4 detection of RNA levels of trV L Ps after trV L Ps antagonized by monoclonal antibody F1B 4;
FIG. 5 detection of trV L Ps GP protein expression following trV L Ps antagonism by monoclonal antibody F1B 4;
FIG. 6 RNA levels of trV L Ps were detected following monoclonal antibody combined use to antagonize trV L Ps;
FIG. 7 detection of trV L Ps GP protein expression following the combined use of monoclonal antibodies antagonizing trV L Ps.
Detailed Description
The invention is further illustrated below with reference to the figures and the specific embodiments.
The invention selects Zaire type Ebola virus VP40 protein as target antigen, designs and synthesizes specific antigen peptide sequence aiming at VP40 protein by utilizing modern bioinformatics and molecular biology technology, establishes hybridoma cell line for stably secreting specific antigen protein monoclonal antibody of anti VP40 protein by adopting fusion hybridoma technology, and prepares, purifies and identifies the monoclonal antibody in large quantity. The successful acquisition of the monoclonal antibody not only lays a foundation for establishing a novel diagnosis method of the Ebola virus disease, namely the diagnosis based on immunological technology, but also provides scientific basis for the research of resisting viruses of the VP40 monoclonal antibody and the VP40 monoclonal antibody and the GP monoclonal antibody in combination and provides a new scheme for the treatment of the anti-Ebola virus. Meanwhile, the kit plays an important role in the research of disease pathogenesis, diagnosis, prognosis, curative effect judgment and the like.
The invention uses hybridoma cell technology. This technique fuses B lymphocytes from immunized mice with myeloma cells to create a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technology. The technology relates to a series of methods such as animal immunization, cell culture, cell fusion, cell clone culture, immunoassay and the like.
Example 1 preparation of monoclonal antibody F1B4 against Ebola VP40 protein
(1) Immunization of mice, namely, the first immunization, namely, the 13 peptide (CHSPAS L PAMVEK) of Ebola VP40 protein of cross-linked K L H and quick antibody-mouse5W are uniformly mixed according to the volume of 1:1, the total volume is 100 ul., 0.1ml (100 ug of the Ebola VP40 protein antigen peptide) of the mice is added to each BA L B/C mouse, the medial thigh is injected intramuscularly, one injection is strengthened in the same way on the 21 st day, trace tail blood is collected on the 35 th day for E L ISA determination, the antibody titer reaches 1:32000, the strengthening immunization is carried out on the tail vein once immediately, and the cell fusion is carried out after 3 days.
(2) Culture of mouse myeloma cell SP2/0 SP2/0 myeloma cell line derived from BA L B/C mouse was cultured in 10% FBS-DMEM mediumInstead, in the presence of 5% CO2Culturing in a 37 ℃ incubator at saturated humidity. The day before fusion was passaged to ensure that cells entered logarithmic growth phase at the time of fusion.
(3) Cell fusion, namely taking BA L B/C mouse peritoneal macrophages as feeder cells, inoculating BA L B/C mouse peritoneal macrophages on a 96-well culture plate one day before fusion, culturing the mouse peritoneal macrophages in HAT culture medium containing 20% FCS for one day, taking spleens the next day, separating splenocytes by adopting a pressure water injection method, centrifugally washing the cells for 1-2 times, then re-suspending the cells by using culture solution, collecting mouse SP2/0 myeloma cells, centrifuging, washing for 2 times, then re-suspending the cells by using the culture solution, taking the cells as SP2/0 cells to be fused, and taking the cells as SP2/0 cells to be fused with 1.0 × 108Spleen lymphocytes of each immunized mouse are mixed with 1.0 × 107Mouse myeloma cells SP2/0 were mixed and fused with 50% PEG 6000. The two cells are mixed and washed once, the supernatant is discarded by centrifugation, the cells are suspended on the wall of a flick tube (without adding liquid) and are added into a cell sediment by 0.8 ml of 50% PEG6000 (pH 8.0) pre-warmed at 37 ℃ within 60-90 seconds, the centrifugal tube is shaken gently but not blown and kept still for 1 minute, then 1ml of serum-free RPMI1640 is added within 1 minute according to the principle of slow and fast, 2ml of serum-free RPMI1640 is added within 2 minutes, 7 ml of serum-free RPMI1640 is added within 3 minutes, and 30-40 ml of serum-free RPMI1640 culture medium pre-warmed at 37 ℃ is added gradually within 1 minute later. Centrifuging at low speed of 800 rpm for 5-10 minutes. Then 20% FCS HAT medium was added, and the cells were individually plated in 96-well feeder cells-containing culture plates, and the cells were plated in 4 plates for each fusion, and placed in a 5% CO atmosphere at 37 ℃ C2Culturing in an incubator.
(4) And (3) screening the hybridoma, namely changing 1/2 culture solution (HAT) once every 4 days, changing HT-containing culture solution after 10 days, continuously culturing the fused hybridoma in HT-containing selective culture solution for about two weeks, sucking culture solution supernatant when cell colonies grow to be in proper size (the cell colonies are preferably full of one visual field when observed under a 10-fold objective lens), diluting the culture solution appropriately to prepare E L ISA, and screening positive clones.
Adopting an E L ISA indirect method to screen positive hybridoma clones, and the main steps are as follows:
1) ebola VP40 egg diluted with 0.01M carbonate buffer solution (pH9.6)White polypeptide with the concentration of 1000ng/ml is added into a 96-well enzyme label plate with a 0.1 ml/well package plate respectively and stays overnight at 4 ℃; 2) 0.01M pH7.4 PBS-Tween 20 plate washing three times; 3) blocking with 2% BSA-0.01M PBS pH 7.2 for 2 hours; 4) washing the plate; 5) adding hybridoma culture supernatant diluted at a ratio of 1:5, 0.1 ml/well, setting positive control (serum of immunized mouse), negative control (SP 2/0 culture supernatant) and blank control, and reacting at room temperature for 2 hr; 6) washing the plate; 7) adding 1:5000 diluted goat anti-mouse Ig (G + M) -HRP, 0.1 ml/hole, reacting for 1 hour at room temperature; 8) washing the plate; 9) adding a substrate (TMB) to react for 5-10 minutes at room temperature in a dark place; 10) 2M H2SO4 terminating the reaction; the OD value is measured at 450nm, and the positive result is that the P/N is more than or equal to 2.1.
(5) Cloning of hybridoma cells: the hybridoma cells were cultured by limiting dilution, and after the hybridoma cells positive for antibody detection (positive for the synthetic Ebola VP40 protein polypeptide) were selected and appropriately proliferated, the cells were counted. Inoculating 10/ml cell suspension diluted with complete 1640 culture medium into 96-well culture plate containing feeder cells, 0.1ml per well, observing cell growth after 7-10 days, detecting antibody level in supernatant, and selecting 5 culture wells with highest antibody titer and capable of growing single clone cells for cloning culture again. The method can be repeated for many times until the positive rate of monoclonal hole antibody detection is 100%.
(6) Inducing ascites by intraperitoneal injection of 0.5ml of pristane into BA L B/C mice one week before inoculation of hybridoma cells, and then inoculation of 5.0 × 106And (4) collecting ascites after 10 days to determine the antibody titer of the positive hybridoma cells.
(7) Purification of monoclonal antibodies: monoclonal antibodies were purified from ascites fluid by affinity purification (Protein G-crosslinked Sepharose). 1) The ascites fluid was diluted 3-fold with cold Binding Buffer and centrifuged at 10000rpm at 4 ℃ for 15 minutes to remove the precipitate. 2) The Sepharose-Protein G-loaded affinity purification column was extensively washed with 10 bed volumes of Binding Buffer. 3) And (4) loading the diluted ascites into a column, and controlling the flow rate to be 8-10 drops/min. 4) The ascites fluid that flowed through was repeatedly applied to the column once. 5) The column was washed well with 20 bed volumes of Binding Buffer until the absorbance of flow-through A280 was less than 0.01. 6) Eluting the bound monoclonal antibody by using an Elution Buffer, controlling the flow rate to be 8-10 drops/min, collecting the eluent in a collecting pipe pre-added with 0.1ml of potassium phosphate Buffer (PH 7.9, 0.5M), collecting 0.5ml of eluent containing the antibody in each pipe, and collecting more than 20 pipes in total. 7) The absorbance of each tube of eluate was measured at A280nm, and the eluate with an absorbance greater than 0.2 was collected. 8) The collected eluate was placed in a dialysis card and dialyzed against 0.1M PBS pH 7.0. The solution was changed every 6 hours for a total of 24 hours. 9) After the antibody solution after dialysis was diluted (1: 100), the protein content was measured at 280 nm. 10) And (4) packaging the purified antibody into small tubes, and placing the small tubes in a low-temperature refrigerator for later use.
(8) The subtype identification of the monoclonal antibody is carried out by adopting a mouse monoclonal antibody immunoglobulin typing kit of Serotec company. And (4) properly diluting the purified monoclonal antibody, and detecting, wherein the operation is strictly carried out according to the kit instruction. The test result shows that the monoclonal antibody secreted by the F1B4 hybridoma cell is IgG1 and kappa type (figure 1), the monoclonal antibody secreted by the A2G7 hybridoma cell is IgG1 and kappa type, and the monoclonal antibody secreted by the G7A6 hybridoma cell is IgG1 and kappa type.
The F1B4 hybridoma cell line is cloned for 3 times, continuously cultured for more than six months, and secreted antibody is stable. The cell strain is frozen and stored by liquid nitrogen, the cell strain grows well after recovery, and the secretion of the antibody is not declined (figure 2). The heavy chain amino acid sequence of the antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
Example 2 study of antiviral Effect of monoclonal antibody F1B4 against Zaire Ebola VP40 protein, F1B4 in combination with anti-GP monoclonal antibody (ZJEB 8-01) and other anti-VP 40 monoclonal antibodies (A2G7 and G7A6)
The experiment is realized by the following ways:
(1) the method has the advantages that a Zaire type Ebola virus-like particle (ZEBOV-trV L Ps) in-vitro replication model (figure 3) is constructed, ZEBOV-trV L Ps can simulate ZEBOV to synthesize miniature filamentous virus-like particles, basic functions of invasion, replication and the like are achieved, biohazard is avoided, and the trV L Ps system is widely applied to research of simulating ZEBOV and has important significance for research of screening virus receptors, antiviral drugs and the like.
(2) trV L Ps particles were collected by ultracentrifugation, incubated with trV L Ps (MIO =3) in vitro with a F1B4 monoclonal antibody dose gradient (3, 5, 10, 15. mu.g/ml) for 1h, and then added to 293T cell culture plates (24-well plates), and trV L Ps without antibody was used as a control group, incubated for 48h, then the supernatant was discarded, PBS was washed 3 times, total RNA of cells was extracted, and then subjected to real-time quantitative PCR with Ebola assay kit (Shanghai, QR-0220-02) to detect the intracellular trV L Ps-RNA level (FIG. 4).
(3) trV L Ps particles were collected by ultracentrifugation, incubated with trV L Ps (MIO =6) in a F1B4 monoclonal antibody concentration gradient (3, 5, 10, 15 μ g/ml) for 1h in vitro with gentle shaking, and then added to 293T cell culture plates (6-well plates), and trV L Ps without antibody was used as a control group, incubated for 48h, the supernatant was discarded, PBS was washed 3 times, total cell proteins were extracted, and intracellular trV L Ps GP protein expression was detected by Western blotting (FIG. 5).
(4) trV L Ps particles were collected by ultracentrifugation, and mixed with ZJEB8-01 (5. mu.g/ml) + F1B4 (5. mu.g/ml), A2G7+ F1B4 (5. mu.g/ml), G7A6 (5. mu.g/ml) + F1B4 (5. mu.g/ml), and incubated with trV L Ps (MIO =3) for 1h in vitro by gentle shaking, 293T cell culture plates (24 well plates) were added, F1B4 (10. mu.g/ml) and trV L Ps without antibody were set as control groups, after incubation for 48h, the supernatant was discarded, PBS was washed 3 times, total RNA was extracted, and intracellular trV L-RNA level was detected by real-time quantitative PCR using Ebola detection kit (Shanghai, Jiang, QR-0220-02) (FIG. 6).
(5) trV L Ps particles were collected by ultracentrifugation, and mixed with ZJEB8-01 (5. mu.g/ml) + F1B4 (5. mu.g/ml), A2G7+ F1B4 (5. mu.g/ml), G7A6 (5. mu.g/ml) + F1B4 (5. mu.g/ml), and incubated with trV L Ps (MIO =6) for 1h in vitro by gentle shaking, 293T cell culture plates (6 well plates) were added, F1B4 (10. mu.g/ml) and antibody-free trV L Ps were used as control groups, and after 48h incubation, the supernatant was discarded, PBS was washed 3 times, total cell proteins were extracted, and intracellular trV L Ps protein expression was detected by Western Blot (FIG. 7).
The F1B4 can effectively reduce the intracellular trV L Ps-RNA level and the GP protein expression when the dosage is 5 mu G/ml, can obviously reduce the expression of trV L Ps-RNA and GP protein when the dosage is 10 and 15 mu G/ml, and has more obvious effect along with the increase of the dosage, and in the research of the combined application of the antibody to resist viruses, the combined antiviral effect of the ZJEB8-01 (5 mu G/ml), F1B4 (5 mu G/ml), A2G7 and F1B4 (5 mu G/ml), G7A6 (5 mu G/ml) and F1B4 (5 mu G/ml) is obviously better than the single antiviral effect of the F1B4 (10 mu G/ml) under the condition that the total dosage of the antibody is 10 mu G/ml.
Experimental results show that F1B4 has an antiviral effect, F1B4, an anti-GP monoclonal antibody (ZJEB 8-01) and other anti-VP 40 monoclonal antibodies (A2G7 and G7A6) have a more remarkable antiviral effect in combined application, and a new reference scheme is provided for treatment of anti-Zaire type Ebola viruses.
Sequence listing
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Claims (1)
1. A monoclonal antibody F1B4 of anti-Ebola virus VP40 protein is characterized in that subtypes IgG1 and kappa of the monoclonal antibody can be specifically combined with C-terminal antigen peptide of ZEBOV-VP40 protein to play an antiviral effect, hybridoma cells for generating the monoclonal antibody are hybridoma cells obtained by fusing, screening, cloning and passaging immune BA L B/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, the hybridoma cells can stably secrete F1B4, the heavy chain amino acid sequence of the antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
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