CN104788544A - Enterovirus 71 type epitope as well as antibody and application and vaccine thereof - Google Patents
Enterovirus 71 type epitope as well as antibody and application and vaccine thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology and immunology and particularly relates to an enterovirus 71 type epitope, an antibody, application and a vaccine thereof. The epitope of the EV71 virus is located on a coat protein VP3 of the EV71 virus and is capable of specifically identifying the EV71 virus; besides, the epitope is presented through a PP carrier to cause a high neutralizing titer, and the serum after immunity is capable of effectively protecting the young rat from EV71 virus infection. The neutrality detection result of the polyclone on each EV71 subtype pseudovirus shows that the polyclonal antibody provided by the invention has high and broad-spectrum neutralizing titer. In addition, the passive protection and the maternal vertical protection experiments prove that the polyclonal antibody provided by the invention can completely protect the young rat from EV71 virus infection.
Description
Technical field
The present invention relates to molecular biology and immunological technique field, particularly relate to Enterovirus 71 antigen epi-position, antibody and application thereof and vaccine.
Background technology
Hand foot mouth disease (Hand, foot and mouth disease, HFMD) be global infectious disease, enterovirns type 71 (Enterovirus 71, and coxsackie virus A 16-type (Coxsackievirus A16 EV71), CA16) be the main pathogens of hand foot mouth disease (Hand, foot and mouth disease, HFMD).From 1969 since the U.S. isolates EV71, in succession report EV71 to infect the HFMD caused popular in countries such as Australia, Japan, Brazil, Holland, Malaysia, TaiWan, Chinas.On May 2nd, 2008, HFMD is classified as Class C transmissible disease by ministry of Health of China.The virion of natural intestinal virus is the globosity of icosahedron cubic symmetry, and without coating and projection, diameter is about 24 ~ 30nm.The infection object of enterovirus is mainly the children of less than 5 years old, pathogeneticing characteristic mainly contains the fever of lasting 3 to 4 days, at palm and vola skin, there is vesica in oral mucosa and tongue, seriously easily induce the nervous system disorders such as encephalitis, aseptic meningitis, acute myelitis, acute cerebellar ataxia, cause serious and death, cause huge burden on society.
EV71 belongs to Picornavirous group, and only containing an open reading frame in genome, be the single-stranded positive RNA containing 7408 Nucleotide, 2194 amino acid whose polyproteins of encoding, polyprotein can be hydrolyzed into P1, P2 and P3 tri-precursor proteins further.P1 precursor protein can be hydrolyzed into VP1, VP2, VP3 and VP4 tetra-virus capsid proteins further; P2 and P3 precursor protein can be biodegradable into seven Nonstructural Proteins, is proteolytic ferment and RNA polymerase, during evolution high conservative.The capsid of virus particle is made up of 60 subunits, the pentamer spline structure that each subunit is assembled into by four kinds of capsid proteins.Wherein, VP4 is embedded in inner side and the nucleoid compact siro spinning technology of virus particle shell, and other three kinds of structural protein are all exposed to the surface of virion, and thus antigenic determinant is generally within VP1, VP2 and VP3.
Epitope (epitope) is the basis of identification, binding antibody in antigen molecule, is therefore the actual active principle detecting antibody.At present for EV71 virus, the relation of antigenic component and protection antibody reaction level is not also established, and namely causes immunoreactive specificity neutralizing epitope also not identify completely.Concrete, the EV71 virus antigen epitope screened at present mainly concentrates on VP1 and VP2, and VP3 epi-position and space conformation epi-position then rarely have report.Therefore, the epitope of further screening, qualification EV71, the epitope particularly for VP3 is very necessary.
Further, because the unique effective measure controlling hand foot mouth disease at present popular are exactly vaccine, and the immunogenicity of vaccine determines that vaccine research and development whether can successful key index.Therefore, the epitope of EV71 should be enriched further, and find the epitope with more high immunogenicity and protectiveness.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide Enterovirus 71 antigen epi-position, antibody and application thereof and vaccine.Enterovirus 71 antigen epi-position provided by the invention is positioned on virus capsid protein VP3, is neutrality epi-position; This epi-position through promise as stronger NAT and defendance level can be caused after P particle delivery, can the infection of the anti-EV71 of protection body of broad spectrum.
The aminoacid sequence of Enterovirus 71 antigen epi-position provided by the invention is as shown in SEQ ID NO:1.
The aminoacid sequence of the epitope of EV71 virus provided by the invention is HYRAHARDGVFDYYT, is positioned on the coat protein VP3 of EV71 virus.Although, current epitope can utilize bioinformatics software to predict, but, increasing research shows, the error probability of software prediction epitope is higher, and the prediction of the former epi-position of software counterwork is only for the total length of object antigen protein, in the process of real application, often there is specific problem, need experiment to confirm.And the present invention is confirmed by test, this epi-position can specific recognition EV71 virus, and this epi-position can cause higher Neutralizing titer after PP carrier is presented, and the serum produced after its immunity can the infection of available protecting children mouse opposing EV71 virus.
Present invention also offers the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:1, it has the nucleotide sequence as shown in SEQ ID NO:2.
The DNA molecular of nucleotide sequence shown in SEQ ID NO:2 can the aminoacid sequence shown in accurate coding SEQ ID NO:1.
Present invention also offers a kind of expression plasmid, comprise the nucleotide sequence as shown in SEQ ID NO:2 and pet28a-P domain carrier.
Pet28a carrier buys precious biotechnology (Dalian) company limited in Dalian, thus basis is constructed pet28a-P domain carrier, can at the P albumen of intestinal bacteria expression in vivo norovirus.The construction process of pet28a-Pdomain carrier is see Zang Y, Bi J, Du D, et al, Development of aNorovirus P particle platform for eliciting neutralizing antibody responses to themembrane proximal external region of human immunodeficiency virus type 1envelope.Protein Pept Lett.2014; 21 (12): 1230 ~ 9.
Gene constructed in the Loop2 of norovirus P particle by by object epi-position, based on its in intestinal bacteria can self-assembly, stable, high expression level advantage, by the surface of target Epitope presentation at P particle, strengthen Proantigen epitope immunogenic.
Present invention also offers a kind of antigen presenting cell, obtained by expression plasmid transformation of E. coli Rosseta provided by the invention.
Expression plasmid transformation of E. coli Rosseta provided by the invention is obtained antigen presenting cell, and can express the epitope through submission after induction, this fusion rotein has space shell structure, becomes the particle of about 20nm.This fusion rotein and adjuvant mixed immunity mouse can be produced specific antibody.
The present invention is claimed fusion rotein of being expressed by antigen presenting cell provided by the invention also.
After antigen presenting cell induction expression protein provided by the invention, inhale post and superdex200 molecular exclusion chromatography post by the affine layer of nickel ion, obtain the fusion rotein of purifying, its aminoacid sequence is as shown in SEQID NO:3.The mode of appearance discerning the size of albumen through protein blot and Electronic Speculum shows, epitope provided by the invention, after norovirus p particle submission, can form certain space shell structure.By neutralizing in immunity and body, external protection experiment display fusion rotein provided by the invention has good immunogenicity and can cause stronger immune response, and presents certain protected effect.The new epi-position EV71/VP3 of EV71 (71-6,176-190aa, HYRAHARDGVFDYYT) in this invention is confirmed as conformation neutralizing epitope.And confirm that promise such as P particle serves booster action to presenting of this epi-position.
The invention provides a kind of polyclonal antibody, obtained by the preparation immune animal comprising fusion rotein provided by the invention.
The preparation comprising fusion rotein provided by the invention is fusion rotein provided by the invention and Al (OH)
3adjuvant mixing is obtained.
As preferably, fusion rotein and Al (OH)
3the mass ratio of adjuvant is 1:8.
Polyclonal antibody provided by the invention is obtained by the preparation immunization. Female BALB/c mouse comprising fusion rotein provided by the invention.
As preferably, immunizing dose be 25 μ g/ only.
As preferably, immune time is 3 times.
Concrete, the preparation method of polyclonal antibody is: by fusion rotein provided by the invention and AL (OH)
3adjuvant mixes, albumen and AL (OH)
3mass ratio be 1:8; Get 6-8 week female BAl BIc/c mouse, at 0 day, 14 days, within 28 days, carry out peritoneal immunity.Immunizing dose is 250 μ g/ time.Respectively at 14,28,42,56 days tail venous blood samplings, venous blood is through 37 DEG C of 1h, and 4 DEG C of 2h, 4 DEG C centrifugal, and reject precipitates, obtained polyclonal antibody.
Polyclonal antibody of the present invention prevents in preparation or treats the application in the medicine of hand foot mouth disease.
The present invention confirms through test, in traditional neutralizing antibody (CPE) and pseudovirus neutralizing antibody (PVLA) detection experiment, polyclone provided by the invention presents with the positive titers value of 64 and 220 respectively, shows that polyclonal antibody provided by the invention can cause higher Neutralizing titer.And polyclone provided by the invention shows each hypotype pseudovirus neutrality detected result of EV71, polyclonal antibody provided by the invention can present the higher and Neutralizing titer of wide spectrum.Further, confirm through passive protection and the vertical Protection of parent, polyclonal antibody provided by the invention can protect young mouse to resist the infection of EV71 completely.
Present invention also offers a kind of enterovirns type 71 detection kit, comprising albumen and antibody;
The polypeptide that albumen is aminoacid sequence shown in SEQ ID NO:1 or fusion rotein provided by the invention;
Antibody is polyclonal antibody provided by the invention.
Test kit provided by the invention can detect enterovirns type 71 quickly and accurately.Antibody wherein and albumen can identify each other, present very high association reaction.
As preferably, in test kit provided by the invention, also comprise enzyme plate.
Proteopexy can be used in enzyme plate the antibody detecting enterovirns type 71 by test kit provided by the invention, also antibody can be coated in enzyme plate, for detecting the antigen of enterovirns type 71.
As preferably, test kit provided by the invention also comprises coating buffer, confining liquid, two anti-, nitrite ion, stop buffers.
Present invention also offers a kind of vaccine, comprise fusion rotein of the present invention and adjuvant.
As preferably, adjuvant is aluminum hydroxide adjuvant, Freund's complete adjuvant or Freund's incomplete adjuvant.
Preferably, adjuvant is aluminum hydroxide adjuvant.
As preferably, the mass ratio of albumen and adjuvant is 1:(8 ~ 10).
Preferably, the mass ratio of albumen and adjuvant is 1:8.
As preferably, the immunity amount of vaccine provided by the invention be 25 μ g/ only, number of times is 3 times.
Fusion rotein provided by the invention can stimulate mouse generation polyclonal antibody, and this antibody can passive protection or vertical protection mouse opposing EV71 infection through test confirmation.Therefore, fusion rotein provided by the invention can make vaccine for preventing the infection of EV71.
The invention provides Enterovirus 71 antigen epi-position, antibody and application thereof and vaccine; the epitope of EV71 virus provided by the invention is positioned on the coat protein VP3 of EV71 virus; this epi-position can specific recognition EV71 virus; and; this epi-position can cause higher Neutralizing titer after PP carrier is presented, and the serum produced after its immunity can the infection of available protecting children mouse opposing EV71 virus.Polyclonal antibody provided by the invention is in traditional neutralizing antibody (CPE) and pseudovirus neutralizing antibody (PVLA) detection experiment, present with the positive titers value of 64 and 220 respectively, show that polyclonal antibody provided by the invention can cause higher Neutralizing titer.And polyclone provided by the invention shows each hypotype pseudovirus neutrality detected result of EV71, polyclonal antibody provided by the invention can present the higher and Neutralizing titer of wide spectrum.Further, confirm through passive protection and the vertical Protection of parent, polyclonal antibody provided by the invention can protect young mouse to resist the infection of EV71 completely.
Accompanying drawing explanation
Fig. 1 shows expression plasmid building process;
Fig. 2-a shows that wherein, swimming lane M is marker with SDS-PAGE electrophoretic method qualification fusion rotein and reference protein; Swimming lane PP shows reference protein electrophoresis result; Swimming lane PP-71-6 shows fusion rotein electrophoresis result;
Fig. 2-b shows that Western bolt identifies fusion rotein and reference protein, and wherein, swimming lane M is marker; Swimming lane PP shows reference protein Western detected result; Swimming lane PP-71-6 shows fusion rotein Western detected result;
Fig. 2-c shows the Odyssey quantitative result of fusion rotein and reference protein; Wherein, swimming lane PP-71-6 shows the detected result of fusion rotein; Swimming lane PP shows the detected result of reference protein; Swimming lane M shows marker; Swimming lane 500 shows that concentration is the detected result of the BSA albumen of 500 μ g/ml; Swimming lane 250 shows that concentration is the detected result of the BSA albumen of 250 μ g/ml; Swimming lane 125 shows that concentration is the detected result of the BSA albumen of 125 μ g/ml; Swimming lane 62.5 shows that concentration is the detected result of the BSA albumen of 62.5 μ g/ml; Swimming lane 31.25 shows that concentration is the detected result of the BSA albumen of 31.25 μ g/ml; Swimming lane 15.625 shows that concentration is the detected result of the BSA albumen of 15.625 μ g/ml;
Fig. 3-a shows the result of electron microscopic observation fusion rotein;
Fig. 3-b shows the form of fusion rotein under Electronic Speculum;
Fig. 4-a shows the result of electron microscopic observation reference protein;
Fig. 4-b shows the form of reference protein under Electronic Speculum;
Fig. 5 shows the position of epi-position provided by the invention VP3 in each hypotype of EV71;
Fig. 6-a shows that the obtained polyclonal antibody of embodiment 5 is to the Immune discrimination of EV71 virus; Wherein, swimming lane 1 shows that the obtained polyclonal antibody of embodiment 5 is to the identification of deactivation EV71 virus; Swimming lane 2 shows that the obtained polyclonal antibody of embodiment 5 is to the identification of EV71 virus-like particle; Swimming lane M shows protein markers;
Fig. 6-b shows that the obtained polyclonal antibody of comparative example 3 is to the Immune discrimination of EV71 virion; Swimming lane 1 shows that the obtained polyclonal antibody of comparative example 3 is to the identification of deactivation EV71 virus; Swimming lane 2 shows that the obtained polyclonal antibody of comparative example 3 is to the identification of EV71 virus-like particle; Swimming lane M shows protein markers;
Fig. 6-c shows the many anti-Immune discrimination to EV71 virion of EV71VP1 rabbit; Swimming lane 1 shows the many anti-identification to deactivation EV71 virus of EV71VP1 rabbit; Swimming lane 2 shows the many anti-identification to EV71 virus-like particle of EV71VP1 rabbit; Swimming lane M shows protein markers;
Fig. 7-a shows suckling mouse passive protection survival rate; Wherein,
show the obtained many anti-passive protection survival rates to suckling mouse of embodiment 5;
show the obtained many anti-passive protection survival rates to suckling mouse of 10 μ g comparative example 3;
show the obtained many anti-passive protection survival rates to suckling mouse of 50 μ g comparative example 3;
the obtained many anti-passive protection survival rates to suckling mouse of comparative example 4;
show only with the suckling mouse survival rate of virus attack;
Fig. 7-b shows that suckling mouse vertically protects survival rate; Wherein,
show the obtained many anti-vertical protection survival rates to suckling mouse of embodiment 5;
show the obtained many anti-vertical protection survival rates to suckling mouse of 10 μ g comparative example 3;
show the obtained many anti-vertical protection survival rates to suckling mouse of 50 μ g comparative example 3;
the obtained many anti-vertical protection survival rates to suckling mouse of comparative example 4;
show only with the suckling mouse survival rate of virus attack;
Fig. 7-c shows suckling mouse passive protection body weight change; Wherein,
show the obtained many anti-passive protection body weight change to suckling mouse of embodiment 5;
the obtained many anti-passive protection body weight change to suckling mouse of comparative example 4;
show only with the suckling mouse body weight change of virus attack;
Fig. 7-d shows that suckling mouse vertically protects body weight change; Wherein,
show the obtained many anti-vertical protection body weight change to suckling mouse of embodiment 5;
the obtained many anti-vertical protection body weight change to suckling mouse of comparative example 4;
show only with the suckling mouse body weight change of virus attack.
Embodiment
The invention provides Enterovirus 71 antigen epi-position, antibody and application thereof and vaccine, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably changes and combination not departing from content of the present invention, spirit and scope, realizes and applies the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
Described in the embodiment of the present invention, as shown in SEQ ID NO:1, the polypeptide of aminoacid sequence is synthetic.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
The fragment of nucleotide sequence shown in SEQ ID NO:2 is building up to promise as in P particle plasmid expression vector PET28a-P domain
Design primer is as follows:
GCGGCCGC is Not I restriction enzyme site, and CCATGG is Nco I restriction enzyme site, and CTCGAG is Xho I restriction enzyme site, and the GC for avoiding phase shift mutation to add.By overlap PCR, obtain goal gene PCR primer fragment, reclaim goal gene PCR primer fragment ligation amplification plasmid Peasy-Blunt, transformation of E. coli, screening positive transformant, after cultivating, extract plasmid, plasmid is through Nco I and the process of Xho I double digestion, connect the original plasmid PET28a-P domain through Nco I and the process of Xho I double digestion with T4 ligase enzyme, obtain expression plasmid.Expression plasmid building process as shown in Figure 1.
Expression plasmid is transformed and expresses bacterial classification Rosseta acquisition antigen presenting cell.
PET28a-P domain empty plasmid is transformed and expresses bacterial classification Rosseta acquisition contrast bacterial classification.
Respectively by antigen presenting cell and contrast strain inoculation in substratum, be specially: 100 μ l preserve bacterial classifications be connected in 100ml LB substratum (containing K+ resistance), 220rpm, 37 DEG C are spent the night.The bacterium that will spend the night next day is transferred in 1L LB substratum (containing K+ resistance) with 1/50 ratio, 220rpm, 37 DEG C, treat that OD600 is 0.6 ~ 0.8, bacterium liquid before staying 1ml to induce, the IPTG adding final concentration 1mM carries out 16 DEG C of inductions of spending the night, bacterium liquid after staying 1ml to induce.Induce forward and backward bacterium liquid to do following same process for 1ml: the centrifugal 2min of 12000rpm, abandon supernatant, 200ul 4 × loading buffer mixes thalline, and 97 DEG C are boiled sample 20min, the centrifugal 20min of 12000rpm.Sample is identified to determine the abduction delivering of albumen through 12%SDS PAGE and is determined that this albumen is expressed for inclusion body.
Embodiment 2
1L antigen presenting cell after Example 1 induction, with 80ml balance liquid (20mM Tris, 0.5MNaCl, [pH 8.0]) blow even, 4 DEG C of ultrasonic 40min, ultrasonic 5s stops 5s, 16000rpm 30min retains precipitation, and 40ml is containing urea balance liquid process (8M urea, 20mM Tris, 0.5M NaCl, [pH 8.0]).4 DEG C of stirrings are spent the night, and next day, 10000rpm 30min, stayed supernatant, and inhale post in passing the affine layer of nickel ion, elutriant is the urea balance liquid containing imidazoles, and the gradient of ultimate aim albumen is 200mM imidazoles, obtains fusion rotein.Purify by identical method and contrast bacterial classification expression reference protein.
Use SDS-PAGE (Fig. 2-a), western blot (Fig. 2-b), Dual band IR laser imaging system to analyze (Fig. 2-c) respectively and identify fusion rotein and reference protein, Dual band IR laser imaging system is quantitatively using BSA albumen as standard substance, concentration is 15.625,31.25,62.5,125,250,500ug/ml.SDS-PAGE after Dual band IR laser imaging, Criterion curve between the signal value excited and respective concentration, y=497.24x-40.495, R
2=0.9947.Result shows, and induction obtains the albumen of expection size.
Electron microscopic observation fusion rotein (Fig. 3 a, Fig. 3 b) and reference protein (Fig. 4 a, Fig. 4 b), form certain similar spatial shell structure both proving, and become diameter to be the particle of about 20nm.Through qualification, the position of epi-position provided by the invention VP3 in each hypotype of EV71 as shown in Figure 5.
Embodiment 3
By fusion rotein obtained for embodiment 2 and AL (OH)
3adjuvant mixes, and the mass ratio of fusion rotein and AL (OH) 3 is 25 μ g:200 μ g/dose, obtained vaccine.
Embodiment 4
By fusion rotein obtained for embodiment 2 and Al (OH)
3adjuvant mixes, and the mass ratio of fusion rotein and Al (OH) 3 is 25 μ g:200 μ g/dose, obtained vaccine.
Comparative example 1
By reference protein obtained for embodiment 2 and Al (OH)
3adjuvant mixes, reference protein and Al (OH)
3mass ratio be 25 μ g:200 μ g/dose, obtained vaccine.
Comparative example 2
By the polypeptide of such as aminoacid sequence shown in SEQ ID NO:1 and Al (OH)
3adjuvant mixes, polypeptide and Al (OH)
3mass ratio be 10 μ g:200 μ g/dose, 50 μ g:200 μ g/dose obtain vaccine.
Embodiment 5
Get 6 ~ 8 weeks female BAl BIc/c mouse, respectively at 0 day, 14 days, the vaccine prepared with embodiment 3 for 28 days carried out peritoneal immunity.Respectively at 14,28,42,56 days tail venous blood samplings.Serum process: 37 DEG C of 1h, 4 DEG C of 2h, 4 DEG C of centrifuging and taking supernatants, obtained polyclonal antibody, antibody is stored in-80 DEG C of refrigerators.
Comparative example 3
Get 6 ~ 8 weeks female BAl BIc/c mouse, respectively at 0 day, 14 days, within 28 days, with contrast, vaccine prepared by comparative example 1 carried out peritoneal immunity.Respectively at 14,28,42,56 days tail venous blood samplings.Serum process: 37 DEG C of 1h, 4 DEG C of 2h, 4 DEG C of centrifuging and taking supernatants, obtained polyclonal antibody, antibody is stored in-80 DEG C of refrigerators.
Comparative example 4
Get 6 ~ 8 weeks female BAl BIc/c mouse, respectively at 0 day, 14 days, within 28 days, with contrast, vaccine prepared by comparative example 2 carried out peritoneal immunity.Respectively at 14,28,42,56 days tail venous blood samplings.Serum process: 37 DEG C of 1h, 4 DEG C of 2h, 4 DEG C of centrifuging and taking supernatants, obtained polyclonal antibody, antibody is stored in-80 DEG C of refrigerators.
Embodiment 6
Western Blot detects the immunogenicity of embodiment 5 gained polyclonal antibody, and antigen is EV71 inactivated vaccine and EV71VLPs vaccine, is market and buys.Antigen is transferred on pvdf membrane after 12%SDS-PAGE, and the polyclonal antibody obtained by immune-blotting method embodiment 5 and comparative example 3 is to the Immune discrimination of EV71 whole virus particles, how anti-as positive control using EV71VP1 rabbit.The results are shown in Figure 6-a ~ Fig. 6-c.
Wherein, Fig. 6-a shows that the obtained polyclonal antibody of embodiment 5 is to the Immune discrimination of EV71 virion; Fig. 6-b shows that the obtained polyclonal antibody of comparative example 3 is to the Immune discrimination of EV71 virion; Fig. 6-c shows the many anti-Immune discrimination to EV71 virion of EV71 VP1 rabbit.
Result shows: the polyclonal antibody that embodiment 5 is obtained and EV71 deactivation, EV71VLPs antigen carry out Western blot experiment, show the positive band of VP3 object size at ~ 28kDa, after showing immune epi-position provided by the invention, have the specific antibody for this epi-position to produce.Resist polyclonal antibody obtained for comparative example 3 and EV71VP1 rabbit as negative control group and positive controls morely simultaneously, all obtain expected results.
Embodiment 7
Elisa detects the immunogenicity of embodiment 5 gained polyclonal antibody:
1. wrap quilt: with coating buffer by fusion rotein obtained to EV71 VLPs, EV71 deactivation, embodiment 2, as shown in SEQ ID NO:1, the polypeptide of aminoacid sequence is diluted to concentration is respectively 3 μ g/ml, 100ul/ hole, 4 DEG C are spent the night.Next day, discard solution in hole, 200ul lavation buffer solution washes 1 time, middle vibration.
2. close: add 200ul confining liquid in above-mentioned wrapped by reacting hole in, put 37 DEG C of incubations 2 hours.Then 200ul lavation buffer solution washes 3 times.
3. application of sample: add antibody diluent 100ul to be checked in reacting hole, in this experiment, antibody to be checked is obtained polyclonal antibody, the PBS of the obtained polyclonal antibody (50ug) of the obtained polyclonal antibody (10ug) of the obtained polyclonal antibody of anti-EV71 VLPs, EV71 deactivation, embodiment 5, comparative example 4, comparative example 4, comparative example 3,37 DEG C of 1.5h.Each sample initial dilution is 1:100, and continuous 10 times are diluted to 10
-5, then 200ul lavation buffer solution washes 3 times.
4. ELIAS secondary antibody is added: add HPP mountain sheep anti-mouse igg (1/10000 dilution) 100ul/ hole.37 DEG C 45 minutes, wash 5 times.
5. substrate solution colour developing is added: add tmb substrate 100ul/ hole, the abundant variable color in 10 ~ 15 minutes of room temperature dark place.
6. termination reaction: add 2M sulfuric acid 50ul/ hole.(the blue flavescence look of tmb substrate).
Result judges: survey OD value: on ELISA detector, uses 450nm to survey each hole OD value.Usually be greater than 2.1 times of the negative control OD value of regulation, be the positive (to calculate after blank control wells zeroing).
The results are shown in Table 1,
Table 1 ELISA detected result
Result is shown: in ELISA experiment, and the obtained polyclonal antibody of EV71VLPs, EV71 deactivation, embodiment 5 polyclonal antibody obtained with comparative example 4 be equal can be identified and in conjunction with corresponding autoantigen.For EV71 VLPs and EV71 inactivation antigen, the polyclonal antibody that embodiment 5 obtains with comparative example 4 all can present very high association reaction, and the immune serum of EV71VLPs also can identify fusion rotein provided by the invention and the antigen with aminoacid sequence shown in SEQ ID NO:1 simultaneously.But the immune serum of EV71 deactivation albumen can not or very weak identification fusion rotein provided by the invention and the antigen with aminoacid sequence shown in SEQ ID NO:1, in this experiment, other otherness the possibility of result of EV71VLPs and EV71 deactivation protein groups causes the microvariations of deactivation protein structure to cause due to the otherness of structure therebetween or formalin.
Embodiment 8
Serum Neutralizing test-carry out traditional neutralizing antibody (CPE) and pseudovirus neutralizing antibody (PVLA) test experience: method is:
Get 96 orifice plates and add the substratum of 100ul DMEM containing 10% foetal calf serum, resist with comparative example 3,4 obtained resist with 1:8 extent of dilution to play continuous 2 times gradient dilutions in 96 orifice plates by obtained for embodiment 5 more more.If good virus control and cell controls.The infection value of EV71 virus strain (C4, KJ508817) is diluted to 100TCID by substratum respectively that contain 10% foetal calf serum with DMEM
50/ 0.05ml/ hole.The pseudovirus diluted is added respectively 0.05ml in 96 orifice plates, 37 DEG C of 5%CO
2hatch 1-1.5h altogether.RD cell is joined in 96 orifice plates, every hole add-on 2 × 10
4/ 100ul, 37 DEG C of 5%CO
2hatch observation of cell pathology situation after 7 days altogether, determine its NAT value.Result judges: in 2 holes of most high dilution serum, have 1 hole to occur cytopathy, cytopathy does not appear in another hole, and this dilution inverse meter is NAT of this serum specimen; When the complete pathology in high dilution 2 hole, adjacent low extent of dilution 2 hole not pathology completely, then the inverse of both Average dilutions is the NAT of this serum specimen; When 1 porocyte pathology all appears in two adjacent extent of dilution serum, there is not cytopathy in another 1 hole, then the inverse of both Average dilutions is the EV71 NAT of this serum specimen.Result is as shown in table 2:
Table 2 CPE detected result
Result display polyclonal antibody tradition neutralizing antibody (CPE) and pseudovirus neutralizing antibody (PVLA) test experience, result shows: detect in the decorum at two kinds of neutralizing antibodies, how anti-embodiment 5 is obtained presents with the positive titers value of 64 and 220 respectively, shows that the epi-position provided by the invention after being presented by PP carrier can cause higher Neutralizing titer.And the obtained how anti-group of comparative example 4 presents identical negative findings with comparative example 3 obtained resist with PBS control group more, imply that epi-position provided by the invention may be the neutralizing epitope of conformation type.
Embodiment 9
To carry out in serum broad spectrum and detections-pseudovirus fluorescent quantitation method (PVLA) with each hypotype pseudovirus of EV71, method is with embodiment 8, and embodiment 5 is obtained resists 20 times of extent of dilution to play continuous 3 times of gradient dilutions in 96 orifice plates more.The infection value of each hypotype pseudovirus of EV71 (B1 – B5, C1 – C5) is diluted to 50000/0.05ml/ hole by substratum respectively that contain 10% foetal calf serum with DMEM.Inoculating cell becomes 293H cell (this laboratory provides) and joins in 96 orifice plates, and add-on is 2 × 10
4/ 100ul/ hole, after 37 DEG C of 5%CO2 hatch 15-20h altogether.The 96 every holes of orifice plate are discarded 190ul, adds luciferase substrate 40ul/ hole, act on 5 minutes, detect its fluorescence radiation by multiple labeling microwell plate detection system and read value, determine its NAT.The results are shown in chart 3:
The anti-wide spectrum NAT detected result of table 3 embodiment more than 5
| B1 | B2 | B3 | B4 | B5 | C1 | C2 | C2like | C3 | C4a | C4b | C5 | |
| EV71NTAb titre | 266.5 | 550.5 | 220.5 | 173 | 110.5 | 562 | 418.5 | 430 | 364.5 | 220 | 151 | 490 |
Result shows: utilize pseudovirus fluorescent quantitation method pseudovirus for each hypotype of EV71 to be detected, and embodiment 5 is obtained manyly anti-ly all presents higher and parallel Neutralizing titer.Show that epi-position provided by the invention is the wide spectrum neutralizing epitope of EV71.
Embodiment 10
Protection has vertically been protected by passive protection and parent.
First, passive protection is tested, and how anti-obtained many anti-, the comparative examples 3 of 15ul embodiment 5 are obtained is 1.5 × 10 with isopyknic infection value respectively
6after the EV71 virus of TCID50 hatches 1.5h altogether, intracerebral injection one age in days suckling mouse, observes suckling mouse survival rate and mean body weight situations in 16 after attacking poison.
Secondly; the vertical Protection of parent; female BAl BIc/c mouse after second time immunity and BALB/c mouse mate; about 19 days female BAl BIc/c mouse fertility suckling mouses after mating; by obtained for immune 15uL embodiment in birth 1 age in days suckling mouse brain 5 many anti-, comparative example 3 is obtained resists more, attacks 15ul respectively containing 1.5 × 10 after 2h
6tCID
50eV71 virus, same to observe 16 days, determine the protection validity (see Fig. 7-a ~ 7-d) of vaccine for suckling mouse.Immunoreactive protection caused by assessment embodiment.
Experimental results show; no matter in passive protection or the vertical Protection of parent; the how anti-infection that the protection opposing EV71 virus of newborn rat 80% ~ 100% all can be provided that embodiment 5 is obtained, and resist that comparative example 3 provides equally presents negative findings with control group more.Meanwhile, body weight record case also shows to increase gradually with the young mouse body weight of the obtained how anti-protection of embodiment 5, otherwise and prepares the young mouse of how anti-protection with comparative example 3.Above result shows that how anti-or immune response that epi-position provided by the invention produces after immunity fully can protect the infection of young mouse-anti EV71.
Below be only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. an Enterovirus 71 antigen epi-position, is characterized in that, this epitope aminoacid sequence is as shown in SEQ ID NO:1.
2. the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:1, it is characterized in that, it has the nucleotide sequence as shown in SEQ ID NO:2.
3. an expression plasmid, is characterized in that, comprises the nucleotide sequence as shown in SEQ ID NO:2 and pet28a-P domain carrier.
4. an antigen presenting cell, is characterized in that, is obtained by expression plasmid transformation of E. coli Rosseta according to claim 3.
5. the fusion rotein of being expressed by antigen presenting cell described in claim 4.
6. a polyclonal antibody, is characterized in that, is obtained by the preparation immune animal comprising fusion rotein described in claim 5.
7. polyclonal antibody as claimed in claim 6 prevents in preparation or treats the application in the medicine of hand foot mouth disease.
8. an enterovirns type 71 detection kit, is characterized in that, comprises albumen and antibody;
The polypeptide that described albumen is aminoacid sequence shown in SEQ ID NO:1 or fusion rotein as claimed in claim 5;
Described antibody is obtained by the preparation immune animal comprising fusion rotein described in claim 5.
9. a vaccine, is characterized in that, comprises fusion rotein as claimed in claim 5 and adjuvant.
10. vaccine according to claim 9, is characterized in that, the mass ratio of described albumen and adjuvant is 1:(8 ~ 10).
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| CN104788544B (en) | 2018-05-08 |
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