CN105085672A - 3D protein specific monoclonal immunoglobulin A antibodies and composition thereof - Google Patents
3D protein specific monoclonal immunoglobulin A antibodies and composition thereof Download PDFInfo
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Abstract
The invention provides 3D protein specific monoclonal immunoglobulin A antibodies capable of specifically binding to polypeptides and a composition thereof. The polypeptide has a consensus sequence shown in the formula of SEQ ID NO: 1 or SEQ ID NO: 2. The invention also provides the composition. The composition comprises at least one first 3D protein specific monoclonal immunoglobulin A antibody which specifically binds to a first polypeptide with a consensus sequence shown in the formula of SEQ ID NO: 1, at least one second 3D protein specific monoclonal immunoglobulin A antibody which specifically binds to a second polypeptide with a consensus sequence shown in the formula of SEQ ID NO: 2, and a pharmaceutically acceptable solution.
Description
Technical field
The present invention relates generally to the prevention and therapy reagent to enterovirus, and 3D protein specific monoclonal immunoglobulin A (IgA) antibody related more specifically to enterovirus, and relate to prevention or therapeutic composition further, comprise 3D protein specific monoclonal igA antibody.
Background technology
Enterovirus is divided into four Main Subtype A, B, C and D, and each hypotype comprises much serotype.Enterovirus causes different diseases.Such as, hand, the main pathogens enough and in stomatosis (HFMD) is the enterovirns type 71 (EV71) and the Coxsackie virus (CV) that belong to picornavirus family.What hand foot mouth disease was day by day serious threatens public health, particularly to infant.EV71 and CV infects and causes serious aseptic meningitis, encephalitis, myocarditis, acute paralysis, pulmonary edema, causes high mortality.
As the member that enterovirus in picornavirus family belongs to, EV71 has typical sense single stranded rna genome, containing single open reading frame, and encode 4 capsid proteins (VP1-4) and 7 Nonstructural Proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D).3D(is also referred to as 3D
pol) albumen as virus RNA dependant RNA polymerase (RdRp), in the uridine acidylate (uridylylation) of the synthesis of viral minus strand and some protein, play Main Function.Find from the research of the crystalline structure of EV71, an EV71 virus particle comprises the nucleocapsid formed by 60 of three virus structural protein VP1-VP3 copies, and the internal surface of capsid adheres to the small protein VP4 of 60 copies.
3D has high sequence homology in all enteroviruses, but has low homology with human protein.The 3D of EV71 and the poliovirus of picornavirus family, Coxsackie virus, the homology RdRps of rhinovirus and foot and mouth disease virus polysaccharase has structure/sequence similarity.
3D has the reactive site of N-end.Kiener etc. with the restructuring 3CD albumen from EV71C4 bacterial strain as immunogen; be separated monoclonal antibody 4B12(IgG1); identify that linear epitope DFEQALFS(corresponds to the 53-60 position of 3D and the 1784-1791 of EV71 polyprotein), close to the avtive spot of 3D polysaccharase; All Enterovirus 71 hypotypes (Kieneretal.Characterizationofamonoclonalantibodyagainstt he3Dpolymeraseofenterovirus71anditsuseforthedetectionofh umanenterovirusAinfection. that 4B12 detects at sex change Dot hybridization
jVirolMethods.2012; 180 (1-2): 75-83).4B12 can detect extensive hypotype strain, and strong prompting 3D-monoclonal antibody specific may be useful to diagnose infections, but does not mention or advise that prevention or treatment use 4B12, says nothing of general 3D specific antibody.
Because vaccine is the best strategy controlling transmissible disease, the candidate vaccine of different EV71 is studied, and comprises EV71 inactivated whole virus vaccines, attenuated live virus vaccines, restructuring VP1 vaccine, based on the DNA vaccination of VP1, and synthetic peptide vaccine and virus sample particle vaccines.Formalin inactivation EV71 vaccine causes gratifying immunoprotection and cross reactivity neutralizing antibody mouse and rhesus monkey; In China, EV71 inactivated vaccine completed phase iii clinical trial in 2013.Because in course of infection, outer capsid contains major antigenic sites, synthetic peptide vaccine only containing nucleocapsid protein, comprises VP1 and VP2(Kungetal.Updateonthedevelopmentofenterovirus71vaccin es.
expertOpinBiolTher.2014; 3:1-10).
Because EV71 is a kind of RNA viruses, sizable heredity and antigen diversity may be caused by the RdRp that easily makes mistakes to such as copying of enteric virus71 type Nucleoprotein Gene.The people such as Chen prove, by one group of monoclonal antibody of anti-VP1, genotype does not reflect its antigenicity, EV71 virus can be divided into different antigen group (Chenetal.AntigenicanalysisofdivergentgenotypeshumanEnter ovirus71virusesbyapanelofneutralizingmonoclonalantibodie s:currentgenotypingofEV71doesnotreflecttheirantigenicity.
vaccine.2013; 31 (2): 425-30).All these show, selecting a desirable strain to develop the vaccine with extensive validity is a challenge.
Owing to not having vaccine and special efficacy antiviral at present, in human vein, injecting immune sphaeroprotein (IVIG) is used for the treatment of serious EV71 infection always clinically.But, the discovery of ADE (ADE) during EV71 infects, the compound action that display antibody infects at control EV71.ADE is a kind of phenomenon, and wherein the neutralizing antibody enhanced virus of the sub-level of preexist enters and copies.The neutralizing antibody display of sub-level, strengthens EV71 and infects the person monocytic cell containing Fc acceptor, and increase the weight of EV71 infecting mouse.In addition, the extensive cross reactivity existed between enterovirus antibody, also may become the potential risk of the ADE in EV71 infects.
The proofs such as Han, the neutralizing antibody (immunoglobulin (Ig)) of the anti-EV71 of lower concentration (being 50 μ g/ml) can strengthen enteric virus71 type and infect monocytic series (Hanetal.Antibodydependentenhancementinfectionofenterovir us71
invitroand
invivo.
virolJ.2011; 8:106).Cao etc. further study each IgG subclass in and and strengthen EV71 infect in effect, and the Neutralization effect of the immunoglobulin (Ig) of finder is mainly mediated by IgG1 subclass, IgG2 subclass is lower, IgG3 part does not have Neutralization effect, but improve enteric virus71 type infection in vitro (Caoetal., HumanIgGsubclassesagainstenterovirusType71:neutralizatio nversusantibodydependentenhancementofinfection.
pLoSOne.2013; 8 (5): e64024).Therefore, inducing antibodies is answered in the design of new generation vaccine, has strong neutralization, but more weak ADE is active.
Because structure capsid protein has antigen diversity because of sudden change, be therefore difficult to find out the universal antibody to them; Up to the present, known two general IgG monoclonal antibody, an anti-VP1(Limetal.Characterizationofanisotype-dependentmonoclo nalantibodyagainstlinearneutralizingepitopeeffectiveforp rophylaxisofenterovirus71infection.
pLoSOne.2012; 7 (1): e29751), another anti-VP3(Kieneretal.Anoveluniversalneutralizingmonoclonalanti bodyagainstenterovirus71thattargetsthehighlyconserved " knob " regionofVP3protein.
pLoSNeglTropDis. 2014; 8 (5): e2895).But the ADE ability of these universal antibodies is not studied.
The safety problem of research to deactivation EV71 virus vaccines of the people such as Jia proposes serious concerns (Jiaetal.Thecross-reactivityoftheenterovirus71tohumanbrai ntissueandidentificationofthecross-reactivityrelatedfrag ments.
virolJ.2010; 7:47).The people such as Jia prove to there is specific IgG in the serum of the patient infected at EV71, have the cross-reactivity to human brain; Then 19 purified polypeptides are used to prepare polyclonal serum, P230-323, P646-755, the antiserum(antisera) of P857-1012 and P1329-1440 shows stronger dyeing to the neurone in the brain of grownup and fetus medullary substance, P1-69, P324-443, P444-565, P566-665, P746-876, the antiserum(antisera) of P1441-1526, P1549-1668, P1732-1851 and P2072-2193 shows weak dyeing, and P70-159, the antiserum(antisera) display not dyeing of P140-249, P1197-1338, P1649-1731 and P1843-1951.
Therefore, in the urgent need to exploitation new tool reply enterovirus, as EV71 and CV causes hand foot mouth disease.
Rapid screening of the present invention, the object reduce analysis cost to reach, improving checkability.
Summary of the invention
The invention provides the monoclonal immunoglobulin A(IgA of specific binding polypeptide) antibody. in one embodiment, described polypeptide is represented by the common recognition sequence be selected from SEQIDNO:1 and SEQIDNO:2.
In another embodiment of described 3D protein specific monoclonal igA antibody, the 3D protein specific monoclonal igA antibody be combined with polypeptide shown in SEQIDNO:1 is 3D-2A10-IgA(CCTCCNO:C2014144), the 3D protein specific monoclonal igA antibody be combined with polypeptide shown in SEQIDNO:2 is 3D-3A12-IgA(CCTCCNO:C2014142).
In another embodiment of described 3D protein specific monoclonal igA antibody, polypeptide shown in SEQIDNO:1 is selected from the polypeptide by shown in SEQIDNOS:3-8, and polypeptide shown in SEQIDNO:2 is selected from the polypeptide by shown in SEQIDNOS:10-14.
Invention also provides composition.In one embodiment, described composition comprises at least one 3D protein specific monoclonal igA antibody, be attached to the first polypeptide representated by common recognition sequence SEQIDNO:1 specifically, at least one the 2nd 3D protein specific monoclonal igA antibody, be attached to the second polypeptide representated by common recognition sequence SEQIDNO:2 specifically, and pharmaceutically acceptable solution.
In another embodiment of described composition, the 3D protein specific monoclonal igA antibody be combined with the first polypeptide shown in SEQIDNO:1 is 3D-2A10-IgA(CCTCCNO:C2014144), the 2nd 3D protein specific monoclonal igA antibody be combined with the second polypeptide shown in SEQIDNO:2 is 3D-3A12-IgA(CCTCCNO:C2014142).
In another embodiment of described composition, described first polypeptide is selected from the polypeptide by shown in SEQIDNOS:3-8, and the second polypeptide is selected from the polypeptide by shown in SEQIDNOS:10-14.
By following detailed description of the preferred embodiment by reference to the accompanying drawings, objects and advantages of the present invention are apparent.
Accompanying drawing explanation
The preferred embodiments of the invention are described referring now to accompanying drawing, and wherein similar Reference numeral represents identical element.
The sign of Fig. 1, EV713D specific monoclonal IgG antibody.(A) picture display uses 3D specific IgG monoclonal antibody (3A12,2A10,7A6G1 and 11F1) to the indirect IF staining of the cell that EV71 infects; Flagellin protein specific monoclonal antibody (5G10) is as negative control, and the mice serum of 3D-immunity is as positive control.(B) use 3D specific IgG monoclonal antibody (3A12,2A10,7A6G1 and 11F1) to the immunoblotting of the VERO-1008 cell pyrolysis liquid that EV71 infects; Flagellin monoclonal antibody specific (5G10) is as negative control, and the mice serum of 3D-immunity is as positive control.
Fig. 2,3D specific IgG monoclonal antibody suppresses copying of EV71 in cell.(A) virus titer under intrabody exists via transfection is shown in.(B) virus titer under the existence of 2A10 or EV-5 of various dose is shown in.EV-5 is a kind of EV71VP2 monoclonal antibody specific.
Fig. 3,3D specific IgG monoclonal antibody suppresses external 3D polymerase activity.(A) signal diagram 3D(RdRp) RNA of mediation extends.(B) picture shows the impact that different IgG monoclonal antibody is extended the RNA that 3D mediates.
Fig. 4, be shown in the antiviral functions of EV713D specific IgG monoclonal antibody in mouse model.
Fig. 5, be shown in 2A10-IgG or EV-5-IgG monoclonal antibody existence under the antibody dependent that copies of EV71 strengthen.
The epi-position of Fig. 6, EV713D specificity 3A12 and 2A10, at EV713D(1RA6) three-dimensional model in spatiality.EV713D(1RA6) position of two identification epi-positions of 3A12 and 2A10 is used to refer to.
Fig. 7, qualification EV713D specificity 3D protein specific monoclonal igA antibody.(A) indirect IF staining of the cell of picture display EV71 infection, 3D specificity 3D protein specific monoclonal igA antibody used is 3A12-IgA and 2A10-IgA, 16CF7IgA (anti-MeV) is as negative control, and the serum of 3D immune mouse is as positive control.(B) EV71 infects the immunoblotting (WB) of VERO-1008 cell, 3D protein specific monoclonal igA antibody used be 3A12-IgA and 2A10-IgA, 16CF7IgA as negative control, the serum of 3D protein immunization mouse is as positive control.
EV71 is suppressed to copy in Fig. 8,3D protein specific monoclonal igA antibody born of the same parents.(A) Transwell basis pontis is shown in when there is IgA antibody, the virus titer in the polarization VERO-pIgR cell that EV71 infects.(B) basis pontis is shown in when there is 3A12-IgA or 16CF7IgA of various dose, the virus titer in the polarization VERO-pIgR cell that EV71 infects.(C) basis pontis is shown in when there is different I gG antibody, the virus titer in the polarization VERO-pIgR cell that EV71 infects.(D) basis pontis is shown in when there is different I gA antibody, the virus titer in the polarization VERO-1008 cell that EV71 infects.
Fig. 9, EV713D-IgA suppress the accumulation of viral protein.(A) immunoblotting (WB) detects the expression of 3D and VP2 in the polarization VERO-pIgR cell infected at EV71, has the existence of IgA antibody in the training period at base portion.(B) illustrate ELISA result, detect the expression as 3D and VP2 in the cell described by (A).
The impact that Figure 10, diagram different I gA monoclonal antibody extend the RNA that 3D mediates.
Figure 11, be shown in the antiviral effect of EV71 3D protein specific monoclonal igA antibody in mouse model.
Figure 12, illustrate the immunoblotting attenuated vaccinia of VTT-3D, the recombinant expressed 3D (virus) of the 3D expressed by VTT-3D expression vector.
Figure 13, diagram immunity and attack malicious program.
3D specific antibody response after exempting from the beginning of Figure 14, diagram: (A) serum IgG; (B) serum IgA; (C) saliva IgA; (D) vagina IgA.
3D specific antibody response after Figure 15, diagram first time booster immunization: (A) serum IgG; (B) serum IgA; (C) saliva IgA; (D) vagina IgA.
3D specific antibody response after Figure 16, diagram second time booster immunization: (A) serum IgG; (B) serum IgA; (C) saliva IgA; (D) vagina IgA.
Figure 17, illustrate and replied by the 3D specific IgG antibodies in the intestines of the newborn mice of being born by immune mother.
Figure 18, illustrate the percentage survival of attacking the newborn mice after poison through mouse adapted strain EV71.
Antibody response after the 3D protein immunization BALB/c mouse of Figure 19, diagram purifying.
Embodiment
The detailed description of some embodiment of the present invention that can be following by reference and more easily understand the present invention.
In this application, in order to describe the field that the invention relates to state more fully, when publication is cited, the whole of disclosure of these publications are incorporated to the application through quoting.
Except as otherwise noted, will adopt molecular biology (comprising recombinant technology), microbiology, cytobiology, biological chemistry in practice of the present invention, nucleic acid chemistry and immunologic prior art, these are within the technical ability of this area.
These technology are existing in the literature to be explained, completely as molecular cloning: laboratory specification sheets, the third edition (Sambrook and Russel, calendar year 2001); Animal cell culture (RIFreshmey edits, version in 1987); Molecular biology program in present age chief editors such as (, 1987, comprise and be supplemented to calendar year 2001) FMAusubel; Immunology program in present age chief editors such as (, 1991) JEColigan; Immunoassay handbook " (D.Wild edits, Stockton Press, New York, 1994); Immunological analysis method (chief editor such as R.Masseyeff, WHAlbert and NAStaines, Weinheim:VCHVERLAGSGESELLSCHAFTMBH, 1993 years).
The present invention finds, 3D protein specific monoclonal igA antibody effectively can suppress enterovirns type 71 (EV71) in a Transwell model, the more important thing is that host can be protected to avoid virus attacks poison.
The 3D albumen (SEQIDNO:15) of EV71BrCr strain is expressed and purifying, and the 3D albumen of purifying is used for the mouse of immunity, in accordance with standard program for generation of monoclonal IgG antibody.Two 3D protein specific monoclonal IgG antibody (hybridoma cell strain 3D-2A10-IgG and hybridoma cell strain 3D-3A12-IgG) have been prepared by the hybridoma technology of routine.Corresponding hybridoma cell line has been deposited in China typical culture collection center (CCTCC), and CCTCCNO:C2014143 is hybridoma cell strain 3D-2A10-IgG and CCTCCNO:C2014141 is hybridoma cell strain 3D-3A12IgG antibody.Depositary institution: China typical culture collection center, preservation address Hubei Wuhan University, preservation date: on July 22nd, 2014.Monoclonal IgG antibody can be the antibody of the Fc structural domain comprised, single-chain antibody, or Fab fragment.The definition of various antibody and production are well-known.
The hybridoma cell line of 3D-2A10-IgG and 3D-3A12-IgG is changed through hypotype, obtains two corresponding 3D protein specific monoclonal igA antibodies, hybridoma cell strain 3D-2A10-IgA and hybridoma cell strain 3D-3A12-IgA.Corresponding hybridoma cell line has been deposited in China typical culture collection center (CCTCC), and CCTCCNO:C2014144 is hybridoma cell strain 3D-2A10-IgA and CCTCCNO:C2014142 is hybridoma cell strain 3D-3A12-IgA antibody.Depositary institution: China typical culture collection center, preservation address Hubei Wuhan University, preservation date: on July 22nd, 2014.
Use the restructuring 3D albumen from arbitrary end consecutive miss, identify the polypeptide of antibodies.Can predict from the binding specificity of corresponding IgG antibody, 3D-3A12IgA antibodies is to KEPAVLTS(SEQIDNO:3), 3D-2A10-IgA in conjunction with YSTYVKDELRSLDKI(SEQIDNO:9).Utilize sequence alignment, the polypeptide identified from enterovirus hypotype A, is high conservative all in the enterovirus strain of B, C and D.As shown in table 1 below, 3D-3A12IgA antibodies to by one common recognition sequence KEPAVLX
7x
8represented polypeptide (SEQIDNO:1), wherein X
7be selected from T, H, R and N, X
8be selected from S, N and K.3D-2A10-IgA antibodies to by one common recognition sequence X
1x
2tX
4vKDELRSX
12x
13kX
15the polypeptide (SEQIDNO:2) be expressed, wherein X
1be selected from Y, M, L and F, X
2be selected from S and V, X
4be selected from Y and F, X
12be selected from L, A, K and R, X
13the D be selected from, E, T and S, X
15be selected from I and V.
Table 1, by the common recognition sequence of 3D-2A10-IgA or 3D-3A12IgA antibody recognition and exemplary sequence
Enterovirus comprises hypotype A, B, C and D.
Enterovirus A hypotype comprises 23 serotypes; Exemplary 3D sequence comprises: (1) human enterovirus 71 (EV71), BrCr strain (GenBankAB204852.1) (SEQIDNO:15), and wherein SEQIDNO:15 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:3 and 9; (2) human coxsackievirus A16 type strain shzh00-1(GenBankAY790926.1) (SEQIDNO:16), wherein SEQIDNO:16 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:3 and 9.
Enterovirus B hypotype comprises 60 kinds of serotypes; Exemplary 3D sequence comprises: (1) human coxsackievirus B3Beijing0811 strain (GenBankGQ141875.1) (SEQIDNO:17), and wherein SEQIDNO:17 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:5 and 10; (2) human Coxsackie A9GRIGGS strain (GenBankD00627.1) (SEQIDNO:18), wherein SEQIDNO:18 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:5 and 10.
Enterovirus C hypotype comprises 23 serotypes; Exemplary 3D sequence comprises: (1) human poliovirus 1 strain CHN-Jiangxi//89-1(GenBank:AF111984.2) (SEQIDNO:19), wherein SEQIDNO:19 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:6 and 11; (2) human coxsackievirus A1KS-ZPH01F/XJ/CHN/2011 strain isolated (GenBank:JX174177.1) (SEQIDNO:20), wherein SEQIDNO:20 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:6 and 12.
Enterovirus D hypotype comprises 5 kinds of serotypes; Exemplary 3D sequence comprises: (1) human enteric virus 68Fermon strain (GenBank:AY426531.1) (SEQIDNO:21), and wherein SEQIDNO:21 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:7 and 13; (2) Human enterovirus virus 94E210 strain isolated (GenBank:DQ916376.1) (SEQIDNO:22), wherein SEQIDNO:22 comprises antibody-binding polypeptide, is represented respectively by SEQIDNOS:8 and 14.
Restructuring 3D albumen can be used as immunogen separately or together with the other oroteins coming from enterovirus in immunogenic composition.As shown in embodiment hereafter, what EV713D protein used energy induction of antibodies to reply and partly protect together with bacterial flagellin attacks poison.Because 3D albumen is guarded at all enterovirus camber, therefore estimate that 3D albumen can be used as immunogen, has extensive protection to various hypotype.In addition, some variant of 3D protein also may be used for immunogenic composition, wherein, these variants compared with corresponding wild-type protein, identity at least 85%, preferably 90%, be more preferably 95%.In addition, adjuvant can be any known, as M59, and aluminium adjuvant.
Because the antibody for 3D albumen does not show ADE effect, the antibody compositions advantageously used is to treat the enterovirus infection of patient.The antibody compositions being used for the treatment of purposes is known, as antibody is used for the treatment of cancer.Antibody compositions comprises pharmaceutically acceptable composition usually, and such as sodium-chlor is dissolved in pharmaceutically acceptable solution.Present invention demonstrates that, 3D-2A10-IgA and 3D-3A12-IgA can partly protect host to avoid attacking poison, and survival rate is 60%(Figure 11).
Embodiment
The sole purpose of embodiment is below provided to be that principle of the present invention is described; They are never intended to restriction or reduce scope of the present invention.
Embodiment 1
EV713D specific IgG monoclonal antibody.
1.1, antigen preparation
The complete 3D gene (protein of coding shown in SEQIDNO:15) of EV71BrCr strain is cloned into carrier pET28a, and restriction enzyme site is NcoI and XhoI.In brief, the e. coli bl21 (DE3) that will transform containing restructuring 3D expression vector, bacterial growth, prepares these recombinant proteins by induction, and by affinity chromatography on Ni-NTA post (Qiagen company) purifying.
1.2, immunity and preparation EV713D specific IgG monoclonal antibody
Preparation EV713D-monoclonal antibody specific method (LiYM as described previously; LiuF, HanCandYanHM.MonoclonalantibodythatblockstheToll-likerec eptor5bindingregionofflagellin.Hybridoma (Larchmt) .2012Feb; 31 (1): 60-62).In brief, 5 week age female SPFBALB/c mouse subcutaneous inoculation 100 μ g3D, the timed interval is 2 weeks.Last reinforcement rear four week and cytogamy first 3 days, mouse 200 μ g3D intraperitoneal inoculations are strengthened.After three days, results mouse boosting cell, and SP2/0 merges, and uses 50% polyoxyethylene glycol (Sigma-Aldrich company, the Missouri State) in fusion.Hybridoma culture supemates ELISA screens.Positive hybridoma cell is cloned by limiting dilution, and stable hybridoma clone is injected into the abdominal cavity of whiteruss pre-treatment BALB/c mouse.Subsequently, results monoclonal antibody, with antibody purification test kit (NAb
tMproteinA/GSpinKit, ThermoScientific, USA), according to the explanation purifying of manufacturers.
1.3, the checking of the binding specificity of 3D specific IgG monoclonal antibody
Vero-1008 cell EV71(MOI=0.1 in 24 orifice plates) infect.Infect after 24 hours, use anhydrous methanol fixed cell, carry out indirect immunofluorescence assay (IFA), first use 3D specific monoclonal antibody (3A12,2A10,7A6G1 and 11F1), the antibody of the goat anti-mouse IgG of then puting together with fluorescein isocyanate; Flagellin monoclonal antibody specific (5G10) is as negative control, and the serum of the mouse of 3D immunity is as positive control.As shown in Fig. 1 (A), 3A12,2A10,7A6G1 and 11F1 display obviously dyeing, reach the level compared favourably with positive control, negative control MAb 5G10 shows dye-free.
Vero-1008 cell cultures and infection are as mentioned above.Cell lysate SDS-PAGE is separated, and is transferred on pvdf membrane, and trace is with specifying antibody conveniently immunoblotting program.As shown in Fig. 1 (B), swimming lane 1,3A12; Swimming lane 2,2A10; Swimming lane 3,7A6G1; Swimming lane 4,11F1; Swimming lane 5,5G10 is as negative control; With swimming lane 6, positive control.Result shows, 3D protein specific IgG monoclonal antibody (3A12,2A10,7A6G1 and 11F1) display corresponds to the particular bands of 3D protein, but negative control (5G10) does not show the combination of 3D.
1.4, suppress in the cell that 3D specific IgG monoclonal antibody copies EV71
Vero-1008 cell is inoculated in 24 orifice plates, every hole 1 × 10
5cell, cultivates after 24 hours, under MOI=0.1, infects EV71.Infect after 1 hour, removing cell conditioned medium liquid, washed cell 3 times.5 μ gIgG are added to the OPTI-MEM of 100 microlitres, and then 5 microlitre liposomes 2000 join the OPTI-MEM of 100 microlitres, finally mixture are joined the cell of virus infection.Transfection is after 3 hours, and the 0.5mlDMEM of cell conditioned medium liquid containing 3%FBS replaces.After eight hours, the cell that results EV71 infects, through 1 freeze-thaw cycle, measures the virus titer (being represented by PFU/ hole) in these cell samples by plaque assay.As shown in Figure 2,3A12 and 2A10 of transfection significantly reduces virus titer; The 7A6G1 of transfection reduces virus titer, but as compared to 3A12 with 2A10 so not remarkable; But the 11F1 of transfection fails to reduce virus titer.As for EV-5, this is a VP2 specific IgG monoclonal antibody; The EV-5 of transfection fails to reduce virus titer; It should be noted that EV-5, when directly joining cell culture, display increases virus titer, shows that its antibody dependent strengthens the effect of (ADE).The data of virus titer show, are very important by the epi-position of different 3D specific IgG monoclonal antibody identification to the ability that they suppress EV71 to copy.
Except the dosage of antibody, the cultivation of Vero-1008 cell, infect and antibody treatment described above.As shown in Fig. 2 (B), the 2A10 of transfection in test dose (0,0.2%, 1 or 5 microgram/hole) scope to virus titer the dependent restraining effect of reduction show dose, but the EV-5 of transfection unrestraint in the dosage range of all tests.Three independent experiments are through repeating, and report is representational data.
1.5,3D specific IgG monoclonal antibody suppresses external 3D polymerase activity
Fig. 3 (A) illustrates 3D(RdRp) RNA that mediates extends.
(50mMHEPES, 75mMKCl, 5mMMgCl in 10 microlitre reactive systems
2, 4mMTCEP, 300 μMs of NTP, and4 μM of RNAComplex), add the functional 3D of 1 microgram, the initial sum that can trigger RNA extends.RNA kind is separated by the polyacrylamide gel electrophoresis of comprise 7 mole of urea 15%, dyes with Stains-All.Suppressing for measuring, adding 2 μ gIgG monoclonal antibodies to each reaction.The Yeast Nucleic Acid of 3D extends active, is determined by the appearance of the RNA band extended.As shown in Fig. 3 (B), swimming lane 1, without the negative control of polysaccharase; Swimming lane 2, positive controls (not getting involved factor); Swimming lane 3,3A12; Swimming lane 4,2A10; Swimming lane 5,7A6G1; Swimming lane 6,11F1; With swimming lane 7, EV-5.3A12,2A10 and 7A6G significantly suppress RNA to extend the appearance of band, but 11F1 and EV-5 fails to suppress RNA to extend.Carried out three independently to test, report be representational data.
1.6, the antiviral effect of 3D monoclonal antibody specific in mouse model
The antiviral efficacy of EV713D specific IgG is studied in mouse model.30 1 age in days newborn mices are divided into 6 groups (often organizing 5 mouse) at random.Give by intraperitoneal inoculation the 3A12 often organizing 100 microgram/50 microlitres respectively, 2A10,7A6G1,11F1 or EV-5IgG antibody, PBS group is as negative control; Then by intraperitoneal inoculation often group give 10
3tCID
50eV71 attacks poison.IgG injects 4 times, is spaced apart 24 hours.Every day collects mouse survival data, collects 2 weeks.As shown in Figure 4,2A10-IgG and 3A12-IgG provides the protection of 20% or 40% respectively.
1.7, the external virus replication under 2A10-IgG or EV-5-IgG existence
By 1 × 10
4pFUEV71 joins the 200 μ l monoclonal antibodies (EV-5 antibody for EV71VP2, or 2A10IgG antibody is for EV713D) of serial dilution.After hatching 1 hour, this mixture infects Caco-2 cell.Collect the Caco-2 cell that EV71 infects, measure the virus titer in these cell samples by plaque assay.As shown in Figure 5, under the concentration of 0.25-16 μ g/ml, compared with baseline (P<0.01), the remarkable enhanced virus of EV-5 infects, but 2A10 does not have enhanced virus to infect under the dosage of all tests.
1.8, survey and draw special 3A12 and 2A10 of EV713D epi-position and at EV713D(1RA6) three-dimensional model in spatial depiction
3D gene (protein representated by coding SEQIDNO:15) and its truncated mutant are cloned into pET28a expression vector, measure the binding activities of IgAs to 3D protein and mutant by western blot.In addition, the exact range of 3A12-IgG and 2A10IgG antibody recognition is determined with the polypeptide of the different lengths of synthesis.As shown in table 1, the polypeptide that 3A12 and 2A10 identifies is respectively: KEPAVLTS(SEQIDNO:3) and YSTYVKDELRSLDKI(SEQIDNO:9).Comparison from the various enterovirus strain of all A, B, C and D hypotypes discloses, the common recognition sequence KEPAVLX identified by 3A12
7x
8(SEQIDNO:1) another common recognition sequence X, and by 2A10 identified
1x
2tX
4vKDELRSX
12x
13kX
15(SEQIDNO:2).As shown in Figure 6, EV713D(1RA6) be used to the position of two epi-positions identified showing 3A12 and 2A10.
Embodiment 2
EV713D protein specific monoclonal igA antibody
2.1,3D protein specific monoclonal igA antibody
2A10-IgA and 3A12-IgA monoclonal antibody is respectively from 2A10-IgG and 3A12-IgG monoclonal antibody, and hypotype conversion is by the hypotype switch technology of routine.
2.2,3D protein specific monoclonal igA antibody dyes 3D albumen in born of the same parents
VERO-1008 cell kind, in 24 orifice plates, infects EV71(MOI=0.1).Infect latter 24 hours, use methyl alcohol fixed cell, carry out indirect IF staining (IFA), the 3D protein specific monoclonal igA antibody of use is 3A12-IgA and 2A10-IgA, the anti-MeV of 16CF7-IgA() be negative control, the serum of 3D immune mouse is positive control.As shown in Fig. 7 (A), 3A12-IgA and 2A10-IgA, and positive serum, the cell response can infected with EV71, but negative control display not dyeing.
2.3, the immunoblotting of 3D protein specific monoclonal igA antibody
The VERO-C1008 cell that EV71 infects is cleaved, and cell lysate separately, is transferred on pvdf membrane on SDS-PAGE, and the antibody that immunoblotting uses is 3A12-IgA, 2A10-IgA, 16CF7-IgA, and positive serum.As shown in Fig. 7 (B), 3A12-IgA, 2A10-IgA and positive serum dye specifically EV71 infect cell, but 16CF7-IgA but can not.These results demonstrate, 3A12-IgA and 2A10-IgA remains the 3D specificity of corresponding 3A12-IgG and 2A10-IgG.
2.4, EV713D specific IgA copies by suppressing EV71 in pIgR born of the same parents
(i) the VERO-pIgR cell polarized, cultivates in containing the Transwell of 0.4 μm of film; Infect EV71, allow (cell adhesion) on the cell of viruses adsorption.After 1 hour, the top of washed cell and pedestal both sides, remove not by the virus of adsorbing.Then, the complete DMEM of 400 μ l is added to limit, top; 3D specificity or irrelevant IgA antibody (30 μ g/100 μ l) is added in susceptor edge.After 24 hours, with the abundant washed cell both sides of PBS, collect the complete DMEM of 400 μ l, the virus titer plaque ethods in cell sample measures.As shown in Fig. 8 (A), 3A12-IgA and 2A10-IgA can suppress EV71 to copy significantly, but 16CF7-IgA does not suppress.
(ii) as (i) operated the VERO-pIgR cell of polarization, but use the IgA antibody (3A12-IgA and 16CF7-IgA) of different amount.As shown in Fig. 8 (B), suppress EV71 to copy in 3A12-IgA born of the same parents, present dose-dependently.
(iii) as (i) operated the VERO-pIgR cell of polarization, but use corresponding IgG antibody (3A12-IgG, 2A10-IgG and 16CF7-IgG).As shown in Fig. 8 (C), all IgG antibody do not have remarkable retarding effect to copying of EV71.
(iv) operation is as (i), but VERO-pIgR cell changes VERO-1008 cell into.As shown in Fig. 8 (D), all three IgA antibody (3A12-IgA, 2A10-IgA and 16CF7-IgA) are copied EV71 does not have remarkable retarding effect, suppresses to depend on the pIgR of VERO cell surface expression in the born of the same parents copied EV71 showing that IgA mediates.
2.5, EV713D specific IgA s suppresses copying of EV71 and CV strain in born of the same parents
Suppress the experiment copied of EV71 and CV strain in born of the same parents described in embodiment 2.4.Result is summarized in hereafter in table 2.
Copying of EV71 and CV strain is suppressed in table 2, born of the same parents
Wherein " a " represents, with the virus titer of substratum group as 100%, after calculating the per-cent of viral survival under the existence of different antibodies, calculates " % virus reduces ".
2.6, EV713DIgA suppresses viral protein expression
(i) the VERO-pIgR cell polarized, cultivates in containing the Transwell of 0.4 μm of film; Infect EV71, allow (cell adhesion) on the cell of viruses adsorption.After 1 hour, the top of washed cell and pedestal both sides, remove not by the virus of adsorbing.Then, the complete DMEM of 400 μ l is added to limit, top; 3D specificity or irrelevant IgA antibody (30 μ g/100 μ l) is added in susceptor edge.After 24 hours, with the abundant washed cell both sides of PBS, collect the complete DMEM of 400 μ l, 3D and the VP2 protein content immunoblotting (WB) in these cell samples measures.As shown in Fig. 9 (A), 3A12-IgA(swimming lane 1) and 2A10-IgA(swimming lane 2) suppress the expression of 3D and VP2 significantly, but 16CF7-IgA(swimming lane 3) and DMEM(swimming lane 4) significantly do not suppress.
(ii) as (i) operated the VERO-pIgR cell of polarization.Cell sample ELISA analyzes.As shown in Fig. 9 (B), 3A12-IgA and 2A10-IgA suppresses the expression of 3D significantly.
2.7,3A12-IgA and 2A10-IgA suppresses the RNA of 3D polysaccharase to extend activity
(50mMHEPES, 75mMKCl, 5mMMgCl in 10 microlitre reactive systems
2, 4mMTCEP, 300 μMs of NTP, and4 μM of RNAComplex), add the functional 3D of 1 microgram, the initial sum triggering RNA extends.RNA kind is separated by the polyacrylamide gel electrophoresis of comprise 7 mole of urea 15%, dyes with Stains-All.As shown in Figure 10, swimming lane 1, without the negative control of polysaccharase; Swimming lane 2, positive controls (not getting involved factor); Swimming lane 3,16CF7-IgA (2 μ g); Swimming lane 4,2A10-IgA (2 μ g); Swimming lane 5,3A12-IgA (2 μ g).Carried out three independently to test, report be representational data.
2.8, the antiviral efficiency of EV713D Specific IgA antibody in neonatal mouse model is evaluated
1 age in days newborn mice gives 100 microgram/50 microlitre IgA antibody respectively by intraperitoneal inoculation; Then by intraperitoneal inoculation often group give 10
3tCID
50eV71 mouse adapted strain attacks poison.IgA antibody injects 4 times, is spaced apart 24 hours.Every day collects mouse survival data, collects 3 weeks.As shown in figure 11,2A10-IgA and 3A12-IgA provides the protection of 40% or 60% respectively.
Embodiment 3
3D is as antigen
3.1, in the vaccinia virus recombinant the Temple of Heaven strain (VTT-3D) expressing 3D, 3D is identified
EV713D is detected in the VERO-1008 cell of recombinant vaccinia virus infection.As shown in Figure 7, swimming lane 1, protein markers, swimming lane 2, the VERO-1008 contrast of simulated infection, the VERO-1008 that swimming lane 3, VTTenv infects, swimming lane 4-7, the VERO-1008 of the recombined vaccinia virus clone infection of 4 purifying.VERO-1008 that is infected or uninfection is separated by SDS-PAGE, and is transferred to pvdf membrane.3D specific IgG monoclonal antibody 2A10IgG antibody is as binding antibody.Result shows, 3D gene inserts vaccinia virus genome at VERO-1008 cell, and correction.
3.2, immune programme for children
Table 3, immune programme for children
Figure 13 is the schematic diagram of immunization protocol.
Antibody response is detected: head exempts from (Figure 14) after each immunity; First time strengthens (Figure 15); Second time strengthens (Figure 16).Every mouse immune 100 microlitre 10 in VTT-3D group
7pFU virus, and every mouse immune 100 μ l30 microgram 3D albumen in 3D group.Collect serum and the mucous membrane sample from vagina and saliva, the titre of the IgA of the anti-3D albumen determined by ELISA.
The antibody response (Figure 17) of the newborn mice detected.
3.3, EV71 attacks poison protection
After three immunity, in VTT-3D group, 3D group, every newborn mice in PBS group and EV71 deactivation group attacks poison 10
3tCID
50virus.Every day observes newborn mice.The immunity of deactivation EV71 protects mouse completely.All mouse in PBS negative control group, death in 3-5 days after attacking poison.As shown in figure 18,3D and VTT-3D provides the protection of 10%-30%.
Embodiment 4
4.1, with the 3D Western Immuno of purifying
The expression and purification of 3D albumen is described in embodiment 1.1.Pure 3D albumen adds from colibacillary flagellin or CTB as adjuvant, then immune mouse, and approach comprises subcutaneous (SC), (IN) or intraperitoneal (IP) in nose.Table 4 summarizes immunization protocol.2 weeks are spaced apart between twice immunity; The volume of SC and IP is 100 microlitres, and the volume of IN is 20 μ L.
The immunization protocol of the 3D albumen of table 4, use purifying
3.2, antibody response
Behind 2 weeks of last immunity, collect serum, small intestine and lung sample, the sample homogenization of small intestine and lung, collect supernatant liquor and be used for test.3D specificity IgA in these samples or IgG antibody response are by ELISA titration.Figure 19 shows 3D-specific IgG antibodies titre: serum (A), lung (b) and small intestine (c); The titre of 3D Specific IgA antibody: serum (d), lung (e) and small intestine (F).
Although the present invention describes with reference to special embodiment, it is to be appreciated that embodiment is illustrative, scope of the present invention is not limited thereto.Alternate embodiment of the present invention will become apparent the those of ordinary skill in the field that the present invention relates to.Such alternate embodiment is all considered to comprise within the spirit and scope of the present invention.Therefore, scope of the present invention is described by appended claim, supported by description above.
Sequence table
Claims (6)
1.3D protein specific monoclonal igA antibody, it is characterized in that, described 3D protein specific monoclonal igA antibody Binding peptide specifically, wherein said polypeptide is represented by the common recognition sequence be selected from SEQIDNO:1 and SEQIDNO:2.
2. 3D protein specific monoclonal igA antibody according to claim 1, it is characterized in that, the 3D protein specific monoclonal igA antibody be combined with polypeptide shown in SEQIDNO:1 is 3D-2A10-IgA(CCTCCNO:C2014144), the 3D protein specific monoclonal igA antibody be combined with polypeptide shown in SEQIDNO:2 is 3D-3A12-IgA(CCTCCNO:C2014142).
3. 3D protein specific monoclonal igA antibody according to claim 1, it is characterized in that, wherein polypeptide shown in SEQIDNO:1 is selected from the polypeptide by shown in SEQIDNOS:3-8, and polypeptide shown in SEQIDNO:2 is selected from the polypeptide by shown in SEQIDNOS:10-14.
4. comprise the composition of 3D protein specific monoclonal igA antibody as claimed in claim 1, it is characterized in that, described composition comprises at least one 3D protein specific monoclonal igA antibody, be attached to the first polypeptide representated by common recognition sequence SEQIDNO:1 specifically, at least one the 2nd 3D protein specific monoclonal igA antibody, be attached to the second polypeptide representated by common recognition sequence SEQIDNO:2 specifically, and pharmaceutically acceptable solution.
5. composition according to claim 4, it is characterized in that, the 3D protein specific monoclonal igA antibody be wherein combined with the first polypeptide shown in SEQIDNO:1 is 3D-2A10-IgA(CCTCCNO:C2014144), the 2nd 3D protein specific monoclonal igA antibody be combined with the second polypeptide shown in SEQIDNO:2 is 3D-3A12-IgA(CCTCCNO:C2014142).
6. composition according to claim 4, it is characterized in that, wherein said first polypeptide is selected from the polypeptide by shown in SEQIDNOS:3-8, and the second polypeptide is selected from the polypeptide by shown in SEQIDNOS:10-14.
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| CN105664166A (en) * | 2016-01-29 | 2016-06-15 | 中国科学院上海巴斯德研究所 | Composition for treating enterovirus infection and drug combination method |
| CN116514945A (en) * | 2023-03-24 | 2023-08-01 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof |
| CN117467015A (en) * | 2023-12-27 | 2024-01-30 | 北京索莱宝科技有限公司 | Antibodies, antibody combinations and uses of human IgA |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105664166A (en) * | 2016-01-29 | 2016-06-15 | 中国科学院上海巴斯德研究所 | Composition for treating enterovirus infection and drug combination method |
| CN105664166B (en) * | 2016-01-29 | 2019-05-10 | 中国科学院上海巴斯德研究所 | A kind of composition that treating enterovirus infection and drug combination method |
| CN116514945A (en) * | 2023-03-24 | 2023-08-01 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof |
| CN116514945B (en) * | 2023-03-24 | 2023-09-22 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof |
| CN117467015A (en) * | 2023-12-27 | 2024-01-30 | 北京索莱宝科技有限公司 | Antibodies, antibody combinations and uses of human IgA |
| CN117467015B (en) * | 2023-12-27 | 2024-03-12 | 北京索莱宝科技有限公司 | Antibodies, antibody combinations and uses of human IgA |
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