CN1746183A - Coronavirus (SARS-CoV) B-cell antigen determinant with extensive cross-immunoreactivity - Google Patents
Coronavirus (SARS-CoV) B-cell antigen determinant with extensive cross-immunoreactivity Download PDFInfo
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Abstract
The present invention relates to the B-cell antigen determinant of one group of coronavirus relevant with severe acute respiratory syndrome, a kind ofly detect the method that whether contains the coronavirus antibody relevant in the testing sample, and/or whether a kind of experimenter of detection infects the method for SARS-CoV with severe acute respiratory syndrome.The invention still further relates to a kind of test kit that whether contains the coronavirus antibody relevant in the testing sample that detects with severe acute respiratory syndrome.The invention further relates to described coronavirus B-cell antigen determinant and be used to prepare the purposes of the medicine that prevents severe acute respiratory syndrome, and the vaccine of a kind of prevention coronavirus infection relevant with severe acute respiratory syndrome.
Description
Invention field
The present invention relates to the B-cell antigen determinant spectrum of one group of coronavirus relevant (SARS-CoV) with severe acute respiratory syndrome, a kind ofly detect the method that whether contains the coronavirus antibody relevant in the testing sample, and/or whether a kind of experimenter of detection infects the method for SARS-CoV with severe acute respiratory syndrome.The invention still further relates to a kind of test kit that whether contains the coronavirus antibody relevant in the testing sample that detects with severe acute respiratory syndrome.The invention further relates to described coronavirus B-cell antigen determinant and compose the purposes that is used to prepare the medicine that prevents severe acute respiratory syndrome, and a kind of vaccine antigen that prevents the coronavirus infection relevant with severe acute respiratory syndrome.
Background technology
The coronavirus relevant with the mankind (Severe Acute Respiratory SyndromeCoronavirus (SARS-CoV)) has been confirmed to be a new viroid, human with the fastest so far velocity determination its gene order, mainly by RNA-RNA-dependent polysaccharase, furcella (S), film (M), tunicle (E), nucleocapsid (N), polysaccharase albumen such as (P) form (1, people such as MarraMA, the genome sequence of SARS associated coronavirus, Sciencexpress, www.sciencexpress.org, on May 1st, 2003; 2, people such as Rota PA, the sign of the novel coronavirus relevant, Sciencexpress, www.sciencexpress.org, on May 1st, 2003 with severe acute respiratory syndrome; 3, Qin E ' de waits the people, the complete sequence of SARS correlated virus and comparative analysis (Isolate BJ01), and Chinese ScienceBulletin 2003,48 (10): 941-948; 4, Peoros JS waits the people, the possible cause of disease-coronavirus of severe acute respiratory syndrome, The Lancet, www.nejm.org, on April 8th, 2003; 5, people such as Ksiazek TG, the novel coronavirus relevant, N Engl J Med, 2003,348 (20): 1953～1966 with severe acute respiratory syndrome; 6, people such as Dorsten C, the evaluation of novel coronavirus among the severe acute respiratory syndrome patient, N Engl J Med, www.nejm.org, on April 10th, 2003; People such as Anand K, the main proteolytic enzyme of coronavirus (3CLpro) structure: design the basis of anti-SARS medicine, Sciencexpress, www.sciencexpress.org, on May 13rd, 2003).Its median spine (S) albumen, film (M) albumen and tunicle (E) albumen have been formed virus coat, are virus identification, combination and the protein that enters host cell.Especially furcella S albumen is key albumen (Fig. 1).
Though it is host receptor bonded position that literature research result shows the proteic 417-546 sequence of HCoV-229E furcella S, point out that also different coronavirus enters host cell by using different acceptors simultaneously.This relevant with the proteic variation of furcella S probably (people such as Marra MA, the source is the same).Nucleocapsid (N) albumen belongs to albumen in the endochylema, and the core and the geneome RNA that are in virion exist with the bonded form.Meeting and N protein binding can be discerned and be wrapped in the virion in conjunction with product by M albumen after viral RNA duplicated in tenuigenin and finishes.So N and M albumen have tangible relation with virus duplicating in host cell.N albumen is one of the main site of host T-cell recognition (see figure 1).Thereby, the polypeptide compound chemical libraries of the synthetic viral protein of development, for the multiple minimum polypeptide antigen determinant (comprising B-cell and T-cell antigen determinant) of seeking virus surface proteins, and then seek and develop synthetic polypeptide vaccine, reagent for clinical diagnosis and serum treatment scheme that extremely important meaning is arranged.Simultaneously, this chemical libraries also can be used for screening and finding the multiple aglucon of viral bind receptor, and then the blocking-up coronavirus enters host cell, and the development medicine pharmacological agent of drug-resistant virus (particularly for) has great reference value.
At present, seeking antigenic determinant method the most commonly used is to utilize various computer softwares, is detained parameters such as coefficient, spirane structure, conservative property and predicts by the wetting ability of reported sequence employing, hydrophobicity, corner structure, HPLC.Our experimental result shows, this Forecasting Methodology and the actual sequence that records only have 30%～50% repeatability (1, ZHANG XM, LIU G and SUN MJ, Brain Research, 2000,868:157-164; 2, ZHANGXM, LIU G and SUN MJ, Brain Research, 2001,895:277-282.).Another kind method is to adopt the research (" juxtaposition " polypeptide compound) of combinatorial chemistry technique to proteantigen determinant spectrum
Combinatorial chemistry is become one to chemosynthesis, Computer Design choosing, the compound that can produce many kinds of structurally associateds simultaneously but change in order, and employing is highly sensitive and high-throughout biological method screens simultaneously to these compounds, therefrom determine the material of biologically active, the technology of perhaps brand-new lead compound.Thereby present technique has related to " like medicine " molecule of numerous species, as small molecules heterogeneous ring compound, natural product, biological oligomer and stand-in thereof etc.The present invention is at aforementioned patent (a kind of peptide library, its synthetic method and the active fragments that screens from this library, application number: 200310101892.6.) the biological oligomer chemiluminescent polypeptide of the synthetic SARS-CoV that makes up " juxtaposition " storehouse basis enterprising confirmed to have the antigenic determinant spectrum of extensive cross-immunoreactivity.The characteristics of this method are that the amino-acid residue peptide (such as 1 to 50 amino-acid residue) with certain number is synthetic " juxtaposition " fragment, protein sequence (is perhaps misplaced at interval by misplacing one by one, comprise from 2 amino acid whose total bits to synthetic peptide fragment) mode all be synthesized, carry out antigen-antibody reaction (perhaps screening reaction of other biological purpose etc. then, as screen and find T-cellular immunization antigenic determinant, and the receptor-ligand of SARS-CoV etc.), once just can obtain all the shortest antigenic determinants, and then plot the antigenic determinant spectrum.These bioactive peptides are carried out large-scale anti-SARS-CoV people's positive serum screening reaction through proper extension or after linear in order the connection, thereby confirmed to can be used for preparing the B-cell polypeptide compound of diagnostic reagent, medicine and vaccine and their collection of illustrative plates.
The inventor adopts solid phase synthesis technique, synthesized a large amount of " juxtaposition " polypeptide compounds in the radio frequency coding techniques short period of time, synthesize the S relevant with SARS-CoV first, M, on the basis of proteic all " juxtaposition " polypeptide of E and N, being linked in sequence of small peptide by gained being had the active coronavirus of cross immunity (SARS-CoV) B cell antigen determinant, use more massive SARS-CoV the infected's positive serum that it is screened, successfully obtain having the active B cell antigen determinant spectrum of extensive cross immunity polypeptide, thereby finished the present invention.
Summary of the invention
One aspect of the present invention relates to one group of B-cell antigen determinant spectrum derived from coronavirus, and it is selected from least a following peptide sequence, or its arbitrary combination, comprising:
S7：
NH2-ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL-CONH2(SEQ ID NO：54)
S9：NH2-TDVSTAIHADQLTPAWRIYSTG-CONH2(SEQ IDNO：56)
S17：NH2-EIDRLNEVAKNLNESLIDLQELGKYEQY-CONH2(SEQ ID NO：64)
N4:NH2-TEPKKDKKKKTDEAQPLPQRQKK-CONH2 (SEQID NO:69) and
MEN-4e6(MEN-441)：NH2-LPQGTTLPKG-CONH2(SEQ IDNO：70)
Of the present invention finish be in chemosynthesis finish on the basis in coronavirus (SARS-CoV) structural protein " juxtaposition " peptide storehouse.
Concrete, the present invention is at first by adopting the physical code method according to disclosed relevant information in the prior art, synthesized " juxtaposition " chemiluminescent polypeptide storehouse,, obtained having the following decapeptide of immunizing antigen by making the reaction of this chemiluminescent polypeptide storehouse and part SARS positive serum:
SEQ ID NO：1 TSGSDLDRCT
SEQ ID NO：2 SGSDLDRCTT
SEQ ID NO：3 SDLDRCTTFD
SEQ ID NO：4 TTFDDVQAPN
SEQ ID NO：5 FDDVQAPNYT
SEQ ID NO：6 MGTQTHTMI
SEQ ID NO：7 MIFDNAFNCT
SEQ ID NO：8 KSGNFKHLRE
SEQ ID NO：9 GNFKHLREFV
SEQ ID NO：10 KDGFLYVYKG
SEQ ID NO：11 SVLYNSTFFS
SEQ ID NO：12 VRQIAPGQTG
SEQ ID NO：13 TRNIDATSTG
SEQ ID NO：14 RNIDATSTGN
SEQ ID NO：15 WPLNDYGFYT
SEQ ID NO：16 YRVVVLSFEL
SEQ ID NO：17 QCVNFNFNGL
SEQ ID NO：18 CVNFNFNGLT
SEQ ID NO：19 NFNGLTGTGV
SEQ ID NO：20 DVSTAIHADQ
SEQ ID NO：21 IGAEHVDTSY
SEQ ID NO：22 SIAYSNNTIA
SEQ ID NO：23 ITTEVMPVSM
SEQ ID NO：24 YGECLGDINA
SEQ ID NO：25 LTVLPPLLTD
SEQ ID NO：26 TALGKLQDVV
SEQ ID NO：27 NFGAISSVLN
SEQ ID NO：28 AISSVLNDIL
SEQ ID NO：29 RLDKVEAEVQ
SEQ ID NO：30 RLITGRLQSL
SEQ ID NO：31 QLIRAAEIRA
SEQ ID NO：32 SANLAATKMS
SEQ ID NO：33 QSKRVDFCGK
SEQ ID NO：34 VPSQERNFTT
SEQ ID NO：35 WFITQRNFFS
SEQ ID NO：36 SGNCDVVIGI
SEQ ID NO：37 FKNHTSPDVD
SEQ ID NO：38 DVDLGDISGI
SEQ ID NO：39 VDLGDISGIN
SEQ ID NO：40 NASVVNIQKE
SEQ ID NO：41 KEIDRLNEVA
SEQ ID NO：42 LQELGKYEQY
SEQ ID NO：43 VVIGIINNTV
SEQ ID NO：44 MVTILLCCMT
SEQ ID NO：45 VTILLCCMTS
SEQ ID NO:46 MEN441:LPQGTTLPKG and
SEQ ID NO：47 MEN533：EASKKPRQKR
In order to obtain to have the B cell antigen determinant spectrum of cross-immunoreactivity more widely, consider that generally having immunogenic polypeptide is greater than 15 amino-acid residues, the inventor is on above-mentioned small peptide basis, 22 the new peptide sequences that obtain by the small peptide antigenic determinant that is linked in sequence, and adopt 42 parts of anti-SARS-CoV antibody positive serum further to screen antigenic determinant and compose with more extensive immune cross-reactivity, therefrom obtain one group and have the active polypeptide of extensive cross-immune reaction, comprising:
S7：
NH2-ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL-CONH2(SEQ ID NO：54)
S9：NH2-TDVSTAIHADQLTPAWRIYSTG-CONH2(SEQ IDNO：56)
S17：NH2-EIDRLNEVAKNLNESLIDLQELGKYEQY-CONH2(SEQ ID NO：64)
N4:NH2-TEPKKDKKKKTDEAQPLPQRQKK-CONH2 (SEQID NO:69) and
MEN-4e6(MEN-441)：NH2-LPQGTTLPKG-CONH2(SEQ IDNO：70)
Above-mentioned 5 polypeptide result of 42 parts of anti-SARS-CoV antibody positive sero-reactions together show that S7 and S9 are positive with the anti-SARS-CoV antibody positive of 30 serum serum respectively, and promptly cross reacting rate is 71.4%; S17 and the anti-SARS-CoV antibody positive of 28 serum serum are positive, and promptly cross reacting rate is 66.7%; N4 and the anti-SARS-CoV antibody positive of 38 serum serum are positive, and promptly cross reacting rate is 90.5%; MEN4e6 and the anti-SARS-CoV antibody positive of 32 serum serum are positive, and promptly cross reacting rate is 76.2%.
In addition, show (Fig. 1) as competitive antigen contrast experiment with the SARS-CoV lysate, 5 synthetic polypeptide competitive inhibition combining of SARS-CoV lysate antigen and anti-SARS-CoV antibody serum, show that these 5 antigens have neutrality.The negative control peptide is respectively contrast 1: peptide at random, contrast 2: be positioned at proteic 945～963 places of S of SARS-CoV, confirm no antigen.
The above results confirms the high cross-immunoreactivity that gained antigenic determinant spectrum all has, show simultaneously, described antigenic determinant spectrum can be used in the detection of the coronavirus antibody relevant with severe acute respiratory syndrome, and preparation prevents the vaccine of the coronavirus relevant with severe acute respiratory syndrome.
Another aspect of the present invention relates to a kind of method that whether contains the coronavirus antibody relevant with severe acute respiratory syndrome in the testing sample that detects, comprise with the coronavirus B-cell antigen determinant at least a of the present invention or its arbitrary combination that detect significant quantity, be suitable under the antigen-antibody bonded condition with testing sample in conjunction with or contact.As the ELISA method of using among the present invention, and horseradish peroxidase of using in the method and substrate thereof.
Accordingly, the invention still further relates to a kind of experimenter of detection and whether infect the method for SARS-CoV, comprise with at least a described coronavirus B-cell antigen determinant or its arbitrary combination that detect significant quantity, be suitable under the antigen-antibody bonded condition with available from experimenter's sample combination or contact.
How those of ordinary skills know the consumption of the polypeptide of the present invention that is adopted according to selected concrete detection mode decision, and the display mode that adapts with selected certification mark.Those of ordinary skills also know the antibody detection method that can be used for described SARS associated coronavirus by the described antigen determinant polypeptide with one or more.
Another aspect of the present invention also relates to a kind ofly detecting whether contain the antigenic test kit of the coronavirus relevant with severe acute respiratory syndrome in the testing sample, wherein contain at least a coronavirus B-cell antigen determinant of the present invention or its arbitrary combination, and suitable developer and damping fluid.
The present invention comprises that also described coronavirus B-cell antigen determinant is used to prepare the purposes of the medicine that prevents severe acute respiratory syndrome.
The present invention also particularly including the vaccine of a kind of prevention coronavirus infection relevant with severe acute respiratory syndrome, wherein contains the described coronavirus B-cell antigen determinant that prevents significant quantity, optional adjuvant, and pharmaceutically acceptable carrier.
Description of drawings
Fig. 1 combines result of experiment with the SARS-CoV employing virus cracking liquid as competitive antigen.
Embodiment
Combinatorial chemistry research divides a three phases: molecular diversity compound library synthetic; Mass scareening (carrying out activity detects); The structure determination of bioactive molecule.
1, the foundation in peptide storehouse
Set up the necessary many aspects of forethought of peptide library: the design template molecule; Select suitable construction unit; Determine synthetic schemes; How to finish semi-automatic or automatically synthetic etc.
Our main purpose is the B-cell line style antigenic determinant that seeks at the people of SARS-CoV virus among the present invention, and draws the antigenic determinant spectrum with cross reactivity.Select the synthesis mode of dislocation by turn can directly find minimum antigenic determinant.Thereby line style 10 peptides are selected as template molecule.Protein is formed with natural L-configuration amino acid, thereby the construction unit natural selection of this chemical libraries is 20 kinds of natural L-configuration amino acid.High-throughput ground synthetic method generally adopts solid phase synthesis technique, and characteristics are that the purifying of intermediate only adopts simple washing and filter operation to finish.Simultaneously, can use excessive greatly construction unit to guarantee to react in the reaction and carry out fully, and easily semi-automation or automatization.Thereby we have selected solid phase synthesis technique, have finished the synthetic of whole polypeptide compounds on resin.When using 20 kinds of natural L-configuration amino acid to synthesize thousands of polypeptide compound as construction unit; identical reaction conditions can carry out under the condition of peptide coding synchronously, carries out reactions steps etc. as identical deprotection steps, different peptide sequence with identical protection amino acid.So just can finish on a large scale, synthesize apace.Wherein coded system have multiple, as chemical code, physical code etc.The characteristics of physical code are not have " chemical pollution ", chemical decoding step that need not be unnecessary etc.Thereby we have selected the sorting technique of IRORI coding-decoding.Once can operate the synthetic of thousands of compounds, we have selected the once scale of synthetic 600～700 polypeptide compounds in this experiment.
2, mass scareening
Generally, screening can be divided into random screening and directed screening.But no matter be random screening or directed screening, all will consider: selected filtering mode is solid phase screening or liquid phase screening, or screen with what method (cell function screening, acceptor screening, antibody screening etc.), with which kind of indicator (isotopic labeling, fluorescent mark, dyeing) etc.Concrete screening method has three kinds: solid phase screening, liquid phase screening and both combinations.The ELISA screening method that uses among the present invention belongs to directed liquid phase screening, and biological respinse is that Ag-Ab is in conjunction with experiment.Contain the anti-SARS-CoV polyclonal antibody of people (comprising IgG and IgM) in the anti-SARS-CoV positive serum of people.Polypeptide compound in being coated on 96 hole Sptting plates is with after the anti-SARS-CoV polyclonal antibody of people combines, through careful washing can flush away not in conjunction with unnecessary or even non-specific binding antibody and serum.At this moment, add and to finish secondary bond, and horseradish peroxidase is retained in the system with the anti-IgG or the anti-IgM antibody of horseradish peroxidase mark.This horseradish peroxidase under certain conditions can the catalytic hydrolysis correspondence substrate, and can under certain wavelengths, record absorption value.The size of this absorption value is strong and weak relevant with what with the anti-SARS-CoV polyclonal antibody of people bonded.
3, confirm the bioactive molecule structure
Coding techniques has been applied to resolve the structure of high-activity compound in the combinatorial libraries.That propose this technology the earliest is Brenner and Lerner, and Nicolaou etc. have proposed radio frequency coding synthesizer and broken through coding form in the past, and it is to be based upon radiofrequency signal and to use on the semiconductor memory basis of multi-functional little reaction instrument.
The radio frequency coding techniques that uses among the present invention can be directly with cracking polypeptide compound coding corresponding aperture number in 96 orifice plates.Thereby the active aperture that screening obtains is crossed and the sub-sequence number of encoding contrasts the sequence that can provide polypeptide.
Concrete, prepared described polypeptide libraries in the following embodiments respectively, and, screened described chemistry of peptides storehouse by adopting anti-SARS-CoV positive serum.Further order is synthesized polypeptide on the basis of gained polypeptide libraries, has obtained the antigenic determinant spectrum at SARS-CoV virus B-cell of the present invention.
Embodiment 1: the preparation process of polypeptide
For synthetic polypeptide of the present invention, the coding instrument of employing is an IRORI Sorting solid phase synthetic instrument, MicroKan microreactor and Rf radio frequency coding, manufacturers: IroriQuantum Microchemistry, 9640Towne Centre Drive, San Diego CA92121, USA)
1. add resin and radio frequency: choose 47 Microkan and be respectively charged into 15 milligrams of Rink resins and radio frequency and cover tight lid;
2. merge Microkan, in a proper container, take off Fmoc protecting group 15min * 2 with 20% piperidines/DMF;
3. washing resin: DMF 3min * 6, DMF3min * 3 are heavily steamed in DCM 3min * 3;
4.IRORI radio frequency coding (or decoding): adopt IRORI Sorting program, Microkan is dispensed in the different aminoacids reaction flask according to the peptide coding order;
5. in each reaction flask, add the Fmoc-protection amino acid, HOBt, the HBTU that are dissolved in calculated amount among the DMF, jolting reaction 3h;
6. merge Microkan and washing resin DMF 3min * 3 times;
7. repeating step 3～7, until connecting whole amino acid;
8. merge Microkan, repeated for 3～4 steps;
9. adopt 15%Ac
2O/CH
2Cl
2The aminoterminal 15min of acetylize polypeptide;
10. use CH
2Cl
2Behind 3min * 3 time, room temperature is dried;
11. employing IRORI Sorting decoding Microkan also is assigned in 15 milliliters of corresponding cracking tubes;
12. each Microkan is with 1mL lysate cracking at room temperature 2h;
13. use 1mL lysate washing by soaking Microkan 5min again;
14. merge lysate and washings;
15. drying up, inert gas flow is concentrated into residual a small amount of lysate;
16. add in advance with the abundant refrigerative methyl tertiary butyl ether/sherwood oil of ice-water bath (v/v:3/1), put into ice-water bath and cool off 20min;
17. centrifugation polypeptide compound (more than the 3000rpm), the supernatant of carefully inclining behind the 10min;
18. add refrigerative methyl tertiary butyl ether/sherwood oil again, thorough washing, centrifugal, the supernatant of inclining, twice totally;
19. the polypeptide resistates is placed under the room temperature, until complete drying;
20. with polypeptide compound 10%HOAc/H
2O or 30%CH
3CN/H
2O or 60%CH
3CN/H
2After the O dissolving, detect purity and correct molecular weight through HPLC-MS.
According to above-mentioned steps, synthesized following decapeptide (SEQ ID NO:1-47) respectively:
SEQ ID NO：1 TSGSDLDRCT
SEQ ID NO：2 SGSDLDRCTT
SEQ ID NO：3 SDLDRCTTFD
SEQ ID NO：4 TTFDDVQAPN
SEQ ID NO：5 FDDVQAPNYT
SEQ ID NO：6 MGTQTHTMI
SEQ ID NO：7 MIFDNAFNCT
SEQ ID NO：8 KSGNFKHLRE
SEQ ID NO：9 GNFKHLREFV
SEQ ID NO：10 KDGFLYVYKG
SEQ ID NO：11 SVLYNSTFFS
SEQ ID NO：12 VRQIAPGQTG
SEQ ID NO：13 TRNIDATSTG
SEQ ID NO：14 RNIDATSTGN
SEQ ID NO：15 WPLNDYGFYT
SEQ ID NO：16 YRVVVLSFEL
SEQ ID NO：17 QCVNFNFNGL
SEQ ID NO：18 CVNFNFNGLT
SEQ ID NO：19 NFNGLTGTGV
SEQ ID NO：20 DVSTAIHADQ
SEQ ID NO：21 IGAEHVDTSY
SEQ ID NO：22 SIAYSNNTIA
SEQ ID NO：23 ITTEVMPVSM
SEQ ID NO：24 YGECLGDINA
SEQ ID NO：25 LTVLPPLLTD
SEQ ID NO：26 TALGKLQDVV
SEQ ID NO：27 NFGAISSVLN
SEQ ID NO：28 AISSVLNDIL
SEQ ID NO：29 RLDKVEAEVQ
SEQ ID NO：30 RLITGRLQSL
SEQ ID NO：31 QLIRAAEIRA
SEQ ID NO：32 SANLAATKMS
SEQ ID NO：33 QSKRVDFCGK
SEQ ID NO：34 VPSQERNFTT
SEQ ID NO：35 WFITQRNFFS
SEQ ID NO：36 SGNCDVVIGI
SEQ ID NO：37 FKNHTSPDVD
SEQ ID NO：38 DVDLGDISGI
SEQ ID NO：39 VDLGDISGIN
SEQ ID NO：40 NASVVNIQKE
SEQ ID NO：41 KEIDRLNEVA
SEQ ID NO：42 LQELGKYEQY
SEQ ID NO：43 VVIGIINNTV
SEQ ID NO：44 MVTILLCCMT
SEQ ID NO：45 VTILLCCMTS
SEQ ID NO:46 MEN441:LPQGTTLPKG and
SEQ ID NO：47 MEN533：EASKKPRQKR。
On above-mentioned synthetic polypeptide basis, connect by further order design, adopt same Code And Decode synthetic route and method, obtain following peptide sequence:
S1：NH
2-SGSDLDRCTTFDDVQAPNYT-CONH
2(12～31，20AA，SEQ ID NO：48)
S2：NH
2-SKPMGTQTHTMIFDNAFNCTFEY-CONH
2(141～163，23AA，SEQ ID NO：49)
S3：NH
2-TAFSPAQDTWGTSAAAYFVGYLKPTTF-CONH
2(236～262，27AA，SEQ ID NO：50)
S4：NH
2-GIYQTSNFRVVPSGD-CONH
2(298～312，15AA，SEQID NO：51)
S5：NH
2-RKKISNCVADYSVLYNSTFFSTFKCYG-CONH
2(342～368，27AA，SEQ ID NO：52)
S6：NH
2-VLAWNTRNIDATSTGNYNYKYRYLRH-CONH
2(420～445，26AA，SEQ ID NO：53)
S7：
NH
2-ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL-CONH
2(471～503，33AA，SEQ ID NO：54)
S8：NH
2-CVNFNFNGLTGTGV-CONH
2(524～537，14AA，SEQID NO：55)
S9：NH
2-TDVSTAIHADQLTPAWRIYSTG-CONH
2(604～625，22AA，SEQ ID NO：56)
S10：NH
2-ITTEVMPVSMAKTSVDCNMY-CONH
2(704～723，20AA，SEQ ID NO：57)
S11：NH
2-AQKFNGLTVLPPLLTDD-CONH
2(834～850，17AA，SEQ ID NO：58)
S12：NH
2-VKQLSSNFGAISSVLNDIL-CON
2(945～963，19AA，SEQ ID NO：59)
S13：NH
2-RLDKVEAEVQIDRLITGRLQSL-CONH
2(965～986，22AA，SEQ ID NO：60)
S14：NH
2-SANLAATKMS-CONH
2(1003～1012，10AA，SEQ IDNO：61)
S15：NH
2-REGVFVFNGTSWFITQRNFFSPQIITTD-CONH
2(1073～1100，28AA，SEQ ID NO：62)
S16：NH
2-TSPDVDLGDISGINASVVNIQKEID-CONH
2(1142～1166，25AA，SEQ ID NO：63)
S17：NH
2-EIDRLNEVAKNLNESLIDLQELGKYEQY-CONH
2(1164～1191，28AA，SEQ ID NO：64)
M1：NH2-A W I M L L Q F A Y S N R N R F L Y I I-CONH2(29～48，20AA，SEQ ID NO：65)
N1：NH2-G R N G A R P K Q R-CONH2(32～41，10AA，SEQ IDNO：66)
N2：NH2-S R G N S P A R M A-CONH2(203～212，10AA，SEQID NO：67)
N3：NH2-E A S K K P R Q K R T A T-CONH2(254～266，13AA，SEQ ID NO：68)
N4：NH2-T E P K K D K K K K T D E A Q P L P Q R QKK-CONH2(367～389，23AA，SEQ ID NO：69)
The contrast polypeptide
MEN 4e6：LPQGTTLPKG(SEQ ID NO：70)
S1-b10：PSGFNTLKPI(SEQ ID NO：71)
The immunological cross-reaction of embodiment 2:ELISA method research B-cell antigen determinant
The polypeptide compound that embodiment 1 is obtained respectively with 42 parts of anti-SARS-CoV antibody positive sero-reactions.Owing to contain anti-SARS-CoV antibody in the SARS-CoV positive serum, measure itself and the combining of polypeptide antigen, its concrete steps are as follows:
1, bag quilt: coating buffer is 0.07M NaHCO
3, 0.03M Na
2CO
3(pH=9.6), the bag by concentration 10 μ g/mL, 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours.
2, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
3, the sealing: confining liquid is 1～3% BSA, 200 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours.
4, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
5, adding one resists: the patients serum dilutes with physiological saline at 1: 100, and hatched 2 hours for 37 ℃ in 100 μ L/ holes.
6, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
7, adding two resists: HRP-rabbit anti-human igg antibody PBS1: 1000～1: 3000 dilutions, hatched 2 hours for 37 ℃ in 100 μ L/ holes.
8, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
9, add enzyme reaction substrate: enzyme reaction substrate is TMB, and hatched 15～30 minutes for 37 ℃ in 100 μ L/ holes.
10, add stop buffer: 2MH
2SO
4, hatched 5～10 minutes for 37 ℃ in 100 μ L/ holes.
11, reading: the OD value of in microplate reader, measuring 450nm and 630nm.
12, neutralization experiment: positive polypeptide compound that screening obtains and anti-SARA-CoV positive serum mixture repeat above-mentioned ELISA experimental procedure at 37 ℃ after hatching 30 minutes.
13, contrast and data processing: three multiple holes of anti-SARA-CoV positive serum reaction got by each polypeptide sample and three multiple holes of normal control serum compare experiment.The OD mean value in three multiple holes of normal control serum is controlled at below 0.1.The OD mean value that the mean value of three the multiple hole of anti-SARA-CoV positive serum reaction OD values deducts the multiple hole of three normal control serum is final result.Positive polypeptide compound through in and experimental verification specificity result.The result as shown in Table 1 and Table 2.
The cross-immune reaction originality result of table 1 synthetic decapeptide and three parts of anti-SARS-CoV antibody positive serum
| SEQ | ID peptide sequence and position | Serum | Serum | Serum |
| No： | No.1 | No.2 | No.3 | |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | TSGSDLDRCT(11-20) SGSDLDRCTT(12-21) SDLDRCTTFD(14-23) TTFDDVQAPN(20-29) FDDVQAPNYT(22-31) PMGTQTHTMI(143-152) MIFDNAFNCT(151-160) KSGNFKHLRE(175-184) GNFKHLREFV(177-186) KDGFLYVYKG(190-199) SVLYNSTFFS(353-362) VRQIAPGQTG(394-403) TRNIDATSTG(425-434) RNIDATSTGN(426-435) WPLNDYGFYT(476-485) YRVVVLSFEL(494-503) QCVNFNFNGL(523-532) CVNFNFNGLT(524-533) NFNGLTGTGV(528-537) DVSTAIHADQ(605-614) IGAEHVDTSY(637-646) SIAYSNNTIA(686-695) ITTEVMPVSM(704-713) | - + - + - - + + - - - + - + - ++ + ++ + +++ - +++ + | +++ ++ ++ ++ - ++ + ++ +++ - +++ ++ - ++ + ++ +++ ++ +++ ++ + ++ +++ | - ++ ++ ++ + - + - - - - + + - - + + ++ ++ + - ++ + |
| 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 | YGECLGDINA(819-820) ITVLPPLITD(840-849) TALGKLQDVV(925-934) NFGAISSVLN(951-960) AISSVLNDIL(954-963) RLDKVEAEVQ(965-974) RLITGRLQSL(977-986) QLIRAAEIRA(993-1002) SANLAATKMS(1003-1112) QSKRVDFCGK(1018-1027) VPSQERNFTT(1050-1059) WFITQRNFFS(1084-1093) SGNCDVVIGI(1105-1114) FKNHTSPDVD(1138-1147) DVDLGDISGI(1145-1154) VDLGDISGIN(1146-1155) NASVVNIQKE(1155-1164) KEIDRLNEVA(1163-1172) LQELGKYEQY(1182-1191) VVIGIINNTV(1210-1219) MVTILLCCMT(1211-1220) VTILLCCMTS(1212-1221) MEN441：LPQGTTLPKG MEN533：EASKKPRQKR | - - - - + ++ - - - + + - - - ++ ++ - + + - - +++ +++ | - - + - + +++ - + - - ++ ++ + ++ ++ +++ +++ + + ++ ++ +++ +++ | + - - ++ + ++ - - - ++ - + - + + + + - - ++ - +++ +++ |
The cross-immunoreactivity of the polypeptide that table 2. newly designs and synthesizes and they and 12 parts of anti-SARS-CoV people's positive serums
*
| Peptide | Ser 1 | Ser 2 | Ser 3 | Ser 4 | Ser5 | Ser 6 | Ser 7 | Ser 8 | Ser9 | Ser1 0 | Ser1 1 | Ser1 2 |
| S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12 S-13 S-14 S-15 S-16 S-17 M-1 N-1 N-2 N-3 N-4 MEN 4e6 | + +- +- +++ ++ +- + ++ +- +- +- +- +++ | +- + +- +- +++ +- + ++ + +- + ++ +- + + +- ++ | +- +- +- +++ + ++ +- +- ++ +- + +- +- +++ + | +- +- +++ ++ + +- + + +- +- +- + + + | +- +- +++ ++ + +- ++ +++ + | +- +- +- +++ +++ + +- +- +- ++ + | + + +++ ++ +- + +- ++ +- + + ++ +++ | +- + +++ + + ++ +- +- + ++ + | + +- +- + +++ + + +- +- ++ +- + +++ + +++ | +- + ++ +++ ++ +++ + + +- +++ + ++ + +++ + | + + + +++ + + + +- +- +- ++ +- +- + + + | +- + +++ +- +- + +- +- +- |
| S1b1 0 | +- | +- | +- | +- | +- | +- | +- |
*Negative control: OD absorbs and to be about 0.2.+-,+, ++, +++and ++ ++, represent the OD absorption region respectively 0.25～0.35,0.4～0.7,0.7～1.0,1.0～2.0 and＞2.0.MEN4e6 and S1-b10 differentiate positive contrast in advance.
Wherein S7S9S17N4 and MEN4e6 are carrying out cross-immune reaction with 30 parts of anti-SARS-CoV people's positive serums respectively, and the result is: S7 and S9 are positive with the anti-SARS-CoV antibody positive of 30 serum serum, and cross reacting rate is 71.4%; S17 and the anti-SARS-CoV antibody positive of 28 serum serum are positive, and cross reacting rate is 66.7%; N4 and the anti-SARS-CoV antibody positive of 38 serum serum are positive, and cross reacting rate is 90.5%; MEN4e6 and the anti-SARS-CoV antibody positive of 32 serum serum are positive, and cross reacting rate is 76.2%.
Embodiment 3:ELISA method is determined the competitiveness experiment of B-cell antigen determinant
With polypeptide compound storehouse and the reaction of SARS-CoV positive serum that obtains in the table 2 of embodiment 2, owing to being synthesized polypeptide, anti-SARS-CoV antibody in the SARS-CoV positive serum neutralizes wholly or in part, thereby in the serum anti-SARS-CoV antibody then with detection kit in standard antigen (be the SARS-CoV employing virus cracking liquid here, the diagnostic ELISA test kit of the big Gibio of China) combination weakens or debond fully, and its concrete steps are as follows:
With excessive polypeptide (or polypeptide mixture) is that antigen adds the sero-fast diluent of patient SARS (1: 100), and final volume is 100 μ l, hatches 60min altogether in 37 ℃.The final concentration of peptide is respectively 2.5,5.0,10.0,20.0 and 40.0 μ mol/L.Two polypeptide----Contrl1 and Contrl2 are as negative control, and Contrl1 is by drawing at random; Contrl2 is the proteic polypeptide that contains the 945th～963 amino-acid residue of S, experimental results show that no antigen.
1, bag quilt: coating buffer is 0.07M NaHCO
3, 0.03M Na
2CO
3(pH=9.6), the bag by concentration 10 μ g/mL (SARS-CoV employing virus cracking liquid), 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours.
2, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
3, the sealing: confining liquid is 1～3% BSA, 200 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours.
4, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
5, add one anti-: in patient's polypeptide and serum, hatched 2 hours for 37 ℃ in 100 μ L/ holes.
6, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
7, adding two resists: HRP-rabbit anti-human igg antibody PBS1: 1000～1: 3000 dilutions, hatched 2 hours for 37 ℃ in 100 μ L/ holes.
8, clean: PBST (pH=7.4 contains 0.05% Tween 20) cleans 5 times, pats dry.
9, add enzyme reaction substrate: enzyme reaction substrate is TMB, and hatched 15～30 minutes for 37 ℃ in 100 μ L/ holes.
10, add stop buffer: 2MH
2SO
4, hatched 5～10 minutes for 37 ℃ in 100 μ L/ holes.
11, reading: the OD value of in microplate reader, measuring 450nm and 630nm.
12, neutralization experiment: positive polypeptide compound that screening obtains and anti-SARA-CoV positive serum mixture repeat above-mentioned ELISA experimental procedure at 37 ℃ after hatching 30 minutes.
13, contrast and data processing: three multiple holes of anti-SARA-CoV positive serum reaction got by each polypeptide sample and three multiple holes of normal control serum compare experiment.The OD mean value in three multiple holes of normal control serum is controlled at below 0.1.The OD mean value that the mean value of three the multiple hole of anti-SARA-CoV positive serum reaction OD values deducts the multiple hole of three normal control serum is final result.Positive polypeptide compound through in and experimental verification specificity result.The result as shown in Figure 1.
Sequence table
＜110〉Chinese Academy of Medical Sciences's medicine bioengineering institute
＜120〉has coronavirus (SARS-CoV) the B-cell antigen determinant of extensive cross-immunoreactivity
<130>IDC040057
<160>71
<170>PatentIn version 3.1
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Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr
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Tyr Arg Val Val Val Leu Ser Phe Glu Leu
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Asp Val Ser Thr Ala Ile His Ala Asp Gln
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Ile Gly Ala Glu His Val Asp Thr Ser Tyr
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Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala
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Ile Thr Thr Glu Val Met Pro Val Ser Met
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Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala
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Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
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Thr Ala Leu Gly Lys Leu Gln Asp Val Val
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Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
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A1a Ile Ser Ser Val Leu Asn Asp Ile Leu
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Arg Leu Asp Lys Val Glu Ala Glu Val Gln
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Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
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Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
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Ser Ala Asn Leu Ala Ala Thr Lys Met Ser
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Gln Ser Lys Arg Val Asp Phe Cys Gly Lys
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Val Pro Ser Gln Glu Arg Asn Phe Thr Thr
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Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser
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Ser Gly Asn Cys Asp Val Val Ile Gly Ile
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Phe Lys Asn His Thr Ser Pro Asp Val Asp
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Asp Val Asp Leu Gly Asp Ile Ser Gly Ile
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Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
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Asn Ala Ser Val Val Asn Ile Gln Lys Glu
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Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
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Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr
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Val Val Ile Gly Ile Ile Asn Asn Thr Val
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Met Val Thr Ile Leu Leu Cys Cys Met Thr
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Val Thr Ile Leu Leu Cys Cys Met Thr Ser
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Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly
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Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg
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Ser Gly Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala
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Pro Asn Tyr Thr
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Ser Lys Pro Met Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala
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Phe Asn Cys Thr Phe Glu Tyr
20
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Thr Ala Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala
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Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe
20 25
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Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp
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Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
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Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly
20 25
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Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn
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Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His
20 25
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Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr
1 5 10 15
Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
20 25 30
Leu
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Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
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Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp
1 5 10 15
Arg Ile Tyr Ser Thr Gly
20
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Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp
1 5 10 15
Cys Asn Met Tyr
20
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Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
1 5 10 15
Asp
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Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
1 5 10 15
Asp Ile Leu
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1 5 10 15
Gly Arg Leu Gln Ser Leu
20
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Ser Ala Asn Leu Ala Ala Thr Lys Met Ser
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Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln
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20 25
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Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
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Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr
20 25
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Ala Trp Ile Met Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe
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Leu Tyr Ile Ile
20
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Gly Arg Asn Gly Ala Arg Pro Lys Gln Arg
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Ser Arg Gly Asn Ser Pro Ala Arg Met Ala
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Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro
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Claims (6)
1. the B-cell antigen determinant of one group of coronavirus relevant with severe acute respiratory syndrome, it is selected from least a following peptide sequence, or its arbitrary combination, comprising:
S7：
NH2-ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL-CONH2(SEQ ID NO：54)
S9：NH2-TDVSTAIHADQLTPAWRIYSTG-CONH2(SEQ IDNO：56)
S17：NH2-EIDRLNEVAKNLNESLIDLQELGKYEQY-CONH2(SEQ ID NO：64)
N4:NH2-TEPKKDKKKKTDEAQPLPQRQKK-CONH2 (SEQID NO:69) and
MEN-4e6(MEN-441)：NH2-LPQGTTLPKG-CONH2(SEQ IDNO：70)
2. one kind is detected the method that whether contains the coronavirus antibody relevant with severe acute respiratory syndrome in the testing sample, comprise with the described coronavirus B-of the claim 1 cell antigen determinant that detects significant quantity be suitable under the antigen-antibody bonded condition with testing sample in conjunction with or contact.
3. one kind is detected the method whether experimenter infects SARS-CoV, comprise with the described coronavirus B-of the claim 1 cell antigen determinant that detects significant quantity be suitable under the antigen-antibody bonded condition with available from experimenter's sample combination or contact.
4. one kind is detected the test kit that whether contains the coronavirus antibody relevant with severe acute respiratory syndrome in the testing sample, wherein contains the described coronavirus B-of claim 1 cell antigen determinant, and suitable developer and damping fluid.
The described coronavirus B-of 5 claims 1 cell antigen determinant is used to prepare the purposes of the medicine that prevents severe acute respiratory syndrome.
6. the vaccine of the coronavirus infection that a prevention is relevant with severe acute respiratory syndrome wherein contains the described coronavirus B cell antigen of the claim 1 of preventing significant quantity determinant, optional adjuvant, and pharmaceutically acceptable carrier.
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| CN112375754A (en) * | 2020-10-27 | 2021-02-19 | 清华大学 | Severe acute respiratory syndrome coronavirus 2 affinity polypeptide based on human angiotensin converting enzyme 2 |
| CN114163523A (en) * | 2020-03-17 | 2022-03-11 | 北京凯因科技股份有限公司 | Single-domain antibody for novel coronavirus and application thereof |
| CN116789767A (en) * | 2023-07-12 | 2023-09-22 | 中国人民解放军总医院第七医学中心 | Polypeptide composition for resisting novel coronavirus disease and application thereof |
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| CN100428254C (en) * | 2006-07-20 | 2008-10-22 | 上海交通大学 | Cross reaction antigen computer-aided screening method |
| CN111458500A (en) * | 2020-03-03 | 2020-07-28 | 苏州百道医疗科技有限公司 | Human respiratory epithelial cell pathogenic cytology detection method and kit |
| CN114163523A (en) * | 2020-03-17 | 2022-03-11 | 北京凯因科技股份有限公司 | Single-domain antibody for novel coronavirus and application thereof |
| CN114163523B (en) * | 2020-03-17 | 2023-07-18 | 北京凯因科技股份有限公司 | Single-domain antibody for novel coronavirus and application thereof |
| CN112375754A (en) * | 2020-10-27 | 2021-02-19 | 清华大学 | Severe acute respiratory syndrome coronavirus 2 affinity polypeptide based on human angiotensin converting enzyme 2 |
| CN112375754B (en) * | 2020-10-27 | 2022-04-22 | 清华大学 | Severe acute respiratory syndrome coronavirus 2 affinity polypeptide based on human angiotensin converting enzyme 2 |
| CN116789767A (en) * | 2023-07-12 | 2023-09-22 | 中国人民解放军总医院第七医学中心 | Polypeptide composition for resisting novel coronavirus disease and application thereof |
| CN116789767B (en) * | 2023-07-12 | 2023-12-22 | 中国人民解放军总医院第七医学中心 | Polypeptide composition for resisting novel coronavirus disease and application thereof |
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