CN104151399B - The shared specificity epitope of bocavirus (HBoV), its application and its antibody - Google Patents
The shared specificity epitope of bocavirus (HBoV), its application and its antibody Download PDFInfo
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Abstract
The invention discloses the shared specificity epitope peptide of bocavirus (HBoV), its application and its antibody.Specifically, the specificity epitope peptide is MSDTDIQDQQPDTVDAPQNT or EHAYPNASHPWDEDVMPDL.
Description
Technical field
The present invention relates to bocavirus, can be especially useful for the epitope of the viral associated diseases diagnosis, and it is applied and its resisted
Body.
Background technology
Human bocavirus (huamn bocavirus, HBoV) is newfound virus in recent years, is the infection people being currently known
Second of parvovirus of class.HBoV has found four kinds of genotype (HBoV1-4), and HBoV1 is acute in one, Sweden in 2005
Found in the nasopharynx sample of children with respiratory tract infections.Then, have again three kinds of bocavirus (HBoV2-4) in 2009~2010 years
It is found successively in fecal sample.
The infection of human bocavirus is in worldwide distribution, and virus can not only detect in respiratory tract, fecal sample, and
There is detection in serum, tonsillotome, saliva and urine.The PCR Analysis of test results of respiratory tract sample is shown, according to state
The difference of family, regional, detection method and crowd, HBoV1 recall rate is about 2-19%, wherein in the infant of 6 months~2 years old
Middle recall rate highest, and recall rate is relatively low in other age groups children and adult.Because HBoV1 is often in upper lower respiratory tract sense
Detected in the sample of dye, it is considered to be the potential virulence factor relevant with respiratory tract infection.It is different from HBoV1, HBoV2-4 warps
Often detect in stool sample, can seldom be detected in respiratory tract infection sample, therefore HBoV2 and HBoV3 may be with gastroenteritis
It is relevant.In children's enteron aisle sample, HBoV2 recall rates are up to 26%, next to that HBoV3 (5%) and HBoV4 (2%).
Bocavirus often with the detection altogether of other viruses, and can persistently exist in pharynx nasalis, thus bocavirus with
The relation of disease is constantly subjected to dispute on.In recent years, serious and lethal bocavirus infection is reported successively.Mitui etc. exists
Detected in the cerebrospinal fluid and serum sample of tetra- severe encephalitis patients of 2009-2010 (wherein two death) HBoV1 and
HBoV2 nucleic acid, and do not detected in cerebrospinal fluid other any pathogen [referring to：Mitui MT, Tabib SM,
Matsumoto T, et al.Detection of human bocavirus in the cerebrospinal fluid of
Children with encephalitis.Clin Infect Dis, 2012,54：964-7.]；2010, Slovenia
One 20 years old ARI patient evolution is acute respiratory failure and pneumothorax, bronchus aspirate, Nasopharyngeal swabs and
The HBoV1DNA of the high copy of detection in plasma sample, bocavirus be certified as the unique pathogen of the patient (referring to：
Ursic T, Steyer A, Kopriva S, et al.Human bocavirus as the cause of a life-
Threatening infection.J Clin Microbiol, 2011,49：1179-81)；2011, one, Germany seriously exhaled
Inhale road infection baby be also certified as acute HBoV1 infection (referring to：RW,
M, van Koningsbruggen-Rietschel S, et al.Severe human bocavirus infection,
Germany.Emerg Infect Dis, 2011,17：2303-5.).These evidences support bocavirus individually to make strongly
For virulence factor.In addition, Holland one to being also further demonstrated that from infant to the longitudinal research of puberty health children
HBoV1 infection and acute respiratory disease and otitis have high correlation (referring to：Meriluoto M, Hedman L,
Tanner L, et al.Association of human bocavirus1infection with respiratory
Disease in childhood follow-up study, Finland.Emerg Infect Dis.2012,18：264-
71.)。
Bocavirus belongs to linear Single-stranded DNA virus, and its Genome Size is about 5kb, contains three ORFs, difference
Coding non-structural protein NS 1, NP1 and two have overlapping capsid protein VP1 and VP2.NS1 genes are transcribed, translated earliest, with
The duplication of viral DNA is related；VP1 and VP2 albumen is the structural proteins of virus, has a good antigenicity, and non-structural protein
NP1 function it is not immediately clear.Major antigen that VP2 albumen contains bocavirus simultaneously can form virus-like particle (VLP),
Bocavirus VLP has the form and antigenicity similar with virion, is successfully used to bocavirus antibody as antigen
Detection.
HBoV1-4 major structural protein VP2 amino acid alignment result is shown, several different bocavirus
Higher homology between VP2 protein sequences be present, for example, HBoV1VP2 and HBoV2-4VP2 sequence homology is about 77-
78%, HBoV2VP2 and HBoV3-4VP2 sequence homology are about 88-90%, HBoV3VP2 and HBoV4VP2 sequence homology
Property is about 90.7%.Recent research also demonstrates that between HBoV1-4VP2VLPs serological cross reaction be present (referring to Kantola
K, Hedman L, Arthur J, et al.Seroepidemiology of human bocaviruses1-4.J Infect
Dis, 2011,204：1403-12；Guo L, Wang Y, Zhou H, et al.Differential seroprevalence of
Human bocavirus species1-4in Beijing, China.PLoS One, 2012,7：e39644).This explanation exists
Common epitope is there may be between HBoV1-4VP2.
At present, the serodiagnosis to bocavirus mainly using VLP as antigen progress elisa assay, but VLP
Preparation need cell cultivation process, relatively arduously, and time-consuming longer, it is inconvenient to prepare, and is not suitable for broad scale research.And
Have from bocavirus major structural protein VP2 epitope well-conserved and specific.Therefore epitope base is come from
The ELISA method of plinth can carry out quick and simple serodiagnosis to bocavirus.However, for bocavirus VP2 eggs
White epitope research is without related report.
In our current research, two highly conserved specificity epitopes in HBoV1-4 of our successful identifications.It is basic herein
On, we establish the bocavirus IgMELISA detection methods based on the specificity epitope, this method and winning based on VLP
Card virus IgM ELISA detection method has good correlation.
The content of the invention
In our current research, we utilize antibody of the bocavirus VP2 genetic fragments phage library to HBoV1-3VP2 albumen
And bocavirus positive human serum is screened, the stronger epitope of four cluster reactivity, binding sequence analysis, synthesis 2 are obtained
Bar dominant antigen epitope polypeptide, mouse is immunized after being coupled with KLH, obtains antiserum.Pass through enzyme linked immunosorbent assay and Diagnosis of Sghistosomiasis
The methods of mark identification, sequence alignment, it is determined that 2 general bocavirus specific antigen epitopes in HBoV1-4, and establish
Bocavirus IgM detection methods based on the specificity epitope.
First aspect present invention provides bocavirus specific antigen epitope peptide general in HBoV1-4, and its sequence is SEQ
ID NO：1 or SEQ ID NO：2.
Second aspect of the present invention provides a kind of non-diagnostic method for detecting bocavirus antibody, including：
1) sample is made to be contacted with the bocavirus specific antigen epitope peptide described in first aspect present invention；
2) antigen-antibody reaction is detected, as a result bocavirus antibody be present for positive explanation.
In one embodiment, the sample is human serum.
In another embodiment, the process of the detection antigen-antibody reaction is：First add horseradish peroxidase
Goat anti human IgM is incubated, and is added TMB colour developings, is finally detected light absorption value.
In still another embodiment, the above-mentioned light absorption value to be detected is OD450.
Third aspect present invention provides a kind of bocavirus detection kit, and it includes rich described in first aspect present invention
Card viral-specific antigens epitope peptide.
In one embodiment, the kit also includes horseradish peroxidase Goat anti human IgM and TMB.
The bocavirus specific antigen epitope peptide that fourth aspect present invention is provided described in first aspect present invention is used to examine
Survey the non-diagnostic purposes of bocavirus antibody.
Fifth aspect present invention provides the bocavirus for the specific antigen epitope peptide described in first aspect present invention
Antibody.
In one embodiment, the bocavirus antibody is by the way that the specificity described in first aspect present invention is resisted
Former epitope peptide is coupled with KLH to be immunized mouse again and prepares.
Brief description of the drawings
Fig. 1 is bocavirus VP2 protein immunizations Dominant Epitopes screening figure.543 amino acid wherein shown in figure the top
Sequence is the amino acid sequence of HBoV1 111-BJ07 strains (GenBank JQ240469) VP2 albumen.
Fig. 2 be synthetic peptide antiserum anti-P1, anti-P2 titre (A) and its with HBoV1-4, human parvovirus B19
With PARV4 VLP reaction (B).
Fig. 3 analyzes for epitope P1 and P2 key amino acid.Wherein " peptide mortifier " refers to each synthesis described in embodiment 3
Small peptide.
Embodiment
The present invention is expanded on further with reference to preferred embodiment.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.Unreceipted specific experimental method in the following example, generally according to normal experiment bar
Part and method, such as Molecular Cloning: A Laboratory room handbook (Sambrook, et al.New York：Cold Spring Harbor
Laboratory Press, 1989) method that method or reagent manufacturer provide described in.
The identification of the bocavirus VP2 protein immunization Dominant Epitopes of embodiment 1.
HBoV1-3VP2 genes used herein are derived from the bocavirus positive diarrheic stools sample 111-BJ07,211-
(No. Genbank is respectively by BJ07 and 46-BJ07：JQ240469, JQ240470 and HM132056).According to method disclosed in document
The random phage storehouse of HBoV1-3VP2 genes is built respectively, is respectively designated as storehouse HBoV1-VP2, HBoV2-VP2 and HBoV3-
VP2.Then be utilized respectively applicant preparation the anti-HBoV1-3VP2 antibody of mouse and bocavirus positive human serum to HBoV1-
3VP2 gene random phages storehouse carry out affine screening (random phage storehouse preparation method and screening technique referring to Khurana S,
Suguitan AL Jr, Rivera Y, et al.Antigenic fingerprinting of H5N1avian influenza
using convalescent sera and monoclonal antibodies reveals potential vaccine
And diagnostic targets.PLoS Med, 20096：e1000049).Fig. 1 is shown respectively with the anti-HBoV1-3VP2 of mouse
The epitope distribution situation for the positive colony that antibody and bocavirus positive human serum are obtained by affine screening, the antigen filtered out
Epitope can be further divided into four clusters (I, II, III, IV).In 1-20aa and 162- it can be seen from result shown in figure
Nearby Dominant Epitopes be present in 180aa.
The immunogenicity and antigen property of the immunodominant epitope of embodiment 2.
The coupling of 2.1 peptides and KLH
According to bocavirus epitope distribution situation and HBoV1VP2 sequential analysis of protein, two advantages of P1 and P2 are synthesized
(sequence is respectively such as SEQ ID NO for epitope peptide：1 and SEQ ID NO：Shown in 2), and be coupled respectively with KLH to increase polypeptide
Immunogenicity (table 1), for mouse to be immunized.The synthesis of peptide and the coupling commission limited public affairs of Shanghai biotechnology with KLH
Department completes.
The sequence of the immune peptide-KLH conjugates used of table 1.
| Title | Position | Sequence |
| P1-KLH | 1-20a | KLH-CMSDTDIQDQQPDTVDAPQNT |
| P2-KLH | 162-180a | KLH-CEHAYPNASHPWDEDVMPDL |
aRepresent polypeptide in HBoV1 111-BJ07 strains (GenBank JQ240469) VP2 albumen (sequence such as SEQ ID
NO：Shown in 3) in relevant position
2.2 immunization protocol
The female Balb/c mouse of 6-8 week old are immunized with above-mentioned KLH coupled peptides.Immunization protocol is as follows：Enter altogether
3 immune, every minor ticks 14 days of row.During first immunisation, isometric peptide-KLH conjugates are mixed with Freund's complete adjuvant, every
The μ g peptide-KLH conjugates of mouse subcutaneous injection 100.During booster immunization, by isometric peptide-KLH conjugates and incomplete Freund's adjuvant
Mixing, every μ g peptide-KLH conjugate of mouse subcutaneous injection 50.Eyeball is taken a blood sample after last time is immune 14 days.Separation serum is put
Saved backup in -20 DEG C.
2.3 immune serum titer determinations
Respectively by P1 and P2 coating buffer (pH9.6 carbonate buffer solutions：Sodium carbonate 1.59g, sodium acid carbonate 2.93g, adds
Water is diluted to 1000mL) (the μ g/ holes of final concentration 1) coated elisa plate (Costar Products) afterwards are diluted, put 4 DEG C overnight,
Addition confining liquid (1%BSA/PBS) is put 37 DEG C and is incubated 2 hours, is washed 5 times with 0.1%PBST buffer solutions, dries cleaning solution.Often
Hole adds the corresponding mouse immune serum that 100 μ l are serially diluted and (proceed by doubling dilution to 1: 160,000 from 1: 2,500),
Non- immune serum is set up simultaneously as control, is put 37 DEG C and is incubated 1 hour, gets rid of liquid, washed with 0.1%PBST buffer solutions
5 times.Then horseradish peroxidase goat anti-mouse IgG (the Sigma companies production of 100 μ l1: 40,000 dilutions is added per hole
Product), put 37 DEG C and be incubated 1 hour, get rid of liquid, washed 5 times with 0.1%PBST buffer solutions.Add 100 μ l3 per hole, 3 ', 5,5 '-
Tetramethyl benzidine (TMB) substrate lucifuge develops the color 15 minutes, adds 50 μ l2M H2SO4Terminating reaction, determine the extinction at 450nm
Value.As a result see Fig. 2A, display P1 and P2 induces stronger antibody, using 2.1 times of mice serum OD450nm before immune as
Cut-off values, antibody titer up to 1: 160,000, prompt P1 and P2 to be respectively provided with stronger immunogenicity.
2.4 immune serums and HBoV1-4 VLP reactivity and the specificity of reaction
HBoV1-4, assays for parvovirus B 19 and PARV4VLPs are prepared according to method disclosed in document, and is surpassed from purifying
[referring to Guo L, Wang Y, Zhou H, et al.Differential seroprevalence of human bocavirus
Species1-4in Beijing, C hina.PLoS One.2012；7(6)：e39644].
100ng is surpassed into the HBoV1-4, assays for parvovirus B 19 and PARV4VLP from purifying, and cell controls are carried out respectively
Nitrocellulose filter (Pall Products) is gone to after 12%SDS-PAGE.2 hours are closed with 5% defatted milk room temperature, and 1: 1,
The immune serum of 000 dilution is incubated overnight.Film washs 5 times through 0.1%PBST, adds 1: 10,000 dilutionThe goat anti-mouse IgG secondary antibody (Li-Cor companies) of Fluor800 marks.After fully washing film with 0.1%PBST, useNear infrared imaging system (Li-Cor companies) sweeps film, is analyzed with Odyssey softwares.As a result see Fig. 2 B, exempt from
For epidemic disease mice serum when carrying out 1: 1000 dilution, P1 and P2 immune serums carry out specific reaction with HBoV1-4VLP,
And do not reacted with assays for parvovirus B 19 and PARV4VLP, this explanation P1 and P2 is shared, specific antigen in HBoV1-4
Epitope.
Embodiment 3.P1 and P2 synthesize the finely positioning of peptide epitopes
In order to further determine that the amino acid residue to be played a crucial role in P1 and P2 peptides, we have synthesized a series of peptide fragments,
It the results are shown in Table 2 and table 3.
Table 2 is used for the peptide fragment of P1 peptide finely positionings
Table 3 is used for the peptide fragment of P2 peptide finely positionings
Competitive ELISA detects：Respectively by P1 and P2 coating buffer (pH9.6 carbonate buffer solutions：Sodium carbonate 1.59g, carbon
Sour hydrogen sodium 2.93g, is diluted with water to 1000mL) (1 μ g/ holes) coated elisa plate (Costar Products) afterwards are diluted, put 4
DEG C overnight, add confining liquid (1%BSA/PBS) put 37 DEG C be incubated 2 hours, washed 5 times with 0.1%PBST buffer solutions, drying is washed
Wash liquid.Simultaneously by various concentrations (30× 10pmol/ holes, 31× 10pmol/ holes, 32× 10pmol/ holes, 33× 10pmol/ holes, 34
× 10pmol/ holes, 35× 10pmol/ holes, 36× 10pmol/ holes, 37× 10pmol/ holes) above-mentioned synthetic peptide (in competitive ELISA
The middle mortifier as full-length peptide) mixed with P1, P2 mouse resisting anteserum (prepared by embodiment 2) of 1: 1000 dilution, and set up not
P1, P2 mouse resisting anteserum of synthetic peptide is added to put 4 DEG C as control and be incubated 2 hours.By peptide-mixtures of antibodies after incubation and right
Added according to the amount in 100 μ l/ holes in the good ELISA Plate of above-mentioned coating, 37 DEG C are incubated 1 hour, get rid of liquid, are delayed with 0.1%PBST
Fliud flushing is washed 5 times.Then the horseradish peroxidase goat anti-mouse IgG of 100 μ l1: 40,000 dilutions is added to every hole
(Sigma Products), put 37 DEG C and be incubated 1 hour, get rid of liquid, washed 5 times with 0.1%PBST buffer solutions.Add again to every hole
Enter 100 μ l3,3 ', 5,5 '-tetramethyl benzidine (TMB) substrate lucifuge develops the color 15 minutes, adds 50 μ l2M H2SO4Terminate anti-
Should, determine the light absorption value at 450nm.To be not added with synthesizing light absorption value of the hole (control) of small peptide at 450nm as standard value
(100%) the antibody binding percentage of each synthesis small peptide of various concentrations, is obtained.
According to different peptide fragments ELISA reaction results (Fig. 3) with P1, P2 mouse resisting anteserum respectively, it is inferred that P1 pass
Key peptide fragment is6IQDQQPDTVD15, key amino acid site is Q7And P11；P2 crucial peptide fragment is161NASHP171With176VMPDL180, key amino acid site is N167, H170, P178, and L180。
The foundation of bocavirus IgM antibody detection method of the embodiment 4. based on epitope and with based on virus-like particle
(VLP) comparison of IgM detection methods
Respectively by P1, P2, P1+P2 or bocavirus VLP coating buffer (pH9.6 carbonate buffer solutions：Sodium carbonate
1.59g, sodium acid carbonate 2.93g, is diluted with water to 1000mL) it is diluted (peptide：1 μ g/ holes, VLP：50ng/ holes) enzyme is coated with afterwards
Target (Costar Products), put 4 DEG C overnight, addition confining liquid (1%BSA/PBS) is put 37 DEG C and is incubated 2 hours, with 0.1%
PBST buffer solutions wash 5 times.The bocavirus positive human serum (coming from BJ Children's Hospital) of 1: 200 dilution is added, puts 37 DEG C
It is incubated 1 hour, gets rid of liquid, washed 5 times with 0.1%PBST buffer solutions.Then the peppery of 100 μ l1: 40,000 dilutions is added per hole
Root peroxidase Goat anti human IgM (Sigma Products), put 37 DEG C and be incubated 1 hour, get rid of liquid, delayed with 0.1%PBST
Fliud flushing is washed 5 times.Add 100 μ l3 per hole, 3 ', 5,5 '-tetramethyl benzidine (TMB) substrate lucifuge develops the color 15 minutes, adds 50
μl2M H2SO4Terminating reaction, determine the light absorption value at 450nm.
Method according to the literature is (referring to Kahn JS, Kesebir D, Cotmore SF, et
al..Seroepidemiology of human bocavirus defined using recombinant virus-like
Particles.J Infect Dis.2008,198：41-50；Hustedt JW, Christie C, Hustedt MM, et
Al.Seroepidemiology of human bocavirus infection in Jamaica.PLoS One.2012,7：
E38206.) determine cut-off values, first using 0.2 as the cut-off values assumed, by it is all be less than 0.2 absorbance meter
Its mean and standard error are calculated, is then used as cut-off values using+3 times of standard errors of mean.For the rich card disease based on virus-like particle
Malicious IgM detection methods, the light absorption value are considered as the positive more than 0.18, are then considered as feminine gender below, for the rich card based on epitope
Virus IgM antibody detection method, the light absorption value are considered as the positive more than 0.15, are then considered as feminine gender below.
P1IgM ELISA, P2IgM ELISA, P1+P2IgM ELISA and VLP IgM ELISA comparison be shown in Table 4 (a, b,
C), as a result show that the IgM detection methods based on epitope peptide have preferable Sensitivity and Specificity, P1IgM ELISA specificity
It is respectively with sensitiveness：97.4% and 72.7%, P2IgM ELISA specificity and sensitiveness are respectively：98.7% and 72.7%,
P1+P2IgM ELISA specificity and sensitiveness are respectively：97.4% and 90.9%, statistical analysis shows that two methods have
Preferable correlation, and utilize the Detection results of two peptides to be better than the Detection results of wall scroll peptide.
The comparison (a) of the synthetic peptide IgM antibody detection method of table 4 and VLP IgM detection methods
Sensitiveness=72.7%, specificity=97.4%；
χ2=40.8, P ＜ 0.01, two methods have correlation
(b)
Sensitiveness=72.7%, specificity=98.7%；
χ2=46.6, P ＜ 0.01, two methods have correlation
(c)
Sensitiveness=90.9%, specificity=97.4%；
χ2=51.2, P ＜ 0.01, two methods have correlation.
Claims (10)
1. the general bocavirus specific antigen epitope peptide in HBoV1-4, its sequence is SEQ ID NO：1 or SEQ ID
NO：2.
2. a kind of non-diagnostic method for detecting bocavirus antibody, including：
1) sample is made to be contacted with the bocavirus specific antigen epitope peptide of claim 1；
2) antigen-antibody reaction is detected, as a result bocavirus antibody be present for positive explanation.
3. the method for claim 2, wherein the sample is human serum.
4. the method for Claims 2 or 3, wherein the process of the detection antigen-antibody reaction is：First add horseradish peroxidase
Enzyme Goat anti human IgM is incubated, and is added TMB colour developings, is finally detected light absorption value.
5. the method for claim 4, wherein the light absorption value is OD450.
6. a kind of bocavirus detection kit, it includes the bocavirus specific antigen epitope peptide described in claim 1.
7. the kit described in claim 6, also comprising horseradish peroxidase Goat anti human IgM and TMB.
8. the bocavirus specific antigen epitope peptide described in claim 1 is used for the non-diagnostic use for detecting bocavirus antibody
On the way.
9. for the bocavirus antibody of the specific antigen epitope peptide described in claim 1.
10. the bocavirus antibody of claim 9, it is by by the specific antigen epitope peptide and KLH described in claim 1
Coupling is immunized mouse again and prepared.
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| CN101094704A (en) * | 2004-11-04 | 2007-12-26 | 麦迪塞弗环球控股有限公司 | Exchange needle retractable safety syringe |
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