CN106749644B - A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG - Google Patents
A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG Download PDFInfo
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- CN106749644B CN106749644B CN201611001112.4A CN201611001112A CN106749644B CN 106749644 B CN106749644 B CN 106749644B CN 201611001112 A CN201611001112 A CN 201611001112A CN 106749644 B CN106749644 B CN 106749644B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
-
- 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
- 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
- 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
- G01N33/577—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
Abstract
The invention discloses a kind of full human monoclonal antibodies and application thereof neutralizing Hepatitis C Virus.The invention particularly discloses a kind of antibody that can be identified and combine hepatitis C virus envelope protein E 2 albumen, its encoding gene, expression vector, purposes.The monoclonal antibody of the present invention can prevent Hepatitis C Virus 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, but also side effect is low.
Description
Technical field
The invention belongs to cellular immunology, molecular biology fields, are related to a kind of monoclonal antibody in full people source, especially relate 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 of the neutralizing antibody and use
On the way.
Background technology
Hepatitis C Virus ((Hepatitis C virus, HCV) be it is known in addition to retrovirus it is only can cause it is slow
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 has infectivity before the onset of usual patient, and can be with 12 years malicious or more.It is estimated that complete at present
Ball is more than 200,000,000 HCV infection persons and is increased every year with the speed of 3,500,000 people, and wherein chronic carriers account for 60%~80%；And
There is HCV pantropic can lead to repeated infection, chronic to make 80% acute infection switch to, and finally develop into hepatic sclerosis/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 generate wide spectrum neutralizing antibody, but HCV has height and becomes
Different, the immunosurveillance for the body that can escape simultaneously inhibits humoral and cellular immune response through a variety of ways.The study found that making for receptor
The antibody that HCV epitopes generate has preferable neutralization activity.In recent years, in relation to HCV infection model such as cell strain and petty action
The foundation of object model so that the further investigation of HCV is rapidly developed, antibody-mediated virus neutralization is expected to become new
Effective HCV-Ab IgG therapy.Huh-7 cells are infected using the HCV pseudovirus of HCVpp different genotypes, can be used for neutralizing
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
It reaches.Hideki etc. constructs the HCVrv that VSV memebrane proteins are replaced with HCV E1 and E2 albumen in human cell line CHO and Huh-7,
Huh-7 can be infected and neutralized by antibody special CD81-E2.Daniel etc. ground for the monoclonal antibody of E2 epitopes
Study carefully, finds the monoclonal antibody mAbs1 for obtaining 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 nucleic acid sequence, HCV points are 7 genotype and dozens of gene hypotype at present.HCV genomes are about
9.6kb, containing single open reading frame.Virus is by envelope protein E1, E2, core protein Core, P7 albumen and non-structural protein
NS3, NS4a, NS4b, NS5a, NS5b are formed.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 are larger differences between different genotype and quasispecies for its sequence.Some domestic scholars, which study, finds choosing
Take the recombinant antigen protein of the representative combined sequence expression comprising different genotype and quasispecies that there is high cross-reactivity.It is external
It is also very active to the research for neutralizing antibody epitope：E1/E2 is immune can to generate higher neutralizing antibody, although neutralizing antibody cannot be protected
Different strain HCV attacks are protected, but the chronicity after different strain virus attack can be prevented；Recombination E1/E2 membrane glycoproteins had in preferable intersect
And activity.The above-mentioned research in relation to neutralizing antibody, to provide thinking further directed to the research of the therapeutic vaccine of HCV.
Studies have shown that 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 function of diagnosing and treating.Therefore, the research based on HCV-Ab IgG neutralizing antibody is found a kind of effective
Method can generate antibody that is strong and widely neutralizing HCV, have important in the immune response of viral infection period excitating organism
Meaning.
Invention content
The object of the present invention is to provide a kind of neutralizations of the monoclonal of full people source HCV-Ab IgG of combination HCV Surface env proteins E2
Antibody, the antibody can be used for preventing hepatitis and can be also used for identification HCV.
To achieve the goals above, present invention employs following technical solutions：
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
Segment includes complementarity determining region CDR1, CDR2, CDR3 of heavy chain variable region and the complementarity-determining region of light chain variable region
Domain CDR1, CDR2, CDR3；The amino acid sequence of heavy chain CDR1, CDR2, CDR3 are 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 are 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 heavy chain variable region of the monoclonal antibody has SEQ IDNO:Amino acid sequence shown in 1 or and its
The light chain variable region of amino acid sequence at least 80% homology, the monoclonal antibody has SEQ ID NO:Shown in 5
Amino acid sequence or with its have at least 80% homology amino acid sequence.
Preferably, the heavy chain variable region of the monoclonal antibody has SEQ IDNO:Amino acid sequence shown in 1；It is described
The light chain variable region of monoclonal antibody has SEQ ID NO:Amino acid sequence shown in 5.
The antibody of conservative sequence variant including preferred antibody amino acid sequence is also included within the scope of the present invention.It protects
It includes not significantly changing the monoclonal neutralizing antibody binding property of the full people source HCV-Ab IgG of the present invention in keep amino acid sequence variation
With the modification of the amino acid sequence of property, the variant such as substituted derived from Similar amino acids well known in the art, the missing of amino acid,
Variant caused by increasing.
The antibody of the present invention further includes people source and non-human source antibodies, and with TRN1001 antibody identical function or change
All antibody made and optimized.
Further, the antigen-binding fragment of the antibody includes Fab, Fab ', F (ab ') 2, Fv or scFv.
The present invention also provides 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 encode foregoing antibody or the nucleic acid molecules of its antigen-binding fragment of the present invention includes having above-mentioned nucleosides
The nucleic acid molecules of the conserved nucleotide sequence variant of acid sequence.So-called conserved nucleotide sequence variant is derived from genetic code degeneration
With the variant of silence, replacement, missing and the increase of nucleotide are also included.
The present invention also provides a kind of DNA fragmentations of foregoing nucleic acid molecules.The DNA fragmentation can be with encoding antibody
Any region of heavy chain or light chain variable region, including heavy chain CDR1, CDR2 or CDR3 or light chain CDR1, CDR2 or CDR3.
The present invention also provides a kind of expression vectors including foregoing nucleic acid molecules, in addition to foregoing nucleic acid
Except molecule, expression vector further 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 albumen is made to obtain expression
Nucleic acid delivery vehicle.Carrier can keep the inhereditary material element that it is carried thin in host by conversion, transduction or transfection host cell
Intracellular is expressed.The type 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.As long as in short, can be replicated in host and steady
Fixed, any plasmid and carrier can be used.In expression vector, other than containing replication orgin, can also contain marker gene and
Other translation control elements.
In specific embodiments of the present invention, the carrier is pCDNA3.3.
The present invention also provides a kind of host containing foregoing nucleic acid molecules or foregoing expression vector is thin
Born of the same parents.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, 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 drosophila S2 or Sf9；CHO, COS, 293 cell or Bowes are black
The zooblast etc. of plain oncocyte.
In specific embodiments of the present invention, the host cell is mammalian cell, more preferable Chinese hamster ovary celI.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion, transfection host cell.
The present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes the antibody of the present invention of therapeutically effective amount
Or its antigen-binding fragment.
Described pharmaceutical composition further includes pharmaceutically acceptable carrier, and the carrier includes but not limited to by the U.S.
That Food and Drug Administration is approved and any adjuvant that can be used for mankind or animal, carrier, excipient, glidant, diluent,
Surfactant, wetting agent, dispersant, suspending agent, stabilizer, isoosmotic pressure agent, solvent or emulsifier etc. are to forming pharmaceutical composition
Object various forms of carriers without side-effects.
The present invention also provides a kind of monoclonal neutralizing antibody of full people source HCV-Ab IgG including the present invention or its antigen bindings
The HCV of segment detects product.
The detection product includes but not limited to detection reagent, kit, chip or test paper.Every includes knot noted earlier
That closes molecule is capable of detecting when that the detection product of HCV is included within the scope of the present invention.
The present invention also provides a kind of methods of the detection HCV levels of non-diagnostic purpose, which is characterized in that the method packet
Include following steps：
(1) sample containing HCV is extracted；
(2) the monoclonal neutralizing antibody or its antigen for the sample and 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.
The present invention also provides a kind of preparation method of the monoclonal neutralizing antibody of foregoing full people source HCV-Ab IgG, institutes
Preparation method is stated using single bone-marrow-derived lymphocyte antibody production techniques.The single bone-marrow-derived lymphocyte antibody production techniques be by
A kind of vitro expression systems for monoclonal antibody that unicellular separation identification technology combines a variety of round pcrs to be formed, i.e., drench from people B
The method that the monoclonal antibody in full people source is directly acquired in bar cell.
Further, described method includes following steps：
(1) detection finds HCV infection patient；
(2) mononuclearcell in HCV infection Venous Blood is detached, carries out cell point using flow cytometry later
Choosing, sub-elects the B cell containing CD235a-/IgD-/CD20+；
(3) antibody light chain and heavy chain variable region in the single B cell of single-cell RT-PCR amplification step (2) acquisition are utilized
Nucleotide fragments；
(4) nucleotide fragments of antibody light chain and heavy chain variable region that step (3) obtains are fused to containing human antibodies
Recombinant expression carrier is formed in the expression vector of constant region, imports host cell expression later；
(5) antibody screening Platform Screening obtains the full people source HCV-Ab IgG with the present invention for combining activity and neutralization activity
Monoclonal neutralizing antibody.
The present invention also provides the monoclonal neutralizing antibodies of foregoing full people source HCV-Ab IgG or its antigen-binding fragment to exist
Prepare the application in HCV detection products.
The detection product includes but not limited to detection reagent, kit, chip or test paper.Every includes knot noted earlier
That closes molecule is capable of detecting when that the detection product of HCV is included within the scope of the present invention.
Antibody according to the present invention can simply and precisely identify 7 kinds of HCV genotype.
The present invention also provides the monoclonal neutralizing antibodies of foregoing full people source HCV-Ab IgG or its antigen-binding fragment to exist
Prepare the application in foregoing pharmaceutical composition.
The present invention also provides the monoclonal neutralizing antibodies of foregoing full people source HCV-Ab IgG or its antigen-binding fragment to exist
Prepare the application inhibited in HCV drugs.
The present invention also provides the monoclonal neutralizing antibodies of foregoing full people source HCV-Ab IgG or its antigen-binding fragment to exist
Prepare the application in the drug for preventing or treating the disease caused by HCV infection.
Further, the disease caused by HCV infection includes hepatopathy, such as hepatitis, hepatic sclerosis or liver cancer and Extrahepatic diseases.
The hepatitis of the present invention refers to acute or chronic hepatitis C.
The antibody TRN1001 of the present invention can be used for treating 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 individual
The dosage of light HCV infection symptom, or effectively reduce the dosage of virion in cycle.The antibody can be used for for example
The neonatal passive immunity of HCV positive carrier childbirth, can be also used for the passive immunity of liver transfer operation to prevent these patients
The HCV infection repeatedly being likely to occur.Particularly, for the estimated not therapeutic effect from interferon therapy, infection gene
The hepatitis C patients of type can mitigate adverse reaction, and can provide the chance for selecting new therapy.
The antibody of disclosure of the invention can include one or more glycosylation sites in heavy chain and light chain variable region, such as originally
Known in field, one or more glycosylation sites present in variable region can lead to the antibody immunogenicity of enhancing,
Or change the pharmacokinetics of antibody due to changing antigen binding.
The antibody of the present invention can be designed as comprising modification in the regions Fc, typically change antibody one or more
Functional characteristic, such as serum half-life, the cytotoxicity that complement combines, Fc receptors combine, and/or antigen relies on.In addition, of the invention
Antibody can be modified by sulphation and (e.g., one or more chemical groups can be connected to antibody), or be modified to change it
Glycosylation, to change the one or more functions characteristic of antibody again.
The present invention full people source HCV-Ab IgG monoclonal neutralizing antibody or its antigen-binding fragment 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 according to the present invention can mark chemically or by genetic engineering, to carry
For detectable antibody.Detectable antibody can be used for for example evaluate object whether oneself through infect hepatitis C virus poison strain or
Generation or progress of the person as the part monitoring infection with hepatitis C virus of clinical trial program are for example to determine designated treatment
The 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 label is dependent on particular detection/analysis/diagnostic techniques for using
And/or method such as immunohistochemical staining (tissue) sample, flow cytometry, laser scanning Cytometry inspection
Survey, fluorescence immunoassay, enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), bioassay (such as phagocytosis make
With measure), Western blotting application etc..Detection/analysis/diagnostic techniques known in the art and/or method are suitably marked
It is denoted as well known to those skilled in the art.
The term as used herein " monoclonal antibody " refers to the antibody obtained from a kind of substantially uniform group, except a small number of possible
Outside the existing mutation naturally occurred, the single antibody for including in the group is identical.Modifier " monoclonal " only indicates anti-
The characteristic of body is obtained from substantially uniform antibody population, this cannot be construed to need to be produced with any specific process anti-
Body.
The term as used herein is " variable " to indicate that certain parts of variable region in antibody are different in sequence, it is formed
Combinations and specificity of the various specific antibodies to its specific antigen.Changeability, which concentrates in light chain and heavy chain variable region, to be known as mutually
It mends in three segments determined in area (CDR) or hypervariable region.Four areas FR are respectively contained 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 of formation connection ring, can shape
At part β-pleated sheet structure.CDR in every chain is by the areas FR firmly against together forming 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 dependent on antibody of antibody.
The advantages of the present invention：
Full people source HCV-Ab IgG neutralize antibody titers height prepared by the present invention, has high cross-reactive, ingredient at high specificity
Clearly, it can be easy with high efficient expression, standardized production, quality control, it is of low cost.
Full people source HCV-Ab IgG neutralizing antibody prepared by the present invention can both cause to avoid HCV polygene types and high variability
Antibody failure, and can to avoid the existing drug in market without high cross-reactivity the problem of.
Description of the drawings
The SDS-PAGE detection figures that Fig. 1 is the antibody TRN1001 of the embodiment of the present invention；
Fig. 2 is in the antibody TRN1001 of the embodiment of the present invention and the envelope glycoprotein of 7 kinds of different subtype HCV euvirus
With determination of activity figure；
Fig. 3 is the antibody TRN1001 and the affine determination of activity figure of HCV virus antigen of the embodiment of the present invention；
Fig. 4 is the neutralization activity measurement chart of the antibody TRN1001 HCV virus strains different from 5 kinds of the embodiment of the present invention,
In, A：h77；B：JFH1；C：SA13；D：S52；E：ED43.
Specific implementation mode
It is further illustrated the present invention below by embodiment.It should be understood that the embodiment of the present invention is for illustrating
The present invention is rather than limiting the invention.The simple modifications that essence according to the present invention carries out the present invention belong to the present invention
Claimed range.
The preparation of the monoclonal neutralizing antibody TRN1001 of 1 full people source HCV-Ab IgG of embodiment
1.PBMC is detached 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, is adhered cell
And dead cell, the cell of CD3-/CD14-/CD16-/IgM- is obtained by fluorescent antibody staining, selection expression CD235a-/
The B cell of IgD-/CD20+ irises out CD27ALL memory B cells, and the bis- 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：The PCR predictions of 96 orifice plates containing single bone-marrow-derived lymphocyte are mixed in liquid and are added
Enter 0.5 μM each subtype heavy chain and light chain constant region primers and Superscript IV reverse transcriptases (Invitrogen,
Carlsbad, CA), while positive and negative control is set；Reverse transcription PCR condition：55 DEG C of 60min, are cooled to 4 DEG C.Product
CDNA-20 DEG C of long-term preservation.
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 made of 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：Then 95 DEG C of 5min of pre-degeneration carry out 35 PCR cycles, each
Cycle is：It 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, is down to
10 DEG C.Second step is using above-mentioned PCR reaction products as template, the PCRa reaction productions containing 3 μ L in 50 μ LPCR reaction systems
Object, HotStarTaq Plus DNA Polymerase and 0.5 μM of each subtype heavy chain and the specific primer of light chain antibody.
PCR reaction conditions：95 DEG C of 5min of pre-degeneration, then carry out 35 PCR cycles, and each cycle is：94 DEG C × 30s, 58/60/64
(H/K/L) DEG C 72 DEG C × 1.5min, 10 DEG C are down to after extending 7min with 72 DEG C by × 30s.PCR product is solidifying with 2.0% agarose
Gel electrophoresis is identified.
3. building the expression vector of recombinant antibodies
With specific primer obtain ELISA test positive heavy chain of antibody and light chain gene segment (including variable region and
Constant region), heavy chain and light chain gene are linked on pcDNA3.3 carriers respectively using the TA methods cloned, connection product is turned
Change in DH5 α competent bacterias, 37 DEG C of overnight incubations on the tablet containing ampicillin, 12 single bacterium colonies of picking are used immediately
Specific primer carries out PCR identifications.It takes 2 μ LPCR products to carry out electrophoresis detection on 1% Ago-Gel, chooses PCR and be 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 large amplification expression positive antibody heavy chain and light chain gene, endotoxin-free extracting in bacillus coli DH 5 alpha
Kit extracts.Transfection reagent Polyetherimide corotation transfected cho cells are used in 10cm culture dishes, 4-6 hours after transfection
Fresh serum-free media is added, is placed in 37 DEG C, 5%CO2It is cultivated 96 hours in constant incubator, collects cell conditioned medium and examined
It surveys.
5. expressing the selective mechanisms of antibody
ELISA is screened：Using different HCV envelope glycoproteins as antigen, coating buffer be used in combination that antigen diluent is a concentration of
100ng/ml is coated in 96 orifice plates of ELISA, and per 100 μ l of hole, 4 DEG C overnight.37 DEG C of 2 hours of closing of confining liquid.Add after closing
Primary antibody, antibody initial concentration are 25 μ g/ml, and 3 times of gradient dilutions are 100 μ l per pore volume, 37 DEG C of 1 hours of incubation, use simultaneously
For HCV positive patients serum as positive control, Rabies virus antibody is negative control.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.100 holes μ L/ substrate developing solution (TMB) are added, after 37 DEG C of avoid light place 5min, use
2M sulfuric acid stopped reactions carry out colorimetric 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 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 a, it is 100 μ l per pore volume, is incubated overnight, cell about 30% is paved with when infection.HCVpp is mixed with antibody
It closes, is placed at room temperature for 30min.HCVpp is added with antibody mixed liquor in 96 orifice plates, infects Huh7 cells.Control group is only added
Liquid is changed in HCVpp, infection afterwards for 24 hours, continues culture 1-2 days.Luciferase activity is measured after infection after 2-3 days, measurement method is
Remove cell conditioned medium, per 30 μ l lysates of hole, fully takes 20 μ l cell pyrolysis liquids to add 30 μ l substrates after cracking, read fluorescence
Value.Compare antibody and control group, calculates and neutralize efficiency.
6. antibody great expression and purifying
The expression vector of having of going out of experimental identification heavy chain of antibody and light chain that the number of neutralization activity is TRN1001 will be neutralized
(wherein, the amino acid sequence of heavy chain variable region 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 90% or more), 37 DEG C, 8%CO2, train in 125rpm shaking tables
It supports 120 hours.Then transfection supernatant is collected, is purified using albumen (Protein) A affinity chromatographies.Pass through SDS-PAGE
Expression and the purifying situation that antibody TRN1001 is examined with western-blot, 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.
2 TRN1001 antibody binding activities of embodiment detect
1, ELISA, which is measured, combines activity
Step：Using different HCV envelope glycoproteins as antigen, it is used in combination coating buffer by a concentration of 100ng/ml of antigen diluent,
96 orifice plates of ELISA are coated in, per 100 μ l of hole, 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, 37 DEG C of 1 hours of incubation, use simultaneously
For HCV positive patients serum as positive control, Rabies virus antibody is negative control.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.100 holes μ L/ substrate developing solution (TMB) are added, after 37 DEG C of avoid light place 5min, use
2M sulfuric acid stopped reactions carry out colorimetric with 450nm wavelength.
The results are shown in Figure 2, and the antibody TRN1001 of expression and purification is carried out TRN1001 antibody after being diluted more than 2000 times
Still can with antigen binding, have extremely strong combination activity (HCV1a, HCV1b, HCV2, HCV3, HCV4, HCV5 in Fig. 2,
HCV6, HCV7 are different HCV envelope glycoproteins).
2, TRN1001 antibody and the specific binding of HCV envelope glycoproteins detect
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 a, it is 100 μ l per pore volume, is incubated overnight, cell about 30% is paved with when infection.The antibody of HCVpp and various concentration
TRN00C018 is mixed, and 75 μ g/ml of initial concentration, 3 times of dilutions are placed at room temperature for 30min.It is mixed that HCVpp and antibody TRN1001 is added
Liquid is closed in 96 orifice plates, infects Huh7 cells.HCVpp is only added in control group, and liquid is changed in infection afterwards for 24 hours, continues culture 1-2 days.Infection
Luciferase activity is measured after 2-3 days afterwards, measurement method is to remove cell conditioned medium, per 30 μ l lysates of hole, is fully cracked
After take 20 μ l cell pyrolysis liquids to add 30 μ l substrates, read fluorescent value.
As a result：TRN1001 energy 100% is specifically bound with HCV envelope glycoproteins, and fluorescent value reduces.
The 3 affine Activity determination of TRN1001 antibody of embodiment
CM5 chips are coupled capture molecule：CM5 sensor core on pieces are fixed on using goat anti-human igg antibody as capture molecule,
It is coupled on CM5 chip gold thin films surface according to the operation of coupling reagent kit.The glucan table of chip is activated with EDC, NHS
Face determines coupling amount with sample injection time, finally uses the remaining activated group of ethanol amine confining surface.Capture molecule on CM5 chips
Capture ligands：Using the full people source HCV-Ab IgG neutralizing antibody of preparation as ligand, the sample introduction of monoclonal antibody is determined with the signal value being calculated
Concentration and time of contact.The affinity and dynamic analysis that monoclonal antibody TRN1001 is combined with HCV-E2 albumen (antigen)：HCV-E2 eggs
White strain uses the dilution of HBS-EP buffer solutions as analyte, and analyte flows successively through chip with the concentration gradually increased, respectively obtains
Signal curve.Each concentration is recycled as 1, is completed 1 time after recycling with the magnesium chloride regeneration chip of 3mol/L to be returned to original
Beginning and end combine the state of antigen.It is analyzed with BiaCore X-100System softwares.
As a result as shown in Figure 3 and Table 1, the affinity of TRN1001 antibody reaches 2.03*10-8mol。
1 TRN1001 of table 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 |
4 TRN1001 antibody of embodiment is detected from the neutralization activity of different HCV virus strains
Huh7 cells are spread in 96 orifice plates, are incubated overnight, cell about 30% is paved with when infection.HCV virus strain (h77,
JFH1, S52, ED43, SA13) it is mixed with the antibody TRN1001 of various concentration, 25 μ g/ml of initial concentration, 3 times of dilutions, room temperature is put
Set 30min.HCV virus strain (h77, JFH1, S52, ED43, SA13) is added with antibody TRN1001 mixed liquors in 96 orifice plates, infection
Huh7 cells.HCV virus strain 2G9 is only added in control group, and liquid is changed in infection afterwards for 24 hours, continues culture 1-2 days.It is surveyed after 2-3 days after infection
Fixed activity, reads fluorescent value.Compare antibody and control group, calculates and neutralize efficiency.
The results are shown in Figure 4, and TRN1001 antibody is shown in Table 2 from the neutralization efficiency of different HCV virus strains.
The neutralization efficiency of table 2 TRN1001 antibody and different HCV virus strains
Although above only describes the specific implementation mode example of the present invention, those skilled in the art should manage
Solution, these are merely examples, and protection scope of the present invention is defined by the appended claims.Those skilled in the art
Without departing from the principle and essence of the present invention, many changes and modifications may be made, but this
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 wheat wins the limited public affairs of biotechnology
Department
<120>A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG
<160> 8
<170> PatentIn version 3.5
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Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
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Gly Arg Ile Ile Gly Ile Val Gly Leu Ala Asn Tyr Ala Gln Lys Phe
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Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Gly Tyr
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Met Glu Leu Ser Thr Leu Lys Ser Asp Asp Thr Ala Met Tyr Tyr Cys
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Claims (15)
1. a kind of the monoclonal neutralizing antibody or its antigen-binding fragment of full people source HCV-Ab IgG, which is characterized in that the antibody or its
Antigen-binding fragment includes complementarity determining region CDR1, CDR2, CDR3 and/or the complementation of light chain variable region of heavy chain variable region
Property determining area CDR1, CDR2, CDR3；The amino acid sequence of heavy chain CDR1, CDR2, CDR3 are 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 are 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, which is characterized in that the heavy chain variable region of the antibody
With SEQ ID NO:Amino acid sequence shown in 1；The light chain variable region of the antibody has SEQ IDNO:Amino shown in 5
Acid sequence.
3. antibody according to claim 1 or its antigen-binding fragment, which is characterized 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, which is characterized in that described in any one of described nucleic acid molecule encoding claim 1-3
Antibody or its antigen-binding fragment.
5. a kind of expression vector including the nucleic acid molecules described in claim 4.
6. a kind of host cell including the nucleic acid molecules described in claim 4 or the expression vector described in claim 5.
7. the drug of the antibody or its antigen-binding fragment described in a kind of any one of claim 1-3 including therapeutically effective amount
Composition.
8. the HCV of a kind of antibody including described in any one of claim 1-3 or its antigen-binding fragment detects product.
9. a kind of method preparing the antibody described in any one of claim 1-3, which is characterized in that the method includes as follows
Step：
（1）Detection finds HCV infection patient；
（2）The mononuclearcell in HCV infection Venous Blood is detached, carries out cell sorting using flow cytometry later, point
Select the B cell containing CD235a-/IgD-/CD20+；
（3）Utilize single-cell RT-PCR amplification step（2）The core of antibody light chain and heavy chain variable region in the single B cell obtained
Acid fragments；
（4）By step（3）The antibody light chain of acquisition and the nucleotide fragments of heavy chain variable region are fused to constant containing human antibodies
Recombinant expression carrier is formed in the expression vector in area, imports host cell expression later；
（5）Antibody screening Platform Screening, which obtains, to be had in conjunction with described in any one of claim 1-3 of activity and neutralization activity
Antibody.
10. a kind of method of the detection HCV levels of non-diagnostic purpose, which is characterized in that described method includes following steps：
（1）Extract the sample containing HCV；
（2）By step（1）Antibody or its antigen-binding fragment described in any one of the sample of acquisition and claim 1-3 connect
It touches；
（3）Detect the immune response of sample and antibody or its antigen-binding fragment.
11. antibody or its antigen-binding fragment the answering in preparing HCV detection products described in any one of claim 1-3
With.
12. antibody or its antigen-binding fragment the answering in preparing the drug for inhibiting HCV described in any one of claim 1-3
With.
13. the antibody or its antigen-binding fragment described in any one of claim 1-3 prevent preparing or treat by HCV infection
Application in the pharmaceutical preparation of caused disease.
14. application according to claim 13, which is characterized in that the disease is hepatopathy, Extrahepatic diseases；The hepatopathy is
Hepatitis, hepatic sclerosis or liver cancer.
15. application according to claim 14, which is characterized in that the hepatitis is acute or chronic hepatitis C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611001112.4A CN106749644B (en) | 2016-11-14 | 2016-11-14 | A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG |
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