CN1661371A - Immune microsphere in use for detecting SARS antigen, preparation method and application - Google Patents
Immune microsphere in use for detecting SARS antigen, preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of immune microsphere in use for detecting SARS antigen, 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 of detecting SARS antigen and its production and use that is used to.
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 SARS detection of antigens method commonly used clinically at present.Use euzymelinked immunosorbent assay (ELISA) (ELISA) to detect antigen, be about on the antibody sandwich ELISA Plate, add the sealing of calf serum or skimmed milk, added behind the test serum incubation again 1 hour, testing sample after washing repeatedly, incubation be flushing again after 1 hour, enzyme-added again target antibody, incubation, flushing adds the substrate of colour developing after repeatedly again.Though this method susceptibility and specificity are fine, finish Total Test needs more than 2 hours.
The method that immune microsphere method in the past detects antigen is a kind of easy, fast (only just can obtain the result in 1 minute) responsive and special method.But no matter the preparation method of the immune latex reagent that adopts is physics or chemistry side, and the immune microsphere that obtains all is not by covalent bonds, so the reagent instability, influenced by temperature and salinity and is easy to occur false positive.
Summary of the invention
The euzymelinked immunosorbent assay (ELISA) that the objective of the invention is to overcome existing detection SARS antigen is easy inadequately, quick; 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 antigen are provided.
Another object of the present invention provides a kind of preparation method who is used to detect the immune microsphere of SARS antigen.
A further object of the present invention provides a kind of purposes that is used to detect the immune microsphere of SARS antigen.
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)) antigen provided by the invention, it is for being kernel with the polystyrene microsphere, its surperficial carboxyl by the poly-D-lysine coupling SARS coronary virus resistant S, N, the antibody of 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 antigen, is to adopt chemical coupling method with SARS coronary virus resistant S, N, and the antibody coupling of M or E albumen comprises following step to microballoon:
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) the sars coronavirus S of usefulness genetic recombination, N, M or E protein immune animal produce antibody, and carry out purifying;
Sars coronavirus S with genetic recombination, N, M or E protein immune animal (Japan large rabbit, available from experimental animal center, Beijing) concrete steps be: by the antigen amount is the 0.5mg/ kg body weight, the immune animal Japan large rabbit, immunity for the first time adds isopyknic complete freund adjuvant with above-mentioned antigen, carries out the immunity second time after two weeks, and immunity for the second time adds isopyknic incomplete freund adjuvant with above-mentioned antigen; Strengthened once immunity in every month later on; Begin to detect antibody titer after the immunity for the second time, treat that antibody titer reaches at 1: 32 o'clock and collects serum with immune double diffusion method; Adopt albumin A affinity column method antibody purification, obtain polyclonal antibody; Adopt conventional ELISA and WesternBlot to identify the polyclonal antibody that obtains;
4) to step 2) add the sars coronavirus S as polyclonal antibody of step 3) in the mixed liquor that obtains, 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;
5) be resuspended in the carbonate buffer solution of pH7.5～8.5 of 1mg/ml cow's serum (BSA) and 0.01～0.05M, after room temperature reaction is complete, the centrifugal solid of telling, the immune microsphere that is used to detect SARS antigen of SARS coronary virus resistant S, M, E and N protein antibodies that obtained coupling.
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
IleGlnLysGluIle 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 HislleAspAlaTyr
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
The purposes that is used to detect the immune microsphere of SARS antigen provided by the invention, this immune microsphere detects SARS antigen as detectable.
The immune microsphere that is used to detect SARS antigen provided by the invention can be used as detectable and detects SARS antigen, 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 antigen provided by the invention, wherein, stir, with testing sample and immune microsphere mixing and be round spot shape with bamboo let, toothpick, sticking plaster or other objects.
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)) antigen provided by the invention is the sars coronavirus S with genetic recombination, N, M, the antibody that E protein immunization rabbit produces is through behind the purifying, antibody is coupled on the 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 antigen 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:
The preparation coupling immune microsphere that is used to detect SARS antigen of the antibody that produces of sars coronavirus S protein immunization rabbit
1.SARS the preparation of coronavirus S proteantigen
Genetic fragment with RT-PCR method amplification sars coronavirus S, with this genetic fragment after order-checking confirms that gene order is correct, it is cloned into expression vector such as prokaryotic expression carrier pQE30, Yeast expression carrier pPICZ α A expresses and protein purification then again.
2, the evaluation of sars coronavirus S proteantigen
Behind the antigen purification, must 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) purity of protein is measured: 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 degree after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 degree 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 degree 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.
3, Polyclonal Antibody Preparation: the S albumen of using purifying is as the immune White Rabbit of antigen difference.The antigen amount is the 0.5mg/ kg body weight, and immunity for the first time adds isopyknic complete freund adjuvant with antigen, carries out the immunity second time after two weeks, and antigen adds isopyknic incomplete freund adjuvant.Strengthened once immunity in every month later on.Immune Hou begins to detect antibody titer with immune double diffusion method for the second time, treats that antibody titer reaches at 1: 32 o'clock and collects serum.Adopt albumin A affinity column method antibody purification.
4, the evaluation of polyclonal antibody: adopt conventional ELISA and Western Blot to identify polyclonal antibody.
5, the preparation of microballoon:
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 the antibody 0.2mg/ml of the SARS coronary virus resistant S albumen that step 3 makes in the mixed liquor that obtains, putting room temperature 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 antibodies, 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 antigen, coupling as the immune microsphere of the sars coronavirus S albumen of antibody.
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 the preparation of immune microsphere of sars coronavirus N protein polyclone antibody
1, the preparation of sars coronavirus N proteantigen:
Genetic fragment with RT-PCR method amplification sars coronavirus N, with this genetic fragment after order-checking confirms that gene order is correct, it is cloned into expression vector such as prokaryotic expression carrier pQE30, Yeast expression carrier pPICZ α A expresses and protein purification then again.
2. the evaluation of antigen:
Behind the antigen purification, must 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 the protein content of antigen with formula 280nmOdx1.45-260nmODx0.74.
(2) purity of protein is measured: 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 degree after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 degree 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 degree 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.Polyclonal Antibody Preparation:
3, distinguish immune animal (animal can be White Rabbit, goat and chicken) with the N albumen of purifying as antigen.The antigen amount is the 0.5mg/ kg body weight, and immunity for the first time adds isopyknic complete freund adjuvant with antigen, carries out the immunity second time after two weeks, and antigen adds isopyknic Freund.Strengthened once immunity in every month later on.Immune Hou begins to detect antibody titer with immune double diffusion method for the second time, treats that antibody titer reaches at 1: 32 o'clock and collects serum.Adopt albumin A affinity column method antibody purification.
4. the evaluation of polyclonal antibody: adopt conventional ELISA and Western Blot to identify polyclonal antibody.
5. the preparation of microballoon:
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 the antibody 0.2mg/ml of SARS coronary virus resistant N albumen in the mixed liquor that obtains, 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 antibodies, 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 antigen, coupling as the immune microsphere of the sars coronavirus N albumen of antibody.
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 the preparation of immune microsphere of sars coronavirus M protein polyclone antibody
1, the preparation of sars coronavirus M proteantigen:
Genetic fragment with RT-PCR method amplification sars coronavirus M, with this genetic fragment after order-checking confirms that gene order is correct, it is cloned into expression vector such as prokaryotic expression carrier pQE30, Yeast expression carrier pPICZ α A expresses and protein purification then again.
2, the evaluation of antigen:
Behind the antigen purification, must 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 the protein content of antigen with formula 280nmOdx1.45-260nmODx0.74.
(2) purity of protein is measured: 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 degree after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 degree 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 degree 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.Polyclonal Antibody Preparation:
3, distinguish immune animal (animal can be White Rabbit, goat and chicken) with the M albumen of purifying as antigen.The antigen amount is the 0.5mg/ kg body weight, and immunity for the first time adds isopyknic complete freund adjuvant with antigen, carries out the immunity second time after two weeks, and antigen adds isopyknic Freund.Strengthened once immunity in every month later on.Immune Hou begins to detect antibody titer with immune double diffusion method for the second time, treats that antibody titer reaches at 1: 32 o'clock and collects serum.Adopt albumin A affinity column method antibody purification.
4, the evaluation of polyclonal antibody: adopt conventional ELISA and Western Blot to identify polyclonal antibody.
5, the preparation of microballoon:
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 the antibody 0.2mg/ml of SARS coronary virus resistant M albumen in the mixed liquor that obtains, 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 antibodies, 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 antigen, coupling as the immune microsphere of the sars coronavirus M albumen of antibody.
This immune microsphere that is used to detect SARS antigen 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 the preparation of immune microsphere of sars coronavirus E protein polyclone antibody
1, the preparation of sars coronavirus E proteantigen:
Genetic fragment with RT-PCR method amplification sars coronavirus E, with this genetic fragment after order-checking confirms that gene order is correct, it is cloned into expression vector such as prokaryotic expression carrier pQE30, Yeast expression carrier pPICZ α A expresses and protein purification then again.
2, the evaluation of antigen:
Behind the antigen purification, must 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 the protein content of antigen with formula 280nmOdx1.45-260nmODx0.74.
(2) purity of protein is measured: 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 degree after 20 hours, discard antigen coated liquid, add 5% calf serum to seal not by antigen coated part, put 37 degree 1 hour, after PBS-tween and the washing of PBS damping fluid, add positive patients serum, put 37 degree 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.Polyclonal Antibody Preparation:
3, distinguish immune animal (animal can be White Rabbit, goat and chicken) with the E albumen of purifying as antigen.The antigen amount is the 0.5mg/ kg body weight, and immunity for the first time adds isopyknic complete freund adjuvant with antigen, carries out the immunity second time after two weeks, and antigen adds isopyknic Freund.Strengthened once immunity in every month later on.Immune Hou begins to detect antibody titer with immune double diffusion method for the second time, treats that antibody titer reaches at 1: 32 o'clock and collects serum.Adopt albumin A affinity column method antibody purification.
4, the evaluation of polyclonal antibody: adopt conventional ELISA and Western Blot to identify polyclonal antibody.
5, the preparation of microballoon:
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 the antibody 0.2mg/ml of SARS coronary virus resistant E albumen in the mixed liquor that obtains, 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 antibodies, 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 antigen, coupling as the immune microsphere of the sars coronavirus E albumen of antibody.
This immune microsphere that is used to detect SARS antigen 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 (SARS) antigen with immune microsphere detectable provided by the invention
Detect the method for (SARS) antigen with the immune microsphere detectable
1, method of operating:
(1) tested serum 10 microlitres is dropped on the clean slide or transparent institute tablet, add 5 microlitre immune microsphere detectable, be the round spot of 1 centimetre of diameter with the bamboo let mixing.
(2) under black background, observe agglutinating reaction.
(3) semiquantitative determination with tested serum doubling dilution, is measured by last method, is antigen titre with the dilutability.
2, the result judges:
(1) naked eyes are judged:
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 represent antigen titre by semiquantitative method.
Observations is seen Fig. 1;
(2) microscopically is judged:
Observe down for 40 times, negative visible microsphere particle uniformly, positive visible microsphere particle aggegation is piece, sees Fig. 2. and Fig. 3.
FPI04185-sequence?list
SEQUENCE?LISTING
＜110〉Institute of Zoology, Academia Sinica
＜120〉be used to immune microsphere of detecting SARS antigen and its production and use
<130>FPI04185
<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
195 200 210
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?Gln?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?Ser?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
1085 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
65 70 75
Claims (3)
1, a kind of immune microsphere that is used to detect SARS antigen, it is for being kernel with the polystyrene microsphere, its surperficial carboxyl by the poly-D-lysine coupling SARS coronary virus resistant S, N, the antibody of 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 antigen of a kind of claim 1 is to adopt chemical coupling method with SARS coronary virus resistant S, N, and the antibody coupling of M or E albumen comprises following step to microballoon:
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) the sars coronavirus S of usefulness genetic recombination, N, M or E protein immune animal produce antibody, and carry out purifying;
4) to step 2) add the SARS coronary virus resistant S of step 3) in the mixed liquor that obtains, N, the antibody 0.2～2.4mg/ml of M or E albumen is 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;
5) be resuspended in the carbonate buffer solution of pH7.5～8.5 of 1mg/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 antigen.
3, the described purposes that is used to detect the immune microsphere of SARS antigen of a kind of claim 1, this immune microsphere detects SARS antigen as detectable.
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| CN109265522A (en) * | 2018-09-29 | 2019-01-25 | 东北农业大学 | For detecting the sensitization Properties of Polystyrene Nano Particles and its preparation method and application of canine distemper virus hemagluttinin proteins H antibody |
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| CN109265522A (en) * | 2018-09-29 | 2019-01-25 | 东北农业大学 | For detecting the sensitization Properties of Polystyrene Nano Particles and its preparation method and application of canine distemper virus hemagluttinin proteins H antibody |
| CN109265522B (en) * | 2018-09-29 | 2022-03-04 | 东北农业大学 | Sensitized polystyrene nano-microsphere for detecting canine distemper virus hemagglutinin H antibody and preparation method and application thereof |
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