CN106749644A

CN106749644A - A kind of neutralizing antibody TRN1001 of full people source HCV-Ab IgG

Info

Publication number: CN106749644A
Application number: CN201611001112.4A
Authority: CN
Inventors: 张远旭; 王月明; 袁晓辉; 廖化新; 昝利鹏; 刘彤; 洪坡
Original assignee: Zhuhai Microlab Biotechnology Co Ltd; GUANGZHOU TAINUODI BIOTECHNOLOGY CO Ltd; Jinan University
Current assignee: Guangzhou Tainuodi Biotechnology Co ltd; Zhuhai Tainuo Maibo Pharmaceutical Co ltd; Jinan University
Priority date: 2016-11-14
Filing date: 2016-11-14
Publication date: 2017-05-31
Anticipated expiration: 2036-11-14
Also published as: CN106749644B

Abstract

The invention discloses a kind of full human monoclonal antibody for neutralizing HCV and application thereof.The invention particularly discloses a kind of antibody, its encoding gene, expression vector, purposes that can be recognized and combine hepatitis C virus envelope protein E 2 albumen.Monoclonal antibody of the invention can prevent HCV from infecting permissive cell, and be full people source, compared with the molecule of other animal derived anti-hepatitis c virus, immunogenicity greatly reduces, and compatibility is good, not only therapeutic effect is good, and side effect is low.

Description

A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG

Technical field

The invention belongs to cellular immunology, biology field, it is related to a kind of monoclonal antibody in full people source, especially relates to And a kind of monoclonal neutralizing antibody of full people source HCV-Ab IgG.In addition, the invention further relates to the preparation method and use of the neutralizing antibody On the way.

Background technology

((Hepatitis C virus, HCV) is known only in addition to retrovirus to cause slow to HCV The RNA virus of sexuality dye.The infection sources of HCV is mainly actual clinical type and asymptomatic subclinical patient, chronic patient and disease Malicious carrier.12 days its blood of usual patient's premorbid is have infectivity, and can be with malicious more than 12 years.According to estimates, it is complete at present Ball increases more than 200,000,000 HCV infection persons and every year with the speed of 3,500,000 people, and wherein chronic carriers account for 60%~80%；And There is HCV pantropic can cause repeated infection, so that 80% acute infection switchs to chronic, finally develop into cirrhosis/liver The probability of cancer is then up to 3.5%~20%.

Control HCV is replicated and the key factor of immune clearance is to produce wide spectrum neutralizing antibody, but HCV has height and becomes Different, the immunosurveillance of the body that can escape simultaneously suppresses humoral and cellular immune response by number of ways.Research finds, makees for acceptor The antibody that HCV epitopes are produced has preferable neutralization activity.In recent years, about HCV infection model such as cell line and petty action The foundation of thing model so that the further investigation to HCV is developed rapidly, antibody-mediated virus neutralization is expected to turn into new Effective HCV-Ab IgG treatment method.Huh-7 cells are infected using the HCV pseudovirus of HCVpp different genotypes, can be used to neutralize The research of antibody activity.Kato etc. realizes the table of HCV Subgenomic replicons in the HeLa and 293 cells of non-liver source property Reach.Hideki etc. is constructed in human cell line CHO and Huh-7 and is replaced the HCVrv of VSV memebrane proteins with HCV E1 and E2 albumen, Huh-7 can be infected and neutralized by CD81-E2 special antibody.Daniel etc. ground for the monoclonal antibody of E2 epitopes Study carefully, discovery obtains the monoclonal antibody mAbs1 of the different genotype of E2 from HCV carrier's serum:7 and A8 to HCVpp and HCVcc infection models show extensive neutralization.

According to the variation of nucleotide sequence, current HCV points is 7 genotype and dozens of gene hypotype.HCV genomes are about 9.6kb, containing single ORF.Virus is by envelope protein E1, E2, core protein Core, P7 albumen, and non-structural protein NS3, NS4a, NS4b, NS5a, NS5b are constituted.The hypervariable region 1 (HVR1) that 27 amino acid residues of N-terminal of E1, E2 are constituted is recognized To be neutralizing epitope, but there is larger difference in its sequence between different genotype and quasispecies.Domestic some scholar's research find choosing The recombinant antigen protein for taking the expression of the representative combined sequence comprising different genotype and quasispecies has cross-reactivity high.It is external Research to neutralizing antibody epitope is also very active：E1/E2 is immune can to produce neutralizing antibody higher, although neutralizing antibody can not be protected Protect different strain HCV to attack, but the chronicity after different strain virus attack can be prevented；Restructuring E1/E2 membrane glycoproteins have in preferably intersection And activity.The above-mentioned research about neutralizing antibody, for the therapeutic vaccine research further directed to HCV provides thinking.

Research shows, the chronicity and subinfection again of HCV infection can be controlled using HCV neutralizing antibodies.Separately there is research table Bright, HCV neutralizing antibodies have the effect of diagnosis and treatment.Therefore, the research based on HCV-Ab IgG neutralizing antibody, finds a kind of effective Method can produce the antibody for strongly and widely neutralizing HCV, with important in the immune response of viral infection period excitating organism Meaning.

The content of the invention

It is an object of the invention to provide a kind of monoclonal neutralization of full people source HCV-Ab IgG of combination HCV Surface env proteins E2 Antibody, the antibody can be used for preventing and treating hepatitis and can be also used for identifying HCV.

To achieve these goals, present invention employs following technical scheme：

A kind of the monoclonal neutralizing antibody or its antigen-binding fragment of full people source HCV-Ab IgG, the antibody or its antigen binding Fragment includes complementarity determining region CDR1, CDR2, CDR3 of weight chain variable district and the complementarity-determining region of light chain variable district Domain CDR1, CDR2, CDR3；The amino acid sequence of heavy chain CDR1, CDR2, CDR3 is respectively such as SEQ ID NO:2、SEQ ID NO:3、 SEQ ID NO:Shown in 4, or respectively with SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:Sequence shown in 4 at least has There is 80% homology；The amino acid sequence of light chain CDR1, CDR2, CDR3 is respectively such as SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:Shown in 8, or respectively with SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:Sequence shown in 8 at least has 80% homology.

Preferably, heavy chain CDR1 has SEQ ID NO:Amino acid sequence shown in 2；Heavy chain CDR3 has SEQ ID NO: Amino acid sequence shown in 3；Heavy chain CDR3 has SEQ ID NO:Amino acid sequence shown in 4；Light chain CDR1 has SEQ ID NO:Amino acid sequence shown in 6；Light chain CDR2 has SEQ ID NO:Amino acid sequence shown in 7；Light chain CDR3 has SEQ ID NO:Amino acid sequence shown in 8.Antibody with above-mentioned preferred sequence is named as TRN1001.

Further, the weight chain variable district of the monoclonal antibody has SEQ IDNO:Amino acid sequence shown in 1 or and its Amino acid sequence with least 80% homology, the light chain variable district of the monoclonal antibody has SEQ ID NO:Shown in 5 Amino acid sequence or there is the amino acid sequence of at least 80% homology with it.

Preferably, the weight chain variable district of the monoclonal antibody has SEQ IDNO:Amino acid sequence shown in 1；It is described The light chain variable district of monoclonal antibody has SEQ ID NO:Amino acid sequence shown in 5.

The antibody of the conservative sequence variant including preferred antibody amino acid sequence is also included within the scope of the present invention.Protect Keeping amino acid sequence variation includes not significantly changing the monoclonal neutralizing antibody binding property of full people source HCV-Ab IgG of the invention with With the modification of the amino acid sequence of property, the variant that Similar amino acids well known in the art are substituted such as is come from, the missing of amino acid, Variant caused by increasing.

Antibody of the invention also includes people source and non-human source antibodies, and with TRN1001 antibody identical function or change All antibody made and optimize.

Further, the antigen-binding fragment of the antibody includes Fab, Fab ', F (ab ') 2, Fv or scFv.

Present invention also offers a kind of nucleic acid molecules of separation, the nucleic acid molecule encoding SEQ ID NO:Ammonia shown in 1 Base acid sequence or SEQ IDNO:Amino acid sequence shown in 5.

The foregoing antibody or the nucleic acid molecules of its antigen-binding fragment of encoding of the invention includes thering is above-mentioned nucleosides The nucleic acid molecules of the conserved nucleotide sequence variant of acid sequence.So-called conserved nucleotide sequence variant comes from genetic code degeneration With the variant of silence, the replacement of nucleotides, missing and increase are also included.

Present invention also offers a kind of DNA fragmentation of foregoing nucleic acid molecules.The DNA fragmentation can be with encoding antibody Any region of heavy chain or light chain variable district, including heavy chain CDR1, CDR2 or CDR3, or light chain CDR1, CDR2 or CDR3.

Present invention also offers a kind of expression vector including foregoing nucleic acid molecules, except foregoing nucleic acid Outside molecule, expression vector also includes the expression regulation sequence being operatively connected with the sequence of nucleic acid molecules.

Expression vector refers to the one kind that can be inserted the polynucleotide for encoding certain albumen and make albumen obtain expression Nucleic acid delivery vehicle.Carrier can be thin in host by conversion, transduction or transfection host cell, the inhereditary material element for carrying it Intracellular is expressed.The species of carrier include bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.In a word, as long as can be replicated in host and steady Fixed, any plasmid and carrier can be used.In expression vector, in addition to containing replication orgin, can also containing marker gene and Other translation control elements.

In specific embodiments of the present invention, the carrier is pCDNA3.3.

It is thin present invention also offers a kind of host containing foregoing nucleic acid molecules or foregoing expression vector Born of the same parents.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or it is high Deng eukaryotic, such as mammalian cell.Representative example has:Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell：Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；CHO, COS, 293 cell or Bowes are black Zooblast of plain oncocyte etc..

In specific embodiments of the present invention, the host cell is mammalian cell, more preferably Chinese hamster ovary celI.

Converted with recombinant DNA, transfection host cell can be carried out with routine techniques well known to those skilled in the art.

The invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes the antibody of the invention of therapeutically effective amount Or its antigen-binding fragment.

Described pharmaceutical composition also includes pharmaceutically acceptable carrier, and the carrier is included but is not limited to by the U.S. FDA's accreditation and can be used for any adjuvant of the mankind or animal, carrier, excipient, glidant, diluent, Surfactant, wetting agent, dispersant, suspending agent, stabilizer, isoosmotic pressure agent, solvent or emulsifying agent etc. are to composition drug regimen The various forms of carriers that thing has no side effect.

Present invention also offers a kind of monoclonal neutralizing antibody including full people source HCV-Ab IgG of the invention or its antigen binding The HCV detection products of fragment.

The detection product includes but is not limited to detection reagent, kit, chip or test paper.It is every including knot noted earlier The detection product for being capable of detecting when HCV for closing molecule is included within the scope of the present invention.

Present invention also offers a kind of method of the detection HCV levels of non-diagnostic purpose, it is characterised in that methods described bag Include following steps：

(1) sample containing HCV is extracted；

(2) the monoclonal neutralizing antibody or its antigen of sample and the foregoing full people source HCV-Ab IgG for obtaining step (1) The contact of binding fragment；

(3) immune response of detection sample and antibody or its antigen-binding fragment.

Present invention also offers a kind of preparation method of the monoclonal neutralizing antibody of foregoing full people source HCV-Ab IgG, institute State preparation method and use single bone-marrow-derived lymphocyte antibody production techniques.The single bone-marrow-derived lymphocyte antibody production techniques be by It is unicellular to separate the vitro expression systems that identification technology combines a kind of monoclonal antibody that various round pcrs are formed, i.e., drenched from people B The method of the monoclonal antibody in the full people source of direct access in bar cell.

Further, methods described comprises the following steps：

(1) detection finds HCV infection patient；

(2) mononuclearcell in HCV infection Venous Blood is separated, cell point is carried out using flow cytometry afterwards Choosing, sub-elects the B cell containing CD235a-/IgD-/CD20+；

(3) using the antibody light chain and weight chain variable district in the single B cell of single-cell RT-PCR amplification step (2) acquisition Nucleotide fragments；

(4) antibody light chain and the nucleotide fragments of weight chain variable district for obtaining step (3) are fused to and contain human antibodies Recombinant expression carrier is formed in the expression vector of constant region, host cell expression is imported afterwards；

(5) antibody screening Platform Screening obtains the of the invention full people source HCV-Ab IgG with binding activity and neutralization activity Monoclonal neutralizing antibody.

Monoclonal neutralizing antibody or its antigen-binding fragment present invention also offers foregoing full people source HCV-Ab IgG exist Prepare the application in HCV detection products.

Antibody of the invention, can simply and precisely identify 7 kinds of HCV genotype.

Monoclonal neutralizing antibody or its antigen-binding fragment present invention also offers foregoing full people source HCV-Ab IgG exist Prepare the application in foregoing pharmaceutical composition.

Monoclonal neutralizing antibody or its antigen-binding fragment present invention also offers foregoing full people source HCV-Ab IgG exist Prepare the application suppressed in HCV medicines.

Monoclonal neutralizing antibody or its antigen-binding fragment present invention also offers foregoing full people source HCV-Ab IgG exist Prepare the application in the medicine of the disease that prevention or treatment are caused by HCV infection.

Further, the disease caused by HCV infection includes hepatopathy, such as hepatitis, cirrhosis or liver cancer and Extrahepatic diseases.

Hepatitis of the invention refers to acute or chronic hepatitis C.

Antibody TRN1001 of the invention can be used for treat acute, patients with chronic hepatitis C, the treatment be by Give the antibody that can combine HCV E2 of these bacteriums." therapeutically effective amount " is to refer to effectively subtract in individuality The dosage of light HCV infection symptom, or effectively reduce the dosage of virion in circulation.The antibody can be used for for example The neonatal passive immunity of HCV positive carrier's childbirths, can be also used for the passive immunity of liver transfer operation to prevent these patients The HCV infection repeatedly being likely to occur.Especially, for being expected therapeutic effect, infection gene not from interferon therapy The hepatitis C patients of type, can mitigate adverse reaction, and can provide the chance of the new treatment method of selection.

The antibody of disclosure of the invention can include one or more glycosylation sites in heavy chain and light chain variable district, such as originally Well known in field, one or more glycosylation sites present in variable region can cause enhanced antibody immunogenicity, Or change the pharmacokinetics of antibody due to changing antigen binding.

Antibody of the invention can be designed as in Fc regions comprising modification, typically change one or more of antibody Functional characteristic, such as serum half-life, the cytotoxicity that complement is combined, Fc acceptors are combined, and/or antigen is relied on.Additionally, of the invention Antibody can be modified by sulphation (e.g., one or more chemical groups can be connected to antibody), or be modified to change it Glycosylation, so as to change the one or more functions characteristic of antibody again.

The monoclonal neutralizing antibody or its antigen-binding fragment of full people source HCV-Ab IgG of the invention can chemically or It is conjugated by genetic engineering and other factors.These factors provide the effect in antibody target required function site or are antibody Improve or provide other performances.

The antibody of full people source HCV-Ab IgG of the invention can be marked chemically or by genetic engineering, to carry For detectable antibody.Detectable antibody can be used for for example evaluation object whether oneself through infect hepatitis C virus poison strain or Person monitors the generation of infection with hepatitis C virus or is in progress for example to determine TA as a part for clinical trial program Effect of scheme.However, they can be used for other detections and/or analysis and/or diagnostic purpose.Detectable part includes But be not limited to enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emitting metal and On-radiation paramagnetic metal ion.

In order to detect and/or analyze and/or diagnostic purpose mark depend on the particular detection/analysis/diagnostic techniques for using And/or the inspection of method such as immunohistochemical staining (tissue) sample, flow cytometry, laser scanning Cytometry (such as phagocytosis is made for survey, FIA, enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), biologicall test With determining), Western blotting application etc..Suitably marked for detection/analysis/diagnostic techniques known in the art and/or method It is designated as well known to those skilled in the art.

Term " monoclonal antibody " used herein refers to the antibody obtained from the substantially uniform colony of a class, except a small number of possible Outside the mutation of the natural generation for existing, the single antibody included in the colony is identical.Modifier " monoclonal " only represents anti- The characteristic of body, is obtained from substantially uniform antibody population, and this can not be construed to need to produce anti-with any specific process Body.

" variable " some parts for representing variable region in antibody of term used herein are different in sequence, and it is formed Combinations and specificity of the various specific antibodies to its specific antigen.Changeability is referred to as mutually in concentrating on light chain and weight chain variable district Benefit is determined in three fragments in area (CDR) or hypervariable region.Each self-contained four FR areas in the variable region of native heavy and light chain (more conservative part in variable region), they are in generally beta sheet configuration, are connected by three CDR for forming connection ring, can shape Into part β-pleated sheet structure.CDR in every chain is by FR areas firmly against together form together and with the CDR of another chain The antigen-binding site of antibody.Constant region does not participate in the combination of antibody and antigen directly, but they show different effects Function, such as participates in the cytotoxicity for depending on antibody of antibody.

The advantages of the present invention：

The present invention prepare full people source HCV-Ab IgG NAT is high, high specificity, with cross-reactive high, composition Clearly, can be with low cost with high efficient expression, standardized production, quality control easily.

Full people source HCV-Ab IgG neutralizing antibody prepared by the present invention can both avoid HCV polygene types and the high variability from causing Antibody failure, market problem of the existing medicine without cross-reactivity high can be avoided again.

Brief description of the drawings

Fig. 1 is the SDS-PAGE detection figures of the antibody TRN1001 of embodiments of the invention；

Fig. 2 is in the antibody TRN1001 and 7 kinds of envelope glycoproteins of different subtype HCV euviruses of embodiments of the invention With determination of activity figure；

Fig. 3 is the antibody TRN1001 and the affine determination of activity figure of HCV virus antigen of embodiments of the invention；

Fig. 4 determines figure for the neutralization activity of the antibody TRN1001 of embodiments of the invention HCV virus strains different from 5 kinds, its In, A：h77；B：JFH1；C：SA13；D：S52；E：ED43.

Specific embodiment

The present invention is further illustrated below by embodiment.It should be understood that embodiments of the invention are for illustrating The present invention is rather than limitation of the present invention.Essence of the invention belongs to the present invention to the simple modifications that the present invention is carried out Claimed scope.

The preparation of the monoclonal neutralizing antibody TRN1001 of the full people source HCV-Ab IgG of embodiment 1

1.PBMC is separated and the sorting of single memory B cells

Cell sorting is carried out from PBMC using flow cytometry after cell count, cell fragment is removed first, cell is adhered And dead cell, obtain the cell of CD3-/CD14-/CD16-/IgM- by fluorescent antibody staining, selection expression CD235a-/ The B cell of IgD-/CD20+, irises out CD27ALL memory B cells, and the double fluorescence marks of E2 are obtained with the antigen of specific marker fluorescein The target cell of note.

2. single-cell RT-PCR separation antibody variable region gene

1) reverse transcription (RT) single celled RNA：To add in the mixed liquid of PCR predictions of 96 orifice plates containing single bone-marrow-derived lymphocyte Enter the constant region primers of 0.5 μM of each subtype heavy chain and light chain and Superscript IV reverse transcriptases (Invitrogen, Carlsbad, CA), while setting positive and negative control；Reverse transcription PCR condition：55 DEG C of 60min, are cooled to 4 DEG C.Product CDNA-20 DEG C long-term preserves.

2) amplification of heavy chain of antibody variable region and chain variable region gene：Using nested PCR method, first with reverse transcription product (cDNA) it is template, antibody variable gene is expanded with the PCRa primer mixtures of antibody variable region.In 50 μ LPCR reaction systems The cDNA by RT-PCR reverse transcriptions containing 5 μ L, HotStarTaq Plus DNA Polymerase and 0.5 μM of each Asia The specific primer of type heavy chain and light chain antibody；Reaction condition：95 DEG C of 5min of predegeneration, then carry out 35 PCR cycles, each Circulate and be：94 DEG C × 30s, 55/56/50 (H/K/L) DEG C × 1mim, 72 DEG C × 1.5min, finally with 72 DEG C of extension 7min, it is down to 10 DEG C.Second step with above-mentioned PCR product as template, produce in 50 μ LPCR reaction systems by the PCRa reactions containing 3 μ L The specific primer of thing, HotStarTaq Plus DNA Polymerase and 0.5 μM of each subtype heavy chain and light chain antibody. PCR reaction conditions：95 DEG C of 5min of predegeneration, then carry out 35 PCR cycles, and each circulation is：94 DEG C × 30s, 58/60/64 DEG C (H/K/L) × 30s, 10 DEG C is down to after extending 7min with 72 DEG C by 72 DEG C × 1.5min.PCR primer is coagulated with 2.0% agarose Gel electrophoresis are identified.

3. the expression vector of recombinant antibodies is built

With specific primer obtain ELISA test positive heavy chain of antibody and light chain gene segment (including variable region and Constant region), be linked on pcDNA3.3 carriers for heavy chain and light chain gene respectively by the method cloned using TA, and connection product is turned In changing DH5 α competence bacteriums, 37 DEG C of overnight incubations on the flat board containing ampicillin, immediately 12 single bacterium colonies of picking use Specific primer enters performing PCR identification.Take 2 μ LPCR products carries out electrophoresis detection on 1% Ago-Gel, chooses PCR and is accredited as Positive bacterium colony carries out gene sequencing, and comparison result is correctly the recombinant expression plasmid of heavy chain of antibody and light chain.

4. antibody expression

The plasmid of a large amount of amplification expression positive antibody heavy chains and light chain gene, endotoxin-free extracting in bacillus coli DH 5 alpha Kit is extracted.Transfection reagent Polyetherimide corotation transfected cho cells are used in 10cm culture dishes, 4-6 hours after transfection Addition fresh serum-free media, is placed in 37 DEG C, 5%CO₂Cultivated 96 hours in constant incubator, collect cell conditioned medium and examined Survey.

5. the selective mechanisms of antibody are expressed

ELISA is screened：With different HCV envelope glycoproteins as antigen, and with coating buffer it is by antigen diluent concentration 100ng/ml, is coated in the orifice plates of ELISA 96, and per the μ l of hole 100,4 DEG C overnight.37 DEG C of 2 hours of closing of confining liquid.Add after closing Primary antibody, antibody initial concentration is 25 μ g/ml, and 3 times of gradient dilutions are 100 μ l per pore volume, and 37 DEG C are incubated 1 hour, while with Used as positive control, Rabies virus antibody is negative control to HCV positive patients serum.The goat anti-human igg (1 marked with HRP:2000 is dilute Release) as 37 DEG C of 1 hours of incubation of secondary antibody.Substrate nitrite ion (TMB) 100 μ L/ holes are added, after 37 DEG C of avoid light place 5min, is used 2M sulfuric acid stopped reactions, colorimetric is carried out with 450nm wavelength.

In antibody and test：By the plasmid that is packaged into of different HCV envelope glycoproteins, fluorescein Luciferase bases are added Because transfection CHO cell is used to pack HCV pseudovirus (HCVpp), supernatant is collected for infecting.Paving Huh7 cells in 96 orifice plates, Per hole cell about 1*10⁴It is individual, it is 100 μ l per pore volume, incubated overnight, cell about 30% is paved with during infection.HCVpp is mixed with antibody Close, room temperature places 30min.Add HCVpp with antibody mixed liquor in 96 orifice plates, infect Huh7 cells.Control group is only added HCVpp, liquid is changed after infection 24h, continues to cultivate 1-2 days.Luciferase activity is determined after infection after 2-3 days, measuring method is Remove cell conditioned medium, per hole with 30 μ l lysates, fully take 20 μ l cell pyrolysis liquids plus 30 μ l substrates after cracking, read fluorescence Value.Compare antibody and control group, calculate and neutralize efficiency.

6. antibody great expression and purifying

The expression vector of heavy chain of antibody that the numbering for having neutralization activity that goes out of experimental identification is TRN1001 and light chain will be neutralized (wherein, the amino acid sequence of weight chain variable district such as SEQ ID NO:Shown in 1；The amino acid sequence of antibody light chain variable region such as SEQ ID NO:Shown in 5) corotation transfected cho cell (cell fusion degree reaches more than 90%), 37 DEG C, 8%CO₂, trained in 125rpm shaking tables Support 120 hours.Then transfection supernatant is collected, is purified using albumen (Protein) A affinity chromatographies.By SDS-PAGE Expression and purifying situation with western-blot inspection antibody TRN1001, as a result confirm to obtain compared with pure protein, and can clearly see Observe the antibody light and heavy chain (Fig. 1) after unwinding.

The TRN1001 antibody binding activities of embodiment 2 are detected

1st, ELISA determines binding activity

Step：With different HCV envelope glycoproteins as antigen, and by antigen diluent concentration it is 100ng/ml with coating buffer, The orifice plates of ELISA 96 are coated in, per the μ l of hole 100,4 DEG C overnight.37 DEG C of 2 hours of closing of confining liquid.Add primary antibody after closing, TRN1001 initial concentrations are 25 μ g/ml, and 3 times of gradient dilutions are 100 μ l per pore volume, and 37 DEG C are incubated 1 hour, while with Used as positive control, Rabies virus antibody is negative control to HCV positive patients serum.The goat anti-human igg (1 marked with HRP:2000 is dilute Release) as 37 DEG C of 1 hours of incubation of secondary antibody.Substrate nitrite ion (TMB) 100 μ L/ holes are added, after 37 DEG C of avoid light place 5min, is used 2M sulfuric acid stopped reactions, colorimetric is carried out with 450nm wavelength.

Result is as shown in Fig. 2 carry out the antibody TRN1001 of expression and purification more than TRN1001 antibody after 2000 times of dilutions Still can with antigen binding, with extremely strong binding activity (HCV1a, HCV1b, HCV2, HCV3, HCV4, HCV5 in Fig. 2, HCV6, HCV7 are different HCV envelope glycoproteins).

2nd, the specific binding detection of TRN1001 antibody and HCV envelope glycoproteins

By the plasmid that is packaged into of different HCV envelope glycoproteins, fluorescein Luciferase gene transfection CHO cells are added For packing HCV pseudovirus (HCVpp), supernatant is collected for infecting.Huh7 cells are spread in 96 orifice plates, per hole cell about 1* 10⁴It is individual, it is 100 μ l per pore volume, incubated overnight, cell about 30% is paved with during infection.The antibody of HCVpp and various concentrations TRN00C018 mixes, and the μ g/ml of initial concentration 75,3 times of dilutions, room temperature places 30min.HCVpp and antibody TRN1001 is added to mix Liquid is closed in 96 orifice plates, Huh7 cells are infected.Control group only adds HCVpp, and liquid is changed after infection 24h, continues to cultivate 1-2 days.Infection Luciferase activity is determined after 2-3 days afterwards, measuring method is to remove cell conditioned medium, per hole with 30 μ l lysates, fully cracking After take 20 μ l cell pyrolysis liquids plus 30 μ l substrates, read fluorescent value.

As a result：TRN1001 energy 100% and the specific binding of HCV envelope glycoproteins, fluorescent value reduction.

The affine Activity determination of TRN1001 antibody of embodiment 3

CM5 chips are coupled capture molecule：It is fixed on goat anti-human igg antibody as capture molecule on CM5 sensor chips, Operation according to coupling reagent kit is coupled on CM5 chip gold thin films surface.The glucan table of chip is activated with EDC, NHS Face, coupling amount is determined with sample injection time, the activated group for finally being remained with monoethanolamine confining surface.Capture molecule on CM5 chips Capture ligands：The full people source HCV-Ab IgG neutralizing antibody that will be prepared determines the sample introduction of monoclonal antibody with the signal value being calculated as part Concentration and time of contact.Affinity and dynamic analysis that monoclonal antibody TRN1001 is combined with HCV-E2 albumen (antigen)：HCV-E2 eggs White strain is diluted as analyte with HBS-EP buffer solutions, and analyte flows successively through chip, respectively obtains with the concentration for gradually increasing Signal curve.Each concentration is completed after 1 circulation with the magnesium chloride regeneration chip of 3mol/L being returned to original as 1 circulation Beginning and end combine the state of antigen.It is analyzed with BiaCore X-100System softwares.

As shown in Figure 3 and Table 1, the affinity of TRN1001 antibody reaches 2.03*10 to result^-8mol。

The TRN1001 of table 1 and the protein bound affinity of HCV-E2 and dynamic analysis result

Ka(1/Ms)	Kd(1/s)	KD(M)	Rmax(RU)
				5.44E+03	1.10E-04	2.03E-08	195.2

The TRN1001 antibody of embodiment 4 is detected from the neutralization activity of different HCV virus strains

Paving Huh7 cells in 96 orifice plates, incubated overnight, cell about 30% is paved with during infection.HCV virus strain (h77, JFH1, S52, ED43, SA13) mix with the antibody TRN1001 of various concentrations, the μ g/ml of initial concentration 25,3 times of dilutions, room temperature is put Put 30min.Add HCV virus strain (h77, JFH1, S52, ED43, SA13) with antibody TRN1001 mixed liquors in 96 orifice plates, infection Huh7 cells.Control group only adds HCV virus strain 2G9, and liquid is changed after infection 24h, continues to cultivate 1-2 days.Surveyed after 2-3 days after infection Fixed activity, reads fluorescent value.Compare antibody and control group, calculate and neutralize efficiency.

Result is as shown in figure 4, TRN1001 antibody is shown in Table 2 from the neutralization efficiency of different HCV virus strains.

The neutralization efficiency of the TRN1001 antibody of table 2 and different HCV virus strains

Although above only describes specific embodiment example of the invention, those skilled in the art should manage Solution, these are merely illustrative of, and protection scope of the present invention is defined by the appended claims.Those skilled in the art On the premise of without departing substantially from principle of the invention and essence, various changes or modifications, but this can be made to these implementation methods A little changes or modification each fall within protection scope of the present invention.

SEQUENCE LISTING

<110>Guangzhou Tylenol enlightening bio tech ltd；Ji'nan University；Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd

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1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Asp

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln Tyr Thr Gly Lys Ala Pro Lys Val

35 40 45

Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Thr Ser Asp

85 90 95

Asn Ser Tyr Val Phe Gly Ser Gly Thr Lys Val Ile Val Leu

100 105 110

<210> 6

<211> 9

<212> PRT

<213>People source

<400> 6

Ser Ser Asp Val Gly Gly Asp Asn Tyr

1 5

<210> 7

<211> 3

<212> PRT

<213>People source

<400> 7

Asp Val Ser

1

<210> 8

<211> 10

<212> PRT

<213>People source

<400> 8

Cys Ser Tyr Thr Ser Asp Asn Ser Tyr Val

1 5 10

Claims

1. a kind of the monoclonal neutralizing antibody or its antigen-binding fragment of full people source HCV-Ab IgG, it is characterised in that the antibody or its Antigen-binding fragment includes the complementation of complementarity determining region CDR1, CDR2, CDR3 and/or light chain variable district of weight chain variable district Property determining area CDR1, CDR2, CDR3；The amino acid sequence of heavy chain CDR1, CDR2, CDR3 is respectively such as SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:Shown in 4；The amino acid sequence of light chain CDR1, CDR2, CDR3 is respectively such as SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:Shown in 8.

2. antibody according to claim 1 or its antigen-binding fragment, it is characterised in that the weight chain variable district of the antibody With SEQ ID NO:Amino acid sequence shown in 1；The light chain variable district of the antibody has SEQ IDNO:Amino shown in 5 Acid sequence.

3. antibody according to claim 1 or its antigen-binding fragment, it is characterised in that the antigen binding fragment of the antibody Section includes Fab, Fab ', F (ab ') 2, Fv or single-chain antibody.

4. a kind of nucleic acid molecules of separation, it is characterised in that any one of the nucleic acid molecule encoding claim 1-3 Antibody or its antigen-binding fragment.

5. the DNA fragmentation of the nucleic acid molecules described in claim 4.

6. a kind of expression vector of the nucleic acid molecules including described in claim 4.

7. the host cell of the expression vector described in a kind of nucleic acid molecules or claim 6 including described in claim 4.

8. antibody or the medicine of its antigen-binding fragment any one of a kind of claim 1-3 including therapeutically effective amount Composition.

9. the HCV of a kind of antibody including any one of claim 1-3 or its antigen-binding fragment detects product.

10. a kind of method for preparing the antibody any one of claim 1-3, it is characterised in that methods described is included such as Lower step：

(1) detection finds HCV infection patient；

(2) mononuclearcell in HCV infection Venous Blood is separated, cell sorting is carried out using flow cytometry afterwards, point Select the B cell containing CD235a-/IgD-/CD20+；

(3) using the antibody light chain and the core of weight chain variable district in the single B cell of single-cell RT-PCR amplification step (2) acquisition Acid fragments；

(4) antibody light chain and the nucleotide fragments of weight chain variable district for obtaining step (3) are fused to constant containing human antibodies Recombinant expression carrier is formed in the expression vector in area, host cell expression is imported afterwards；

(5) antibody screening Platform Screening obtains the Dan Ke of the of the invention full people source HCV-Ab IgG with binding activity and neutralization activity Grand neutralizing antibody.

The method of the detection HCV levels of 11. a kind of non-diagnostic purposes, it is characterised in that methods described comprises the following steps：

(1) sample containing HCV is extracted；

(2) sample that step (1) is obtained is connect with the antibody any one of claim 1-3 or its antigen-binding fragment Touch；

Antibody or its antigen-binding fragment answering in HCV detection products are prepared any one of 12. claim 1-3 With.

Antibody or its antigen-binding fragment answering in the medicine for suppressing HCV is prepared any one of 13. claim 1-3 With.

Antibody or its antigen-binding fragment any one of 14. claim 1-3 are preparing prevention or are treating by HCV infection Application in the pharmaceutical preparation of the disease for causing.

15. applications according to claim 14, it is characterised in that the disease includes hepatopathy, such as hepatitis, cirrhosis or liver Cancer, also including Extrahepatic diseases.

16. applications according to claim 15, it is characterised in that the hepatitis is acute or chronic hepatitis C.