CN1661372A - Immunization microsphere in use for detecting SARS antibody, preparation method and application - Google Patents
Immunization microsphere in use for detecting SARS antibody, preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of immunization microsphere in use for detecting SARS antibody, preparation method and application.This immune microsphere takes polystyrene microsphere which surface is modified with carboxyl as kernel,the carboxyl couples with the antibodyies which resists SARS coronavirus of S N M or E proteini through multi polylysine chemically.The immune microsphere can be used for detecting SARS antigen as detecing reagent.Comparing to the existing technology,the immune microsphere using for detecting SARS antigen possesses better sensitivity and specificity,higher accuracy,avoiding the disadvantage of false positive of PCR disgnosis kit.the process is fast.on the other hand,detection needs no instrument equipment,so this immune microsphere is suitable for the largeness base sanitary and anti-epidemic units to use.
Description
Technical field
The present invention relates to a kind of immune microsphere that is used to detect SARS antibody, and its production and use.
Technical background
SARS (SARS (Severe Acute Respiratory Syndrome)) coronavirus is a kind of newfound coronavirus, and this novel disease incidence that it causes is fast, and velocity of propagation is fast, if untimely diagnoses and treatment, case fatality rate is very high.
Be euzymelinked immunosorbent assay (ELISA) (ELISA) in scientific research and detection method commonly used clinically at present.Use euzymelinked immunosorbent assay (ELISA) (ELISA) to detect antibody, be about on the antigen coated ELISA Plate, add the sealing of calf serum or skimmed milk, added behind the test serum incubation again 1 hour,, add the substrate of colour developing after washing repeatedly again through the enzyme-added mark second antibody in flushing back repeatedly, incubation.Though this method susceptibility and specificity are fine, finish Total Test needs more than 2 hours.
Be a kind of easy, method fast, only needed 1 minute just can obtain the result and use the immune microsphere method to detect antibody.This method is with the antigen coupling or is linked on the microballoon of organic polymer preparation, makes immune microsphere reagent and detect antibody.But the preparation method of existing immune microsphere reagent, no matter be physics or chemical method, the immune microsphere that obtains is not all by covalent bonds, so immune microsphere itself is very unstable, it is very big influenced by temperature and salinity, causes false-positive experimental error easily.
Summary of the invention
It is easy inadequately, quick to the objective of the invention is to overcome existing euzymelinked immunosorbent assay (ELISA); And the immune microsphere of immune microsphere method itself is very unstable, causes the defective of false positive experimental error easily, thereby a kind of detection that both can be quick, easy, the very stable again immune microsphere that is used to detect SARS antibody are provided.
Another object of the present invention is to provide a kind of described preparation method who is used to detect the immune microsphere of SARS antibody.
A further object of the present invention is to provide a kind of described purposes that is used to detect the immune microsphere of SARS antibody.
The objective of the invention is to realize by the following technical solutions:
The immune microsphere that is used to detect SARS (SARS (Severe Acute Respiratory Syndrome)) antibody provided by the invention, it is for being kernel with the polystyrene microsphere, its surperficial carboxyl by the poly-D-lysine coupling as the sars coronavirus S of antigen, N, M or E albumen, described polystyrene microsphere is that surface diameter is 200～900 nanometers, and there is carboxyl modified on its surface.
The invention provides a kind of described preparation method who is used to detect the immune microsphere of SARS antibody, is to adopt chemical coupling method with sars coronavirus S, N, and M or E albumen are coupled to microballoon as antigen, comprise following step:
1) with polystyrene microsphere 15～20mg/ml, after the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the phosphate buffer of pH4～5 of 0.01～0.05M, the carbodiimide that adds 2～4mg/ml then, after room temperature reaction is complete, centrifugal, with the carbonate buffer solution washing of pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH7.5～8.5 of 0.01～0.05M; Described polystyrene microsphere diameter is 200～900 nanometers, and there is carboxyl modified on its surface;
2) in the mixed liquor that step 1) obtains, add the poly-D-lysine of 0.2～2.4mg/ml, complete in room temperature reaction; After the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the phosphate buffer of pH4～5 of 0.01～0.05M, add the carbodiimide of 2～4mg/ml then, complete in room temperature reaction; With the carbonate buffer solution washing of pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH7.5～8.5 of 0.01～0.05M;
3) to step 2) add sars coronavirus S in the mixed liquor that obtains as antigen, N, M or E albumen 0.2～2.4mg/ml are complete in room temperature reaction; After the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH8.8～9.8 of 0.1～0.5M; Add 50～200 microlitre 0.25M acetamides, after room temperature reaction is complete, the centrifugal solid of telling;
4) be resuspended in the carbonate buffer solution of pH7.5～8.5 of 1g/ml cow's serum (BSA) and 0.01～0.05M, in room temperature reaction fully after, the centrifugal solid of telling obtains being used to detect the immune microsphere of SARS antibody.
The amino acid sequence of described sars coronavirus S albumen is:
MetPheIlePheLeu LeuPheLeuThrLeu ThrSerGlySerAsp LeuAspArgCysThr
ThrPheAspAspVal GlnAlaProAsnTyr ThrGlnHisThrSer SerMetArgGlyVal
TyrTyrProAspGlu IlePheArgSerAsp ThrLeuTyrLeuThr GlnAspLeuPheLeu
ProPheTyrSerAsn ValThrGlyPheHis ThrIleAsnHisThr PheAspAsnProVal
IleProPheLysAsp GlyIleTyrPheAla AlaThrGluLysSer AsnValValArgGly
TrpValPheGlySer ThrMetAsnAsnLys SerGlnSerValIle IleIleAsnAsnSer
ThrAsnValValIle ArgAlaCysAsnPhe GluLeuCysAspAsn ProPhePheAlaVal
SerLysProMetGly ThrGlnThrHisThr MetIlePheAspAsn AlaPheAsnCysThr
PheGluTyrIleSer AspAlaPheSerLeu AspValSerGluLys SerGlyAsnPheLys
HisLeuArgGluPhe ValPheLysAsnLys AspGlyPheLeuTyr ValTyrLysGlyTyr
GlnProIleAspVal ValArgAspLeuPro SerGlyPheAsnThr LeuLysProIlePhe
LysLeuProLeuGly IleAsnIleThrAsn PheArgAlaIleLeu ThrAlaPheSerPro
AlaGlnAspThrTrp GlyThrSerAlaAla AlaTyrPheValGly TyrLeuLysProThr
ThrPheMetLeuLys TyrAspGluAsnGly ThrIleThrAspAla ValAspCysSerGln
AsnProLeuAlaGlu LeuLysCysSerVal LysSerPheGluIle AspLysGlyIleTyr
GlnThrSerAsnPhe ArgValValProSer GlyAspValValArg PheProAsnIleThr
AsnLeuCysProPhe GlyGluValPheAsn AlaThrLysPhePro SerValTyrAlaTrp
GluArgLysLysIle SerAsnCysValAla AspTyrSerValLeu TyrAsnSerThrPhe
PheSerThrPheLys CysTyrGlyValSer AlaThrLysLeuAsn AspLeuCysPheSer
AsnValTyrAlaAsp SerPheValValLys GlyAspAspValArg GlnIleAlaProGly
GlnThrGlyValIle AlaAspTyrAsnTyr LysLeuProAspAsp PheMetGlyCysVal
LeuAlaTrpAsnThr ArgAsnIleAspAla ThrSerThrGlyAsn TyrAsnTyrLysTyr
ArgTyrLeuArgHis GlyLysLeuArgPro PheGluArgAspIle SerAsnValProPhe
SerProAspGlyLys ProCysThrProPro AlaLeuAsnCysTyr TrpProLeuAsnAsp
TyrGlyPheTyrThr ThrThrGlyIleGly TyrGlnProTyrArg ValValValLeuSer
PheGluLeuLeuAsn AlaProAlaThrVal CysGlyProLysLeu SerThrAspLeuIle
LysAsnGlnCysVal AsnPheAsnPheAsn GlyLeuThrGlyThr GlyValLeuThrPro
SerSerLysArgPhe GlnProPheGlnGln PheGlyArgAspVal SerAspPheThrAsp
SerValArgAspPro LysThrSerGluIle LeuAspIleSerPro CysSerPheGlyGly
ValSerValIleThr ProGlyThrAsnAla SerSerGluValAla ValLeuTyrGlnAsp
ValAsnCysThrAsp ValSerThrAlaIle HisAlaAspGlnLeu ThrProAlaTrpArg
IleTyrSerThrGly AsnAsnValPheGln ThrGlnAlaGlyCys LeuIleGlyAlaGlu
HisValAspThrSer TyrGluCysAspIle ProIleGlyAlaGly IleCysAlaSerTyr
HisThrValSerLeu LeuArgSerThrSer GlnLysSerIleVal AlaTyrThrMetSer
LeuGlyAlaAspSer SerIleAlaTyrSer AsnAsnThrIleAla IleProThrAsnPhe
SerIleSerIleThr ThrGluValMetPro ValSerMetAlaLys ThrSerValAspCys
AsnMetTyrIleCys GlyAspSerThrGlu CysAlaAsnLeuLeu LeuGlnTyrGlySer
PheCysThrGlnLeu AsnArgAlaLeuSer GlyIleAlaAlaGlu GlnAspArgAsnThr
ArgGluValPheAla GlnValLysGlnMet TyrLysThrProThr LeuLysTyrPheGly
GlyPheAsnPheSer GlnIleLeuProAsp ProLeuLysProThr LysArgSerPheIle
GluAspLeuLeuPhe AsnLysValThrLeu AlaAspAlaGlyPhe MetLysGlnTyrGly
GluCysLeuGlyAsp IleAsnAlaArgAsp LeuIleCysAlaGln LysPheAsnGlyLeu
ThrValLeuProPro LeuLeuThrAspAsp MetIleAlaAlaTyr ThrAlaAlaLeuVal
SerGlyThrAlaThr AlaGlyTrpThrPhe GlyAlaGlyAlaAla LeuGlnIleProPhe
AlaMetGlnMetAla TyrArgPheAsnGly IleGlyValThrGln AsnValLeuTyrGlu
AsnGlnLysGlnIle AlaAsnGlnPheAsn LysAlaIleSerGln IleGlnGluSerLeu
ThrThrThrSerThr AlaLeuGlyLysLeu GlnAspValValAsn GlnAsnAlaGlnAla
LeuAsnThrLeuVal LysGlnLeuSerSer AsnPheGlyAlaIle SerSerValLeuAsn
AspIleLeuSerArg LeuAspLysValGlu AlaGluValGlnIle AspArgLeuIleThr
GlyArgLeuGlnSer LeuGlnThrTyrVal ThrGlnGlnLeuIle ArgAlaAlaGluIle
ArgAlaSerAlaAsn LeuAlaAlaThrLys MetSerGluCysVal LeuGlyGlnSerLys
ArgValAspPheCys GlyLysGlyTyrHis LeuMetSerPhePro GlnAlaAlaProHis
GlyValValPheLeu HisValThrTyrVal ProSerGlnGluArg AsnPheThrThrAla
ProAlaIleCysHis GluGlyLysAlaTyr PheProArgGluGly ValPheValPheAsn
GlyThrSerTrpPhe IleThrGlnArgAsn PhePheSerProGln IleIleThrThrAsp
AsnThrPheValSer GlyAsnCysAspVal ValIleGlyIleIle AsnAsnThrValTyr
AspProLeuGlnPro GluLeuAspSerPhe LysGluGluLeuAsp LysTyrPheLysAsn
HisThrSerProAsp ValAspLeuGlyAsp IleSerGlyIleAsn AlaSerValValAsn
IleGInLysGluIle AspArgLeuAsnGlu ValAlaLysAsnLeu AsnGluSerLeuIle
AspLeuGlnGluLeu GlyLysTyrGluGln TyrIleLysTrpPro TrpTyrValTrpLeu
GlyPheIleAlaGly LeuIleAlaIleVal MetValThrIleLeu LeuCysCysMetThr
SerCysCysSerCys LeuLysGlyAlaCys SerCysGlySerCys CysLysPheAspGlu
AspAspSerGluPro?ValLeuLysGly?ValLysLeuHisTyr?Thr
The amino acid sequence of described sars coronavirus N albumen is:
MetSerAspAsnGly ProGlnSerAsnGln ArgSerAlaProArg IleThrPheGlyGly
ProThrAspSerThr AspAsnAsnGlnAsn GlyGlyArgAsnGly AlaArgProLysGln
ArgArgProGlnGly LeuProAsnAsnThr AlaSerTrpPheThr AlaLeuThrGlnHis
GlyLysGluGluLeu ArgPheProArgGly GlnGlyValProIle AsnThrAsnSerGly
ProAspAspGlnIle GlyTyrTyrArgArg AlaThrArgArgVal ArgGlyGlyAspGly
LysMetLysGluLeu SerProArgTrpTyr PheTyrTyrLeuGly ThrGlyProGluAla
SerLeuProTyrGly AlaAsnLysGluGly IleValTrpValAla ThrGluGlyAlaLeu
AsnThrProLysAsp HisIleGlyThrArgAsn?ProAsnAsnAsnAla AlaThrValLeuGln
LeuProGlnGlyThr ThrLeuProLysGly PheTyrAlaGluGly SerArgGlyGlySer
GlnAlaSerSerArg SerSerSerArgSer ArgGlyAsnSerArg AsnSerThrProGly
SerSerArgGlyAsn SerProAlaArgMet AlaSerGlyGlyGly GluThrAlaLeuAla
LeuLeuLeuLeuAsp ArgLeuAsnGlnLeu GluSerLysValSer GlyLysGlyGlnGln
GlnGlnGlyGlnThr ValThrLysLysSer AlaAlaGluAlaSer LysLysProArgGln
LysArgThrAlaThr LysGlnTyrAsnVal ThrGlnAlaPheGly ArgArgGlyProGlu
GlnThrGlnGlyAsn PheGlyAspGlnAsp LeuIleArgGlnGly ThrAspTyrLysHis
TrpProGlnIleAla GlnPheAlaProSer AlaSerAlaPhePhe GlyMetSerArgIle
GlyMetGluValThr ProSerGlyThrTrp LeuThrTyrHisGly AlaIleLysLeuAsp
AspLysAspProGln PheLysAspAsnVal IleLeuLeuAsnLys HisIleAspAlaTyr
LysThrPheProPro ThrGluProLysLys AspLysLysLysLys ThrAspGluAlaGln
ProLeuProGlnArg GlnLysLysGlnPro ThrValThrLeuLeu ProAlaAlaAspMet
AspAspPheSerArg?GlnLeuGlnAsnSer?MetSerGlyAlaSer?AlaAspSerThrGln?Ala
The amino acid sequence of described sars coronavirus M albumen is:
MetAlaAspAsnGly ThrIleThrValGlu GluLeuLysGlnLeu LeuGluGlnTrpAsn
LeuValIleGlyPhe LeuPheLeuAlaTrp IleMetLeuLeuGln PheAlaTyrSerAsn
ArgAsnArgPheLeu TyrIleIleLysLeu ValPheLeuTrpLeu LeuTrpProValThr
LeuAlaCysPheVal LeuAlaAlaValTyr ArgIleAsnTrpVal ThrGlyGlyIleAla
IleAlaMetAlaCys IleValGlyLeuMet TrpLeuSerTyrPhe ValAlaSerPheArg
LeuPheAlaArgThr ArgSerMetTrpSer PheAsnProGluThr AsnIleLeuLeuAsn
ValProLeuArgGly ThrIleValThrArg ProLeuMetGluSer GluLeuValIleGly
AlaValIleIleArg GlyHisLeuArgMet AlaGlyHisSerLeu GlyArgCysAspIle
LysAspLeuProLys GluIleThrValAla ThrSerArgThrLeu SerTyrTyrLysLeu
GlyAlaSerGlnArg ValGlyThrAspSer GlyPheAlaAlaTyr AsnArgTyrArgIle
GlyAsnTyrLysLeu?AsnThrAspHisAla?GlySerAsnAspAsn?IleAlaLeuLeuVal?Gln
The amino acid sequence of described sars coronavirus E albumen is:
MetTyrSerPheVal SerGluGluThrGly ThrLeuIleValAsn SerValLeuLeuPhe
LeuAlaPheValVal PheLeuLeuValThr LeuAlaIleLeuThr AlaLeuArgLeuCys
AlaTyrCysCysAsn IleValAsnValSer LeuValLysProThr ValTyrValTyrSer
ArgValLysAsnLeu?AsnSerSerGluGly?ValProAspLeuLeu?Val
This immune microsphere that is used to detect SARS antibody is used 1%BSA, 5% glycerine, 0.01%NaN successively respectively
3, 0.01M the PBS damping fluid of pH8.0 resuspended, and with 400w ultrasonic Treatment 20 seconds, standby.
The invention provides a kind of described purposes that is used to detect the immune microsphere of SARS antibody.The detectable that can be used as this immune microsphere detects anti-(SARS) antibody, and its detection method is as follows:
Tested blood serum sample is dropped on the clean slide or transparent plastic sheet, add the immune microsphere that is used to detect SARS antibody provided by the invention, wherein, coupling as the sars coronavirus S of antigen, N, the immune microsphere of M or E albumen respectively accounts for 1/4, stirs with bamboo let, toothpick, sticking plaster or other objects, with testing sample and immune microsphere mixing and be round spot shape.
Under black background, with the naked eye or in the agglutinating reaction of microscopically Direct observation, and with following symbol record result:
The visible agglutinating particle of (-) no naked eyes;
The visible thin agglutinating particle of (+) naked eyes, the background muddiness;
The visible big agglutinating particle of (++) naked eyes, background is muddy slightly;
(+++) the visible big and clear agglutinating particle of naked eyes, background is clear;
(++ ++) naked eyes are aggegation piece greatly and clearly as seen, and background is clear.
Also can carry out the semiquantitative determination antibody titer, with tested serum doubling dilution, measure by last method, be antibody titer with the dilutability.
The SARS disease is a kind of serious extremely strong deadly infectious disease of infectiousness, need diagnose quickly and accurately.The immune microsphere that is used to detect SARS (SARS (Severe Acute Respiratory Syndrome)) antibody provided by the invention is the sars coronavirus S with genetic recombination, N, and M, E albumen is coupled on (latex) polystyrene microsphere by chemical method.This immune microsphere compared with prior art, its beneficial effect is:
The first, the accuracy height.Because the genetic engineering recombinant antigen that uses, its purity reaches 99%, for this reason, has avoided the false-positive shortcoming of PCR diagnostic kit;
The second, fast.Whole testing process only needs several minutes, has avoided enzyme-linked immunologic diagnosis kit to need the time-consuming shortcoming of a few hours;
The 3rd, economical and practical.Detection is without any need for instrument and equipment, is fit to vast basic health epidemic prevention unit and uses.
In a word, this immune microsphere can the anti-SARS antibody of fast detecting serum, and susceptibility and specificity are all good.
Description of drawings
Fig. 1 detects SARS antibody for immune microsphere: a left side (feminine gender), right (positive);
Fig. 2 is the gene recombinant protein immune microsphere reaction (40X) of immune forward and backward rabbit anteserum and SARS CoV; Wherein: rabbit anteserum before the A. immunity; B. immune back rabbit anteserum;
Fig. 3 is normal person and patients serum and the reaction of SARS CoV gene recombinant protein immune microsphere; Wherein: A. normal human serum (10X); B.SARS patients serum (strong reaction (10X); C.SARS patients serum (reacting 40X by force); D.SARS patients serum (1: 200 dilution 40X).
Embodiment
Embodiment 1, coupling as the preparation of the immune microsphere of the sars coronavirus S albumen of antigen
The preparation and the evaluation of sars coronavirus S proteantigen:
Preparation---with gene recombination technology clone, expression and purifying SARS S albumen as antigen: with the genetic fragment of RT-PCR method amplification sars coronavirus S, with this genetic fragment after order-checking confirms that gene order is correct, again it is cloned into expression vector such as prokaryotic expression carrier pQE30, or Yeast expression carrier pPICZ α A, express then and protein purification.
Identify---behind the antigen purification, identify by following test:
1) antigen protein assay: get the antigen 1 milliliter with its protein content of spectrophotometric instrumentation, after its 260nm and 280nm place survey the OD value respectively, can calculate with formula 280nmOdx1.45-260nmODx0.74 about the protein content 1mg/ml of antigen;
2) antigen protein purity testing: getting antigen 20 microlitres (containing protein content 5 micrograms), to walk protein electrophoresis be the SDS-PAGE electrophoresis, and the result is shown as a band.
3) enzyme linked immune assay (ELISA): marked microwell plate by enzyme with 2.5 mcg/ml antigens, 200 microlitre bags, put 4 ℃ after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 ℃ 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 ℃ 2 hours, after PBS-tween and the washing of PBS damping fluid, add goat anti-human igg's 200 microlitres of the suitableeest working concentration HRP mark, put room temperature after 2 hours, wash each 3 times with PBS-tween and PBS damping fluid respectively, add reaction substrate 200 microlitres, 495nm measures absorbance again.
4) control experiment: add 10 microlitre antibody positive serum and negative serums with immune microsphere 10 microlitres, behind the mixing, positive serum has agglutinating particle, and negative serum does not then have aggegation.
The preparation of immune microsphere:
1) with diameter is the polystyrene microsphere 15mg/ml of 200 nanometers, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml then, putting room temperature fluctuated 4 hours, with 12000 rev/mins centrifugal 10 minutes, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
2) poly-D-lysine of adding 0.1mg/ml in the mixed liquor that step 1) obtains, putting room temperature fluctuated 12 hours, with the carbonate buffer solution washing of the pH8.8 of 0.5M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml is then put room temperature and was fluctuated 4 hours; With the carbonate buffer solution washing of the pH8.8 of 0.5M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
3) to step 2) add sars coronavirus S albumen 0.2mg/ml in the mixed liquor that obtains as antigen, put room temperature and fluctuated 12 hours, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH8.8 of 0.1M; Add on the 50 microlitre 0.25M acetamides sealings poly-D-lysine not by the carboxy moiety of antigen combination, add acetamide after, put room temperature and fluctuated 30 fens, with 13000 rev/mins centrifugal 10 minutes, tell solid;
4) be resuspended in the carbonate buffer solution of the pH7.5 of 1g/ml cow's serum (BSA) and 0.015M, put room temperature and fluctuated 30 fens, the centrifugal solid of telling, obtain being used to detecting SARS antibody, coupling as the immune microsphere of the sars coronavirus S albumen of antigen.
This immune microsphere that is used to detect SARS antibody is used 1%BSA, 5% glycerine, 0.01%NaN successively respectively
3, 0.01M the PBS damping fluid of pH8.0 resuspended, and with 400w ultrasonic Treatment 20 seconds, standby.
Embodiment 2, coupling as the preparation of the immune microsphere of the sars coronavirus N albumen of antigen
The preparation and the evaluation of sars coronavirus N proteantigen:
Preparation---with gene recombination technology clone, expression and purifying SARS N albumen as antigen: with the genetic fragment of RT-PCR method amplification sars coronavirus N, with this genetic fragment after order-checking confirms that gene order is correct, again it is cloned into expression vector such as prokaryotic expression carrier pQE30, or Yeast expression carrier pPICZ α A, express then and protein purification.
Identify---behind the antigen purification, identify by following test:
1) antigen protein assay: get the antigen 1 milliliter with its protein content of spectrophotometric instrumentation, after its 260nm and 280nm place survey the OD value respectively, can calculate with formula 280nmOdx1.45-260nmODx0.74 about the protein content 1mg/ml of antigen;
2) antigen protein purity testing: getting antigen 20 microlitres (containing protein content 5 micrograms), to walk protein electrophoresis be the SDS-PAGE electrophoresis, and the result is shown as a band.
3) enzyme linked immune assay (ELISA): marked microwell plate by enzyme with 2.5 mcg/ml antigens, 200 microlitre bags, put 4 ℃ after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 ℃ 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 ℃ 2 hours, after PBS-tween and the washing of PBS damping fluid, add goat anti-human igg's 200 microlitres of the suitableeest working concentration HRP mark, put room temperature after 2 hours, wash each 3 times with PBS-tween and PBS damping fluid respectively, add reaction substrate 200 microlitres, 495nm measures absorbance again.
4) control experiment: add 10 microlitre antibody positive serum and negative serums with immune microsphere 10 microlitres, behind the mixing, positive serum has agglutinating particle, and negative serum does not then have aggegation.
The preparation of immune microsphere:
1) with diameter is the polystyrene microsphere 15mg/ml of 200 nanometers, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml then, putting room temperature fluctuated 4 hours, with 12000 rev/mins centrifugal 10 minutes, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
2) poly-D-lysine of adding 0.1mg/ml in the mixed liquor that step 1) obtains, putting room temperature fluctuated 12 hours, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml is then put room temperature and was fluctuated 4 hours; With the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
3) to step 2) add sars coronavirus N albumen 0.2mg/ml in the mixed liquor that obtains as antigen, put room temperature and fluctuated 12 hours, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH8.8 of 0.1M; Add on the 50 microlitre 0.25M acetamides sealings poly-D-lysine not by the carboxy moiety of antigen combination, add acetamide after, put room temperature and fluctuated 30 fens, with 13000 rev/mins centrifugal 10 minutes, tell solid;
4) be resuspended in the carbonate buffer solution of the pH7.5 of 1g/ml cow's serum (BSA) and 0.015M, put room temperature and fluctuated 30 fens, the centrifugal solid of telling, obtain being used to detecting SARS antibody, coupling as the immune microsphere of the sars coronavirus N albumen of antigen.
This immune microsphere that is used to detect SARS antibody is used 1%BSA, 5% glycerine, 0.01%NaN successively respectively
3, 0.01M the PBS damping fluid of pH8.0 resuspended, and with 400w ultrasonic Treatment 20 seconds, standby.
Embodiment 3, coupling as the preparation of the immune microsphere of the sars coronavirus M albumen of antigen
The preparation and the evaluation of sars coronavirus M proteantigen:
Preparation---with gene recombination technology clone, expression and purifying SARS M albumen as antigen: with the genetic fragment of RT-PCR method amplification sars coronavirus M, with this genetic fragment after order-checking confirms that gene order is correct, again it is cloned into expression vector such as prokaryotic expression carrier pQE30, or Yeast expression carrier pPICZ α A, express then and protein purification.
Identify---behind the antigen purification, identify by following test:
1) antigen protein assay: get the antigen 1 milliliter with its protein content of spectrophotometric instrumentation, after its 260nm and 280nm place survey the OD value respectively, can calculate with formula 280nmOdx1.45-260nmODx0.74 about the protein content 1mg/ml of antigen;
2) antigen protein purity testing: getting antigen 20 microlitres (containing protein content 5 micrograms), to walk protein electrophoresis be the SDS-PAGE electrophoresis, and the result is shown as a band.
3) enzyme linked immune assay (ELISA): marked microwell plate by enzyme with 2.5 mcg/ml antigens, 200 microlitre bags, put 4 ℃ after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 ℃ 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 ℃ 2 hours, after PBS-tween and the washing of PBS damping fluid, add goat anti-human igg's 200 microlitres of the suitableeest working concentration HRP mark, put room temperature after 2 hours, wash each 3 times with PBS-tween and PBS damping fluid respectively, add reaction substrate 200 microlitres, 495nm measures absorbance again.
4) control experiment: add 10 microlitre antibody positive serum and negative serums with immune microsphere 10 microlitres, behind the mixing, positive serum has agglutinating particle, and negative serum does not then have aggegation.
The preparation of immune microsphere:
1) with diameter is the polystyrene microsphere 15mg/ml of 200 nanometers, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml then, putting room temperature fluctuated 4 hours, with 12000 rev/mins centrifugal 10 minutes, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
2) poly-D-lysine of adding 0.1mg/ml in the mixed liquor that step 1) obtains, putting room temperature fluctuated 12 hours, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml is then put room temperature and was fluctuated 4 hours; With the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
3) to step 2) add sars coronavirus M albumen 0.2mg/ml in the mixed liquor that obtains as antigen, put room temperature and fluctuated 12 hours, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH8.8 of 0.1M; Add on the 50 microlitre 0.25M acetamides sealings poly-D-lysine not by the carboxy moiety of antigen combination, add acetamide after, put room temperature and fluctuated 30 fens, with 13000 rev/mins centrifugal 10 minutes, tell solid;
4) be resuspended in the carbonate buffer solution of the pH7.5 of 1g/ml cow's serum (BSA) and 0.015M, put room temperature and fluctuated 30 fens, the centrifugal solid of telling, obtain being used to detecting SARS antibody, coupling as the immune microsphere of the sars coronavirus M albumen of antigen.
This immune microsphere that is used to detect SARS antibody is used 1%BSA, 5% glycerine, 0.01%NaN successively respectively
3, 0.01M the PBS damping fluid of pH8.0 resuspended, and with 400w ultrasonic Treatment 20 seconds, standby.
Embodiment 4, coupling as the preparation of the immune microsphere of the sars coronavirus E albumen of antigen
The preparation and the evaluation of sars coronavirus E proteantigen:
Preparation---with gene recombination technology clone, expression and purifying SARS E albumen as antigen: with the genetic fragment of RT-PCR method amplification sars coronavirus E, with this genetic fragment after order-checking confirms that gene order is correct, again it is cloned into expression vector such as prokaryotic expression carrier pQE30, or Yeast expression carrier pPICZ α A, express then and protein purification.
Identify---behind the antigen purification, identify by following test:
1) antigen protein assay: get the antigen 1 milliliter with its protein content of spectrophotometric instrumentation, after its 260nm and 280nm place survey the OD value respectively, can calculate with formula 280nmOdx1.45-260nmODx0.74 about the protein content 1mg/ml of antigen;
2) antigen protein purity testing: getting antigen 20 microlitres (containing protein content 5 micrograms), to walk protein electrophoresis be the SDS-PAGE electrophoresis, and the result is shown as a band.
3) enzyme linked immune assay (ELISA): marked microwell plate by enzyme with 2.5 mcg/ml antigens, 200 microlitre bags, put 4 ℃ after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 ℃ 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 ℃ 2 hours, after PBS-tween and the washing of PBS damping fluid, add goat anti-human igg's 200 microlitres of the suitableeest working concentration HRP mark, put room temperature after 2 hours, wash each 3 times with PBS-tween and PBS damping fluid respectively, add reaction substrate 200 microlitres, 495nm measures absorbance again.
4) control experiment: add 10 microlitre antibody positive serum and negative serums with immune microsphere 10 microlitres, behind the mixing, positive serum has agglutinating particle, and negative serum does not then have aggegation.
The preparation of immune microsphere:
1) with diameter is the polystyrene microsphere 15mg/ml of 200 nanometers, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml then, putting room temperature fluctuated 4 hours, with 12000 rev/mins centrifugal 10 minutes, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
2) poly-D-lysine of adding 0.1mg/ml in the mixed liquor that step 1) obtains, putting room temperature fluctuated 12 hours, with the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the phosphate buffer of the pH4 of 0.01M, the carbodiimide that adds 0.2mg/ml is then put room temperature and was fluctuated 4 hours; With the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH7.5 of 0.01M;
3) to step 2) add sars coronavirus E albumen 0.2mg/ml in the mixed liquor that obtains as antigen, put room temperature and fluctuated 12 hours, after the carbonate buffer solution washing of the pH8.8 of 0.1M 3 times, be resuspended in the carbonate buffer solution of the pH8.8 of 0.1M; Add on the 50 microlitre 0.25M acetamides sealings poly-D-lysine not by the carboxy moiety of antigen combination, add acetamide after, put room temperature and fluctuated 30 fens, with 13000 rev/mins centrifugal 10 minutes, tell solid;
4) be resuspended in the carbonate buffer solution of the pH7.5 of 1g/ml cow's serum (BSA) and 0.015M, put room temperature and fluctuated 30 fens, the centrifugal solid of telling, obtain being used to detecting SARS antibody, coupling as the immune microsphere of the sars coronavirus E albumen of antigen.
This immune microsphere that is used to detect SARS antibody is used 1%BSA, 5% glycerine, 0.01%NaN successively respectively
3, 0.01M the PBS damping fluid of pH8.0 resuspended, and with 400w ultrasonic Treatment 20 seconds, standby.
Embodiment 5, detect anti-(SARS) antibody with immune microsphere detectable provided by the invention
Experimental technique:
Tested blood serum sample 10 microlitres are dropped on the clean slide or transparent plastic sheet, add the 5 microlitres immune microsphere that is used to detect SARS antibody provided by the invention, wherein, the coupling of embodiment 1～4 preparation as the sars coronavirus S of antigen, N, the immune microsphere of M or E albumen respectively accounts for 1/4, stirs with bamboo let, toothpick, sticking plaster or other objects, with testing sample and immune microsphere mixing and be the round spot shape of 1 centimetre of diameter.
Under black background, with the naked eye or in the agglutinating reaction of microscopically Direct observation, and with following symbol record result:
The visible agglutinating particle of (-) no naked eyes;
The visible thin agglutinating particle of (+) naked eyes, the background muddiness;
The visible big agglutinating particle of (++) naked eyes, background is muddy slightly;
(+++) the visible big and clear agglutinating particle of naked eyes, background is clear;
(++ ++) naked eyes are aggegation piece greatly and clearly as seen, and background is clear.
Laboratory sample:
1) normal rabbit serum and S, N, M, the E albumen rabbit anteserum of immune SARS virus respectively;
2) normal human serum and SARS patients serum;
Experimental result:
As shown in Figure 2, under 40 power microscopes, observe the reaction that is negative of normal rabbit anteserum and immune microsphere, as seen uniform microsphere particle; The rabbit anteserum and the immune microsphere that have infected SARS virus are positive, and visible microsphere particle aggegation is piece.
As shown in Figure 3, under 10 power microscopes, observe normal person's serum and immune microsphere (SARS CoV gene recombinant protein immune microsphere the be coupling SARS-S albumen) reaction that is negative, visible microsphere particle uniformly; Patient's SARS serum and immune microsphere are positive, and visible microsphere particle aggegation is piece.
SARS patient's sample being carried out the semiquantitative determination antibody titer, with tested serum doubling dilution, measure by last method, is antibody titer with the dilutability.Observations is seen Fig. 3, wherein, and when dilutability is 200 times, still positive (as the right figure of Fig. 1), dilutability is 1: 200 o'clock, be positive (as the left figure of Fig. 1), therefore, the antibody titer of this 1: 200 sample (being dilutability) is 1: 20.
SEQUENCE?LISTING
＜110〉Institute of Zoology, Academia Sinica
＜120〉a kind of immune microsphere of detecting SARS antibody and its production and use that is used to
<130>FPI04040
<150>CN?200410003420.1
<151>2004-02-25
<160>4
<170>PatentIn?version?3.1
<210>1
<211>1255
<212>PRT
<213>corona?virus
<400>1
Met?Phe?Ile?Phe?Leu?Leu?Phe?Leu?Thr?Leu?Thr?Ser?Gly?Ser?Asp?Leu
1 5 10 15
Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln
20 25 30
His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg
35 40 45
Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser
50 55 60
Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Asp?Asn?Pro?Val
65 70 75 80
Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn
85 90 95
Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln
100 105 110
Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys
115 120 125
Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met
130 135 140
Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr
145 150 155 160
Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser
165 170 175
Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly
180 185 190
Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp
195 200 205
Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu
210 215 220
Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro
225 230 235 240
Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
245 250 255
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile
260 265 270
Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys
275 280 285
Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn
290 295 300
Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr
305 310 315 320
Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser
325 330 335
Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr
340 345 350
Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly
355 360 365
Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala
370 375 380
Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly
385 390 395 400
Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe
405 410 415
Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
420 425 430
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu
435 440 445
Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly
450 455 460
Lys?Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp
465 470 475 480
Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val
485 490 495
Val?Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly
500 505 510
Pro?Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn
515 520 525
Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg
530 535 540
Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp
545 550 555 560
Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys
565 570 575
Ser?Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser
580 585 590
Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys?Thr?Asp?Val?Ser?Thr
595 600 605
Ala?Ile?His?Ala?Asp?Gln?Leu?Thr?Pro?Ala?Trp?Arg?Ile?Tyr?Ser?Thr
610 615 620
Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln?Ala?Gly?Cys?Leu?Ile?Gly?Ala?Glu
625 630 635 640
His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile
645 650 655
Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys
660 665 670
Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala
675 680 685
Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile
690 695 700
Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys?Thr?Ser?Val?Asp?Cys
705 710 715 720
Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu
725 730 735
Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile
740 745 750
Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys
755 760 765
Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe
770 775 780
Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile
785 790 795 800
Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met
805 810 815
Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile
820 825 830
Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr
835 840 845
Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala
850 855 860
Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe
865 870 875 880
Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn
885 890 895
Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala
900 905 910
Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly
915 920 925
Lys?Leu?Glu?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu
930 935 940
Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn
945 950 955 960
Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp
965 970 975
Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln
980 985 990
Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser?Ala?Asn?Leu?Ala?Ala
995 1000 1005
Thr?Lys?Met?Set?Glu?Cys?Val?Leu?Gly?Gln?Ser?Lys?Arg?Val?Asp
1010 1015 1020
Phe?Cys?Gly?Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala
1025 1030 1035
Pro?His?Gly?Val?Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln
1040 1045 1050
Glu?Arg?Asn?Phe?Thr?Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys
1055 1060 1065
Ala?Tyr?Phe?Pro?Arg?Glu?Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser
1070 1075 1080
Trp?Phe?Ile?Thr?Gln?Arg?Asn?Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr
1075 1090 1095
Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly?Asn?Cys?Asp?Val?Val?Ile?Gly
1100 1105 1110
Ile?Ile Asn?Asn?Thr?Val?Tyr Asp?Pro?Leu?Gln?Pro Glu?Leu?Asp
1115 1120 1125
Ser?Phe Lys?Glu?Glu?Leu?Asp Lys?Tyr?Phe?Lys?Asn His?Thr?Ser
1130 1135 1140
Pro?Asp Val?Asp?Leu?Gly?Asp Ile?Ser?Gly?Ile?Asn Ala?Ser?Val
1145 1150 1155
Val?Asn Ile?Gln?Lys?Glu?Ile Asp?Arg?Leu?Asn?Glu Val?Ala?Lys
1160 1165 1170
Asn?Leu Asn?Glu?Ser?Leu?Ile Asp?Leu?Gln?Glu?Leu Gly?Lys?Tyr
1175 1180 1185
Glu?Gln Tyr?Ile?Lys?Trp?Pro Trp?Tyr?Val?Trp?Leu Gly?Phe?Ile
1190 1195 1200
Ala?Gly Leu?Ile?Ala?Ile?Val Met?Val?Thr?Ile?Leu Leu?Cys?Cys
1205 1210 1215
Met?Thr Ser?Cys?Cys?Ser?Cys Leu?Lys?Gly?Ala?Cys Ser?Cys?Gly
1220 1225 1230
Ser?Cys Cys?Lys?Phe?Asp?Glu Asp?Asp?Ser?Glu?Pro Val?Leu?Lys
1235 1240 1245
Gly?Val Lys?Leu?His?Tyr?Thr
1250 1255
<210>2
<211>422
<212>PRT
<213>corona?virus
<400>2
Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile
1 5 10 15
Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly
20 25 30
Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn
35 40 45
Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu?Glu
50 55 60
Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser?Gly
65 70 75 80
Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val?Arg
85 90 95
Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr
100 105 110
Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys
115 120 125
Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys
130 135 140
Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu
145 150 155 160
Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu?Gly
165 170 175
Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser?Arg
180 185 190
Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro
195 200 205
Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu
210 215 220
Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln
225 230 235 240
Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser
245 250 255
Lys?Lys?Pro?Arg?Gln?Lys?Arg?Thr?Ala?Thr?Lys?Gln?Tyr?Asn?Val?Thr
260 265 270
Gln?Ala?Phe?Gly?Arg?Arg?Gly?Pro?Glu?Gln?Thr?Gln?Gly?Asn?Phe?Gly
275 280 285
Asp?Gln?Asp?Leu?Ile?Arg?Gln?Gly?Thr?Asp?Tyr?Lys?His?Trp?Pro?Gln
290 295 300
Ile?Ala?Gln?Phe?Ala?Pro?Ser?Ala?Ser?Ala?Phe?Phe?Gly?Met?Ser?Arg
305 310 315 320
Ile?Gly?Met?Glu?Val?Thr?Pro?Ser?Gly?Thr?Trp?Leu?Thr?Tyr?His?Gly
325 330 335
Ala?Ile?Lys?Leu?Asp?Asp?Lys?Asp?Pro?Gln?Phe?Lys?Asp?Asn?Val?Ile
340 345 350
Leu?Leu?Asn?Lys?His?Ile?Asp?Ala?Tyr?Lys?Thr?Phe?Pro?Pro?Thr?Glu
355 360 365
Pro?Lys?Lys?Asp?Lys?Lys?Lys?Lys?Thr?Asp?Glu?Ala?Gln?Pro?Leu?Pro
370 375 380
Gln?Arg?Gln?Lys?Lys?Gln?Pro?Thr?Val?Thr?Leu?Leu?Pro?Ala?Ala?Asp
385 390 395 400
Met?Asp?Asp?Phe?Ser?Arg?Gln?Leu?Gln?Asn?Ser?Met?Ser?Gly?Ala?Ser
405 410 415
Ala?Asp?Ser?Thr?Gln?Ala
420
<210>3
<211>221
<212>PRT
<213>corona?virus
<400>3
Met?Ala?Asp?Asn?Gly?Thr?Ile?Thr?Val?Glu?Glu?Leu?Lys?Gln?Leu?Leu
1 5 10 15
Glu?Gln?Trp?Asn?Leu?Val?Ile?Gly?Phe?Leu?Phe?Leu?Ala?Trp?Ile?Met
20 25 30
Leu?Leu?Gln?Phe?Ala?Tyr?Ser?Asn?Arg?Asn?Arg?Phe?Leu?Tyr?Ile?Ile
35 40 45
Lys?Leu?Val?Phe?Leu?Trp?Leu?Leu?Trp?Pro?Val?Thr?Leu?Ala?Cys?Phe
50 55 60
Val?Leu?Ala?Ala?Val?Tyr?Arg?Ile?Asn?Trp?Val?Thr?Gly?Gly?Ile?Ala
65 70 75 80
Ile?Ala?Met?Ala?Cys?Ile?Val?Gly?Leu?Met?Trp?Leu?Ser?Tyr?Phe?Val
85 90 95
Ala?Ser?Phe?Arg?Leu?Phe?Ala?Arg?Thr?Arg?Ser?Met?Trp?Ser?Phe?Asn
100 105 110
Pro?Glu?Thr?Asn?Ile?Leu?Leu?Asn?Val?Pro?Leu?Arg?Gly?Thr?Ile?Val
115 120 125
Thr?Arg?Pro?Leu?Met?Glu?Ser?Glu?Leu?Val?Ile?Gly?Ala?Val?Ile?Ile
130 135 140
Arg?Gly?His?Leu?Arg?Met?Ala?Gly?His?Ser?Leu?Gly?Arg?Cys?Asp?Ile
145 150 155 160
Lys?Asp?Leu?Pro?Lys?Glu?Ile?Thr?Val?Ala?Thr?Ser?Arg?Thr?Leu?Ser
165 170 175
Tyr?Tyr?Lys?Leu?Gly?Ala?Ser?Gln?Arg?Val?Gly?Thr?Asp?Ser?Gly?Phe
180 185 190
Ala?Ala?Tyr?Asn?Arg?Tyr?Arg?Ile?Gly?Asn?Tyr?Lys?Leu?Asn?Thr?Asp
195 200 205
His?Ala?Gly?Ser?Asn?Asp?Asn?Ile?Ala?Leu?Leu?Val?Gln
210 215 220
<210>4
<211>76
<212>PRT
<213>corona?virus
<400>4
Met?Tyr?Ser?Phe?Val?Ser?Glu?Glu?Thr?Gly?Thr?Leu?Ile?Val?Asn?Ser
1 5 10 15
Val?Leu?Leu?Phe?Leu?Ala?Phe?Val?Val?Phe?Leu?Leu?Val?Thr?Leu?Ala
20 25 30
Ile?Leu?Thr?Ala?Leu?Arg?Leu?Cys?Ala?Tyr?Cys?Cys?Asn?Ile?Val?Asn
35 40 45
Val?Ser?Leu?Val?Lys?Pro?Thr?Val?Tyr?Val?Tyr?Ser?Arg?Val?Lys?Asn
50 55 60
Leu?Asn?Ser?Ser?Glu?Gly?Val?Pro?Asp?Leu?Leu?Val
62 70 75
Claims (3)
1, a kind of immune microsphere that is used to detect SARS antibody, it is for being kernel with the polystyrene microsphere, its surperficial carboxyl by the poly-D-lysine coupling as the sars coronavirus S of antigen, N, M or E albumen, described polystyrene microsphere is that surface diameter is 200～900 nanometers, and there is carboxyl modified on its surface.
2, the described preparation method who is used to detect the immune microsphere of SARS antibody of a kind of claim 1 is to adopt chemical coupling method with sars coronavirus S, N, and M or E albumen are coupled to microballoon as antigen, comprise following step:
1) with polystyrene microsphere 15～20mg/ml, after the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the phosphate buffer of pH4～5 of 0.01～0.05M, the carbodiimide that adds 2～4mg/ml then, after room temperature reaction is complete, centrifugal, with the carbonate buffer solution washing of pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH7.5～8.5 of 0.01～0.05M; Described polystyrene microsphere diameter is 200～900 nanometers, and there is carboxyl modified on its surface;
2) in the mixed liquor that step 1) obtains, add the poly-D-lysine of 0.2～2.4mg/ml, complete in room temperature reaction; After the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the phosphate buffer of pH4～5 of 0.01～0.05M, add the carbodiimide of 2～4mg/ml then, complete in room temperature reaction; With the carbonate buffer solution washing of pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH7.5～8.5 of 0.01～0.05M;
3) to step 2) add sars coronavirus S in the mixed liquor that obtains as antigen, N, M or E albumen 0.2～2.4mg/ml are complete in room temperature reaction; After the carbonate buffer solution washing with pH8.8～9.8 of 0.1～0.5M, be resuspended in the carbonate buffer solution of pH8.8～9.8 of 0.1～0.5M; Add 50～200 microlitre 0.25M acetamides, after room temperature reaction is complete, the centrifugal solid of telling;
4) be resuspended in the carbonate buffer solution of pH7.5～8.5 of 1g/ml cow's serum (BSA) and 0.01～0.05M, in room temperature reaction fully after, the centrifugal solid of telling obtains being used to detect the immune microsphere of SARS antibody.
3, the described purposes that is used to detect the immune microsphere of SARS antibody of a kind of claim 1, this immune microsphere can be used as detectable and detects anti-SARS antibody.
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