CN115028711A - Fully human monoclonal antibody with high affinity for resisting hepatitis C virus and application thereof - Google Patents

Fully human monoclonal antibody with high affinity for resisting hepatitis C virus and application thereof Download PDF

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CN115028711A
CN115028711A CN202210676544.4A CN202210676544A CN115028711A CN 115028711 A CN115028711 A CN 115028711A CN 202210676544 A CN202210676544 A CN 202210676544A CN 115028711 A CN115028711 A CN 115028711A
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CN115028711B (en
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胡萍
辛洪波
尧志峰
刘苏俊
张文华
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Nanchang University
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    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
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    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The invention relates to a fully human monoclonal antibody with high affinity for resisting hepatitis C virus and application thereof, belonging to the technical field of biological engineering. The amino acid sequence of the heavy chain variable region of the monoclonal antibody or the fragment thereof is shown as SEQ ID NO 12; the amino acid sequence of the light chain variable region is shown as SEQ ID NO 18. The invention also provides a coding gene, an expression vector, application and a composition of the antibody. The binding molecule of the invention can prevent hepatitis C virus from infecting susceptible cells, is fully human, has low immunogenicity and good affinity compared with other animal-derived (such as murine-derived) anti-hepatitis C virus molecules, has good treatment effect and low side effect, and simultaneously provides great guarantee for the standardized production of patent drugs of the antibody.

Description

Fully human monoclonal antibody of high affinity anti-hepatitis C virus and application thereof
Technical Field
The invention relates to the technical field of biological engineering, in particular to a fully human monoclonal antibody with high affinity for resisting hepatitis C virus and application thereof.
Background
In the eighties of the last century, researchers first isolated a hepatitis virus, distinct from hepatitis a b, from the blood of chimpanzees, and this virus was subsequently named Hepatitis C Virus (HCV). Hepatitis c infection has become a global health problem and is susceptible to HCV in different sexes, ages and ethnic groups. Currently, the worldwide number of patients infected with HCV is estimated to exceed 1.84 million people, and the global HCV infection rate estimated by the seropositive rate is about 1.5% to 3.5%. According to the estimation, the prevalence rate of hepatitis C in China is 1%, more than 1000 ten thousand HCV carriers exist, and the number of hepatitis C patients is the first in the world.
The prevalence distribution in various regions varies widely around the globe due to the high degree of variation of HCV and immune escape mechanisms. At present, the hepatitis C virus strains which are mainly prevalent all over the world are mainly divided into 11 genotypes and more than 70 subtypes, and HCV viruses with different genotypes have different virulence and great difference in pathogenic capability. By analyzing the results of sample 32030 patients in 29 provinces and cities in China, the hepatitis C subtypes which are mainly popular in China are 1b (n ═ 16713, 52.18%), 2a (n ═ 9188, 28.69%), 3b (n ═ 2261, 7.06%), 6a (n ═ 2052, 6.41%) and 3a (n ═ 1479, 4.62%), and mixed infection of the main subtypes is found in other small part samples, and the combination is as follows: 1b-2a, 1b-3b, 1b-6a, 3a-3b, 1b-3a and 2a-6a, and the distribution of these HCV gene subtypes is closely related to the gender, age and geographical location of the population.
According to long-term clinical practice and laboratory technical tests, the clinical diagnosis standard of hepatitis c can be as follows: the clinical manifestations are general hypodynamia, anorexia, right costal discomfort, hepatosplenomegaly and so on; the laboratory tests show that glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase are slightly and moderately increased, and the anti-HCV antibody is positive and the HCV RNA is positive; further diagnosing according to the duration of the disease course of the patient and the existence of the corresponding epidemiological history.
The hepatitis C treatment mainly aims at eliminating viruses, keeping the replication level of HCV in vivo at an extremely low load for a long time, fully reducing liver injury lesion and the like caused by HCV infection, and improving the long-term survival rate and the survival quality of patients. In the long-term hepatitis C treatment, the clinical classical treatment method is interferon combined with ribavirin, and the method has large side effect, is easy to generate drug resistance and has large effect difference on patients with different genotypes. With the advent of international novel drugs such as DAAs (Direct Anti-viral Agents) such as Dalatavir hydrochloride tablets and Soft Ashbyr capsules, most DAAs have been approved in China for clinical use, the main action mechanism of DAA drugs is to inhibit NS3/4A protease and NS5A/B polymerase in HCV particles, and the HCV infection can be effectively treated by identifying the HCV genotype infected by the patient before the Direct antiviral drugs are used and selecting a proper antiviral scheme in combination with other conditions of liver injury of the patient. In 2018, the 'propansand' (Sofosbuviretavir tablets) produced by Gilidide corporation formally enters the medicine catalog of China, marks the coming of a new hepatitis C treatment era, and the new hepatitis C treatment medicine which is orally taken, genotyped and single tablet brings good news to patients, but inevitable drug resistance and higher price thereof are forbidden to some patients.
1975, study of university of CambridgeMilstein and
Figure BDA0003694897750000021
the hybridoma technology is invented for the first time, and the technical principle is that immunized mouse spleen cells and myeloma cells are fused, and hybridoma cells capable of stably secreting single antibodies are screened out after fusion. Meanwhile, the concept of the monoclonal antibody is also developed, and the antibody is an antibody which is highly uniform and only aims at a certain specific epitope and is generated by a single B cell clone, and has the advanced advantages of high purity, strong specificity, less cross reaction and the like. However, since the method produces the mouse-derived protein, the method has considerable immunogenicity to human bodies and cannot meet the requirement of long-term treatment. With the development of modern molecular biology and protein engineering techniques, the limitations of the techniques have been addressed to varying degrees. Initially, researchers achieved the production of novel "chimeric" antibodies by replacing most murine Fc sequences with human crystallizable (Fc) sequences. Later, the degree of humanization of monoclonal antibodies has been improved by further bioengineering murine antigen binding regions (fabs) grafted onto human immunoglobulin (IgG) frameworks, and today mature technology has enabled the production of fully human monoclonal antibodies.
At present, the technology of preparing the monoclonal antibody of the whole human source is various. There are mainly antibody library technologies, such as phage display antibody library technology, ribosome display antibody library technology, single cell cloning expression technology, etc. These techniques are superior and inferior, the antibody library technique can directly prepare specific and stable monoclonal antibodies in vitro, however, the previous library construction workload is very large, the required library capacity needs to cover the antibody diversity of certain animals, high-throughput screening can only obtain partial fragments of the antibodies, and the high affinity of the antibodies cannot be ensured at the later stage. The single cell cloning expression method is to obtain certain specific B cell through flow cytometry and to separate the heavy and light chain variable region gene of its homologous antibody through gene technology for expression in eukaryotic system. In this method, the sorting of the precursor B cells is particularly critical, and highly specific and stable antigens are required as probes for efficient sorting.
Monoclonal antibody drugs have been a hot spot for drug development in the last decade. As a novel macromolecular protein medicine, the monoclonal antibody has the advantages of strong specificity and obvious effect, and simultaneously, because the humanization level of the monoclonal antibody medicine is higher, the immunogenicity is relatively lower, the human body is not easy to generate rejection reaction, and the safety is greatly ensured. With the wide application of monoclonal antibodies in the tumor field, research and development of monoclonal antibody drugs corresponding to infectious diseases are also actively promoted, palivizumab for respiratory syncytial virus infection and ibazumab for resisting HIV infection are successively appeared, and monoclonal antibody drugs for treating hepatitis C are not yet appeared.
Disclosure of Invention
The inventor selects E2 protein with good immunogenicity on the HCV surface as an antigen epitope through intensive research, extracts memory B cells from whole blood of rehabilitation people infected with hepatitis C virus pathogens, activates the cells into plasma cells in vitro, screens the plasma cells secreting specific antibodies by adopting a certain specific antigen, and screens specific antibody genes by utilizing the technologies of efficient extraction of a small amount of cell RNA, nested PCR and the like. The monoclonal antibody is fully human, and the variable regions and the constant regions of the heavy chain and the light chain of the monoclonal antibody are all derived from human genes, so the monoclonal antibody has the characteristics of low immunogenicity and high safety.
HCV is a small, enveloped, single-stranded RNA virus with a genome of 9.6kb in length. The HCV particle consists of a positive RNA genome with 5 'and 3' untranslated regions (UTRs) and an Open Reading Frame (ORF) encoding a protein precursor of approximately 3000 amino acids. The UTR constitutes a highly conserved cis-acting RNA element that regulates translation and replication of the viral genome. After assembly processing of the protein precursors encoded by open reading frames, 10 structural and nonstructural proteins were formed (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins). The outer envelope of HCV particle mainly comprises E1 and E2 glycoprotein, the coding regions of the two proteins are respectively positioned at residue 192-383 site (E1) and 384-746 site (E2) at the N end of the HCV protein structure, and the two proteins are taken as highly glycosylated type I transmembrane proteins which can be combined to form a non-covalent heterodimer structure and have important functions on virus recognition and invasion of host cells. The (core) core protein of HCV particles, a multifunctional protein, has a major role in forming viral capsids, and plays a role in encapsulating and protecting genomic RNA when viruses are transferred between host cells. Other nonstructural proteins such as NS2 and NS3 have been shown to have RNA helicase activity and serine protease activity, and can assist in replication and assembly of viral genome together with other nonstructural proteins. The E1 and E2 proteins, which are envelope proteins on the surface of the virus, are the major viral antigens that elicit a protective immune response. The molecular weight of the two proteins is greatly different, the number of amino acids of the E2 protein is far more than that of the E1 protein, but compared with the E1 protein, the number of hypervariable regions (HVRs) of the E2 protein is less, and the protein structure is more stable, so that when an antigen target is selected, the E2 protein is selected as an epitope, and finally, the fully human monoclonal antibody with High safety and High affinity for the hepatitis C virus is obtained.
In one aspect, the present invention provides a high affinity fully human monoclonal antibody against hepatitis c virus, having a heavy chain variable region and a light chain variable region:
(I) the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO 10-13; or has an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO 10-13;
(II) the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO 14-18; or an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO 14-18.
Preferably, the monoclonal antibody has any one of the following groups of heavy chain variable regions and light chain variable regions:
(i) heavy chainThe amino acid sequence of the variable region is shown as SEQ ID NO:10 (5H) 12 ) (ii) a The amino acid sequence of the light chain variable region is shown in SEQ ID NO:14 (7 kappa) 1 )；
(ii) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 10 (5H) 12 ) (ii) a The amino acid sequence of the light chain variable region is shown in SEQ ID NO:17 (7 kappa) 13 )；
(iii) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11 (5H) 17 ) (ii) a The amino acid sequence of the light chain variable region is shown in SEQ ID NO:15 (7 kappa) 2 )；
(iv) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12 (5H) 20 ) (ii) a The amino acid sequence of the light chain variable region is shown in SEQ ID NO:18 (7 kappa) 30 )；
(v) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13 (5H) 24 ) (ii) a The amino acid sequence of the light chain variable region is shown in SEQ ID NO:16 (7 kappa) 9 )。
The monoclonal antibody composed of the heavy chain variable region and the light chain variable region can be specifically combined with hepatitis C virus E2 protein.
In another aspect of the present invention, there is provided a nucleic acid molecule encoding the monoclonal antibody described above.
Preferably, the nucleic acid molecule has the nucleotide sequence of the heavy chain variable region as shown in any one of SEQ ID NO 1-4; and the variable region of the light chain as set forth in any one of SEQ ID Nos 5 to 9.
More preferably, the nucleic acid molecule has any one of the following sets of heavy chain variable region and light chain variable region:
(i) the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 1 (5H) 12 ) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO. 5 (7 kappa) 1 )；
(ii) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO:1 (5H) 12 ) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO:8 (7 kappa) 13 )；
(iii) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:2 (5H) 17 ) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO:6 (7 kappa) 2 )；
(iv) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO:3 (5H) 20 ) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO:9 (7 kappa) 30 )；
(v) The variable region of the heavy chain has the nucleotide sequence shown in SEQ ID NO:4 (5H) 24 ) (ii) a The variable region in the light chain has the nucleotide sequence shown in SEQ ID NO:7 (7 kappa) 9 )。
In another aspect of the invention, there is provided an expression vector comprising the nucleic acid molecule described above.
The expression vector may contain, in addition to the nucleic acid molecule described above, a suitable promoter or control sequence. The vector may be used to transform an appropriate host cell so that it can express the protein.
In another aspect of the present invention, there is provided a host cell comprising the above-described expression vector.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells such as E.coli, Streptomyces; salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. Particularly suitable host cells for use in the present invention are eukaryotic host cells, especially mammalian cells, such as 293 cells.
In another aspect of the present invention, there is provided a method for preparing the monoclonal antibody, comprising the steps of:
(1) memory B cell sorting: separating peripheral blood mononuclear cells from a blood sample of a hepatitis C rehabilitation patient, and sorting the peripheral blood mononuclear cells to obtain memory B cells;
(2) in-vitro activation culture and positive hole screening of memory B cells: activating the memory B cells into plasma cells, detecting secretory IgG antibodies of the memory B cells after the memory B cells are activated into the plasma cells in vitro by ELISA, and screening to obtain positive holes;
(3) constructing and screening an antibody variable region gene library: carrying out reverse transcription on the positive hole cells to obtain cDNA, and amplifying genes of heavy chain variable regions and light chain variable regions of the antibodies;
(4) constructing a library of antibody gene heavy chain variable regions and light chain variable regions: respectively connecting the amplified genes of the heavy chain variable region and the light chain variable region of the antibody to an expression vector;
(5) screening of antibody heavy chain variable region and light chain variable region genes: the recombinant plasmid was co-transfected into HEK293T cells for expression.
In another aspect of the invention, the monoclonal antibody or the binding fragment thereof is provided for use in the preparation of a medicament for detecting, treating and preventing hepatitis c virus infection.
In another aspect of the present invention, there is provided a composition comprising an antibody mixture of one or more of said monoclonal antibodies in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the monoclonal antibody of the invention or fragment thereof without substantially reducing the effectiveness of the composition as is often the case.
Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.
The compositions of the present invention may be formulated into various dosage forms as desired, and may be administered by a physician in a dosage amount beneficial to the patient, depending on such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like. The administration may be, for example, by injection or other therapeutic means.
The pharmaceutical composition may comprise two or more monoclonal antibodies or fragments thereof having neutralizing activity against hepatitis c virus.
In another aspect of the present invention, there is provided a kit for detecting hepatitis c virus, which comprises the monoclonal antibody or fragment thereof.
Based on the monoclonal antibody or the fragment thereof, a kit for conveniently, rapidly and accurately detecting the hepatitis C virus can be prepared. As a detection method of the present invention, an indirect ELISA method is used, in which an antigen to be detected is coated on a solid phase carrier, and detection is performed using the monoclonal antibody or the fragment thereof of the present invention. In a preferred embodiment of the present invention, the monoclonal antibody or a fragment thereof is an antibody, and is detected according to the principle of double antibody sandwich method. The double antibody sandwich method is conventionally performed by immobilizing a primary antibody (e.g., the monoclonal antibody of the present invention) on a carrier, reacting the primary antibody with an antigen, washing, reacting with a secondary antibody (the secondary antibody carries a detectable signal or can bind to a substance carrying a detectable signal), and detecting a signal by a chemiluminescent or enzyme-linked chromogenic reaction. The double antibody sandwich method is particularly suitable for the detection of antigens having two or more epitopes.
For convenience in detection, the kit may further comprise, in addition to the monoclonal antibody or fragment thereof of the present invention, other detection reagents or auxiliary reagents, such as those conventionally used in ELISA kits, the properties of which and their formulation methods are well known to those skilled in the art, such as color developing agents, labels, secondary antibodies, anti-antibodies, sensitizers, and the like. It will be understood by those skilled in the art that various modifications of the detection kit are encompassed in the present invention as long as the monoclonal antibody or fragment thereof of the present invention is utilized as a reagent for recognizing hepatitis C virus therein.
In addition, instructions for use may be included in the kit to instruct the method of use of the reagents loaded therein.
After obtaining the monoclonal antibody or the fragment thereof provided by the present invention, various immunology-related methods can be used to detect the HCV-E2 protein in the sample, so as to determine whether the donor of the sample to be tested is infected with the hepatitis C virus, and these methods are all included in the present invention. Preferably, the method is for the purpose of non-disease diagnosis.
In another aspect of the invention, there is provided a method of non-therapeutically inhibiting hepatitis c virus, said method comprising administering to a patient an effective amount of said monoclonal antibody or fragment thereof.
In another aspect of the present invention, there is provided a method for non-therapeutic detection of hepatitis c virus, wherein said monoclonal antibody or fragment thereof is contacted with a test sample, and the presence and amount of hepatitis c virus is detected by detecting the binding of said monoclonal antibody or fragment thereof to the test sample.
As used herein, the term "test sample" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. The term also includes samples that have been treated by any means after they have been obtained, for example by treating with reagents, solubilizing, or enriching certain components such as proteins or polynucleotides. The term encompasses various clinical samples obtained from any species, also including cultured cells, cell supernatants and cell lysates.
Has the advantages that: compared with the prior art, the invention successfully screens the fully human monoclonal antibody for resisting the hepatitis C virus, the monoclonal antibody has the characteristic of high affinity, and the monoclonal antibody can be specifically combined with the hepatitis C virus and can obviously resist the hepatitis C virus. Compared with a mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no other species of components, does not generate toxic and side effects such as anti-mouse anti-antibody and the like in a human body, has better biocompatibility, is more suitable and has more potential to become a macromolecular drug for treating hepatitis C virus, and provides great guarantee for the standardized production of finished drugs of the antibodies.
Drawings
Figure 1ELISA screen positive memory B cell wells.
FIG. 2 is the electrophoresis diagram of the PCR amplification of the heavy chain variable region gene H, H' and the light chain variable region gene kappa, lambda chain of the antibody.
FIG. 3 shows the results of electrophoretic verification of the heavy chain variable region gene, the light chain variable region gene and the vector.
FIG. 4 shows the results of electrophoretic verification of the recombinant plasmid.
FIG. 5 shows a screening scheme for heavy and light chain variable region genes of antibodies.
FIG. 6 shows the results of screening the combination of heavy and light chain variable region genes of antibodies.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1: preparation of fully human monoclonal antibody of hepatitis C virus
1. Sorting of memory B cells
1.1 acquisition of peripheral blood mononuclear cells
Blood is collected by professionals in a sterile environment, and fresh whole blood of hepatitis C recovery volunteers is extracted into disposable vacuum blood collection tubes, wherein the whole blood volume required by each person is 50 ml. The pre-cooled PBS solution after filtration through a 0.22 μm filter membrane was diluted 1:1 with fresh anticoagulated whole blood. The diluted blood is slowly added into the lymphocyte separating solution with the same volume for centrifugation, the centrifugation condition is 2000rpm, Acc4, Dec4, and the centrifugation is carried out for 30min at room temperature. After the centrifugation, the centrifuge tube was slowly removed, and the centrifuged second cloudy mononuclear cell layer was carefully aspirated into a 15ml centrifuge tube previously filled with a pre-cooled PBS solution. Centrifuging the sucked mononuclear cells at 1200rpm for 5min at room temperature, discarding the supernatant, and gently blowing and washing with a precooled PBS solution. The resulting solution was centrifuged again, and the collected cells were Peripheral Blood Mononuclear Cells (PBMC) and counted.
1.2 memory B cell sorting
(after cell counting, specific MS or LS columns were selected based on the PBMC obtained from the separation) the cells were centrifuged at 300g for 10min and the supernatant was removed completely. At a rate of 1 × 10 7 The number of cells is 40. mu.l buffer, and the appropriate amount of buffer is proportionally aspirated to resuspend the cell pellet. At a rate of 1 × 10 7 The cell pellet was mixed well with 10. mu.l of nasal B cell Biotin-Antibody Cocktail, and incubated in a refrigerator at 4 ℃ for 5 min. At a rate of 1 × 10 7 The cell pellet was mixed well with 30. mu.l of buffer and 20. mu.l of Anti-Biotin Micro Beads, and left in a refrigerator at 4 ℃ for 10 min. Adding 1-2 ml buffer solution to wash the cells, centrifuging for 10min at 300g, and discarding the supernatant. The cell pellet was resuspended with 500. mu.l buffer. The column was placed on a magnet and the column was washed with the appropriate volume of buffer (LS column with 3ml buffer and MS column with 500. mu.l buffer). The cell suspension was slowly added to the column and the eluted unlabeled cells were collected. The column was washed 3 times with the appropriate amount of buffer solution (3 ml volume buffer in LS column and 500. mu.l volume buffer in MS column) and the wash solution was added after the column was drained during the wash. Collecting all the eluent to obtain the suspension containing B cells. The column was removed from the separator, placed on a collection tube, and buffer was pipetted into the column and the plunger immediately forced to wash out the magnetic bead labeled cells. Counting cells, centrifuging at 300g for 10min, and completely removing supernatant every 10min 7 Total cells were resuspended in cell pellet with 80. mu.l buffer. At a rate of 1 × 10 7 Adding 20 mul of CD27 Microbeads as unit into the cell number, adding a proper amount of labeled magnetic beads, and fully mixing the solutionIncubate at 4 ℃ for 15min in a refrigerator. At a rate of 1 × 10 7 The cells were washed in the unit of the number of cells plus 1ml of buffer, centrifuged at 300g for 10min, and the supernatant was completely removed. When the number of cells is less than 1X 10 8 At this time, the cells were resuspended in 500. mu.l of buffer (if more cells are present, the buffer is doubled). The post was placed on the magnet. The column was washed with the appropriate volume of buffer. The cell suspension was applied to the column. The flow-through unlabeled cells were collected and the column was washed 3 times with the appropriate volume of buffer (3 ml for LS column and 500. mu.l for MS column). Collecting all the eluent, namely the suspension containing the unlabeled cells, and adding the washing liquid after the liquid in the column is drained in the washing process. The column was removed from the separator, immediately placed on a collection tube, an appropriate volume of buffer was pipetted into the column, and the plunger was immediately forced to wash out the magnetic bead labeled cells. The cell is a memory B cell. The sorted memory B cells were counted and cryopreserved.
2. In-vitro activation culture and positive hole screening of memory B cells
And (4) inoculating the sorted memory B cells into a 96-well cell culture plate according to different cell density gradients. Mu.l of 10% FBS IMDM was added to each well as a base medium containing an appropriate concentration of IL-21 as a B cell activating factor. On the basis, a stable cell line of Co-60 inactivated 3T3-CD40L cells is added into each well to be used as a feeder layer cell. Culturing at 37 deg.C under 5% CO2 for several days to activate memory B cell to become plasma cell and secrete antibody; ELISA detection of secreted IgG antibodies after activation of memory B cells into plasma cells in vitro: cell culture supernatants were collected, coated with a microplate containing Goat Anti-Human Kappa protein and Goat Anti-Human Lambda protein, and the culture supernatants of memory B cells were assayed by ELISA to determine the amount of IgG antibody secreted.
Dissolving Goat Anti-Human Kappa protein and Goat Anti-Human Lambda protein in 100 ng/well of 50 mul/well of coating solution to coat an enzyme label plate respectively, standing overnight at 4 ℃, washing the plate for 5 times by a plate washing machine, wherein the washing solution is PBS solution containing 0.05% Tween-20; sealing with 200 μ l/hole of sealing solution at 37 deg.C for 2h, and washing for 5 times; respectively adding cell culture supernatant or IgG standard substance diluted in gradient into corresponding wells, simultaneously using water or PBS as negative control, standing at 50 μ l/well at 37 deg.C for 1h, and washing for 5 times; adding 1:5000 diluted peroxidase-labeled Goat Anti-Human IgG antibody and 1:10000 diluted Goat x-Human IgG-Fc Fragment HRP Conjugated 50 μ l/well into corresponding wells, standing at 37 deg.C for 45min, and washing for 5 times; adding 100 μ l/hole of color developing solution, and developing at room temperature in dark place for 15 min; adding 2mol/L sulfuric acid 50 mu L/hole, stopping the color reaction, and detecting the OD value of the absorbance of each hole under the condition that the wavelength is 450 nm. And finally, determining positive memory B cell wells by comparing and analyzing the OD value of each well and the corresponding inoculation number of the memory B cells of each well. As can be seen from FIG. 1, in which A1 and A2 are feeder cells as control wells and the OD value is 0.0613, 12 positive wells were finally determined to be numbered A3-A14, wherein the OD values of three positive wells numbered A3, A6 and A7 are significantly higher than those of the other wells, and the OD values are 0.731, 0.4003 and 0.4179, respectively. The other positive wells were also raised to some extent, and the OD values were all around 0.13.
3. Construction and screening of antibody variable region gene library
3.1 Positive well B cell RNA extraction
After microscopic observation, the cell culture supernatant was carefully aspirated away, washed once with PBS, digested with 40. mu.l of 0.25% trypsin per well, and the digestion was observed under a microscope, then the digestion was stopped with 5. mu.l of fetal calf serum per well, the digested cells were blown down and scraped with a pipette tip, and finally 2.25. mu.l of RNase inhibitor (40U/. mu.l) was added per tube. Quickly putting the digested cells into liquid nitrogen for more than 2 min; placing in a PCR instrument at 98 deg.C for 3min, and immediately placing in liquid nitrogen; after the quick freezing, 0.5. mu.l of proteinase K (20mg/ml) and 2.25. mu.l of RNase inhibitor (40U/. mu.l) were added to each tube, and the mixture was placed in a PCR apparatus at 53 ℃ for 1 hour. After the concentration of the extracted RNA was measured, cDNA was obtained by reverse transcription using Promega reverse transcription kit.
3.2 amplification of genes for heavy and light chain variable regions of the antibody
Amplifying antibody heavy chain variable region gene VH by a two-step RT-PCR method (in order to improve the experimental efficiency of each step, heavy chain variable region gene VH primers are divided into two categories of H and H', then PCR amplification and subsequent experiments are respectively carried out, and the two categories are mixed together to pick out single clone when recombinant plasmid is transformed) and light chain variable region gene V kappa or V lambda: taking the cDNA after reverse transcription as a PCR template to carry out a first reaction of nested PCR; then, the first reaction product of the nested PCR is used as a template to perform the second reaction of the nested PCR. Specific primer sequences, PCR reaction procedures, and PCR reaction system references (Nat Protoc. 2009; 4(3): 372-84.).
The PCR products of the heavy chain VH and light chains Vkappa and Vlambda were detected by agarose gel electrophoresis, the results are shown in FIG. 2, FIG. 2A: the results of two rounds of nested PCR amplification of antibody heavy chain (H, H' chain) variable region sequences; b, performing nested PCR two-round amplification on variable region sequences of antibody light chains (kappa chains and lambda chains); c: the results of nested PCR amplification verification of the heavy chain and light chain variable region sequences of samples A3, A4 and A7. M: marker DL 2000. The gel recovery target strip is specifically operated according to the instruction of the OMEGA gel recovery kit. Finally, the concentration is measured by using Nanodrop2000, and the product is stored for a long time at the temperature of-20 ℃. The result shows that the samples A3, A4 and A7 are subjected to two nested PCR amplifications (the kappa chain of the sample A3 is not amplified) to detect obvious target bands, and the positions of the target bands are between 250bp and 500bp, so that the samples A3, A4 and A7 are determined to be selected for subsequent experiments.
3.3 construction of antibody Gene heavy chain variable region and light chain variable region libraries
The amplified heavy chain variable region gene VH and light chain variable region genes Vkappa and Vlambda were ligated to expression vectors pIgH (AbVec-hIgG1 for VH), pIgK (AbVec-hIgKappa for Vkappa) and pIg lambda (IG-lambda expression vector for V lambda), respectively (NCBI GenBank accession Nos.: FJ475055, FJ475056, FJ 517647).
Sequentially using AgeI-HF and Sal I-HF to carry out enzyme digestion on VH and pIgH; cutting V lambda and pIg lambda by AgeI-HF and Xho I; the V kappa and the pIg kappa were digested with AgeI-HF, and then with BsiW I. The enzyme digestion product is detected by 1.2 percent agarose gel electrophoresis, the band is observed, the target gene band is at the position of 300bp-500bp, and the experimental result is shown in figure 3. The gel recovery target strip is specifically operated according to the instruction of the OMEGA gel recovery kit.
FIG. 3 shows a heavy chain variable region gene, a light chain variable region gene and a vectorAnd (5) electrophoresis verification results. Wherein VH, VH', V lambda and V kappa are all enzyme digestion verification. A: the enzyme digestion results of the variable region genes of sample heavy chains (H chain, H' chain) A3, A4 and A7; b: the results of the enzyme digestion of the variable region genes of No. A3, A4 and A7 light chain (kappa chain and lambda chain); c: cleavage of the vector plasmid (IgH, Ig κ, Ig λ), M: marker DL2000, M 2 : Marker DL10000。
According to the size of the target fragment, namely the heavy chain and light chain variable region gene fragment of the antibody is 400bp, and the corresponding heavy chain and light chain carrier plasmid is 5000bp, the successful enzyme digestion of the heavy chain and light chain variable region gene of the A3, A4 and A7 sample antibody and the carrier plasmid can be judged.
The target gene fragment after digestion is ligated with the corresponding constant region vector pIgH (AbVec-hIgG1 for VH), pIgkK (AbVec-hIgKappa for V kappa) or pIg lambda (IG-lambda expression vector for V lambda) after digestion, and enzymatically ligated at 16 ℃ overnight.
The ligated recombinant plasmid was transformed into TOP10 E.coli cells. The next day, positive clone colonies were picked up in Amp-containing cells + After shaking culture at 37 ℃ for 12h at 200rpm in LB medium, 50% sterilized glycerol was used for conservation of bacteria, and plasmids were extracted (see the OMEGA plasmid extraction kit for details). And (3) enzyme digestion verification of the positive clonobacterium antibody vector plasmids of the heavy chain H-IgH, the H' -IgH and the light chain lambda-Ig lambda. The experimental results are shown in FIG. 4, after the recombinant plasmids A3, A4 and A7 are digested, two bands can be observed at the positions of the band above 2000bp (5000bp) and the band 400bp, namely two bands of the plasmid gene and the antibody heavy-light chain variable region gene formed after the recombinant plasmids are digested, which shows that the early-stage A3, A4 and A7 sample heavy-chain plasmid is successfully enzymatically linked (M: DL 2000).
4. Screening of antibody heavy chain variable region and light chain variable region genes
After the recombinant plasmids are transformed, monoclonal plasmids are picked, a certain plasmid of an antibody heavy chain and an antibody lambda light chain or an antibody kappa light chain (two light chains are provided, namely lambda and kappa; only one heavy chain is H, and any one of H, lambda or kappa can be combined to form an antibody) are cotransfected to HEK293T cells, an enzyme-linked immunosorbent assay method (Elisa method) is adopted to detect the specific antibody secretion amount of HCV-E2 antigen protein (self-made), and an antibody recombinant plasmid combination with high expression of HCV-E2 specific IgG antibody is screened.
The specific steps of cell transfection are as follows:
(1) inoculating cells: 0.25% of cells after termination of the digestion with pancreatin-EDTA were placed at 1X 10/well 4 Each cell was inoculated into a 96-well plate and supplemented with culture medium to 190. mu.L. Meanwhile, a control group is set, wherein the control group 1 is added with a transfection reagent and cells but not plasmids; control 2 was cell only, without transfection reagent and plasmid.
(2) The plasmid and transfection reagent were diluted separately with opti-MEM such that 0.15. mu.L of transfection reagent was added per well and 100ng of plasmid was added per well.
(3) According to the following steps: 1, mixing the diluted plasmid and diluted transfection reagent, adding the mixture to the cell-seeded wells after 5min, and adding 5% CO at 37 deg.C 2 Culturing in an incubator for 24-72h, and detecting the content of the antibody.
(4) Elisa assay cell culture supernatant IgG secretion: collecting the cell culture supernatant after transfection in the step (3), and coating an enzyme label plate with HCV-E2 antigen protein (self-made) according to the coating amount of 100 ng/hole by using a coating solution (50 mu l/hole); the microplate was additionally coated with Goat Anti-Human Kappa + Goat Anti-Human Lambda (100 ng/well each) protein and incubated overnight at 4 ℃. The plate washing machine washes the plate 5 times. The wash solution was a PBS solution containing 0.05% Tween. Blocking with 200. mu.l of blocking solution/well at 37 ℃ for 2h, and washing the plate 5 times. Cell culture supernatants, IgG standards and anti-HCV-E2 protein antibodies (primary antibodies) were added to the corresponding wells, respectively, and the wells were incubated at 37 ℃ for 1 hour with water or PBS solution as a negative control for 5 times at 50. mu.l/well. Add 1:5000 diluted Goat Anti-Human IgG and Goat Anti-Mouse IgG labeled with HRP and 50. mu.l/well of 1:10000 diluted Goat x-Human IgG-Fc Fragment HRP Conjugated to the corresponding wells, leave them at 37 ℃ for 45min, and wash the plate 5 times. Adding TMB color developing agent 100 μ l/hole, and developing at room temperature in dark for 20 min. Adding 50 mu L/hole of 2mol/L sulfuric acid, stopping the color reaction, and detecting OD/450nm with the reference wavelength of 630 nm.
Using the screening format shown in FIG. 5, we screened positive memory B cell wells specifically by ELISA. Screening against in different batchesRanking the antibody affinity as a standard, selecting the optimal heavy-light chain sequence for next batch screening after ranking, grading the mixed sequence to the monoclonal sequence layer by layer, finally screening the obtained monoclonal antibody with high affinity, and selecting the effective monoclonal heavy chain and light chain combination for detecting the monoclonal antibody after identifying and analyzing (sequencing is completed by Jiangsu Kingsry Biotech company). Screening to obtain heavy and light chain monoclonal sequences, selecting 5 heavy and light chain monoclonal sequences respectively, and performing in-vitro transfection expression in a one-to-one matching way, wherein the heavy chain monoclonal sequences of the 5 heavy chains: 5H 12 ,5H 16 ,5H 17 ,5H 20 ,5H 24 And 5 light chain monoclonal sequences: 7 kappa 1 ,7κ 2 ,7κ 9 ,7κ 13 , 7κ 30 And finally obtaining 5 monoclonal antibodies with high affinity after ELISA screening, wherein the antibody combination is as follows: 5H 12 +7κ 1 ， 5H 12 +7κ 13 ，5H 17 +7κ 2 ，5H 20 +7κ 30 ，5H 24+ 7κ 9 (the results correspond to FIG. 6).
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
SEQUENCE LISTING
<110> university of Nanchang
<120> a fully human monoclonal antibody with high affinity against hepatitis C virus and use thereof
<130> 1
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 356
<212> DNA
<213> 5H12 heavy chain variable region nucleotide sequence
<400> 1
gtacattccg aggtgcagct ggtgcagtcg gggggaacct tggtacagcc tggggggtcc 60
ctgagactct cttgtgaagc ctctggattc acctttagcg actatgccat gggctgggtc 120
cgccagactc caggaaaggg gctggagtgg ctgtcggcta ttcgtaaaag tggcactacc 180
acatactacg cggactccgt gaagggccgg ttcatcatct ccagagacaa ttccaagaac 240
accctgtatc tgcaaatgaa taggctgagg gtcggcgaca cggccactta ttactgtgcg 300
actcacccca tcgcgggcta ctggggccag ggaaccacgg tcaccgtctc ctcagc 356
<210> 2
<211> 356
<212> DNA
<213> 5H17 heavy chain variable region nucleotide sequence
<400> 2
gtacattccc aggtccagct ggtacagtcg gggggaacct tggtacagcc tggggggtcc 60
ctgagactct cttgtgaagc ctctggattc acctttagca actatgccat gggctgggtc 120
cgccagactc caggaaaggg gctggagtgg ctgtcggcta ttcgtaaaag tggcactacc 180
acatactacg cggactccgt gaagggccgg ttcatcatct ccagagacaa ttccaagaac 240
accctgtatc tgcaaatgaa taggctgagg gtcggcgaca cggccactta ttactgtgcg 300
actcacccca tcgcgggcta ctggggccag ggaaccacgg tcaccgtctc ctcagc 356
<210> 3
<211> 374
<212> DNA
<213> 5H20 heavy chain variable region nucleotide sequence
<400> 3
gtacattcct cccaggtcca gctggtacag tcggggggaa ccttggtaca gcctgggggg 60
tccctgagac tctcttgtga agcctctgga ttcaccttta gcaactatgc catgggctgg 120
gtccgccaga ctccaggaaa ggggctggag tggctgtcgg ctattcgttc caaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagggtcg gcgacacggc ctcggggact 300
tattactgtg cgactcaccc catcgcgtcg gggggctact ggggccaggg aaccacggtc 360
accgtctcct cagc 374
<210> 4
<211> 380
<212> DNA
<213> 5H24 heavy chain variable region nucleotide sequence
<400> 4
gtacattccg aggtgcagct ggtgcagtcg gcttcggggg gattgacctt ggtacagcct 60
ggggggtccc tgagactctc ttgtgaagcc tctggattca cctttagcaa ctatgccatg 120
ggctcggctt gggtccgcca gactccagga aaggggctgg agtggctgtc ggcttcggct 180
attcgtaaaa gtggcactac cacatactac gcggactccg tgaagggccg gttcatcatc 240
tccagagaca attccaagaa caccctgtat ctgcaaatga ataggctgag ggtcggcgac 300
acggccactt attactgtgc gactcacccc atcgcgggct actggggcca gggaaccacg 360
gtcaccgtct ttgcctcagc 380
<210> 5
<211> 357
<212> DNA
<213> 7 kappa 1 light chain variable region nucleotide sequence
<400> 5
gtacattcag aaatagtgat gacgcagtct ccagccaccc tgtctgtgtc tagggaccca 60
ggggaaagag ccaccctctc cttagggtgc aggttagggg ccagtcagag tgttagcagc 120
aacttagcct ggtaccggca gaaacctggc caggctccca ggctcctcat ctatggtgca 180
tccaccaggg ccactggtat cccagccagg ttcagtggca gtgggtctgg gacagagttc 240
actctcacca tcagcagcct gcagtctgaa gattttgcag tttattactg tcagcagtat 300
aataactggc ctccgtggac gttagggacc ggccaaggga ccaaggtgga aatcaaa 357
<210> 6
<211> 345
<212> DNA
<213> 7 kappa 2 light chain variable region nucleotide sequence
<400> 6
gtacattcag aaatagtgat gacgcagtct ccagccaccc tgtctgtgtc tccaggggaa 60
agagccaccc tctcctgcag ggccagtcag agtgttagca gcaacttagc ctggtaccgg 120
cagaaacctg gccaggctcc caggctcctc atctatggtg catccaccag ggccactatc 180
tatggtgcag gtatcccagc caggttcagt ggcagtgggt ctgggacaga gttcactctc 240
accatcagca gcctgcagtc tgaagatttt gcagtttatt actgtcagca gtataataac 300
tggcctccgt ggacgttcgg ccaagggacc aaggtggaaa tcaaa 345
<210> 7
<211> 339
<212> DNA
<213> 7 kappa 9 light chain variable region nucleotide sequence
<400> 7
gtacattcag aaatagtgat gacgcagtct ccactgtctg tgtctccagg ggaaagagcc 60
accctctcct gcagggccag tagggccact cagagtgtta gcagcaactt agcctggtac 120
cggcagaaac ctggccaggc tcccaggctc ctcatctatg gtgcatccac cagggccact 180
ccagccaggt tcagtggcag tgggtctggg acagagttca ctctcaccat cagcagcctg 240
cagtctgaag attttgcagt ttattactgt cagcagtata ataactggcc tccgtggacg 300
ttcggccaag ggaccaaggt gagggccact gaaatcaaa 339
<210> 8
<211> 345
<212> DNA
<213> 7 kappa 13 light chain variable region nucleotide sequence
<400> 8
gtacattcag aaatagtgat gacgcagtct tctccaccag ccaccctgtc tgtgtctcca 60
ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagcagcaa cttagcctgg 120
taccggcaga aacctggcca ggctcccagg ctcctcatct atggtgcatc caccagggcc 180
actggtatcc cagccaggtt cagtggcagt gggtctggga cagagttcac tctcaccatc 240
agcagcctgc agtctgaaga ttttgcagtt tattactgtc agcagtataa taactggcct 300
ccgtggacgt tcggccaagg gaccaaggtg tctccagaaa tcaaa 345
<210> 9
<211> 363
<212> DNA
<213> 7 kappa 30 light chain variable region nucleotide sequence
<400> 9
gtacattcag aaatagtgat gacgcagtct ccagccccac tggtaaccct gtctgtgtct 60
ccaggggaaa gagccaccct ctcctgcagg gccagtcaga gtgccagtgt tagcagcaac 120
ttagcctggt accggcagaa acctggccag gctcccaggc tcctcatcta tggtgcatcc 180
accagggcca ctggtatccc accactggta gccaggttca gtggcagtgg gtctgggaca 240
gagttcactc tcaccatcag cagcctgcag tctgaagatt ttgcagttta ttactgtcag 300
cagtataata actggcctcc gtggacgttc ggccaaggga ccaaggtgga agccagtatc 360
aaa 363
<210> 10
<211> 115
<212> PRT
<213> 5H12 heavy chain variable region amino acid sequence
<400> 10
Val His Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
1 5 10 15
Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ala
20 25 30
Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala
35 40 45
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
50 55 60
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
85 90 95
Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ala
100 105 110
Ser Ile Lys
115
<210> 11
<211> 118
<212> PRT
<213> 5H17 heavy chain variable region amino acid sequence
<400> 11
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Thr Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe
20 25 30
Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys Gly Leu
35 40 45
Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn
65 70 75 80
Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr Ala Thr
85 90 95
Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 12
<211> 124
<212> PRT
<213> 5H20 heavy chain variable region amino acid sequence
<400> 12
Val His Ser Ser Gln Val Gln Leu Val Gln Ser Gly Gly Thr Leu Val
1 5 10 15
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr
20 25 30
Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys Gly
35 40 45
Leu Glu Trp Leu Ser Ala Ile Arg Ser Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Ser Gly Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Ser Gly Gly
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 13
<211> 126
<212> PRT
<213> 5H24 heavy chain variable region amino acid sequence
<400> 13
Val His Ser Glu Val Gln Leu Val Gln Ser Ala Ser Gly Gly Leu Thr
1 5 10 15
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly
20 25 30
Phe Thr Phe Ser Asn Tyr Ala Met Gly Ser Ala Trp Val Arg Gln Thr
35 40 45
Pro Gly Lys Gly Leu Glu Trp Leu Ser Ala Ser Ala Ile Arg Lys Ser
50 55 60
Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile
65 70 75 80
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu
85 90 95
Arg Val Gly Asp Thr Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Phe Ala Ser
115 120 125
<210> 14
<211> 119
<212> PRT
<213> 7 kappa 1 light chain variable region amino acid sequence
<400> 14
Val His Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
1 5 10 15
Ser Arg Asp Pro Gly Glu Arg Ala Thr Leu Ser Leu Gly Cys Arg Leu
20 25 30
Gly Ala Ser Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys
35 40 45
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala
50 55 60
Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe
65 70 75 80
Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr
85 90 95
Cys Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr Leu Gly Thr Gly Gln
100 105 110
Gly Thr Lys Val Glu Ile Lys
115
<210> 15
<211> 115
<212> PRT
<213> 7 kappa 2 light chain variable region amino acid sequence
<400> 15
Val His Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
1 5 10 15
Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
20 25 30
Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg
35 40 45
Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Ile Tyr Gly Ala Gly
50 55 60
Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu
65 70 75 80
Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln
85 90 95
Gln Tyr Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
100 105 110
Glu Ile Lys
115
<210> 16
<211> 113
<212> PRT
<213> 7 kappa 9 light chain variable region amino acid sequence
<400> 16
Val His Ser Glu Ile Val Met Thr Gln Ser Pro Leu Ser Val Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ala Thr Gln Ser
20 25 30
Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp
85 90 95
Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Arg Ala Thr Glu Ile
100 105 110
Lys
<210> 17
<211> 115
<212> PRT
<213> 7 kappa 13 light chain variable region amino acid sequence
<400> 17
Val His Ser Glu Ile Val Met Thr Gln Ser Ser Pro Pro Ala Thr Leu
1 5 10 15
Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
20 25 30
Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala
35 40 45
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
50 55 60
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
85 90 95
Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Ser Pro
100 105 110
Glu Ile Lys
115
<210> 18
<211> 121
<212> PRT
<213> 7 kappa 30 light chain variable region amino acid sequence
<400> 18
Val His Ser Glu Ile Val Met Thr Gln Ser Pro Ala Pro Leu Val Thr
1 5 10 15
Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
20 25 30
Gln Ser Ala Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro
35 40 45
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
50 55 60
Gly Ile Pro Pro Leu Val Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
65 70 75 80
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val
85 90 95
Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln
100 105 110
Gly Thr Lys Val Glu Ala Ser Ile Lys
115 120

Claims (7)

1. A high affinity fully human monoclonal antibody against hepatitis C virus having the following heavy and light chain variable regions:
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region is shown in SEQ ID NO 18.
2. The fully human monoclonal antibody of claim 1, wherein the antibody specifically binds hepatitis C virus E2 protein.
3. A nucleic acid molecule encoding the monoclonal antibody of claim 1, having the following heavy chain variable region and light chain variable region:
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 3; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 9.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the expression vector of claim 4.
6. Use of the monoclonal antibody or binding fragment thereof of claim 1 or 2 in the preparation of a medicament for detecting, treating, or preventing hepatitis c virus infection.
7. A composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1, and a pharmaceutically acceptable carrier.
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