CN115028711B - High-affinity anti-hepatitis C virus fully-humanized monoclonal antibody and application thereof - Google Patents

High-affinity anti-hepatitis C virus fully-humanized monoclonal antibody and application thereof Download PDF

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CN115028711B
CN115028711B CN202210676544.4A CN202210676544A CN115028711B CN 115028711 B CN115028711 B CN 115028711B CN 202210676544 A CN202210676544 A CN 202210676544A CN 115028711 B CN115028711 B CN 115028711B
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CN115028711A (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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    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G—PHYSICS
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    • 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
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Abstract

The invention relates to a high-affinity anti-hepatitis C virus fully-humanized monoclonal antibody and application thereof, belonging to the technical field of bioengineering. 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 the infection of the hepatitis C virus to the susceptible cells, is fully human, has low immunogenicity, good affinity, good treatment effect and low side effect compared with other animal-derived (such as murine) anti-hepatitis C virus molecules, and simultaneously provides great guarantee for the standardized production of the antibody.

Description

High-affinity anti-hepatitis C virus fully-humanized monoclonal antibody and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to a high-affinity fully human monoclonal antibody against hepatitis C virus and application thereof.
Background
In the eighties of the last century, researchers isolated for the first time a hepatitis virus, which was identified later as Hepatitis C Virus (HCV), from the blood of chimpanzees, as distinguished from hepatitis a and b. Hepatitis C infection has become a global health problem, and people of different sexes, ages and ethnicities are susceptible to HCV. Patients currently worldwide infected with HCV have been estimated to be over 1.84 million people, with a global HCV infection rate estimated by seropositive rates of about 1.5% -3.5%.
There are large differences in epidemic distribution among regions due to the high degree of variation and immune escape mechanisms of HCV worldwide. Currently, the hepatitis C virus strains which are mainly popular worldwide are mainly divided into 11 genotypes, more than 70 subtypes, and HCV viruses with different genotypes have different virulence and pathogenic abilities which are also greatly different. Analysis of the results of the 32030 patient samples from 29 provinces in our country revealed that the main popular hepatitis c subtypes in our country were 1b (n=16 713, 52.18%), 2a (n=9188, 28.69%), 3b (n= 2261,7.06%), 6a (n= 2052,6.41%), and 3a (n= 1479,4.62%), whereas mixed infections of the main subtypes were found in other small fractions of samples, the combination of which is as follows: 1b-2a,1b-3b,1b-6a,3a-3b,1b-3a and 2a-6a, the distribution of these HCV genotypes is closely related to the sex, age and geographical location of the population.
Clinical diagnostic criteria for hepatitis c, based on long-term clinical experience and laboratory techniques, are as follows: clinically, the Chinese medicinal composition has the characteristics of hypodynamia, anorexia, uncomfortable rib in the right season, hepatosplenomegaly and the like; laboratory examination shows that glutamic pyruvic transaminase is slightly and moderately elevated, anti-HCV antibody is positive, and HCV RNA is positive; further combining the duration of the patient's course with the existence of the corresponding epidemiological history.
The main purpose of hepatitis C treatment is to remove viruses, keep the replication level of HCV in vivo at extremely low loading for a long time, fully relieve liver injury lesions caused by HCV infection, and the like, and improve the long-term survival rate and the survival quality of patients. In the long-term hepatitis C treatment, the classical treatment method in clinic is interferon combined with ribavirin, and the method has larger side effects, is easy to generate drug resistance and has larger difference in effects on patients with different genotypes. With the advent of international new drugs such as dartavir hydrochloride tablets and asunavir soft capsules, direct Anti-viral Agents (DAAs), most of DAAs have been approved for clinical use in China, and the main mechanism of action of DAA drugs is to inhibit NS3/4A protease and NS5A/B polymerase in HCV particles, and before using the Direct antiviral drugs, the identification is performed by the genotype of the hepatitis c infected by a patient, and other conditions of liver injury of the patient are combined, so that HCV infection can be effectively treated by selecting a suitable antiviral scheme. In 2018, "propion sand" (Sofos cloth Wei Weipa tavir tablet) produced by Jilidean company formally enters the catalogue of medicines in China to mark the coming of new times of hepatitis C treatment, and the new drugs of the full-oral, genetype and single tablet of hepatitis C treatment bring good news to patients, but the unavoidable drug resistance and higher price of the new drugs make part of patients prohibitive.
In 1975, two researchers at university of Cambridge, milstein andthe hybridoma technology is disclosed for the first time, the principle of the technology is that immunized mouse spleen cells and myeloma cells are fused, and hybridoma cells capable of stably secreting single antibodies are screened after fusion. Meanwhile, the concept of monoclonal antibodies is also derived, and the antibodies are highly uniform and only aimed at a specific epitope, and have the advanced advantages of high purity, strong specificity, less cross reaction and the like. However, since such methods produce murine proteins, they are quite immunogenic in humans and do not meet long-term treatment. With the development of modern molecular biology and protein engineering techniques, the limitations of this technique are addressed to varying degrees. Initially, researchers have achieved the production of novel "chimeric" antibodies by substituting a human crystallizable fragment (Fragment crystallizable, fc) sequence for most murine Fc sequences. Thereafter, the murine antigen binding region was further engineered (Fragment of antigenbinding, fab) is grafted onto the human immunoglobulin (IgG) framework to increase the degree of humanization of monoclonal antibodies, and today's mature technology has been able to produce fully human monoclonal antibodies.
At present, the technology for preparing fully human monoclonal antibodies is various. There are mainly antibody library techniques such as phage display antibody library techniques and ribosome display antibody library techniques, monoclonal expression techniques, and the like. The techniques have advantages and disadvantages, the antibody library technique can directly prepare specific and stable monoclonal antibodies in vitro, however, the library construction workload in the early stage is quite huge, the required library capacity needs to contain antibody diversity of a certain animal, and high-throughput screening can only obtain partial fragments of the antibodies, and the high affinity of the antibodies cannot be ensured in the later stage. The monoclonal expression method is to separate specific B cell with flow cytometry and separate the heavy and light chain variable region gene of the homologous antibody through gene technology, so as to express in eukaryotic system. In this method, the sorting of the pre-B cells is particularly critical, and highly specific and stable antigens are required as probes to be efficiently sorted.
Monoclonal antibody drugs have become a hotspot in 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 meanwhile, the humanized level of the monoclonal antibody medicine is higher, the immunogenicity is relatively lower, rejection reaction is not easy to occur to a human body, and the safety is also greatly ensured. With the wide application of monoclonal antibodies in the tumor field, the development of monoclonal antibody drugs corresponding to infectious diseases is also actively advanced, and panivizumab for respiratory syncytial virus infection and ibalizumab for anti-HIV infection are sequentially developed, but monoclonal antibody drugs for treating hepatitis C are not yet developed.
Disclosure of Invention
Through intensive researches, the inventor selects E2 protein with good immunogenicity on the HCV surface as an epitope, extracts memory B cells from whole blood of a recovered population after infection of hepatitis C virus pathogens, activates plasma cells in vitro, screens plasma cells secreted with specific antibodies by adopting a specific antigen, and screens specific antibody genes by utilizing technologies such as efficient extraction of a small amount of cellular RNA, nested PCR and the like. The monoclonal antibody is fully human, and the heavy chain, the light chain variable region and the constant region are all derived from human genes, so that the monoclonal antibody has the characteristics of low immunogenicity and high safety, and test results prove that the monoclonal antibody has the characteristic of high affinity, can obviously resist hepatitis C virus, and provides great guarantee for the standardized production of the antibody.
HCV is a small enveloped single stranded RNA virus with a genome of 9.6kb in length. The HCV particles consist of a positive polarity RNA genome with 5 'and 3' untranslated regions (UTRs), and an Open Reading Frame (ORF) encoding a protein precursor of approximately 3000 amino acids. UTRs constitute highly conserved cis-acting RNA elements that regulate translation and replication of the viral genome. After assembly processing of the protein precursors encoded by the open reading frames, 10 structural and non-structural proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) are mainly formed. The outer envelope of HCV particle mainly comprises E1 and E2 glycoprotein, the coding regions of these two proteins are respectively located at the 192-383 locus (E1) and 384-746 locus (E2) of the N-terminal residue of HCV protein structure, and they can combine to form a non-covalent heterodimer structure as a highly glycosylated type I transmembrane protein, which plays an important role in virus recognition and invasion of host cells. The (core) core protein of HCV particles serves as a multifunctional protein, which has a main role in forming viral capsids, and plays a role in encapsulating and protecting genomic RNAs when viruses are transferred between host cells. Other nonstructural proteins such as NS2 and NS3 have been studied to demonstrate RNA helicase activity and serine protease activity that can assist in the replication and assembly of viral genomes with other nonstructural proteins. The E1 and E2 proteins, which are envelope proteins on the viral surface, are the major viral antigens that elicit a protective immune response. The two proteins have larger molecular weight difference, 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 hypervariable region (High Variable Region, HVR) 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 antigen epitope, and finally, the fully human monoclonal antibody with high safety and high affinity for resisting the hepatitis C virus is obtained.
In one aspect, the invention provides a high affinity anti-hepatitis c virus fully human monoclonal antibody 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 in any one of SEQ ID NO 10-13; or an amino acid sequence with the same function, which is 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;
the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs 14 to 18; or an amino acid sequence with the same function, which is 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 sets of heavy chain variable regions and light chain variable regions:
(i) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 10 (5H 12 ) The method comprises the steps of carrying out a first treatment on the surface of the 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 in SEQ ID NO. 10 (5H 12 ) The method comprises the steps of carrying out a first treatment on the surface of the 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 ) The method comprises the steps of carrying out a first treatment on the surface of the 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 ) The method comprises the steps of carrying out a first treatment on the surface of the 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 ) The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16 (7. Kappa.) 9 )。
The monoclonal antibodies of the heavy chain variable region and the light chain variable region can specifically bind to hepatitis C virus E2 protein.
In another aspect of the invention, there is provided a nucleic acid molecule encoding the monoclonal antibody described above.
Preferably, the nucleic acid molecule has a nucleotide sequence of the heavy chain variable region as set forth in any one of SEQ ID NOs 1 to 4; and the nucleotide sequence of the light chain variable region 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 regions and light chain variable regions:
(i) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 1 (5H 12 ) The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 5 (7 kappa) 1 )；
(ii) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 1 (5H 12 ) The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the light chain variable region is 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 ) The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 6 (7 kappa) 2 )；
(iv) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 3 (5H 20 ) The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 9 (7. Kappa 30 )；
(v) The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 4 (5H 24 ) The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the light chain variable region is 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 comprise, in addition to the nucleic acid molecules described above, suitable promoter or control sequences. The vector may be used to transform an appropriate host cell to enable expression of the protein.
In another aspect of the invention, a host cell is provided, said host cell comprising an expression vector as described above.
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 Sf9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. Host cells particularly suitable for use in the present invention are eukaryotic host cells, particularly mammalian cells, such as 293 cells.
In another aspect of the present invention, there is provided a method for preparing the above monoclonal antibody, comprising the steps of:
(1) Memory B cell sorting: separating peripheral blood mononuclear cells from a blood sample of a patient with hepatitis C rehabilitation, and separating memory B cells from the peripheral blood mononuclear cells;
(2) In vitro activation culture of memory B cells and positive well selection: activating the memory B cells into plasma cells, detecting secreted IgG antibodies of the memory B cells after in-vitro activation of the plasma cells by ELISA, and screening to obtain positive holes;
(3) Construction and screening of antibody variable region gene library: carrying out reverse transcription on the positive hole cells to obtain cDNA, and amplifying antibody heavy chain variable region and light chain variable region genes;
(4) Construction of antibody Gene heavy chain variable region and light chain variable region libraries: ligating the amplified antibody heavy chain variable region and light chain variable region genes to expression vectors, respectively;
(5) Screening of antibody heavy chain variable region and light chain variable region genes: the recombinant plasmid is co-transfected into HEK293T cells for expression.
In another aspect of the invention, the use of the monoclonal antibody or binding fragment thereof in the manufacture of a medicament for detecting, treating, preventing hepatitis C virus infection is provided.
In another aspect of the invention, there is provided a composition comprising a therapeutically effective amount of an antibody mixture of one or more of said monoclonal antibodies, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means that the molecular entity and composition 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 antibodies or fragments thereof of the present invention without substantially reducing the efficacy of the composition in the usual manner.
Specific examples of some substances which may be 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 carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; 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; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.
The composition of the present invention may be formulated into various dosage forms as required, and the dosage beneficial to the patient may be determined by the physician according to the type, age, weight and general condition of the patient, the mode of administration, etc. The administration may be, for example, 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, comprising 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 mode of the invention, an indirect ELISA method is adopted, an antigen to be detected is coated on a solid phase carrier, and the monoclonal antibody or the fragment thereof is utilized for detection. As a preferred mode of the present invention, the monoclonal antibody or a fragment thereof is an antibody, which can be detected according to the principle of the double antibody sandwich method. The conventional method of double antibody sandwich method is to fix the primary antibody (such as monoclonal antibody of the invention) on the carrier, then make the primary antibody react with antigen, then react with the secondary antibody (the secondary antibody carries detectable signal or can combine with the substance carrying detectable signal) after washing, finally make chemiluminescence or enzyme-linked chromogenic reaction to detect signal. The double antibody sandwich method is particularly suitable for detection of antigens having two or more epitopes.
For convenience in detection, the kit may contain, 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 nature of which and the methods of formulating them are well known to those skilled in the art, such as color-developing agents, labels, secondary antibodies, anti-antibodies, sensitizers, etc. It will be appreciated by those skilled in the art that various variants of the detection kit are encompassed by the present invention, provided that the monoclonal antibody or fragment thereof of the present invention is utilized therein as an agent for recognizing hepatitis c virus.
Further, instructions for use may be included in the kit for use in describing the method of use of the reagents loaded therein.
After obtaining the monoclonal antibodies or fragments thereof provided by the present invention, various immunological related methods may be used to detect HCV-E2 protein in the sample, thereby determining whether the donor of the sample to be tested is infected with hepatitis C virus, and all such methods are included in the present invention. Preferably, the method is for non-disease diagnosis purposes.
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 the monoclonal antibody or fragment thereof is contacted with a sample to be tested, and the presence and amount of hepatitis C virus are obtained by detecting the binding of the monoclonal antibody or fragment thereof to the sample to be tested.
As used herein, the term "sample to be tested" encompasses a variety of sample types, including blood and other body fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or their progeny. The term also includes samples that have been treated by any means after they have been obtained, for example by treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides. The term encompasses various clinical samples from any species, as well as cultured cells, cell supernatants, and cell lysates.
The beneficial effects are that: compared with the prior art, the invention successfully screens the fully human monoclonal antibody against the hepatitis C virus, and the monoclonal antibody has the characteristic of high affinity, can specifically bind the hepatitis C virus, and can obviously resist the hepatitis C virus. Compared with the murine antibody, the gene of the fully human antibody is fully derived from human genes, has no other species components, does not generate toxic or side effects such as anti-mouse anti-antibody and the like in human body, has better biocompatibility, is more suitable and has better potential to become a macromolecular medicament for treating hepatitis C virus, and provides great guarantee for the standardized production patent medicine of the antibody.
Drawings
FIG. 1ELISA screens positive memory B cell wells.
FIG. 2 is an electropherogram of an antibody after PCR amplification of the heavy chain variable region genes H, H' and light chain variable region genes kappa, lambda.
FIG. 3 shows the results of electrophoresis verification of the heavy chain variable region gene, the light chain variable region gene and the vector.
FIG. 4 shows the results of the electrophoresis verification of recombinant plasmids.
FIG. 5 is a screening pattern of antibody heavy and light chain variable region genes.
FIG. 6 shows the results of a gene combination screen for the heavy and light chain variable regions of an antibody.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The experimental procedure, without specific conditions noted in the examples, is generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring HarborLaboratoryPress, 1989) or as recommended by the manufacturer.
Example 1: preparation of fully human monoclonal antibodies to 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, fresh whole blood of the hepatitis C rehabilitation volunteer is extracted into a disposable vacuum blood collection tube, and the whole blood volume required by each person is 50ml. The pre-chilled PBS solution after filtration through a 0.22 μm filter was diluted 1:1 with fresh anti-coagulated whole blood. Slowly adding diluted blood into lymphocyte separation solution with equal volume, centrifuging at 2000rpm under Acc4 and Dec4 conditions, and centrifuging at room temperature for 30min. After centrifugation, the tube was slowly removed and the second cloudy mononuclear cell layer after centrifugation was carefully aspirated into a 15ml tube pre-filled with pre-chilled PBS. The aspirated monocytes were centrifuged at 1200rpm at room temperature for 5min, the supernatant discarded, and washed gently with pre-chilled PBS. The resulting solution was centrifuged again and the collected cells were peripheral blood mononuclear cells (periphery blood mononuclear cell, PBMC) and counted.
1.2 memory B cell sorting
(after cell counting, specific MS or LS columns were selected based on the isolated PBMC) cells were centrifuged at 300g for 10min and the supernatant was removed thoroughly. At every 1×10 7 The amount of cells was 40. Mu.l buffer as a unit and appropriate amounts of buffer were taken up in proportion to resuspend the cell pellet. At every 1×10 7 The cells were added in an amount of 10 mu l Naive B cell Biotin-Antibody Cocktail units, the cell pellet was thoroughly mixed and incubated in a refrigerator at 4℃for 5min. At every 1×10 7 Cell count 30. Mu.l buffer and 20. Mu.l Anti-Biotin MicroThe Beads were used as a unit, and the cell pellet was thoroughly mixed and placed in a refrigerator at 4℃for 10min. The cells were washed with 1-2 ml buffer, centrifuged at 300g for 10min, and the supernatant was discarded. The cell pellet was resuspended with 500. Mu.l buffer. The column was placed on a magnet and the column was rinsed 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 exuded unlabeled cells were collected. The column was washed 3 times with an appropriate amount of buffer (3 ml of buffer was added to the LS column, 500. Mu.l of buffer was added to the MS column), and the washing was performed after the column had drained. Collecting all eluates to obtain suspension containing B cells. The column was removed from the separator, placed on a collection tube, and the pipetting buffer was added to the column and immediately pushed by force against the piston to wash out the magnetically labeled cells. Cell count, 300g centrifugation for 10min, complete removal of supernatant, every 10 7 The total cells were resuspended in cell pellet with 80. Mu.l buffer. At every 1×10 7 Cell number was added with 20 mu lCD and 27 MicroBeads as units, a proper amount of labeled magnetic beads was added, the solution was thoroughly mixed, and incubated in a refrigerator at 4℃for 15min. At every 1×10 7 Cell number cells were washed in 1ml buffer, centrifuged at 300g for 10min, and the supernatant was removed thoroughly. When the number of cells is less than 1X 10 8 At this time, the cells were resuspended with 500. Mu.l buffer (if more cells were present, the buffer doubled). The post is placed onto 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 (LS column with 3ml buffer and MS column with 500. Mu.l buffer). Collecting all the eluent, namely the suspension containing 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, a suitable volume of buffer was aspirated into the column, and immediately the piston was forced to wash out the magnetic bead labeled cells. The cell is a memory B cell. The selected memory B cells were counted and frozen.
2. In vitro activation culture and positive hole screening of memory B cells
The memory B cells obtained by sorting are inoculated into a 96-well cell culture plate according to different cell density gradients. 200 μl of IMDM of 10% FBS was added per well as basal medium containing IL-21 as B cell activating factor at appropriate concentration. On the basis of the above, co-60 inactivated 3T3-CD40L cell stable cell line was added to each well as feeder cells. Culturing for several days at 37 ℃ under the condition of 5% CO2, so that the memory B cells can be activated into plasma cells and secrete antibodies; ELISA detection of secreted IgG antibodies after in vitro activation of memory B cells into plasma cells: cell culture supernatants were collected, ELISA was used to detect culture supernatants of memory B cells using the Goat Anti-Human Kappa (Goat Anti-Human Kappa) protein and the Goat Anti-Human Lambda (Goat Anti-Human Lambda) protein coated ELISA plates, and the secretion amount of IgG antibodies was determined.
Respectively coating 100 ng/Kong Rong of the Goat Anti-Human Kappa protein and the Goat Anti-Human Lambda protein on an ELISA plate at 50 μl/hole coating liquid, and washing the plate for 5 times at 4 ℃ with PBS solution containing 0.05% Tween-20; blocking with blocking solution 200 μl/hole at 37deg.C for 2h, and washing the plate for 5 times; adding cell culture supernatant or gradient diluted IgG standard into corresponding well, respectively, and washing the plate with water or PBS as negative control, 50 μl/well, and 37deg.C for 1 hr for 5 times; adding a peroxide horseradish enzyme-labeled Goat Anti-Human IgG antibody diluted 1:5000 and a diluted Goat x-Human IgG-Fc Fragment HRP Conjugated mu l/hole diluted 1:10000 into corresponding holes respectively, standing at 37 ℃ for 45min, and washing the plates for 5 times; adding 100 μl/hole of the color development liquid, and developing at room temperature in dark place for 15min; the chromogenic reaction was terminated by adding 50. Mu.l/well of 2mol/L sulfuric acid, and the OD value of the absorbance of each well was measured at a wavelength of 450 nm. Finally, comparing and analyzing the OD value of each hole and the corresponding memory B cell inoculation number of each hole to determine the positive memory B cell hole. As can be seen from FIG. 1, wherein A1 and A2 are feeder cells as control wells, the OD value is 0.0613, and finally 12 positive wells are numbered A3-A14, wherein the OD values of three positive wells numbered A3, A6 and A7 are significantly higher than those of other wells, and the OD values are 0.731,0.4003,0.4179, respectively. The remaining positive wells were also raised to some extent, with OD values of about 0.13.
3. Construction and screening of antibody variable region Gene library
3.1 extraction of B cell RNA from Positive well
After microscopic observation, the cell culture supernatant was carefully aspirated, washed once with PBS, digested with 40. Mu.l of 0.25% trypsin per well, stopped with 5. Mu.l of fetal bovine serum per well, blown off with a gun, scraped off, and finally 2.25. Mu.l of RNase inhibitor (40U/. Mu.l) was added per tube. Rapidly placing the digested cells into liquid nitrogen for more than 2 min; placing the sample in a PCR instrument at 98 ℃ for 3min, and immediately placing the sample in liquid nitrogen; after quick-freezing, 0.5. Mu.l proteinase K (20 mg/ml) and 2.25. Mu.l RNase inhibitor (40U/. Mu.l) were added to each tube and placed in a PCR apparatus at 53℃for 1h. The concentration of the extracted RNA was measured, and reverse transcription was performed using a Promega reverse transcription kit to obtain cDNA.
3.2 amplification of antibody heavy chain variable region and light chain variable region genes
The two-step RT-PCR method is used for amplifying antibody heavy chain variable region gene VH (in order to improve the efficiency of each step of experiment, heavy chain variable region gene VH primers are divided into two main categories of H and H', PCR amplification and subsequent experiment are respectively carried out, and when recombinant plasmid is transformed, the two main categories are mixed together to select monoclonal) and light chain variable region gene V kappa or V lambda: taking the cDNA after reverse transcription as a PCR template, and performing nested PCR first round reaction; and then taking the first round reaction product of the nested PCR as a template to perform the second round reaction of the nested PCR. Specific primer sequences, PCR reaction procedures and PCR reaction system references (Nat Protoc.2009;4 (3): 372-84.).
PCR products of heavy chain VH and light chain V kappa and V lambda were detected by agarose gel electrophoresis, and the results are shown in FIG. 2, FIG. 2A: nested PCR two-round amplification results of antibody heavy chain (H, H' chain) variable region sequences; b, nested PCR two-round amplification results of antibody light chain (kappa, lambda chain) variable region sequences; c: a3 And (3) performing nested PCR amplification on the heavy chain and light chain variable region sequences of A4 and A7 samples to verify the results. M: marker DL2000. The target strip is recovered by the gel, and the specific operation is referred to the specification of the OMEGA gel recovery kit. Finally, the concentration is measured by using a Nanodrop2000, and the product is stored for a long time at the temperature of minus 20 ℃. The results show that obvious target bands are detected by the samples A3, A4 and A7 through two times of nested PCR amplification (the kappa chain of the sample A3 is not amplified), and the positions of the target bands are between 250bp and 500bp, namely 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
Amplified antibody heavy chain variable region genes VH and light chain variable region genes Vκ, Vλ were ligated to expression vectors pIgH (AbVec-hIgG 1 for VH), pIgκ (AbVec-hIgkappa for Vκ) and pIgλ (IG-lambda expression vector for V λ), respectively (NCBI GenBank accession numbers: FJ475055, FJ475056, FJ517647, respectively.
Sequentially using AgeI-HF and Sal I-HF to cleave VH and pIgH; cutting V lambda and pIg lambda by AgeI-HF and Xho I; cutting V kappa, pIgkappa with AgeI-HF, and cutting V kappa, pIgkappa with BsiW I. The enzyme digestion products are detected by 1.2% agarose gel electrophoresis, the bands are observed, the target gene bands are at 300bp-500bp positions, and the experimental results are shown in figure 3. The target strip is recovered by the gel, and the specific operation is referred to the specification of the OMEGA gel recovery kit.
FIG. 3 shows the results of electrophoresis verification of the heavy chain variable region gene, the light chain variable region gene and the vector. Wherein VH, VH' and V lambda, V kappa are all verified by digestion. A: a3, A4 and A7 sample heavy chain (H chain and H' chain) variable region gene digestion results; b: a3, A4, A7-like light chain (kappa chain, lambda chain) variable region gene cleavage results; c: cleavage of the vector plasmid (IgH, igkappa, iglambda), 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 fragments of the antibody are 400bp, and the corresponding heavy chain and light chain vector plasmids are 5000bp, the enzyme digestion success of the heavy chain and light chain variable region genes of the A3, A4 and A7 sample antibodies and the vector plasmids can be judged.
The target gene fragment after cleavage was ligated with the corresponding constant region vector pIgH (AbVec-hIgG 1 for VH), pIgκ (AbVec-hIgkappa for V κ) or pIgλ (IG-lambda expression vector for V λ) after cleavage, and the ligation was performed enzymatically overnight at 16 ℃.
The ligated recombinant plasmid was transformed into TOP10 E.coli cells. The positive clone colonies were picked the next day on the Amp + In LB medium of (A), shaking culture was carried out at 200rpm and 37℃for 12 hours, and then the resultant was sterilized with 50% sterilized glycerol to extract a plasmid (see O for detailsMEGA plasmid extraction kit instructions). And (3) enzyme cutting to verify positive clone antibody vector plasmid of heavy chain H-IgH, H' -IgH and light chain lambda-Iglambda. The experimental results are shown in FIG. 4, after the recombinant plasmids A3, A4 and A7 are subjected to enzyme digestion, two bands can be observed at the positions of more than 2000bp bands (5000 bp) and 400bp bands, namely two bands of a plasmid gene and an antibody heavy and light chain variable region gene formed after the enzyme digestion of the recombinant plasmids, which shows that the recombinant plasmids A3, A4 and A7 are successfully subjected to enzyme digestion in the earlier stage (M: DL 2000).
4. Screening of antibody heavy chain variable region and light chain variable region genes
After transforming the recombinant plasmid, selecting a monoclonal plasmid, co-transfecting HEK293T cells with a plasmid of an antibody heavy chain and an antibody lambda light chain or an antibody kappa light chain (the light chain is lambda and kappa, the heavy chain is only H, and any combination of H and lambda or kappa can form an antibody), detecting the secretion of a specific antibody of HCV-E2 antigen protein (homemade) by adopting an enzyme-linked immunosorbent assay (Elisa method), and screening the antibody recombinant plasmid combination of the high-expression HCV-E2 specific IgG antibody.
The specific steps of cell transfection are as follows:
(1) Inoculating cells: the cells after termination of 0.25% pancreatin-EDTA digestion were 1X 10 per well 4 Each cell was seeded in a 96-well plate, and the culture medium was further supplemented to 190. Mu.L. Meanwhile, a control group is arranged, wherein the control group 1 is a transfection reagent and cells, but no plasmid is added; control group 2 was cells only, no transfection reagent and plasmid.
(2) The plasmid and transfection reagent were diluted with opti-MEM, respectively, such that 0.15. Mu.L of transfection reagent was added per well and 100ng of plasmid per well.
(3) According to the following steps of 1:1 mixing the diluted plasmid and the diluted transfection reagent, adding into the cell-inoculated well after 5min at 37deg.C with 5% CO 2 Culturing in an incubator for 24-72h, and detecting the antibody content.
(4) Elisa detects cell culture supernatant IgG secretion: collecting the cell culture supernatant after transfection in the step (3), and coating the ELISA plate with a coating solution (50 μl/hole) according to a coating amount of 100 ng/hole for HCV-E2 antigen protein (self-made); the ELISA plates were additionally coated with the Goat Anti-Human kappa+Goat Anti-Human Lambda (100 ng/well each) proteins overnight at 4 ℃. The plate washer washes the plate 5 times. The wash solution was a PBS solution containing 0.05% Tween. The plate was washed 5 times with 200. Mu.l of blocking solution per well at 37℃for 2 h. Cell culture supernatants, gradient diluted IgG standards and anti-HCV-E2 protein antibodies (primary antibodies) were added to the corresponding wells, respectively, while using water or PBS solution as a negative control, 50. Mu.l/well, and left at 37℃for 1h, and the plates were washed 5 times. HRP-labeled Goat Anti-Human IgG and Goat Anti-Mouse IgG were added at 1:5000 dilution and Goat x-Human IgG-Fc Fragment HRP Conjugated μl/well at 1:10000 dilution, respectively, and the wells were left at 37deg.C for 45min and washed 5 times. 100 μl/well of TMB developer was added and developed at room temperature in the dark for 20min. The chromogenic reaction was terminated by adding 50. Mu.l/well of 2mol/L sulfuric acid, OD/450nm was detected, and the reference wavelength was 630nm.
Using the screening pattern shown in FIG. 5, we performed specific screening of positive memory B cell wells by ELISA. Ranking the screening of different batches by taking the affinity of the antibodies as a standard, selecting the optimal heavy and light chain sequence for the next batch of screening after ranking, grading the mixed sequence to the monoclonal sequence layer by layer, finally screening the obtained high-affinity monoclonal antibody, and selecting the effective monoclonal heavy chain and light chain combination for monoclonal antibody detection after identification and analysis (sequencing is completed by Jiangsu Style biotechnology company). 5 heavy and light chain monoclonal sequences obtained by screening are respectively selected to carry out 'one-to-one' pairing in vitro transfection expression, and 5 heavy chain monoclonal sequences are obtained: 5H 12 ,5H 16 ,5H 17 ,5H 20 ,5H 24 And 5 light chain monoclonal sequences: 7K 1 ,7κ 2 ,7κ 9 ,7κ 13 ,7κ 30 Finally obtaining 5 strains of high-affinity monoclonal antibodies after ELISA screening, and combining the antibodies: 5H 12 +7κ 1 ，5H 12 +7κ 13 ，5H 17 +7κ 2 ，5H 20 +7κ 30 ，5H 24+ 7κ 9 (the result corresponds to FIG. 6).
The foregoing descriptions of specific exemplary embodiments of the present invention are 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 the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various 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 against hepatitis C virus with high affinity 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> 7k9 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 (5)

1. A fully human monoclonal antibody having high affinity against hepatitis c virus, characterized in that it has the following heavy chain variable regions 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 as SEQ ID NO. 18.
2. A nucleic acid molecule encoding the monoclonal antibody of claim 1, having a heavy chain variable region and a light chain variable region as follows:
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.
3. An expression vector comprising the nucleic acid molecule of claim 2.
4. Use of the monoclonal antibody of claim 1 in the preparation of a kit for detecting hepatitis c virus infection.
5. A composition comprising the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier.
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