CN116693666B - Monoclonal antibody of anti-hepatitis B core antigen and application thereof - Google Patents
Monoclonal antibody of anti-hepatitis B core antigen and application thereof Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Abstract
The invention relates to a monoclonal antibody of an anti-hepatitis B core antigen and application thereof, belonging to the technical field of biology. The invention provides a monoclonal antibody of an anti-hepatitis B core antigen, wherein a heavy chain variable region comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.1, a CDR2 with an amino acid sequence shown as SEQ ID NO.2 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO.3, and a light chain variable region comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO. 10. The monoclonal antibody can obviously inhibit the expression of HBsAg, HBeAg and HBV DNA of HepG2.2.15 cells and HepAD38 cells.
Description
Technical Field
The invention relates to a monoclonal antibody of an anti-hepatitis B core antigen and application thereof, belonging to the technical field of biology.
Background
The hepatitis B virus (Hepatitis B virus, HBV) is a hepadnavirus, consists of an envelope and a nucleocapsid, has strong infectivity, mainly invades human liver, and can cause related diseases such as acute viral hepatitis B (Acute Hepatitis B, AHB), chronic viral hepatitis B (Chronic Hepatitis B, CHB), liver fibrosis, liver cancer and the like after infection.
At present, nucleoside (nucleotide) antiviral drugs are generally used for inhibiting the replication of hepatitis B virus, so as to treat hepatitis B virus infection and related diseases caused by the hepatitis B virus infection. However, nucleoside (nucleotide) antiviral drugs generally have the problems of low clinical cure rate and easy virus rebound and even liver failure after drug withdrawal. Interferon may also be used for antiviral treatment of hepatitis b virus infection. However, most patients have poor tolerance to interferon and have more adverse reactions, and only 3% -7% of chronic viral hepatitis b patients treated for 1 year get hepatitis b surface antigen (Hepatitis B surface antigen, HBsAg) clearance (hepatitis b surface antigen clearance is a sign of clinical cure or functional cure of chronic viral hepatitis b).
Monoclonal antibodies (Monoclonal antibody) are highly homogeneous antibodies raised against only one specific epitope by a single B cell clone. Compared with interferon and nucleotide antiviral drugs, the monoclonal antibody drug has the advantages of strong specificity, high sensitivity, high effectiveness and good safety in treatment. The monoclonal antibody medicine capable of effectively inhibiting the replication of the hepatitis B virus is expected to overcome the defects of the existing anti-hepatitis B virus infection medicine.
The genome of hepatitis b virus consists of an incomplete circular double-stranded DNA, comprising four open reading frames (open reading frame, ORF) encoding proteins required for viral replication and packaging, the S, C, P and X regions. Several proteins encoded by the open reading frame elicit specific anti-hepatitis b virus immune responses, such as the hepatitis b surface antigen (Hepatitis B surface antigen, HBsAg) mentioned above, as well as hepatitis b e antigen (Hepatitis B e antigen, HBeAg) and hepatitis b core antigen (Hepatitis B core antigen, HBcAg).
Among them, hepatitis b core antigen has strong immunogenicity, and hepatitis b core antibodies (anti-HBc) generated by immune reaction of hepatitis b core antigen can be detected almost by hepatitis b virus infected persons, but it is widely thought that hepatitis b core antibodies are not protective antibodies, have only a certain epidemiological investigation significance, and, because hepatitis b core antibodies exist in serum for a long time, are a sign of acute infection and later chronic infection of hepatitis b virus, hepatitis b core antibodies have been widely used in clinical screening and diagnosis of chronic hepatitis b virus (CHB). At present, no monoclonal antibody against hepatitis B core antigen capable of effectively inhibiting replication of hepatitis B virus exists.
Disclosure of Invention
In order to solve the problems, the invention provides a monoclonal antibody of an anti-hepatitis B core antigen, wherein a heavy chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.1, a CDR2 with an amino acid sequence shown as SEQ ID NO.2 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO.3, and a light chain variable region comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO. 10.
In one embodiment of the present invention, the heavy chain variable region of the monoclonal antibody comprises a CDR1 having an amino acid sequence shown as SEQ ID NO.1, a CDR2 having an amino acid sequence shown as SEQ ID NO.2 and a CDR3 having an amino acid sequence shown as SEQ ID NO.3, and the light chain variable region comprises a CDR1 having an amino acid sequence shown as SEQ ID NO.8, a CDR2 having an amino acid sequence shown as SEQ ID NO.9 and a CDR3 having an amino acid sequence shown as SEQ ID NO. 10.
In one embodiment of the invention, the heavy chain variable region of the monoclonal antibody comprises FR1 with an amino acid sequence shown as SEQ ID NO.4, FR2 with an amino acid sequence shown as SEQ ID NO.5, FR3 with an amino acid sequence shown as SEQ ID NO.6 and/or FR4 with an amino acid sequence shown as SEQ ID NO.7, and the light chain variable region comprises FR1 with an amino acid sequence shown as SEQ ID NO.11, FR2 with an amino acid sequence shown as SEQ ID NO.12, FR3 with an amino acid sequence shown as SEQ ID NO.13 and/or FR4 with an amino acid sequence shown as SEQ ID NO. 14.
In one embodiment of the invention, the heavy chain variable region of the monoclonal antibody comprises FR1 with an amino acid sequence shown as SEQ ID NO.4, FR2 with an amino acid sequence shown as SEQ ID NO.5, FR3 with an amino acid sequence shown as SEQ ID NO.6 and FR4 with an amino acid sequence shown as SEQ ID NO.7, and the light chain variable region comprises FR1 with an amino acid sequence shown as SEQ ID NO.11, FR2 with an amino acid sequence shown as SEQ ID NO.12, FR3 with an amino acid sequence shown as SEQ ID NO.13 and FR4 with an amino acid sequence shown as SEQ ID NO. 14.
In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
In one embodiment of the present invention, the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.17 and the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO. 18.
In one embodiment of the invention, the light chain and the heavy chain are linked by disulfide bonds.
The invention also provides a gene which codes for the monoclonal antibody.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment of the invention, the recombinant plasmid vector is a pET-30a (+) expression vector, a pcDNA3.1/GS expression vector, a pXMJ19 expression vector, a pBAD24 expression vector or a pMAL-c2x expression vector.
The invention also provides a host cell transfected with the recombinant plasmid; alternatively, the genome of the host cell integrates the above-described genes.
In one embodiment of the invention, the host cell is a HepG2 cell or a HepG2.2.15 cell.
The invention also provides a method for preparing the monoclonal antibody, which comprises the following steps: inoculating the host cells into a cell culture medium for culture to obtain a culture solution; the monoclonal antibody is separated and extracted from the culture solution.
The invention also provides a medicament containing the monoclonal antibody, and the medicament has at least one of the following uses:
(a) Inhibiting hepatitis b virus replication;
(b) Preventing and/or treating hepatitis b virus infection; and/or the number of the groups of groups,
(c) Preventing and/or treating diseases caused by hepatitis B virus infection.
In one embodiment of the present invention, the disease associated with the hepatitis b virus infection includes one or more of acute viral hepatitis b, chronic viral hepatitis b, liver fibrosis or liver cancer.
In one embodiment of the invention, the medicament further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the pharmaceutical carrier comprises one or more of a microcapsule, microsphere, nanoparticle or liposome.
In one embodiment of the invention, the pharmaceutical excipients comprise one or more of solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retarders.
In one embodiment of the invention, the medicament is in the form of a powder, tablet, granule, capsule, solution, emulsion, suspension or injection.
The invention also provides the use of the monoclonal antibody or the gene or the recombinant plasmid or the host cell or the method in the preparation of a medicament having at least one of the following uses:
(a) Inhibiting hepatitis b virus replication;
(b) Preventing and/or treating hepatitis b virus infection; and/or the number of the groups of groups,
(c) Preventing and/or treating diseases caused by hepatitis B virus infection.
In one embodiment of the present invention, the disease associated with the hepatitis b virus infection includes one or more of acute viral hepatitis b, chronic viral hepatitis b, liver fibrosis or liver cancer.
In one embodiment of the invention, the medicament further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the pharmaceutical carrier comprises one or more of a microcapsule, microsphere, nanoparticle or liposome.
In one embodiment of the invention, the pharmaceutical excipients comprise one or more of solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retarders.
In one embodiment of the invention, the medicament is in the form of a powder, tablet, granule, capsule, solution, emulsion, suspension or injection.
The technical scheme of the invention has the following advantages:
the invention provides a monoclonal antibody of an anti-hepatitis B core antigen, wherein a heavy chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.1, a CDR2 with an amino acid sequence shown as SEQ ID NO.2 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO.3, and a light chain variable region comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and/or a CDR3 with an amino acid sequence shown as SEQ ID NO. 10. In vitro experiments show that the monoclonal antibody can remarkably inhibit the expression of HBsAg, HBeAg and HBV DNA of HepG2.2.15 cells and HepAD38 cells, has the function of inhibiting the replication of hepatitis B virus, and can be prepared in a large quantity in a short time, so that the monoclonal antibody has great application prospect in preparing medicines for inhibiting the replication of hepatitis B virus, preventing and/or treating hepatitis B virus infection and/or preventing and/or treating related diseases caused by the hepatitis B virus infection.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Experimental example 1: preparation, extraction and identification of monoclonal antibodies against hepatitis B core antigen
1. Hepatitis B core antigen immunized mice
Balb/c mice (females, 8-10 weeks old, purchased from Guangdong province medical laboratory animal center) were immunized with HBcAg protein antigen (Orb 384353, biorbyt) having a purity of greater than 90% and an amino acid sequence shown in SEQ ID NO. 19. The specific immunization procedure is as follows:
performing primary immunization, primary boosting immunization and secondary boosting immunization respectively at 0d, 14d and 21d, taking blood for 24d, separating serum, detecting the specific antibody titer in the serum by ELISA (enzyme-linked immunosorbent assay), and performing tertiary boosting immunization when the titer reaches more than 10000 to obtain a hepatitis B core antigen immunized mouse;
in the first immunization, HBcAg protein antigen is diluted to a concentration of 1mg/mL by using phosphate buffer (PBS buffer, concentration 0.01M, pH 7.2) to obtain a diluted solution, and then the diluted solution is mixed with Freund's complete adjuvant (F5881, sigma-Aldrich) according to the Freund's complete adjuvant: dilution = 1:1, obtaining an immune preparation, and finally injecting the immune preparation into Balb/c mice subcutaneously in a multi-point manner, wherein the immune dosage is 200 mu L/mouse; the first boost and the second boost were identical to the first boost except that the adjuvant was replaced with Freund's incomplete adjuvant F5506, sigma-Aldrich); third boost the remaining steps were consistent with the first boost and the second boost except that subcutaneous multipoint injection was changed to tail vein injection.
2. Mouse spleen cell and myeloma cell fusion
(1) Hepatitis b core antigen immunized mice were anesthetized and then blood was collected from the eyeballs (serum was subsequently collected as a positive control for later screening, or other in vivo and in vitro experiments);
(2) After killing the hepatitis B core antigen immune mice by neck breaking, putting the mice into 75% (v/v) alcohol for soaking and sterilizing for 5 minutes;
(3) Dissecting and taking out the spleen of the mouse in a sterile super clean bench, putting the spleen into a 70 mu m filter screen arranged on a 50mL centrifuge tube, lightly grinding the spleen into single cells by using a pestle end of a 5mL sterile syringe, adding 30mL serum-free DMEM (purchased from Gibco) for washing 1 time, centrifuging for 8min at 300g, discarding the supernatant, and finally adding 20mL serum-free DMEM to resuspend the spleen cells and counting under a microscope to obtain spleen cell suspension; placing NS-1 cells (mouse myeloma cells purchased from ATCC) into a 50mL centrifuge tube, adding 30mL of serum-free DMEM to wash for 1 time, centrifuging for 8min at 300g, discarding the supernatant, adding 40mL of serum-free DMEM to resuspend the NS-1 cells, and counting under a microscope to obtain an NS-1 cell suspension;
(4) The NS-1 cell suspension was added to the spleen cell suspension to 25X 10 cells in each mouse 6 Mixing NS-1 cells, centrifuging for 8min at 300g, discarding the supernatant to obtain mixed cells, then adding 40mL serum-free DMEM for washing for 1 time, centrifuging for 8min at 300g, and discarding the supernatant to obtain washed mixed cells;
(5) After beating the bottom of a 50mL centrifuge tube to disperse the washed mixed cells, 1mL of 50% (w/v, g/100 mL) polyethylene glycol 3350 (P-3640, sigma) aqueous solution (prepared in advance and filtered for sterilization) is added dropwise in 1min, then a 1mL straw is used for uniformly stirring for 1min, then 4mL of serum-free DMEM is added dropwise in 4min, then 10mL of serum-free DMEM is added dropwise in 2min, and finally water bath is carried out at 37 ℃ for 10min;
(6) 30mL of DMEM containing 10% (v/v) fetal bovine serum (purchased from Gibco) is added into a 50mL centrifuge tube, uniformly mixed, 300g is centrifuged for 8min, the supernatant is discarded, and then 20mL of DMEM containing 20% (v/v) fetal bovine serum is added to resuspend the mixed cells, so as to obtain a mixed cell suspension;
(7) Firstly, respectively adding 10mL of mixed cell suspension into two new 50mL centrifuge tubes, then adding 40mL of DMEM containing 20% (v/v) fetal bovine serum into each centrifuge tube, and uniformly mixing to obtain mixed solution;
(8) Pouring the mixed solution in two 50mL centrifuge tubes into a sterile sample adding tank, adding the mixed solution into a 96-well plate by using a row gun according to the addition amount of 100 mu L/hole for plating, and paving 5 96-well plates for every 50mL cell suspension, wherein 10 96-well plates are paved in total;
(9) After the completion of the plate laying, the mixture was put into 5% (v/v) CO at 37 DEG C 2 Is cultured for 20 hours;
(10) 2 XHAT medium (YZ-H0262-1 VL, solarbio) was added to a 96-well plate at an addition rate of 100. Mu.L/well using a lance, and returned to 37℃at 5% (v/v) CO 2 During the culture, cell culture supernatants having wells grown from the clones were collected by observation at intervals.
3. ELISA screening monoclonal antibody against hepatitis B core antigen
(1) Collecting 80 mu L of cell culture supernatant from which cloning holes grow in a 96-well plate, and marking positions;
(2) The quantitative and qualitative kit (purchased from Beijing Wantai) for detecting the core antibody is taken out and rewarmed to room temperature (25 ℃) 30min in advance;
(3) 50 mu L of sample diluent is firstly added into each well of a 96-well plate, and 50 mu L of cell culture supernatant to be detected, 50 mu L of standard substance and 50 mu L of negative sample are respectively added;
(4) Pasting a film on a 96-well plate, and incubating for 30min at room temperature (25 ℃);
(5) Preparing a washing Buffer solution (Wash Buffer,30mL of concentrated solution and 570mL of deionized water), adding 300 mu L of the washing Buffer solution into each well of a 96-well plate, and washing the plate on a plate washer for 5 times;
(6) Removing liquid in the wells, then beating the wells to dry, and adding 100 mu L of enzyme-labeled reagent into each well of the 96-well plate;
(7) Pasting a film on a 96-well plate, and incubating for 30min at room temperature (25 ℃) under the dark condition;
(8) Adding 300 mu L of washing buffer solution into each well of a 96-well plate, and washing the plate on a plate washer for 5 times;
(9) Removing liquid in the wells, then beating to dry, adding 50 mu L of liquid A and 50 mu L of liquid B into each well of the 96-well plate, shaking and mixing uniformly, and developing for 15min at 37 ℃ in dark places;
(10) Adding 50 mu L of stop solution into each well of a 96-well plate, vibrating and uniformly mixing, using an enzyme-labeled analyzer within 10min, and setting a detection absorbance value with a wavelength of 450nm (630 nm is used as a control wavelength);
(11) Cells in positive cloning wells were selected based on OD values of standard wells and negative wells.
4. ELISA positive cloning hole cell cloning
(1) Mixing cells in the positive cloning holes uniformly by using a sample gun, and counting under a microscope to calculate the cell concentration;
(2) Adding cells in 100 positive cloning holes into 10mL 1 XHT culture medium (YZ-H0262-1 VL, solarbio), and uniformly mixing to obtain a mixed solution;
(3) Pouring the mixed solution into a sterile tank, adding into 96-well plate with a discharge gun at an addition rate of 100 μl/well for plating, and adding 5% (v/v) CO at 37deg.C after plating 2 During the culture, observing cells every other day, and changing the liquid by half;
(4) When new cell clones appear in the 96-well plate, collecting cell culture supernatant of the well from which the clones grow, referring to step 3, performing ELISA detection by using a core antibody quantitative qualitative kit;
(5) After selection of cells from the still positive clone wells (i.e.the hybridoma cells selected) transferred to 48 wells, 1 XHT was used to culture cells at 37℃on the basis of 5% (v/v) CO 2 Is cultured in an incubator;
(6) And (3) continuing to enlarge and culture positive cloned hole cells to 24-hole plates, 12-hole plates, 6-hole plates, 25-T culture flasks and 75-T culture flasks, and simultaneously freezing and storing the screened hybridoma cells for later use.
In the step, 3 hybridoma cells producing the monoclonal antibodies of the murine hepatitis B core antigen are screened, the 3 hybridoma cells producing the monoclonal antibodies of the murine hepatitis B core antigen are respectively named as 1G9, 2H4 and 6F8, and the monoclonal antibodies of the hepatitis B core antigen produced by 1G9, 2H4 and 6F8 are sent to the Kirschner company for subtype identification and light chain identification, and the identification results are shown in Table 1.
TABLE 1 subtype identification results of monoclonal antibodies produced by 1G9, 2H4 and 6F8
5. Large-scale preparation and purification of monoclonal antibody against hepatitis B core antigen
(1) Collecting the hybridoma cells obtained by expansion culture in a 50mL centrifuge tube, counting under a microscope, centrifuging for 10min at 800g after counting is completed, and discarding the supernatant;
(2) Cells were resuspended to 4X 10 by adding PBS buffer to a 50mL centrifuge tube 6 Obtaining a cell heavy suspension by using the total concentration of the cell heavy suspension per mL;
(3) The cell resuspension is subjected to intraperitoneal injection on Balb/c mice, and the injection dosage is 500 mu L/mouse;
(4) After 12d injection, aseptically collecting ascites in the abdominal cavity of the mouse, centrifuging at 4 ℃ for 10min in a 15mL centrifuge tube with 500 g;
(5) Collecting ascites supernatant in 1.5mL EP tube, 1 mL/tube, writing a label, and storing at-20deg.C to obtain ascites specimen;
(6) Preparing acetate buffer solution: after 59mL of solution A and 41mL of solution B were mixed, the pH was adjusted to 4.80 using hydrochloric acid (solution A: 0.06M aqueous sodium acetate; solution B: 0.06M aqueous glacial acetic acid);
(7) Taking out the ascites specimen 30min in advance, re-warming to room temperature (25 ℃) and centrifuging at 4 ℃ and 12000rpm for 10min, and taking the supernatant;
(8) The supernatant was mixed with acetate buffer according to the supernatant: acetate buffer = 1:2, dropwise adding n-octanoic acid to the mixture containing 33 mu L of n-octanoic acid in each mL of supernatant under stirring at room temperature (25 ℃), and obtaining a mixed solution;
(9) Standing the mixed solution at 4deg.C for more than 2 hr to allow it to fully precipitate, centrifuging at 12000rpm at 4deg.C for 30min, and collecting supernatant;
(10) Adding ammonium sulfate to the supernatant in 30min under ice bath until the content of ammonium sulfate in the supernatant is 0.277 g/mL to reach 45% saturation, to obtain saturated solution;
(11) Standing the saturated solution at 4deg.C for more than 2 hr to allow it to fully precipitate, centrifuging at 12000rpm at 4deg.C for 30min, and collecting protein precipitate;
(12) Dissolving protein precipitate with 1.5mL PBS buffer to obtain monoclonal antibody solution;
(13) After 2. Mu.L of the monoclonal antibody lysate was used to detect the protein concentration using Nanodrop 2000A 280, the protein concentration was labeled and frozen at-80 ℃.
The protein concentration of the monoclonal antibody solution obtained by culturing 1G9 in this step is 3.269mg/mL, the protein concentration of the monoclonal antibody solution obtained by culturing 2H4 is 3.700mg/mL, and the protein concentration of the monoclonal antibody solution obtained by culturing 6F8 is 3.333mg/mL.
Experimental example 2: effect of monoclonal antibodies against hepatitis B core antigen on expression of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells
The experimental procedure was as follows:
(1) HepG2.2.15 cells (purchased from ATCC) were used at 0.05X10 per well 6 Seed amount of each was inoculated into a 96-well plate with 200. Mu.L of DMEM medium containing 10% (v/v) fetal bovine serum per well at 37℃in 5% (v/v) CO 2 Culturing for 16h to enable cells to grow on the wall;
(2) After 16h of incubation, fresh medium was changed, and monoclonal antibody 1E10 (obtained by substituting HBcAg protein antigen with HbA2 protein antigen based on monoclonal antibody lysate prepared in Experimental example 1, hbA2 protein antigen amino acid sequence shown in SEQ ID NO. 20) was used as control protein, and monoclonal antibody lysate prepared in experimental example 1 was added to 96-well plates to protein concentrations of 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, CO at 37℃and 5% (v/v) respectively in each well 2 Is cultured for 3d in an incubator;
(4) After 3d of culture, the cell culture supernatants from each well were collected, and the levels of HBsAg and HBeAg were detected using a hepatitis b virus surface antigen quantitative assay kit (purchased from beijing vantai) and a hepatitis b virus e antigen quantitative assay kit (purchased from beijing vantai), respectively, and HBV DNA levels were detected using a hepatitis b virus nucleic acid quantitative assay kit (purchased from hensan-xiang, henna), the detection results of which are shown in tables 2 to 4.
The results in Table 2 show that the monoclonal antibodies produced by 1G9, 2H4 and 6F8 all have a significant inhibitory effect on HBsAg in HepG2.2.15 cells, and that the more significant the effect on the secretion of HBsAg in cells is with increasing antibody concentration, wherein the effect of 1G9 is most pronounced at 200. Mu.g/mL (P < 0.001).
The results in Table 3 show that monoclonal antibodies produced by 1G9, 2H4 and 6F8 also have a significant inhibitory effect on HBeAg secretion by HepG2.2.15 cells, and that the greater the inhibitory effect, the more pronounced the 1G9 effect at 200. Mu.g/mL (P < 0.001).
The results in Table 4 show that the monoclonal antibodies produced by 1G9, 2H4 and 6F8 also have inhibitory effects on HBV DNA expression levels in HepG2.2.15 cells, and that there is a concentration effect, with the effect of 1G9 being most pronounced at 200. Mu.g/mL (P < 0.001).
TABLE 2 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on the expression level of HBsAg in HepG2.2.15 cells
TABLE 3 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on the expression level of HBeAg in HepG2.2.15 cells
TABLE 4 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on HBV DNA expression in HepG2.2.15 cells
Experimental example 3: effect of monoclonal antibodies against hepatitis B core antigen on expression of HBsAg, HBeAg and HBV DNA in HepAD38 cells
The experimental procedure was as follows:
referring to the method of experimental example 2, the effect of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on the expression of HBsAg, HBeAg and HBV DNA in HepAD38 cells (purchased from ATCC) was examined, and the results of the examination are shown in tables 5 to 7. Monoclonal antibodies produced by 1G9 were sent to Kirschner for gene sequencing, and the sequencing results are shown in Table 8.
The results in Table 5 show that the monoclonal antibodies produced by 1G9, 2H4 and 6F8 all have a significant inhibitory effect on HBsAg in HepAD38 cells, and that the more significant the effect of inhibiting secretion of HBsAg in cells with increasing antibody concentration, the more significant the effect of 1G9 at 200. Mu.g/mL (P < 0.001).
The results in Table 6 show that monoclonal antibodies produced by 1G9, 2H4 and 6F8 also have a significant inhibitory effect on HBeAg secretion by HepAD38 cells, and that the greater the inhibitory effect, the more pronounced the effect of 1G9 at 200. Mu.g/mL (P < 0.001) was.
The results in Table 7 show that the monoclonal antibodies produced by 1G9, 2H4 and 6F8 also have inhibitory effects on HBV DNA expression levels in HepAD38 cells, and that there is a concentration effect, with the effect of 1G9 being most pronounced at 200. Mu.g/mL (P < 0.001).
TABLE 5 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on the expression level of HBsAg in HepAD38 cells
TABLE 6 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on the expression level of HBeAg in HepAD38 cells
TABLE 7 influence of monoclonal antibodies produced by 1G9, 2H4 and 6F8 on HBV DNA expression in HepAD38 cells
TABLE 8 heavy and light chains of monoclonal antibodies produced by 1G9
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A monoclonal antibody against hepatitis b core antigen, characterized in that the heavy chain variable region of the monoclonal antibody comprises CDR1 with the amino acid sequence shown in SEQ ID No.1, CDR2 with the amino acid sequence shown in SEQ ID No.2 and CDR3 with the amino acid sequence shown in SEQ ID No.3, and the light chain variable region comprises CDR1 with the amino acid sequence shown in SEQ ID No.8, CDR2 with the amino acid sequence shown in SEQ ID No.9 and CDR3 with the amino acid sequence shown in SEQ ID No. 10.
2. The monoclonal antibody of claim 1, wherein the heavy chain variable region of the monoclonal antibody comprises FR1 having the amino acid sequence shown in SEQ ID No.4, FR2 having the amino acid sequence shown in SEQ ID No.5, FR3 having the amino acid sequence shown in SEQ ID No.6 and/or FR4 having the amino acid sequence shown in SEQ ID No.7, and the light chain variable region comprises FR1 having the amino acid sequence shown in SEQ ID No.11, FR2 having the amino acid sequence shown in SEQ ID No.12, FR3 having the amino acid sequence shown in SEQ ID No.13 and/or FR4 having the amino acid sequence shown in SEQ ID No. 14.
3. The monoclonal antibody of claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.15 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 16.
4. A gene encoding the monoclonal antibody of any one of claims 1 to 3.
5. A recombinant plasmid carrying the gene according to claim 4.
6. A host cell transfected with the recombinant plasmid of claim 5; alternatively, the genome of the host cell has integrated therein the gene of claim 4.
7. A method of preparing the monoclonal antibody of any one of claims 1-3, comprising: inoculating the host cell of claim 6 into a cell culture medium for culturing to obtain a culture solution; the monoclonal antibody is separated and extracted from the culture solution.
8. A medicament comprising the monoclonal antibody of any one of claims 1 to 3.
9. The monoclonal antibody of any one of claims 1 to 3 or the gene of claim 4 or the recombinant plasmid of claim 5 or the host cell of claim 6 or the monoclonal antibody prepared by the method of claim 7 is applied to the preparation of a medicament for preventing and/or treating acute viral hepatitis b, chronic viral hepatitis b, liver fibrosis or liver cancer caused by hepatitis b virus infection.
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