CN111234012A

CN111234012A - Hepatitis B virus neutralizing antibody B432 and application thereof

Info

Publication number: CN111234012A
Application number: CN201811442220.4A
Authority: CN
Inventors: 张林琦; 贲海静; 史宣玲
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2020-06-05
Anticipated expiration: 2038-11-29
Also published as: CN111234012B

Abstract

The invention discloses a hepatitis B virus neutralizing antibody B432 and application thereof. The invention provides an IgG antibody, which is named as a monoclonal antibody B432 and consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are amino acid residues at positions 44-51, 69-76 and 115-131 from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-48 th amino acid residue, the 66 th-68 th amino acid residue and the 105 th-113 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence. The invention can be used for treating hepatitis B chronic infection patients and clinically replacing serum purified polyclonal antibody to prevent mother-infant transmission. The invention has important application prospect in treating and preventing hepatitis B virus infection.

Description

Hepatitis B virus neutralizing antibody B432 and application thereof

Technical Field

The invention relates to a hepatitis B virus neutralizing antibody B432 and application thereof.

Background

Hepatitis B Virus (HBV) infection is widespread throughout the world and is particularly severe in china. Hepatitis B virus belongs to a member of hepadnaviridae, and the infection of hepatitis B virus induces hepatitis, about 8000 ten thousand to 1 hundred million people in China have chronic hepatitis B virus infection, and about 2.4 hundred million chronic hepatitis B patients in the world. Hepatitis B virus infectors add up more if considered already recovered acute hepatitis B virus infectors. Research shows that 35-62% of Chinese people are infected by hepatitis B virus, 56-98% of sub-sahara south Africa people are infected by hepatitis B virus, about 20 hundred million people are infected by hepatitis B virus all over the world, and hepatitis B virus infection is a global sanitary problem.

Hepatitis B virus induces immune inflammation to damage the liver of a patient during the infection process. Most of Chinese hepatitis B patients are infected with hepatitis B virus by mother and infant transmission or children, most of the Chinese hepatitis B patients are infected with chronic hepatitis B virus, and the Chinese hepatitis B patients carry the hepatitis B virus throughout the life. The adult infected with hepatitis B virus can eliminate hepatitis B virus by means of autoimmune system, and is acute infection of hepatitis B virus. Hepatitis B virus infection causes acute and chronic hepatitis, cirrhosis and even liver cancer, the mechanism of inducing hepatitis is mainly that hepatitis B virus in liver cells induces liver inflammation of the immune system of the organism, the replication template covalent closed circular DNA (cccDNA) of the hepatitis B virus is used as a virus storage bank to be stably and timely supplemented and is very difficult to remove, and the replication of the hepatitis B virus induces liver inflammation in the continuous and repeated immune killing process.

At present, the hepatitis B virus vaccine has good prevention effect, can only control the replication of hepatitis B virus in clinical treatment, and can not cure hepatitis B virus infection. The hepatitis B vaccine controls the wide spread of hepatitis B virus in China and the mother-infant spread caused by the hepatitis B mother, clinically reduces the hepatitis B virus replication level of the hepatitis B mother during pregnancy through nucleoside medicaments such as entecavir and tenofovir, and comprehensively intervenes by measures of injecting Hepatitis B Immunoglobulin (HBIG) after the birth of a newborn and timely immunizing the hepatitis B vaccine, thereby effectively reducing the mother-infant spread of the hepatitis B virus. Although hepatitis B vaccines control the transmission of hepatitis B virus, the hepatitis B virus infection is not cured by clinical treatments such as nucleoside drugs and interferon for patients with a large number of infected hepatitis B viruses. The nucleoside medicine can effectively control the DNA replication of hepatitis B virus, but can not completely eliminate hepatitis B virus, and is difficult to reduce hepatitis B surface antigen, and hepatitis B virus still can relapse after a hepatitis B patient stops taking the medicine. The interferon has long treatment period and great side effect, can only act on a relatively small number of hepatitis B patients during the treatment period, has low cost performance of treatment effect, and basically treats hepatitis B infection by mainly taking nucleoside medicaments at present.

Disclosure of Invention

The invention aims to provide a hepatitis B virus neutralizing antibody B432 and application thereof.

The invention provides an IgG antibody, which is named as a monoclonal antibody B432 and consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are amino acid residues at positions 44-51, 69-76 and 115-131 from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-48 th amino acid residue, the 66 th-68 th amino acid residue and the 105 th-113 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence.

The heavy chain variable region consists of 20 th-142 th amino acid residues from the tail end of N of a sequence 1 in a sequence table.

The light chain variable region consists of 17 th to 123 th amino acid residues from the tail end of N of a sequence 3 in a sequence table.

The heavy chain is (a) or (b) as follows: (a) protein consisting of 20 th-472 th amino acid residues from the tail end of N in a sequence 1 of a sequence table; (b) a protein shown in a sequence 1 in a sequence table.

The light chain is (c) or (d) as follows: (c) protein consisting of 17 th-230 th amino acid residues from the N tail end of the sequence 3 in the sequence table; (d) a protein shown in sequence 3 of the sequence table.

The gene encoding the IgG antibody also belongs to the protection scope of the invention.

The gene encoding the heavy chain is (1) or (2) or (3) as follows:

(1) a DNA molecule shown as nucleotide 972-2330 from the 5' end of the sequence 2 in the sequence table;

(2) a DNA molecule shown as the 915-2333 nucleotide from the 5' end of the sequence 2 in the sequence table;

(3) a DNA molecule shown in a sequence 2 of a sequence table.

The genes encoding the light chain are as follows (4) or (5) or (6):

(4) DNA molecule shown by the 963-1604 nucleotide from the 5' end of the sequence 4 in the sequence table;

(5) DNA molecule shown by 915-1607 nucleotides from the 5' end of the sequence 4 in the sequence table;

(6) DNA molecule shown in sequence 4 of the sequence table.

The invention also protects the application of any IgG antibody in preparing a medicament for inhibiting hepatitis B virus.

The invention also provides a medicament for inhibiting hepatitis B virus, and the active ingredient of the medicament is the IgG antibody.

The invention also protects the application of any IgG antibody in the preparation of a medicament for neutralizing hepatitis B virus.

The invention also provides a medicament for neutralizing hepatitis B virus, and the active ingredient of the medicament is the IgG antibody.

The invention also protects the application of any IgG antibody in the preparation of a medicament for preventing and/or treating hepatitis B.

The invention also provides a medicament for preventing and/or treating hepatitis B, which comprises the IgG antibody as an active ingredient.

The monoclonal antibody B432 can identify different genotype hepatitis B viruses, and ensures the broad spectrum of antibody neutralization.

The hepatitis B virus is of each genotype. For example, hepatitis B virus of genotype A, hepatitis B virus of genotype B, hepatitis B virus of genotype C, and hepatitis B virus of genotype D. The A genotype hepatitis B virus is the hepatitis B virus with genotype A. The B genotype hepatitis B virus is the hepatitis B virus with genotype B. Hepatitis B virus of C genotype, i.e., the hepatitis B virus of which the genotype is C type. D genotype hepatitis B virus, i.e., hepatitis B virus with D genotype.

Compared with serum polyclonal antibody, the monoclonal antibody has great advantages in neutralizing activity and source convenience. The inventor of the invention utilizes the culture technology of humanized memory B cells, and the monoclonal antibody B432 is separated from a hepatitis B vaccine immunized person, and can be used for treating hepatitis B chronic infection patients and clinically replacing serum purified polyclonal antibody to prevent mother-infant transmission. The invention has important application prospect in treating and preventing hepatitis B virus infection.

Drawings

FIG. 1 shows the result of step one in example 3.

FIG. 2 shows the results of step two of example 3.

FIG. 3 shows the result of step one in example 4.

FIG. 4 shows the results of step two of example 4.

FIG. 5 shows the results of example 5.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, the PBS buffer used in the examples was 10mM PBS buffer at pH 7.2. PBST solution: 0.5% (volume percent) Triton X-100 in PBS buffer. Hepatitis b immunoglobulin solution (HBIG solution): beijing Tiantan organism with concentration of about 120 mg/ml. Unless otherwise specified, the cell culture conditions were 37 ℃ and 5% CO₂And (4) environment. JC126 vector contains HDV replicon, references to JC126 vector: li Ying et al, construction of hepatitis D virus replication packaging vector, China journal of modern medicine, 2017(11), pages 31-35. The expression plasmid pHBV1.5 for type A hepatitis B envelope protein is described in the following documents: bruss, V.and D.Ganem, The roll of envelope proteins in hepatitis B viral analysis, Proc Natl Acad Sci U S A,1991.88(3): p.1059-63. The expression plasmid for type B hepatitis B envelope protein, pHBV1.18-B, is described in the following documents: hu, W, et al, CpG oligodeoxynuclotide inhibitsHBVreproduction in a dynamic information music model, animal Ther,2014. The expression plasmid of the type C hepatitis B envelope protein is pHBV1.18-C. The following documents are described: wang, X.J., et al, A simple and effective strategy for the de novo constraint of great-this-genome-length syndromes B viruses reptilities.J., Methods of J vitamins, 2014.207: p.158-62. The expression plasmid of hepatitis B envelope protein type D, namely pCMV-HBV, is described in the following documents: j Virol,2010.84(13): p.6387-99.J., and methods of inhibiting of hepatitis B virus replication by MyD 88. J.. HBs antigen: GENBANK ACCESSION NO: P30019.1, VRL 07-NOV-2018. HBV transgenic mice (high replication HBV transgenic mice): the following documents are described: liu Lu luster, etc., the research on the effect of a high-replication HBV transgenic mouse model on anti-hepatitis B virus drugs, and 99-102 of Chinese pathophysiology journal, 2007,23 (1). Rabbit anti-human hepatitis delta antigen polyclonal antibody (HDV delta antibody for short): beijing Zexi Yuan GmbH. The monoclonal antibody MERS-27 and the preparation method thereof are disclosed in patent 201310565893.X (publication No. CN 104628848A, publication No. CN 104628848B). The hepatitis B virus surface antigen diagnostic kit (HBsAg, cat # 30811010101) and the hepatitis B virus e antigen detection kit (HBeAg, cat # 30811010103) are both Shanghai Kehua products.

Example 1 discovery of antibodies

The monoclonal antibody of hepatitis B virus is searched from the blood of hepatitis B vaccine immune persons by utilizing human memory B cell culture, and then the obtained antibodies are compared in effect.

The method for screening and cloning the hepatitis B small membrane protein specific B cell antibody gene by B cell culture comprises the following steps: separating peripheral blood lymphocyte PBMC from peripheral blood of normal people immunized by hepatitis B vaccine, separating memory B cells from the PBMC by a flow cytometer, co-culturing the memory B cells and supporting cells 3T3-CD40L for 10 days, detecting whether secreted cell supernatant contains hepatitis B virus small membrane protein HBs antibody by Elisa, cracking B cells reverse transcription cDNA by antibody positive holes, cloning all antibody variable region genes of the positive holes by using antibody variable region specific primers, and screening a pair of hepatitis B small membrane protein specific antibody genes by light-heavy chain pairing.

A monoclonal antibody (binding antibody) having a good effect was found, and this monoclonal antibody was designated as monoclonal antibody B432, and abbreviated as antibody B432.

The monoclonal antibody B432 is an IgG antibody, the heavy chain is shown as sequence 1 in the sequence table (the amino acid residues at the 1 st to 19 th positions form a signal peptide, the amino acid residues at the 20 th to 142 th positions form a variable region, the amino acid residues at the 143 st and the 472 th positions form a constant region; the CDR1, the CDR2 and the CDR3 are sequentially shown as the 44 th to 51 th positions, the 69 th to 76 th positions and the 115 th and the 131 th positions), and the light chain is shown as sequence 3 in the sequence table (the amino acid residues at the 1 st to 16 th positions form a signal peptide, the amino acid residues at the 17 th to 123 th positions form a variable region, the amino acid residues at the 124 th and the 230 th positions form a constant region; and the CDR1, the CDR2 and the CDR3 are sequentially shown as the 43 th to 48 th.

Example 2 preparation of monoclonal antibody B432

Firstly, preparing recombinant plasmid

Inserting the DNA molecule shown in the sequence 2 of the sequence table into pLB-simple Vector to obtain the recombinant plasmid. The recombinant plasmid has been verified by sequencing. This plasmid was in turn designated as heavy chain expression plasmid.

In the sequence 2 of the sequence table, the 1 st to 666 th nucleotides are CMV promoters, the 915 nd and 971 st nucleotides are signal peptide coding regions, the 972 nd 1340 th nucleotides are heavy chain variable region coding regions, the 1341 st 2330 th nucleotides are heavy chain constant region coding regions, the 2331 st 2333 th nucleotides are stop codons, and the 2392 nd 2540 th nucleotides are polyA terminators. The DNA molecule shown in the sequence 2 of the sequence table expresses the heavy chain shown in the sequence 1 of the sequence table.

Inserting the DNA molecule shown in the sequence 4 of the sequence table into pLB-simple Vector to obtain the recombinant plasmid. The recombinant plasmid has been verified by sequencing. This plasmid was in turn designated as light chain expression plasmid.

In the sequence 4 of the sequence table, the 1 st-666 th nucleotide is CMV promoter, the 915 nd-962 th nucleotide is signal peptide coding region, the 963 th-1283 th nucleotide is light chain variable region coding region, the 1284 nd-1604 nucleotide is light chain constant region coding region, the 1605 nd-1607 th nucleotide is stop codon, and the 1614 nd-1762 nd nucleotide is polyA terminator. The DNA molecule shown in the sequence 4 of the sequence table expresses the light chain shown in the sequence 3 of the sequence table.

pLB-simple Vector is a component of pLB zero background quick connect kit with the biological cargo number of Tiangen VT206, http:// www.tiangen.com/? productive show/t1/6/id/308. html.

II, preparing the antibody

1. The 293T cells are cultured by adopting serum-free DMEM medium, the heavy chain expression plasmid and the light chain expression plasmid are cotransfected by virtue of PEI transfection reagent and cultured for 8 hours, then the culture medium is replaced by DMEM medium containing 2% fetal calf serum and cultured for 72 hours, then the cells are centrifuged at 4 ℃ and 4000rpm for 30min, and supernatant is collected.

2. And (3) taking the supernatant obtained in the step 1, and purifying by using an Econo-Pac polypropylene chromatographic column.

Polypropylene chromatography column (Biorad, cat # 7321010), 1.5X 12cm, bed volume about 20 ml.

The procedure was carried out by mixing ① -500mL of supernatant with about 1mL of protein A beads (Thermo, cat # 10006D), shaking the mixture at 4 ℃ for 10 hours, then introducing the whole system into a column, washing ② with 60mL of binding buffer, eluting ③ with 30mL of elution buffer, and collecting the post-column solution.

Binding buffer (ph 8.0): each liter contains 112.6g of glycine, 175.2g of sodium chloride and the balance of water.

Elution buffer (ph 3.0): every 500ml contains 7.5g of glycine and the balance of water.

3. And (3) taking the solution after the column passing obtained in the step (2), concentrating by using an ultrafiltration concentration tube, and replacing the system by PBS buffer solution to obtain an antibody solution with the antibody concentration of about 2mg/ml, which is named as B432 antibody solution.

Example 3 binding ability of antibody to hepatitis B Virus

First, ELISA detection

1. The microplate was removed and the coating solution was added for coating (50ng coating antigen/well).

The coating antigen is HBs antigen. The coating stock solution was prepared using PBS buffer as a solvent.

2. After completion of step 1, the microplate was removed, 200. mu.l of PBS buffer containing 2% BSA was added to each well, incubated at 37 ℃ for 2 hours for blocking, and then the supernatant was aspirated and washed twice with PBST solution.

3. After completing step 2, the microplate was taken, 200. mu.l of antibody diluent was added to each well, incubated at 37 ℃ for 1 hour, and then the supernatant was aspirated and washed twice with PBST solution. The antibody dilution was obtained by diluting the B432 antibody solution or HBIG solution or MERS-27 antibody solution prepared in example 2 with PBS buffer. Each antibody dilution was set with 5 replicate wells.

4. After completion of step 3, the microplate was taken, anti-human IgG-HRP (goat anti-human IgG-HRP, Promega, cat # W4038) was added, incubated at 37 ℃ for 1 hour, and then the supernatant was aspirated and washed 6 times with PBST solution.

5. And (4) after the step (4) is finished, adding a developing solution into the ELISA plate for developing, stopping developing at a proper time, and reading the absorbance value of 450 nm.

The results are shown in FIG. 1. The abscissa of fig. 1 shows the concentration of B432 antibody in the antibody dilution, HBIG concentration is 1000 times higher than the concentration of B432 antibody, and MERS-27 antibody concentration is the same as B432 antibody. For example, when the concentration of the B432 antibody in the antibody dilution is 0.1. mu.g/ml, the concentration of HBIG is 0.1mg/ml, and the concentration of MERS-27 antibody is 0.1. mu.g/ml. The results indicate that the B432 antibody can efficiently bind to HBs antigen.

Second, immunofluorescence assay

The test plasmid is an A-type hepatitis B envelope protein expression plasmid or a B-type hepatitis B envelope protein expression plasmid or a C-type hepatitis B envelope protein expression plasmid or a D-type hepatitis B envelope protein expression plasmid.

The test antibody solution was the B432 antibody solution or MERS-27 antibody solution prepared in example 2.

1. Taking 6-well plate, placing alcohol-sterilized cover glass in each well before plate spreading, and adding about 10 per well⁶Huh7 cells were cultured for 15 hours.

2. After completion of step 1, the 6-well plate was taken, and 2. mu.g of the plasmid to be tested and 2. mu.g of PEI transfection reagent were added to each well and cultured for 72 hours.

3. After step 2, the coverslip in the 6-well plate was taken, washed 3 times with PBS buffer, then fixing for 15min by 4% paraformaldehyde solution, then washing for 3 times by PBS buffer solution, then the membrane was penetrated with PBST solution at room temperature for 20min, and then washed 3 times with PBS buffer, then adding goat serum dropwise, sealing at room temperature for 30min, adding primary antibody working solution (diluting the test antibody solution to 1000 times volume) dropwise, incubating at 4 deg.C for 15 hr, then washed 3 times with PBST solution, then added dropwise with a working solution of a fluorescent secondary antibody (goat anti-human IgG-Dylight 594, cat # CW0248, kang century) and incubated at 37 ℃ for 1h, then 3 times of immersion washing with PBST solution, then counterstaining with DAPI, then 4 times of washing with PBST solution, then sealing the film by using a sealing liquid containing an anti-fluorescence quenching agent, and collecting an analysis image under a laser confocal microscope.

The results are shown in FIG. 2. The results indicate that the B432 antibody can bind A, B, C, D genotype of the membrane protein expressed by hepatitis B virus.

Example 4 neutralizing Activity of antibodies

HBV infection and antibody neutralization test

The test antibody solution is the B432 antibody solution or MERS-27 antibody solution or HBIG solution prepared in example 2.

1. Culturing HepAD38 cells, collecting supernatant, adding PEG8000 into the supernatant to make the concentration of the supernatant in the system be 8%, centrifuging to precipitate virus, and dissolving with PMM culture medium to obtain HBV virus solution.

2. HepG2-NTCP cells were seeded in 48-well plates and after approximately 5 hours of attachment, culture was continued for 16 hours by replacing with PMM medium.

3. After completion of step 2, the 48-well plate was taken, the supernatant was aspirated and added with the HBV virus solution (MOI ═ 200), test antibody solution and PEG8000 prepared in step 1 (to make the final concentration of PEG8000 5%), cultured for 16-20 hours, then the supernatant was aspirated and discarded, the cells were washed 2 times with PBS buffer, and then cultured for 7-8 days with PMM medium (every two days).

3. And (3) after the step 2 is completed, sucking the supernatant, detecting the content of HBe by adopting a hepatitis B virus e antigen detection kit, and comparing the neutralizing activity of the antibody by utilizing a Graphpad fitting curve. IC50 of the antibody was calculated using Prism 5 software.

2 replicate wells were set for each concentration of each test antibody.

The results are shown in FIG. 3. The abscissa of FIG. 3 shows the concentration of B432 antibody in the system, HBIG concentration is 1000 times higher than that of B432 antibody, and MERS-27 antibody concentration is the same as that of B432 antibody. For example, when the concentration of the B432 antibody in the system is 0.1. mu.g/ml, the concentration of HBIG is 0.1mg/ml, and the concentration of MERS-27 antibody is 0.1. mu.g/ml. The results show that the B432 antibody can efficiently neutralize HBV virus.

The IC50 value for the B432 antibody was 0.025. mu.g/ml.

HBIG had an IC50 value of 15. mu.g/ml.

Second, HDV infection and antibody neutralization test

The test antibody solution is the B432 antibody solution or MERS-27 antibody solution or HBIG solution prepared in example 2. A blank control (NC) was set with an equal volume of PBS buffer instead of test antibody solution.

The test plasmid is C-type hepatitis B envelope protein expression plasmid or D-type hepatitis B envelope protein expression plasmid.

1. Huh7 cells were cultured to 80% -90% confluency in 10cm diameter dishes.

2. And (3) after the step 1 is completed, taking the culture dishes, adding 20 mu g of test plasmid, 10 mu g of JC126 vector and 30 mu g of PEI transfection reagent into each culture dish, culturing for 8 days, collecting supernatant, adding PEG8000 to enable the concentration of the PEG8000 in a system to be 5%, centrifuging to precipitate viruses, and dissolving by using a PMM culture medium to obtain the HDV virus solution.

3. HepG2-NTCP cells were seeded in 48-well plates and after approximately 5 hours of attachment, culture was continued for 16 hours by replacing with PMM medium.

4. After completion of step 3, the 48-well plate was taken out, the supernatant was aspirated and the virus solution (MOI ═ 500), test antibody solution and PEG8000 prepared in step 2 (final concentration of PEG8000 in the system was 5%) were added, and cultured for 16 to 20 hours, then the supernatant was aspirated and discarded, the cells were washed 2 times with PBS buffer, and then cultured for 7 days with PMM medium (every two days).

In the system, the concentration of the B432 antibody was 0.5. mu.g/ml. In the system, the concentration of HBIG is 0.5mg/ml, which is 1000 times of the concentration of monoclonal antibody. In the system, the concentration of MERS-27 antibody was 0.5. mu.g/ml.

5. After completion of step 4, the 48-well plate was taken, washed 3 times with PBS buffer, then fixed with 4% paraformaldehyde solution for 15min, then washed 3 times with PBS buffer, then penetrated 20min at room temperature with PBST solution, then washed 3 times with PBS buffer, then goat serum was added dropwise and blocked at room temperature for 30min, then primary anti-working solution (primary anti-HDV δ antibody) was added dropwise and incubated at 4 ℃ for 15 hours, then washed 3 times with PBST solution, then fluorescent secondary anti-working solution (secondary anti-donkey anti-rabbit AlexaFluor TM 594 labeled secondary antibody, cat # a-21207, Invitrogen usa) was added dropwise and incubated at 37 ℃ for 1h, then washed 3 times with PBST solution, then counterstained with DAPI for nuclei, then washed 4 times with PBST solution, then mounted with mounting solution containing an anti-fluorescent agent, and an analysis image was collected under a laser confocal microscope.

The results are shown in FIG. 4. Compared with a blank control or the Mers27 antibody, the B432 antibody can effectively block the infection of the HDV virus packaged by the hepatitis B virus type C envelope plasmid and the HDV virus packaged by the hepatitis B virus type D envelope plasmid.

Example 5 evaluation of antibody for the depletion of hepatitis B virus surface antigen in mice

HBV transgenic mice were divided into three groups of 4 mice each, half male and female.

Group B432: the B432 antibody solution prepared in example 2 was injected into mice tail vein, and the immunization dose of the B432 antibody was 20mg/kg body weight.

MERS-27 group: the MERS-27 antibody solution was injected into mice tail vein, and the immunizing dose of the MERS-27 antibody was 20mg/kg body weight.

Blank control group (NC group): mice were injected tail vein with equal volume of PBS solution.

Blood is collected from the retroorbital venous plexus after 1 day of injection, 3 days of injection, 7 days of injection, 14 days of injection, 21 days of injection and 28 days of injection respectively, and the content of hepatitis B surface antigen HBs in serum is detected by adopting a hepatitis B virus surface antigen diagnostic kit.

The results are shown in FIG. 5. The B432 antibody can effectively eliminate hepatitis B virus surface antigen in a mouse body.

SEQUENCE LISTING

<110> Qinghua university

<120>CGGNQAYX186104

<130> hepatitis B virus neutralizing antibody B432 and application thereof

<160>4

<170>PatentIn version 3.5

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Gly

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Asp Tyr Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

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Trp Val Ser Phe Ile Gly Ala Asp Gly Ala Thr Ile Tyr Tyr Ala Asp

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Ser Val Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser

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Leu Phe Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Phe Tyr

100 105 110

Tyr Cys Ala Lys Glu Met Gly Val Arg Asp Arg Tyr Tyr Tyr Tyr Ala

115120 125

Ile Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

145 150 155 160

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

210 215 220

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280285

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

340 345 350

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

370 375 380

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

420 425 430

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

435 440445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

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Ser Leu Ser Leu Ser Pro Gly Lys

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agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300

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agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480

gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540

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cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt 720

aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa 780

ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc 840

cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc 900

gattgaattc caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg 960

gtgtacattc tgtgcagctg gtgcagtctg ggggaggcgt ggttcagcct ggggggtccc 1020

tgagactctc ctgtgcagcc tctggattca tatttggtga ttatgccatg tactgggtcc 1080

gtcaagctcc agggaagggt ctggagtggg tctcttttat tggtgcggat ggtgccacta 1140

tatactatgc agactctgtg acgggccgat tcaccatctc cagagacaac agcaaaaact 1200

ccctgtttct gcaaatgaac agtctgagaa ctgaggacac cgccttctat tactgtgcaa 1260

aagagatggg cgtccgtgac cgttattatt attatgctat agatgtctgg ggccaaggga 1320

ccacggtcac cgtctcctca gcgtcgacca agggcccatc ggtcttcccc ctggcaccct 1380

cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag gactacttcc 1440

ccgaacctgt gacggtctcg tggaactcag gcgccctgac cagcggcgtg cacaccttcc 1500

cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc gtgccctcca 1560

gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc aacaccaagg 1620

tggacaagag agttgagccc aaatcttgtg acaaaactca cacatgccca ccgtgcccag 1680

cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 1740

tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 1800

ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc 1860

cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc 1920

aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 1980

ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc 2040

tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc ctggtcaaag 2100

gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact 2160

acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctat agcaagctca 2220

ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg 2280

ctctgcacaa ccactacacg cagaagagcc tctccctgtc cccgggtaaa tgagtgcgac 2340

ggccggcaag cccccgctcc ccgggctctc gcggtcgtac gaggaaagct tggccgccat 2400

ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 2460

tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 2520

tatcttatca tgtctggatc 2540

<210>3

<211>230

<212>PRT

<213>Artificial sequence

<400>3

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

20 2530

Glu Arg Ala Ser Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Gly Tyr

35 40 45

Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile

50 55 60

Tyr His Ser Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

85 90 95

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Lys Trp Pro Leu

100 105 110

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

115 120 125

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

130 135 140

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

145 150 155 160

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

165 170 175

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

180 185190

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

195 200 205

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

210 215 220

Phe Asn Arg Gly Glu Cys

225 230

<210>4

<211>1762

<212>DNA

<213>Artificial sequence

<400>4

acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480

gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540

gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 600

gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc 660

cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt 720

aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa 780

ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc 840

cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc 900

gattgaattc caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg 960

gtgaaattgt gctgacacag tctccaggca ccctgtcttt gtctccaggg gaaagagcct 1020

ccctctcctg cagggccagt cagagcgttg gcggctactt agcctggtac cagcagacgc 1080

ctggccaggc tcccaggctc ctcatctatc attcgtccac cagggccact ggcatcccag 1140

ccaggttcag tggcagtggg tctgggacag acttcactct caccatcagt aggctagaac 1200

ctgaagattt tgcagtttac tactgtcaac agcgtaccaa atggccgctc actttcggcg 1260

gagggaccaa ggtggagatc aaacgtacgg tggctgcacc atctgtcttc atcttcccgc 1320

catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg aataacttct 1380

atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc 1440

aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga 1500

cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg 1560

gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttagaag cttggccgcc 1620

atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 1680

tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 1740

tgtatcttat catgtctgga tc 1762

Claims

1. An IgG antibody consisting of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are amino acid residues at positions 44-51, 69-76 and 115-131 from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-48 th amino acid residue, the 66 th-68 th amino acid residue and the 105 th-113 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence.

2. The IgG antibody of claim 1, wherein:

the heavy chain variable region consists of 20 th-142 th amino acid residues from the tail end of N of a sequence 1 in a sequence table;

3. The IgG antibody of claim 2, wherein:

the heavy chain is (a) or (b) as follows: (a) protein consisting of 20 th-472 th amino acid residues from the tail end of N in a sequence 1 of a sequence table; (b) protein shown in a sequence 1 in a sequence table;

4. A gene encoding the IgG antibody of claim 3, wherein:

the gene encoding the heavy chain is (1) or (2) or (3) as follows:

(3) a DNA molecule shown in a sequence 2 of a sequence table;

the genes encoding the light chain are as follows (4) or (5) or (6):

(6) DNA molecule shown in sequence 4 of the sequence table.

5. Use of the IgG antibody of claim 1 or 2 or 3 in the manufacture of a medicament for inhibiting hepatitis b virus.

6. A medicament for inhibiting hepatitis b virus, which comprises as an active ingredient the IgG antibody according to claim 1 or 2 or 3.

7. Use of the IgG antibody of claim 1 or 2 or 3 in the manufacture of a medicament for neutralizing hepatitis b virus.

8. An agent for neutralizing hepatitis B virus, which comprises as an active ingredient the IgG antibody according to claim 1, 2 or 3.

9. Use of an IgG antibody according to claim 1 or 2 or 3 for the manufacture of a medicament for the prevention and/or treatment of hepatitis b.

10. A pharmaceutical agent for preventing and/or treating hepatitis B, which comprises as an active ingredient the IgG antibody of claim 1, 2 or 3.