CN111548412B - Hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence - Google Patents
Hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence Download PDFInfo
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Abstract
The invention relates to the technical field of immunology, and particularly discloses a hepatitis B surface antigen monoclonal antibody, a preparation method, application and an amino acid sequence thereof, wherein the monoclonal antibody comprises the following components: an amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution. The invention has the characteristics of higher sensitivity, better specificity and better stability.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to a hepatitis B surface antigen monoclonal antibody, a preparation method, application and an amino acid sequence.
Background
The outer shell part of the hepatitis B virus contains surface antigen (HBsAg), the core part contains core antigen (HBcAg), E antigen (HBEAg), and DNA of hepatitis B virus (HBV-DNA, DNA-P). After hepatitis B virus infection, a great amount of surface antigens are usually left in blood, and surface antigenemia is formed. The surface antigen itself is not whole hepatitis B virus, but rather the outer shell of hepatitis B virus, it is itself non-infectious but antigenic, it is just one of the markers of hepatitis B virus infection. It may indicate that the hepatitis B virus has been infected in the past, or is currently being infected with hepatitis B virus.
The existing HBcAb detection method mainly comprises a Chemiluminescent Microparticle Immunoassay (CMIA), an enzyme-linked immunosorbent assay (ELISA) one-step method and a colloidal gold method. Regardless of the detection method used, the quality of the detection result is closely related to the sensitivity, specificity and stability of the monoclonal antibody used in the assay. The gel Jin Mian fatigue chromatography is a detection technology developed on the basis of ELISA, all reagents used in the method are dry reagents, a plurality of reagents are combined on a plastic strip with the thickness of about 6mm multiplied by 70mm, water absorbing materials are attached to two ends of the test strip to form a single reagent strip, the experimental process is short, and the method is one of the main methods of bedside detection. The method has the outstanding advantages that the method is rapid, the reagent is convenient to store and detect, no special equipment is needed, single detection can be realized, and the method is suitable for emergency detection; but has slightly poorer sensitivity and specificity, and is more suitable for primary screening of basic medical units and specimens. At present, mature colloidal gold test strips, such as HCG gold label strips, are widely applied to clinical detection. In the detection of HBsAg of one of the important markers of hepatitis B, the detection sensitivity of the gold-labeled test paper with good foreign property is similar to ELISA, and the colloidal gold test paper with good maturity is widely applied in China, and the detection sensitivity of the domestic HBsAg colloidal test paper has reached 1ng/ml, but a certain gap still exists between the detection sensitivity and the foreign product to be further improved. If the detection sensitivity and the specificity can be further improved, the detection diversification is realized, the quantitative and semi-quantitative detection is carried out, and the colloidal gold paper strip can be more widely applied to various fields.
Therefore, in order to obtain a better HBsAg detection method, it is a constantly sought-after goal to find HBsAg monoclonal antibodies with excellent sensitivity, specificity and stability. In addition, since the storage conditions of the test reagents and test strips are sometimes unsatisfactory, it is generally desired that the test reagents and test strips maintain high stability and reliability after severe environmental changes.
Disclosure of Invention
The invention provides a hepatitis B surface antigen monoclonal antibody with higher sensitivity, better specificity and better stability, a preparation method, application and an amino acid sequence for solving the technical problems existing in the existing clinical HBsAg detection.
The first technical scheme of the invention is as follows: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from histidine to alanine, the heavy chain obtained after substitution being SEQ ID N0:3, a step of;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from arginine to glutamic acid, the heavy chain obtained after substitution being SEQ ID N0:5, a step of;
the SEQ ID NO:2 from threonine to aspartic acid, the light chain obtained after substitution being SEQ ID N0:6, preparing a base material;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from serine to alanine, the heavy chain obtained after substitution being SEQ ID N0:7, preparing a base material;
the SEQ ID NO:2 from threonine to arginine, the light chain obtained after substitution being SEQ ID N0:8, 8;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from phenylalanine to valine, said SEQ ID NO:1 from alanine to glycine, the heavy chain obtained after substitution being SEQ ID N0:9, a step of performing the process;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from arginine to tyrosine, said amino acid 77 of SEQ ID NO:1 by phenylalanine to arginine, the heavy chain obtained after substitution is SEQ ID N0:11;
the SEQ ID NO:2 from glycine to valine, the light chain obtained after substitution being SEQ ID N0:12;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from tryptophan to threonine, the heavy chain obtained after substitution being SEQ ID N0:13;
and the antibody binds to hepatitis b surface antigen.
Preferably, the substitution is selected from the group,
the SEQ ID N0:1 from leucine to phenylalanine, the heavy chain obtained after substitution is SEQ ID N0: 15;
the SEQ ID NO:2 from threonine to glycine, said amino acid sequence of SEQ ID NO:2 from glycine to glutamine, the light chain obtained after substitution being SEQ ID N0:16;
and the antibody binds to hepatitis b surface antigen.
The second technical scheme of the invention: a monoclonal antibody of hepatitis B surface antigen includes amino acid sequence.
The third technical scheme of the invention: the preparation process of monoclonal antibody against hepatitis B surface antigen includes the following steps,
(A) Obtaining a variable region gene;
(B) Inserting the sequence qualified variable region gene sequence into a heavy-light chain expression plasmid by using a total gene synthesis means;
(C) Transforming the expression plasmid into competent cells;
(D) Extracting endotoxin-removed expression plasmid from resuscitated bacteria;
(E) Selecting HEK293 cells, and subculturing the HEK293 cells;
(F) Transiently transfecting HEK293 cells;
(G) Purifying the recombinant antibody.
Preferably, the obtaining in step (A) comprises the steps of,
(A1) Designing heavy chain primers and light chain primers, and synthesizing corresponding immunoglobulin G gene sequences;
(A2) Fishing variable region genes of a monoclonal antibody of hepatitis B surface antigen from a total RNA reverse transcription template of the hybridoma cells;
(A3) The sequence of the fished gene is inserted into a T vector and then sequenced.
Preferably, in the step (A1), the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the monoclonal antibody binding to hepatitis b surface antigen are selected, and the amino acid sequence of the light chain variable region comprises SEQ ID NO:2, said heavy chain amino acid sequence may comprise the sequence of SEQ ID NO:1 according to SEQ ID NO:2 and SEQ ID NO:1, and the sequence of the heavy chain primer and the light chain primer are designed respectively by using the conserved sequence characteristics of the V gene, the CH region and the CL region of the sequence variable region, and the corresponding immunoglobulin G gene sequence is synthesized by using hybridoma cells.
Preferably, the fishing in the step (A2) includes the steps of,
(A21) Taking a 0.2mL centrifuge tube, adding 10 mu L of 5 х Taq Buffer reagent, 4 mu L of dNTP mix (10 mM each) reagent, 1 mu L of hybridoma cell total RNA reverse transcription template, 5 mu L of heavy chain primer, 5 mu L of light chain primer, 24.75 mu L of ddH20 reagent and 0.25 mu L of Taq DNA polymerase (5 u/. Mu.L) reagent into the centrifuge tube respectively, shaking and mixing the Mixture in the centrifuge tube uniformly, and placing the Mixture on a centrifuge for centrifugation;
(A22) Placing the centrifuge tube in the step (A21) in a gene amplification instrument module for amplification.
Preferably, the amplification in step (A22) is performed in the following order,
(a1) Amplifying for 90min at 94 ℃;
(a2) Cycling amplification for 30 times in the order of 30s at 94 ℃, 30s at 56 ℃ and 10min at 72 ℃;
(a3) Amplifying for 1min at 72 ℃;
(a4) Amplification was carried out at a temperature of 4℃until completion.
Preferably, the inserting and sequencing in step (A3) comprises the steps of,
(A31) Adding a T carrier, a fished gene sequence fragment, 1 mu L of a 10× DNA ligase buffer reagent, 0.5-1 mu L of ligase and water into a centrifuge tube, uniformly mixing the mixed solution in the centrifuge tube, and centrifuging on the centrifuge to enable the sample to be completely submerged at the bottom of the tube;
(A32) After centrifugation, the centrifuge tube is incubated for 1 to 3 hours at the temperature of 16 ℃ or the centrifuge tube is incubated for overnight at the temperature of 4 ℃;
(A33) And (3) sequencing the sample after incubation, analyzing the sequence fed back by sequencing by using an antibody database and/or NCBI (NCBI), determining whether the sample belongs to a required antibody sequence, if so, continuing to carry out the subsequent steps, and if not, re-fishing.
Preferably, the molar ratio of the T vector to the fragment of the extracted gene sequence is 1: (3-5).
Preferably, the molar ratio of the T vector to the fragment of the extracted gene sequence is determined by electrophoresis.
Preferably, in the step (B),
the gene sequences of the variable regions of the heavy and light chains of the antibody after correct sequencing and bioinformatics analysis optimization are respectively inserted into an expression plasmid pcDNA3.1 with the heavy and light chain constant regions.
Preferably, the conversion in step (C) comprises the steps of,
(C1) Taking out the competent cells prepared by the multiple tubes, and putting the competent cells on ice for melting;
(C2) Three tubes were taken out and a control group was formed therein in such a manner that the expression plasmid DNA was added, the standard supercoiled plasmid DNA was added, and no DNA was added, and then blotted uniformly using a pipette and left on ice for 30 minutes;
(C3) Respectively placing the three pipes in water at 42 ℃, and performing water bath heat shock for 90 seconds; then the three pipes are quickly transferred into ice water for ice bath for 1-2 min;
(C4) After ice bath, 900ul of antibiotic-free LB medium was added to each tube, and the mixture was gently shaken on a shaker at 37℃and incubated for 45 minutes to resuscitate the bacteria;
(C5) Taking a proper volume of resuscitated bacteria, uniformly coating the resuscitated bacteria on an LB plate containing antibiotics, inverting the LB plate, and culturing the resuscitated bacteria at 37 ℃ for 12 to 16 hours to finish the transformation.
Preferably, the tubes are filled with the corresponding plasmid DNA in a ratio of 100ul competent cells to 20ng plasmid DNA.
Preferably, the extraction in the step (D) comprises the steps of,
(D1) Centrifuging to obtain 13ml of thallus in resuscitated bacteria, adding 260ul Buffer N3 reagent into thallus, and pre-cooling on ice for 30-60 min;
(D2) Adding 500ul of solution I/RNAseA mixed reagent after precooling, and transferring the thallus to an EP pipe after completely suspending by vortex oscillation;
(D3) Adding 500ul of solution II reagent into an EP pipe, mixing the mixture upside down, and cooling the mixture on ice for 2min;
(D4) Adding 250ul of ice pre-cooled buffer N3 reagent into an EP tube, mixing the mixture upside down, cooling the mixture on ice for 1 to 5 minutes, and centrifuging the mixture at 15000 Xg for 30 minutes at the temperature of 4 ℃;
(D5) Transferring the supernatant in the EP tube in the step (D4) to a new EP tube, adding ETR of which the volume is 0.1 times that of the supernatant, mixing, placing the mixed solution on ice for 10min, and reversing for many times during the placing;
(D6) After the EP tube in step (D5) was left in a temperature environment of 42℃for 5min, it was centrifuged at 12000 Xg for 3min at 25 ℃;
(D7) Adding 200ul of GPS reagent into the EP tube in the step (D6), standing for 3-5 min at room temperature, centrifuging for 3min at 12000 Xg, and discarding the waste liquid;
(D8) Transferring the supernatant in the step (D7) into 2 new EP pipes, adding 0.5 times of ethanol with the volume of the supernatant into each EP pipe, mixing, and standing at room temperature for 2min;
(D9) Transferring all the mixed liquid in the 2 EP pipes in the step (D8) into an adsorption column, centrifuging 10000 Xg for 1min, and circulating for multiple times;
(D10) Adding 500ul buffer HB reagent into the adsorption column in the step (D9), and centrifuging for 1min at 10000 Xg;
(D11) Adding 700ul DNA wash buffer reagent into the adsorption column in the step (D10), centrifuging for 1min at 10000 Xg, and repeating twice;
(D12) Centrifuging 13000 Xg of the adsorption column in the step (D11) for 5min;
(D13) Placing the adsorption column in the step (D12) in a new EP pipe, adding a 125ul Elution buffer reagent into the adsorption column, standing for 5min, and centrifuging and eluting 10000 Xg of the EP pipe for 2-3 min;
(D14) Repeating the step (D13) without replacing the EP tube;
(D15) Adding 3M NaAC 25ul of 1/10 of the liquid volume in the tube into the EP tube in the step (D14), and mixing the mixture upside down;
(D16) 193ul of 0.7 times isopropanol was added to the EP tube in step (D15), mixed upside down, left at room temperature for 5min, and centrifuged at 15000 Xg for 30min at 4℃and the supernatant discarded;
(D17) Adding 70% ethanol into the EP tube in the step (D16), reversing the process upside down, centrifuging at 15000 Xg for 10-30 min at 4 ℃, discarding the supernatant in a sterile environment, and then drying for 15-20 min;
(D18) 50ul of sterile water was added to the dried product in step (D17), and the mixture was redissolved overnight at 4℃to complete the extraction.
Preferably, the subculture in the step (E) comprises the steps of,
(E1) Taking out HEK293 cells from a liquid nitrogen tank or taking out HEK293 cells transported by dry ice, and carrying out cell resuscitating and culturing;
(E2) Firstly, counting cells, adding the cell suspension into a culture solution according to a required proportion, and starting subculture;
(E3) The cell density after passage was controlled at 0.3X10 6 Every 4 days, passage 1 time per milliliter; or controlling the cell density after passage to 0.6X10 6 Every 3 days, passage 1 time per milliliter; cell viability was controlled above 95% until the entire subculture process was completed.
Preferably, the culture solution is supportable at the mostHigh cell density of 1.3X10 7 Individual/ml; if the number of dead cells is excessive in the shake culture process, inoculating low cell density for static culture, and performing shake culture after the cells are recovered to the normal survival rate.
Preferably, the transient transfection in step (F) comprises the steps of,
(F1) Taking two sterile centrifuge tubes with 15ml, adding 5ml KPM and 100 mug sterile plasmid DNA into one sterile centrifuge tube, lightly blowing and mixing; adding 5ml KPM and 500 μl TA-293 transfection reagent into another sterile centrifuge tube, gently stirring, and mixing;
(F2) Transferring all the liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the sterile plasmid DNA, gently blowing and uniformly mixing, and standing for 10min at room temperature to prepare a plasmid-carrier compound;
(F3) From CO 2 Removing HEK293 cells from the constant temperature shaker, adding the plasmid-vector complex of step (F2) while shaking, and then returning to CO 2 Shake culturing in a constant temperature shaking table; after 3 hours, an appropriate amount of antibiotics was added.
Preferably, the purification in step (G) comprises the steps of,
(G1) Adding filler into a chromatographic column according to the proportion of 1ml protein A reagent for each 100ml cell culture solution supernatant, and slowly stirring at 4 ℃ for 24 hours;
(G2) Rinsing the chromatographic packing obtained in the step (G1) in 100mM Tris-HCl buffer solution with pH of 8.2 for 4 times, eluting with the same buffer solution containing 250mM sodium chloride, then carrying out fractional precipitation on the liquid by using ammonium sulfate, treating at the temperature of 4 ℃ for 12 hours, centrifuging at a high speed, and taking the precipitate;
(G3) The precipitate was redissolved in the buffer without sodium chloride in step (G2) and concentrated 10-fold after dialysis overnight in the same buffer to give a sample for use.
Preferably, the starting concentration of the chromatographic liquid in the step (G2) before the precipitation of ammonium sulfate is 35% saturation, and the ending concentration after the precipitation is 63% saturation; assaying the purity of the sample in step (G3) using HPLC; the protein content of the sample in step (G3) was quantitatively determined using Follin phenol.
Preferably, the method further comprises the step (H) of detecting the antibody titer, wherein the antibody titer detection comprises the following steps,
(H1) The coating is carried out by the method,
diluting hepatitis B surface antigen to 0.5 mug/ml with CB, adding 100 mu l of the diluted hepatitis B surface antigen to an enzyme-labeled plate hole, incubating for 2 hours at 37 ℃ or overnight at 4 ℃;
(H2) The sealing-off is carried out,
the ELISA plate in the step (H1) is dried by beating, 200 mu l of sealing liquid is added into each hole, the sealing is carried out for 1 to 2 hours at room temperature, and the sealing liquid is preserved for standby at the temperature of 4 ℃ after the drying;
(H3) Adding a primary antibody to the mixture,
diluting the sample by PBS for 5 times, adding 100 mu l of diluted sample into each hole of the ELISA plate in the step (H2), setting a blank control group and a compound hole, incubating for 30min at 37 ℃, washing the plate for 3-4 times by a plate washing machine, and then beating to dry;
(H4) Adding a secondary antibody into the mixture,
diluting the secondary antibody into 5000 times of working solution by using a washing solution, adding 100 mu l of working solution into each hole of the ELISA plate in the step 3, incubating for 30min at 37 ℃, washing the plate for 3-4 times by using a plate washing machine, and then beating to dry;
(H5) The color of the color is developed,
restoring the color development liquid A and the color development liquid B to room temperature, mixing the color development liquid A and the color development liquid B into working liquid according to the proportion of 32:1, adding 100 μl into each hole of the ELISA plate in the step 4, and incubating for 10min at 37 ℃;
(H6) The termination is performed such that the first and second channels are terminated,
adding 50 μl of stop solution to each well of the ELISA plate in step (H5);
(H7) The reading is made and the data is read,
the microplate in step (H6) was measured and the OD was read at 450nm using a microplate reader.
Preferably, the blocking solution is prepared by adding 1g of BSA reagent into 100ml of PBS reagent for dissolution; the washing solution used for washing the plate is prepared by adding 0.5ml of Tween-20 reagent into 1 LPBS.
The fourth technical scheme of the invention: the application of hepatitis B surface antigen monoclonal antibody in detecting hepatitis B surface antigen in serum.
The invention has the following beneficial effects:
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability through the traditional hybridoma fusion technology, and provides a related sequence, wherein the antibody is defined as an antibody No. 1, the heavy chain sequence of the antibody comprises SEQ ID NO. 1, and the light chain sequence of the antibody comprises SEQ ID NO. 2;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting the 101 th amino acid in the heavy chain sequence of SEQ ID NO. 1; the heavy chain sequence of the antibody No. 2 comprises SEQ ID NO. 3, and the light chain sequence comprises SEQ ID NO. 4; SEQ ID NO. 4 is the same as SEQ ID NO. 2;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting the 9 th amino acid in the heavy chain sequence of SEQ ID NO. 1 and the 31 st amino acid in the light chain sequence of SEQ ID NO. 2; the heavy chain sequence of the antibody No. 3 comprises SEQ ID NO. 5, and the light chain sequence comprises SEQ ID NO. 6;
the invention replaces the 97 th amino acid in the heavy chain sequence of SEQ ID NO. 1 and replaces the 31 st amino acid in the light chain sequence of SEQ ID NO. 2 to obtain a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability; the heavy chain sequence of the antibody No. 4 comprises SEQ ID NO. 7, and the light chain sequence comprises SEQ ID NO. 8;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting the 104 th amino acid and the 105 th amino acid in the heavy chain sequence of SEQ ID NO. 1; the heavy chain sequence of antibody No. 5 comprises SEQ ID NO. 9, and the light chain sequence comprises SEQ ID NO. 10; SEQ ID NO. 10 is the same as SEQ ID NO. 2;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting 77 th amino acid and 104 th amino acid in the heavy chain sequence of SEQ ID NO. 1 and 76 th amino acid in the light chain sequence of SEQ ID NO. 2; the heavy chain sequence of the antibody No. 6 comprises SEQ ID NO. 11, and the light chain sequence comprises SEQ ID NO. 12;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting the 107 th amino acid in the heavy chain sequence of SEQ ID NO. 1; the heavy chain sequence of antibody No. 7 comprises SEQ ID NO. 13, and the light chain sequence comprises SEQ ID NO. 14; SEQ ID NO. 14 is the same as SEQ ID NO. 2;
the invention obtains a monoclonal recombinant antibody of anti-hepatitis B surface antigen with strong specificity, high sensitivity and good stability by substituting 54 th amino acid in the heavy chain sequence of SEQ ID NO. 1 and substituting 53 th amino acid and 68 th amino acid in the light chain sequence of SEQ ID NO. 2; the heavy chain sequence of the antibody No. 8 comprises SEQ ID NO. 15, and the light chain sequence comprises SEQ ID NO. 16;
after diluting antibody No. 1, antibody No. 2, antibody No. 3, antibody No. 4, antibody No. 5, antibody No. 6, antibody No. 7, antibody No. 8 and the main stream commercial antibody by different factors, respectively, it acted on hepatitis b surface antigen, and then measured at 450nm using a microplate reader and the OD value was read, and the measurement results are shown in table 1:
from the results of the antibody titer detection in table 1, the titer of the original antibody No. 1 is slightly worse than that of the main stream commercial antibody, and the affinity of the antibodies No. 2, no. 3, no. 4, no. 7 and No. 8 is not improved but is reduced after optimization; however, the antibodies modified by the No. 5 and the No. 6 antibodies are determined and obtained through the optimization, and the affinity of the antibodies is higher than that of the original sequence antibodies and the main commercial antibodies.
Compared with the similar antibodies in the current domestic market, the hepatitis B surface antigen (HBsAg) monoclonal antibody prepared by the invention has better specificity and sensitivity, and can ensure that the detection sensitivity of the hepatitis B surface antigen (HBsAg) on a colloidal gold detection platform is more than that of the hepatitis B surface antigen (HBsAg) in abroad, so that the clinical detection of the HBsAg has higher accuracy, and the occurrence probability of false negative detection results is greatly reduced.
The invention relates to an affinity-optimized monoclonal recombinant antibody against hepatitis B virus surface antigen, and provides an amino acid sequence with optimized heavy and light chain variable region amino acid, and high-yield secretory expression and performance evaluation of the monoclonal antibody in eukaryotic HEK293 cells; the high-affinity and high-specificity hepatitis B surface antigen (HBsAg) antibody obtained by the invention has the potential of being developed into a humanized neutralizing antibody.
On the basis of the traditional hybridoma monoclonal antibody technology, the invention uses molecular biology technology to call the antibody heavy and light chain variable region of hybridoma cells secreting hepatitis B surface antigen, the company utilizes bioinformatics and protein structure simulation analysis means to simulate and optimize the variable region, finally designs 8 optimization schemes which are feasible in bioinformatics analysis level, constructs a vector through a whole gene synthesis means, uses HEK293 cells to carry out recombinant expression, then prepares purified antibodies, and carries out indirect ELISA to detect the titer, so that each group of optimization schemes carries out activity detection by using a hepatitis B virus core antibody detection kit of Shanghai department China biological engineering Limited, the affinity of the optimized sequences is verified to be uneven, and finally, the hepatitis B surface antigen (HBsAg) monoclonal antibody sequence with strong specificity, high sensitivity and good stability is obtained, and the HBsAg in serum can be detected more accurately and rapidly on a colloidal gold platform.
Drawings
FIG. 1 is a flow chart of the preparation method of the hepatitis B surface antigen monoclonal antibody.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not intended to be limiting.
Example 1:
the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from histidine to alanine, the heavy chain obtained after substitution being SEQ ID N0:3, a step of;
and the antibody binds to hepatitis b surface antigen.
Example 2:
the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from arginine to glutamic acid, the heavy chain obtained after substitution being SEQ ID N0:5, a step of;
the SEQ ID NO:2 from threonine to aspartic acid, the light chain obtained after substitution being SEQ ID N0:6, preparing a base material;
and the antibody binds to hepatitis b surface antigen.
Example 3: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from serine to alanine, the heavy chain obtained after substitution being SEQ ID N0:7, preparing a base material;
the SEQ ID NO:2 from threonine to arginine, the light chain obtained after substitution being SEQ ID N0:8, 8;
and the antibody binds to hepatitis b surface antigen.
Example 4: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from phenylalanine to valine, said SEQ ID NO:1 from alanine to glycine, the heavy chain obtained after substitution being SEQ ID N0:9, a step of performing the process;
and the antibody binds to hepatitis b surface antigen.
Example 5: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from arginine to tyrosine, said amino acid 77 of SEQ ID NO:1 by phenylalanine to arginine, the heavy chain obtained after substitution is SEQ ID N0:11;
the SEQ ID NO:2 from glycine to valine, the light chain obtained after substitution being SEQ ID N0:12;
and the antibody binds to hepatitis b surface antigen.
Example 6: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from tryptophan to threonine, the heavy chain obtained after substitution being SEQ ID N0:13;
and the antibody binds to hepatitis b surface antigen.
Example 7: the amino acid sequence of the monoclonal antibody of hepatitis B surface antigen comprises
An amino acid sequence of the variable region of a heavy chain comprising SEQ ID NO: 1; and
an amino acid sequence of a variable region of a light chain comprising SEQ ID NO:2, the sequence of which is set forth in seq id no,
the SEQ ID NO:1 and SEQ ID NO:2 has at least one substitution.
The substitution is selected from the group consisting of,
the SEQ ID N0:1 from leucine to phenylalanine, the heavy chain obtained after substitution is SEQ ID N0: 15;
the SEQ ID NO:2 from threonine to glycine, said amino acid sequence of SEQ ID NO:2 from glycine to glutamine, the light chain obtained after substitution being SEQ ID N0:16;
and the antibody binds to hepatitis b surface antigen.
Specific amino acid sequence substitutions are shown in Table 2:
example 8:
the hepatitis B surface antigen monoclonal antibody comprises an amino acid sequence of the hepatitis B surface antigen monoclonal antibody.
Example 9:
firstly, obtaining the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of a non-human monoclonal antibody combined with hepatitis B surface antigen; the non-human monoclonal antibody which binds to hepatitis B surface antigen may be selected from a murine monoclonal antibody comprising the amino acid sequence of the light chain of SEQ ID NO:2, the heavy chain amino acid sequence may comprise the sequence of SEQ ID NO:1, a sequence of 1; after the IgG gene of the mouse antibody is analyzed, heavy chain primers and light chain primers are respectively designed according to the conserved sequence characteristics of a V gene region, a CH1 region and a CL1 region of a variable region of a heavy chain gene and a light chain gene of the mouse antibody, and the heavy chain primers and the light chain primers are sent to Shanghai biological company, inc. for synthesis, so that a mouse antibody immunoglobulin G gene sequence is obtained;
the antibody is an immunoglobulin G antibody, and the amino acid sequence is that of the immunoglobulin G antibody;
the antibody is a monoclonal antibody.
The preparation method of the hepatitis B surface antigen monoclonal antibody shown in figure 1 comprises the following steps,
(A) Obtaining a variable region gene;
the obtaining in step (a) comprises the steps of,
(A1) Designing a heavy chain primer and a light chain primer, and synthesizing corresponding immunoglobulin G gene sequences;
in the step (A1), the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the monoclonal antibody combined with the hepatitis B surface antigen are selected, wherein the amino acid sequence of the light chain variable region comprises SEQ ID NO:2, the heavy chain amino acid sequence may comprise the sequence of SEQ ID NO:1 according to SEQ ID NO:2 and SEQ ID NO:1, and the sequence of the CH region and the CL region, respectively designing a heavy chain primer and a light chain primer, wherein the heavy chain primer is a heavy chain fishing primer, the light chain primer is a light chain fishing primer, as shown in table 3, and synthesizing the corresponding immunoglobulin G gene sequence by using hybridoma cells.
Table 3: after the mouse antibody IgG genes are analyzed, heavy and light chain primers are respectively designed according to the conserved sequence characteristics of the V gene region and the CH1 and CL1 regions of the variable region of the mouse antibody heavy and light chain genes,
(A2) Fishing variable region genes of a monoclonal antibody of hepatitis B surface antigen from a total RNA reverse transcription template of the hybridoma cells;
the fishing in step (A2) includes the steps of,
(A21) Taking a 0.2mL centrifuge tube, adding 10 mu L of 5 х Taq Buffer reagent, 4 mu L of dNTP mix (10 mM each) reagent, 1 mu L of hybridoma cell total RNA reverse transcription template, 5 mu L of heavy chain primer, 5 mu L of light chain primer, 24.75 mu L of ddH20 reagent and 0.25 mu L of Taq DNA polymerase (5 u/. Mu.L) reagent into the centrifuge tube respectively, shaking and mixing the Mixture in the centrifuge tube uniformly, and placing the Mixture on a centrifuge for centrifugation;
(A22) Placing the centrifuge tube in the step (A21) in a gene amplification instrument module for amplification.
The amplification in step (A22) is performed in the following order,
(a1) Amplifying for 90min at 94 ℃;
(a2) Cycling amplification for 30 times in the order of 30s at 94 ℃, 30s at 56 ℃ and 10min at 72 ℃;
(a3) Amplifying for 1min at 72 ℃;
(a4) Amplification was carried out at a temperature of 4℃until completion.
(A3) The sequence of the fished gene is inserted into a T vector and then sequenced.
The inserting and sequencing in step (A3) comprises the steps of,
(A31) Adding a T carrier, a fished gene sequence fragment, 1 mu L of a 10× DNA ligase buffer reagent, 0.5-1 mu L of ligase and water into a centrifuge tube, uniformly mixing the mixed solution in the centrifuge tube, and centrifuging on the centrifuge to enable the sample to be completely submerged at the bottom of the tube;
(A32) After centrifugation, the centrifuge tube is incubated for 1 to 3 hours at the temperature of 16 ℃ or the centrifuge tube is incubated for overnight at the temperature of 4 ℃;
(A33) And (3) sequencing the sample after incubation, analyzing the sequence fed back by sequencing by using an antibody database and/or NCBI (NCBI), determining whether the sample belongs to a required antibody sequence, if so, continuing to carry out the subsequent steps, and if not, re-fishing.
The molar ratio of the T vector to the fragment of the extracted gene sequence is 1: (3-5).
The molar ratio of the T vector to the fragment of the extracted gene sequence is determined by electrophoresis.
(B) Inserting the sequence qualified variable region gene sequence into a heavy-light chain expression plasmid by using a total gene synthesis means;
in the step (B), the variable region gene sequences of the heavy and light chain of the antibody after the correct sequencing and the variable region gene sequences of the heavy and light chain of the antibody after the bioinformatic analysis and optimization are respectively inserted into an expression plasmid pcDNA3.1 with a heavy and light chain constant region.
(C) Transforming the expression plasmid into competent cells;
the conversion in step (C) comprises the steps of,
(C1) Taking out the competent cells prepared by the multiple tubes, and putting the competent cells on ice for melting;
(C2) Three tubes were taken out and a control group was formed therein in such a manner that the expression plasmid DNA was added, the standard supercoiled plasmid DNA was added, and no DNA was added, and then blotted uniformly using a pipette and left on ice for 30 minutes;
(C3) Respectively placing the three pipes in water at 42 ℃, and performing water bath heat shock for 90 seconds; then the three pipes are quickly transferred into ice water for ice bath for 1-2 min;
(C4) After ice bath, 900ul of antibiotic-free LB medium was added to each tube, and the mixture was gently shaken on a shaker at 37℃and incubated for 45 minutes to resuscitate the bacteria;
(C5) Taking a proper volume of resuscitated bacteria, uniformly coating the resuscitated bacteria on an LB plate containing antibiotics, inverting the LB plate, and culturing the resuscitated bacteria at 37 ℃ for 12 to 16 hours to finish the transformation.
The tubes were filled with the corresponding plasmid DNA in a proportion of 100ul competent cells to 20ng plasmid DNA.
(D) Extracting endotoxin-removed expression plasmid from resuscitated bacteria;
the extraction in step (D) comprises the steps of,
(D1) Centrifuging to obtain 13ml of thallus in resuscitated bacteria, adding 260ul Buffer N3 reagent into thallus, and pre-cooling on ice for 30-60 min;
(D2) Adding 500ul of solution I/RNAseA mixed reagent after precooling, and transferring the thallus to an EP pipe after completely suspending by vortex oscillation;
(D3) Adding 500ul of solution II reagent into an EP pipe, mixing the mixture upside down, and cooling the mixture on ice for 2min;
(D4) Adding 250ul of ice pre-cooled buffer N3 reagent into an EP tube, mixing the mixture upside down, cooling the mixture on ice for 1 to 5 minutes, and centrifuging the mixture at 15000 Xg for 30 minutes at the temperature of 4 ℃;
(D5) Transferring the supernatant in the EP tube in the step (D4) to a new EP tube, adding ETR of which the volume is 0.1 times that of the supernatant, mixing, placing the mixed solution on ice for 10min, and reversing for many times during the placing;
(D6) After the EP tube in step (D5) was left in a temperature environment of 42℃for 5min, it was centrifuged at 12000 Xg for 3min at 25 ℃;
(D7) Adding 200ul of GPS reagent into the EP tube in the step (D6), standing for 3-5 min at room temperature, centrifuging for 3min at 12000 Xg, and discarding the waste liquid;
(D8) Transferring the supernatant in the step (D7) into 2 new EP pipes, adding 0.5 times of ethanol with the volume of the supernatant into each EP pipe, mixing, and standing at room temperature for 2min;
(D9) Transferring all the mixed liquid in the 2 EP pipes in the step (D8) into an adsorption column, centrifuging 10000 Xg for 1min, and circulating for multiple times;
(D10) Adding 500ul buffer HB reagent into the adsorption column in the step (D9), and centrifuging for 1min at 10000 Xg;
(D11) Adding 700ul DNA wash buffer reagent into the adsorption column in the step (D10), centrifuging for 1min at 10000 Xg, and repeating twice;
(D12) Centrifuging 13000 Xg of the adsorption column in the step (D11) for 5min;
(D13) Placing the adsorption column in the step (D12) in a new EP pipe, adding a 125ul Elution buffer reagent into the adsorption column, standing for 5min, and centrifuging and eluting 10000 Xg of the EP pipe for 2-3 min;
(D14) Repeating step (D13) without replacing the EP tube;
(D15) Adding 3M NaAC 25ul of 1/10 of the liquid volume in the tube into the EP tube in the step (D14), and mixing the mixture upside down;
(D16) 193ul of 0.7 times isopropanol was added to the EP tube in step (D15), mixed upside down, left at room temperature for 5min, and centrifuged at 15000 Xg for 30min at 4℃and the supernatant discarded;
(D17) Adding 70% ethanol into the EP tube in the step (D16), reversing the process upside down, centrifuging at 15000 Xg for 10-30 min at 4 ℃, discarding the supernatant in a sterile environment, and then drying for 15-20 min;
(D18) 50ul of sterile water was added to the dried product in step (D17), and the mixture was redissolved overnight at 4℃to complete the extraction.
(E) Selecting HEK293 cells, and subculturing the HEK293 cells;
the subculture in step (E) comprises the steps of,
(E1) Taking out HEK293 cells from a liquid nitrogen tank or taking out HEK293 cells transported by dry ice, and carrying out cell resuscitating and culturing;
(E2) Firstly, counting cells, adding the cell suspension into a culture solution according to a required proportion, and starting subculture;
(E3) The cell density after passage was controlled at 0.3X10 6 Every 4 days, passage 1 time per milliliter; or controlling the cell density after passage to 0.6X10 6 Every 3 days, passage 1 time per milliliter; cell viability was controlled above 95% until the entire subculture process was completed.
The maximum cell density supportable by the culture solution is 1.3X10 7 Individual/ml; if the number of dead cells is excessive in the shake culture process, inoculating low cell density for static culture, and performing shake culture after the cells are recovered to the normal survival rate.
Because the growth rate of cells per culture is still different, the cell density after passage is controlled to be 0.3X10 6 And each milliliter. E3 is a supplementary explanation of E2.
(F) Transiently transfecting HEK293 cells;
transient transfection in step (F) comprises the steps of,
(F1) Taking two sterile centrifuge tubes with 15ml, adding 5ml KPM and 100 mug sterile plasmid DNA into one sterile centrifuge tube, lightly blowing and mixing; adding 5ml KPM and 500 μl TA-293 transfection reagent into another sterile centrifuge tube, gently stirring, and mixing;
(F2) Transferring all the liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the sterile plasmid DNA, gently blowing and uniformly mixing, and standing for 10min at room temperature to prepare a plasmid-carrier compound;
(F3) Taking out HEK293 cells from the CO2 constant temperature shaking table, adding the plasmid-carrier complex in the step (F2) while shaking, and then putting the mixture back into the CO2 constant temperature shaking table for shake culture; after 3 hours, an appropriate amount of antibiotics was added.
(G) Purifying the recombinant antibody;
the purification in step (G) comprises the steps of,
(G1) Filling materials are added into a chromatographic column according to the proportion of 1ml protein A reagent for each 100ml cell culture solution supernatant, and then stirring is carried out slowly for 24 hours at the temperature of 4 ℃;
(G2) Rinsing the chromatographic liquid obtained in the step (G1) in 100mM Tris-HCl buffer solution with pH of 8.2 for 4 times, eluting with the same buffer solution containing 250mM sodium chloride, then carrying out fractional precipitation on the liquid by using ammonium sulfate, treating at the temperature of 4 ℃ for 12 hours, centrifuging at a high speed, and taking the precipitate;
the starting concentration of the chromatographic liquid in the step (G2) before the ammonium sulfate precipitation is 35% saturation, and the ending concentration after the precipitation is 63% saturation; assaying the purity of the sample in step (G3) using HPLC; the protein content of the sample in step (G3) was quantitatively determined using Follin phenol.
(G3) The precipitate was redissolved in the buffer without sodium chloride in step (G2) and concentrated 10-fold after dialysis overnight in the same buffer to give a sample for use.
(H) Detecting antibody titer;
the antibody titer detection comprises the steps of,
(H1) Coating, diluting the hepatitis B surface antigen to 0.5 mug/ml by CB, adding 100 mu l of the surface antigen per well into a plate hole of an enzyme label, and incubating for 2 hours at 37 ℃ or overnight at 4 ℃;
(H2) Sealing, namely drying the ELISA plate in the step (H1) in a beating way, adding 200 mu l of sealing liquid into each hole, sealing for 1-2H at room temperature, and preserving at the temperature of 4 ℃ for later use after drying in a beating way;
(H3) Adding primary antibody, diluting the sample by PBS for 5 times, adding 100 mu l of diluted sample into each hole of the ELISA plate in the step (H2), setting a blank control group and a compound hole, incubating for 30min at 37 ℃, washing the plate for 3-4 times by a plate washing machine, and then beating to dry; wherein the blank control group refers to a negative control; the compound holes are used for ensuring the accuracy of experimental data, each hole needs to be repeated in a certain number, and the average value of the holes is taken during measurement;
(H4) Adding a secondary antibody, diluting the secondary antibody into 5000 times of working solution by using a washing liquid, adding 100 mu l of the working solution into each hole of the ELISA plate in the step 3, incubating for 30min at 37 ℃, washing the plate for 3-4 times by using a plate washing machine, and then beating to dry;
(H5) Developing, recovering the developing solution A and the developing solution B to room temperature, mixing the developing solution A and the developing solution B into working solutions in a ratio of 35:1, adding 100 μl into each hole of the ELISA plate in the step 4, and incubating for 10min at 37 ℃; the color development liquid A is prepared by taking 8.2g of anhydrous sodium acetate, 2.5g of beta-dextrin and 428.6mg of urea hydrogen peroxide, adding double distilled water to 1000mL, and regulating the pH to 5.0; the color development liquid B is prepared by dissolving 100mgTMB in 10mL DMSO and preserving in a brown bottle;
(H6) Terminating, adding 50 μl of a termination solution into each well of the ELISA plate in step (H5); the formula of the stop solution is H2SO4 solution with the concentration of 1.25 mol/L;
(H7) Reading, the ELISA plate in the step (H6) is measured at 450nm by using an ELISA reader, and OD value is read.
Adding 1g BSA reagent into 100ml PBS reagent to dissolve the blocking solution to obtain the product; the washing solution used for washing the plate was 1LPBS, and 0.5ml of Tween-20 reagent was added thereto.
Example 10:
the application of hepatitis B surface antigen monoclonal antibody in detecting hepatitis B surface antigen in serum.
Sequence listing
<110> Hangzhou Boyue biotechnology Co., ltd
<120> hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence
<140> 2020104791483
<141> 2020-05-29
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<213> mouse (Artificial Sequence)
<400> 12
Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Thr Tyr
20 25 30
Gly Val Gly Trp Tyr Gln Gln Leu Pro Gly Ser Gly Leu Arg Thr Ile
35 40 45
Ile Tyr Gly Thr Thr Ser Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Cys Gly Ser Val Asp Ser Ser Ser Tyr Val Val Phe Gly Ser
65 70 75 80
Gly Thr Arg Leu Thr Val Leu
85
<210> 13
<211> 117
<212> PRT
<213> mouse (Artificial Sequence)
<400> 13
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe
20 25 30
Ala Ile His Trp Val Lys Gln Ala Pro Gly Lys Asn Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Arg Asn Asn Lys Ser Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Ile Ala Tyr
65 70 75 80
Leu Glu Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ser Arg Ser Arg His Asp Trp Phe Ala Tyr Thr Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 14
<211> 87
<212> PRT
<213> mouse (Artificial Sequence)
<400> 14
Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Thr Tyr
20 25 30
Gly Val Gly Trp Tyr Gln Gln Leu Pro Gly Ser Gly Leu Arg Thr Ile
35 40 45
Ile Tyr Gly Thr Thr Ser Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Cys Gly Ser Val Asp Ser Ser Ser Tyr Gly Val Phe Gly Ser
65 70 75 80
Gly Thr Arg Leu Thr Val Leu
85
<210> 15
<211> 117
<212> PRT
<213> mouse (Artificial Sequence)
<400> 15
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe
20 25 30
Ala Ile His Trp Val Lys Gln Ala Pro Gly Lys Asn Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Phe Asn Asn Lys Ser Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Ile Ala Tyr
65 70 75 80
Leu Glu Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ser Arg Ser Arg His Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 16
<211> 87
<212> PRT
<213> mouse (Artificial Sequence)
<400> 16
Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Thr Tyr
20 25 30
Gly Val Gly Trp Tyr Gln Gln Leu Pro Gly Ser Gly Leu Arg Thr Ile
35 40 45
Ile Tyr Gly Thr Gly Ser Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Cys Gln Ser Val Asp Ser Ser Ser Tyr Gly Val Phe Gly Ser
65 70 75 80
Gly Thr Arg Leu Thr Val Leu
85
<210> 17
<211> 323
<212> PRT
<213> mouse (Artificial Sequence)
<400> 17
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly
<210> 18
<211> 106
<212> PRT
<213> mouse (Artificial Sequence)
<400> 18
Arg Gly Gln Pro Lys Ser Thr Pro Thr Leu Thr Val Phe Pro Pro Ser
1 5 10 15
Ser Glu Glu Leu Lys Glu Asn Lys Ala Thr Leu Val Cys Leu Ile Ser
20 25 30
Asn Phe Ser Pro Ser Gly Val Thr Val Ala Trp Lys Ala Asn Gly Thr
35 40 45
Pro Ile Thr Gln Gly Val Asp Thr Ser Asn Pro Thr Lys Glu Gly Asn
50 55 60
Lys Phe Met Ala Ser Ser Phe Leu His Leu Thr Ser Asp Gln Trp Arg
65 70 75 80
Ser His Asn Ser Phe Thr Cys Gln Val Thr His Glu Gly Asp Thr Val
85 90 95
Glu Lys Ser Leu Ser Pro Ala Glu Cys Leu
100 105
Claims (3)
1. The hepatitis B surface antigen monoclonal antibody comprises an amino acid sequence and is characterized in that: the amino acid sequence consists of a heavy chain amino acid sequence and a light chain amino acid sequence;
the heavy chain amino acid sequence and the light chain amino acid sequence are any one of the following combination forms:
one of the combination forms is as follows:
the heavy chain amino acid sequence is SEQ ID N0:3, a sequence shown in 3;
the light chain amino acid sequence is SEQ ID NO:4;
the second combination form is:
the heavy chain amino acid sequence is SEQ ID N0:5, a sequence shown in seq id no;
the light chain amino acid sequence is SEQ ID NO:6;
the third combination form is:
the heavy chain amino acid sequence is SEQ ID N0:7;
the light chain amino acid sequence is SEQ ID NO:8;
the fourth combination form is as follows:
the heavy chain amino acid sequence is SEQ ID N0:9;
the light chain amino acid sequence is SEQ ID NO:10, a sequence shown in seq id no;
the fifth combination form is:
the heavy chain amino acid sequence is SEQ ID N0:11, a sequence shown in seq id no;
the light chain amino acid sequence is SEQ ID NO:12, a sequence shown in seq id no;
the sixth combination form is:
the heavy chain amino acid sequence is SEQ ID N0:13, a sequence shown in seq id no;
the light chain amino acid sequence is SEQ ID NO:14, a sequence shown in seq id no;
the seventh combination form is:
the heavy chain amino acid sequence is SEQ ID N0:15, a sequence shown in seq id no;
the light chain amino acid sequence is SEQ ID NO: 16.
2. The preparation method of the hepatitis B surface antigen monoclonal antibody is characterized by comprising the following steps: comprises the steps of,
(A) Obtaining the heavy and light chain gene sequences of claim 1;
(B) Inserting the heavy chain and light chain gene sequences qualified in sequence into a heavy-light chain expression plasmid by using a total gene synthesis means;
(C) Transforming the expression plasmid into competent cells;
(D) Extracting endotoxin-removed expression plasmid from resuscitated bacteria;
(E) Selecting HEK293 cells, and subculturing the HEK293 cells;
(F) Transiently transfecting HEK293 cells;
(G) Purifying the recombinant antibody.
3. Use of the hepatitis b surface antigen monoclonal antibody of claim 1 for detecting hepatitis b surface antigen in serum.
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