CN105319361B - Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip and preparation method thereof, application method - Google Patents
Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip and preparation method thereof, application method Download PDFInfo
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Abstract
A kind of Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip of the present invention, belong to field of biological detection, including SSRV recombinant antigens p29 expression, purifying, it is prepared by monoclonal antibody, it is prepared by polyclonal antibody, colloid gold immune layer test strips prepare and its detect the sensitivity and specificity of SSRV antigens, the invention further relates to the preparation method and application method of the colloidal gold immunochromatographimethod reagent strip, ground with traditional Viral extraction, the methods such as centrifugation are compared, it is convenient that the colloidal gold immuno-chromatography test paper strip set up with the present invention has, quickly, sensitive and special the advantages of, clear and definite diagnostic result can be obtained in 10 minutes, it is particularly suitable for clinical and grass-roots unit to use.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of Scylla paramamosain reovirus colloidal gold immunochromatographimethod examination
Agent bar and preparation method thereof and the application in the detection of Scylla paramamosain reovirus.
Background technology
Scylla paramamosain reovirus (Scylla serrata reovirus, SsRV) is (hereinafter referred to as blue or green Scylla paramamosain
Crab) one of zymad of significant damage is caused in aquaculture, the mud crab communicable disease that SsRV causes-" clear water disease " gives mud crab
Aquaculture causes huge economic loss.Currently, due to the epidemic disease background is unclear, control technology Recent Progresses In The Development slow,
Usually seem helpless in the control of the disease.Therefore, detection SsRV viruses infection early, the propagation of preventing virus turns into
The primary means of current prevention and control " clear water disease ".And SsRV virus-infected animals are found to the full extent, it is desirable to provide easy,
Effective detection method.To the medical diagnosis on disease one carried out by Symptom Observation at present, disease has occurred and that when occurring etc. symptom,
And it is sick without effective treatment method for this, its economic loss is inevitable.Two is to bear animal tissue's ultra-thin section
Virus after dye in the direct tissues observed of electron microscope is, it is necessary to the instrument of costliness and the knack people by strict training
Member, while structure observation is bred to virion during virus is a large amount of in the tissue, animal or i.e. will appear from symptom, economical
Loss is inevitable.Three is the RT-PCR technology based on nucleic acid amplification, though the technology sensitivity is high, complex operation, it is necessary to
Supporting instrument and equipment and more skilled professional and technical personnel, be unfavorable for the scene of the disease, rural area and grass-roots unit use and
Daily monitoring.Therefore, in order to accurately detect and prevent and treat mud crab " clear water is sick " in time, foundation one kind is conveniently, simply, quickly, accurately
Detection SsRV methods, the early stage monitoring to mud crab " clear water disease " has important practical significance.
Colloidal gold immunochromatographimethod technology is quick, convenient due to its, is not required to special installation, as a result judges the advantage such as directly perceived, more
It is valued by people to get over.So far, Scylla paramamosain reovirus immunity colloidal gold test paper strip method is yet there are no to build
Vertical report.
The content of the invention
A technical problem to be solved by this invention is directed to above-mentioned state of the art and provides a kind of by immune
Colloidal gold-labeled method develops a kind of rapidly detection Scylla paramamosain reovirus colloid simple to operate, with low cost, quick
Golden immunochromatography reagent bar.
Another technical problem to be solved by this invention is directed to above-mentioned state of the art and provides one kind and prepare
The method for stating Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip.
Another technical problem to be solved by this invention is directed to above-mentioned state of the art and provides a kind of quick, letter
Just, the application method of accurate detection Scylla paramamosain reovirus immunochromatography reagent bar.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：The Scylla paramamosain reovirus collaurum is exempted from
Epidemic disease chromatographs reagent strip, it is characterised in that：The test strips are by sample pad, pad, nitrocellulose membrane, absorption pad, hard polychlorostyrene second
Alkene liner plate is constituted, and the anti-Scylla paramamosain reovirus p29 albumen lists of immuno-gold labeling thing are coated with the pad
Clonal antibody, the nitrocellulose membrane is coated with the detection of anti-Scylla paramamosain reovirus p29 protein polyclone antibodies
Line and the nature controlling line for being coated with rabbit anti-mouse igg antibody；Wherein, the nitrocellulose membrane is placed in the middle of RPVC liner plate,
The sample pad is arranged on the side of the RPVC liner plate, and the sample pad is arranged on the RPVC
The opposite side of liner plate, the pad is smooth between sample pad and nitrocellulose membrane.
The invention provides a kind of method for preparing Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip, it is special
Levy and be：The method is comprised the following steps：
(1) acquisition of antigen：Polymerase chain reaction, as primer, is used with sequence shown in SEQ ID NO.1 and SEQ ID NO.2
The corresponding genetic fragments of anti-Scylla paramamosain reovirus p29 should be expanded, prokaryotic expression carrier pET28a (+) is cloned into, used
IPTG induces expression of recombinant e. coli, affinity chromatography purified recombinant antigens is used, to obtain the Escherichia coli restructuring table of purifying
The histidine-tagged p29 gene proteins His-p29 of the band that reaches；
(2) monoclonal antibody of anti-Scylla paramamosain reovirus SsRV p29 albumen is prepared：With step (1) purifying
His-p29 protein immunization Balb/c mouse, through cell fusion, screening is the hybridoma of CGMCC No.10888 by preserving number
Strain 1M9 produces the monoclonal antibody for obtaining secreting anti-Scylla paramamosain reovirus p29 albumen；
(3) preparation of colloid gold particle：Colloidal gold solution is prepared as reducing agent using trisodium citrate, and adjusts colloid
The pH of gold solution is standby to 8；
(4) the colloid gold label thing of anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies is prepared：By step (2)
The monoclonal antibody of obtained anti-Scylla paramamosain reovirus p29 albumen is added in standby colloidal gold solution, and anti-plan cave is blue or green
The quality of the monoclonal antibody of crab reovirus p29 albumen and the volume ratio of colloidal gold solution are 1：100, purified and concentration
The anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies of collaurum binding marker are formed afterwards；
(5) preparation of pad：The anti-Scylla paramamosain of the collaurum binding marker sprayed on the pad exhales the lonely disease of intestines
The concentration of malicious p29 protein monoclonal antibodies is 1.5mg/ml, and quantity for spray is 40-50ul/cm；
(6) anti-Scylla paramamosain reovirus p29 protein polyclone antibodies and rabbit anti-mouse igg antibody are prepared；
(7) preparation of antibody solid phase nitrocellulose membrane：By anti-Scylla paramamosain reovirus p29 protein polyclone antibody bags
Antigen is captured as detection line to the detection line position on nitrocellulose membrane, concentration is 1mg/ml, and quantity for spray is 2-3ul/cm；
The Quality Control line position that the rabbit anti-mouse igg secondary antibody of anti-binding antibody is sprayed on nitrocellulose membrane captures antigen as nature controlling line, dense
It is 0.75mg/ml to spend, and coating parameter is 1-3ul/cm；
(8) by absorption pad, the nitrocellulose membrane with detection line and nature controlling line, pad, sample pad, RPVC
Liner plate is assembled into the colloidal gold immunochromatographydetection detection test paper bar that can detect anti-Scylla paramamosain reovirus p29.
Further, the preparation method of colloid gold particle is as follows in the step (3)：Gold chloride is pressed with trisodium citrate
According to 1：2 ratio is mixed and heated and is made colloidal gold solution, and the particle size diameter that collaurum is obtained is 10-20nm.
Further, in the step (4) anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies collaurum mark
Remember that the preparation method of thing is as follows:Monoclonal antibody is added in the colloidal gold solution in step (3), even, addition ox is mixed slowly
Haemocyanin, the precipitation resuspended precipitation of the phosphate buffer containing 1% bovine serum albumin after centrifugation is added and overlaps nitrogen, system
Into the monoclonal antibody of colloid gold label.
Further, the method for preparing anti-Scylla paramamosain reovirus p29 protein polyclone antibodies in the step (6)
It is as follows：Purifying His-p29 albumen prepared by the step (1) is made into antigen immune new zealand white rabbit：Initial immunity takes antigen
With the fully emulsified mixing of equivalent complete Freund's adjuvant, multiple spot hypodermic injection on the inside of the neck, back, back leg in rabbit, after 14 days
Mixed with incomplete Freund's adjuvant with antigen, subcutaneous multi-point injection；1 time is carried out after 7 days again to be immunized, immunizing dose and immunization route
It is immune with the 2nd time identical, gather a small amount of serum and be used as ELISA detection antibody potency；Carry out the 4th after 14 days to be immunized, immunizing agent
Amount and immunization route are immune with the 3rd time identical, Animal Anesthesia are carried out after 14 days, by surgically separating arteria carotis, nothing
Bacterium collects blood, and 4 DEG C of the blood being collected into places 2h, and then 4 DEG C, 3000 revs/min are centrifuged 15 minutes, collect supernatant and are system
Standby anti-mud crab reovirus polyvalent antibody；Polyvalent antibody caprylic acid-ammonium initial purification, and G- albumen affinity purifications
Afterwards, the anti-mud crab reovirus p29 protein polyclone antibodies of high-purity are obtained.
The present invention also provides a kind of method of use Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip, and it is special
Levy and be：Comprise the following steps：
(1) pretreatment of mud crab sample：Take appropriate mud crab cheek tissue sample to be placed in lapping apparatus, add the TEN of equivalent
Grinding buffer solution is Tris-EDTA-NaCl, is fully ground homogenised tissue and is made suspension, and suspension stands 5 minutes or centrifugation
Afterwards, supernatant is taken as trial-production bar sample solution；
(2) detect：The μ L of sample solution 100 for taking step (1) are dropped in test paper sample pad of the present invention, and knot is observed after 10 minutes
Really, while taking appropriate mud crab reovirus liquid and TEN grinding buffer solutions respectively respectively as positive and negative control；
(3) result judgement：It is mud crab when the detection line is presented red line when detection sample is added in sample pad
Reovirus is positive, i.e., contain mud crab reovirus in sample；It is that mud crab exhales intestines lonely if detection line occurs without red line
It is viral negative, i.e., mud crab reovirus is not contained in sample；If red line does not occur in nature controlling line, test paper is invalid.
The Cleaning Principle of Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip of the present invention：
By the water accepting layer insertion of sample-adding end or dropwise addition TEN grindings thereon of Scylla paramamosain reovirus quick detection test paper
Buffer solution, the detection sample of virion is contained from mud crab cheek tissue extraction, according to double-antibody sandwich immunochromatography principle, i.e., by
The anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies of collaurum binding marker with detection sample liquid in antigen it is anti-
Should, formed by gold mark monoclonal antibody-Scylla paramamosain reovirus compound, wrapped in advance to nitrocellulose membrane when the compound is chromatographed
During by the polyclonal antibody of the anti-Scylla paramamosain reovirus p29 albumen in detection line, antibody herein can recognize this
Antigen in compound, the result of reaction just forms gold mark monoclonal antibody+Scylla paramamosain reovirus antigen+anti-Scylla paramamosain and exhales intestines
The interlayer structure of the polyclonal antibody of lonely virus p29 albumen, is finally that anti-Scylla paramamosain reovirus p29 protein monoclonals resist
The colloid gold particle marked on body is fixed herein and accumulates the macroscopic red line of appearance, unreacted gold mark monoclonal antibody then still after
Subsequent layers analysis moves ahead, and when the point of arrival is coated on nature controlling line in advance exempts from anti-mouse IgG, Antibody types are the gold mark monoclonal antibody of IgG by it
It is combined with, so as to also occur being fixed by colloid gold particle and being accumulated and show macroscopic red line herein, result is：
Detection line display is red to represent that anti-Scylla paramamosain reovirus is positive；Detection line does not show red, represents that anti-Scylla paramamosain is exhaled
The lonely virus of intestines is negative, i.e., be free of anti-Scylla paramamosain reovirus or anti-Scylla paramamosain reovirus content in detection sample
Extremely low, nature controlling line display is red, represents that test paper is effective；Or anti-plan cave red is not shown at nature controlling line, illustrates that test paper fails, i.e.,
Mud crab reovirus p29 protein monoclonal antibodies or exempt from anti-mouse IgG inactivation, testing result is invalid.
Compared with prior art, the advantage of the invention is that：
1st, Scylla paramamosain reovirus quick detection test paper of the invention is quick, easy, accurate, sensitive with detecting
The features such as, and with stability higher；
2nd, in Scylla paramamosain reovirus quick detection test paper application method of the invention, without adding special cracking
Liquid；Only need to be by careless mud crab cheek tissue mashing, compared with the methods such as the grinding of traditional Viral extraction, centrifugation, the convenient letter of the method
It is single, can rapid extraction Viral diagnosis；
3rd, influenceed smaller by extraneous factor, detection sensitivity and accuracy are high；
4th, can stablize and preserve, it is easy to carry, instrument and equipment is not required to, integrated cost is relatively low, is especially suitable for clinical and basic unit's list
Position uses.
5th, test paper of the invention easy control simple to operate, without professional's operation.
Preservation explanation
1st, hybridoma cell strain Mab-1M9, Classification And Nomenclature is：Anti- mud crab exhales orphan's virus p29 protein monoclonal antibodies long miscellaneous
Tumor cell strain is handed over, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 16th, 2015
(CGMCC), deposit number is CGMCC No.10888, depositary institution address：The institute 3 of Chaoyang District Beijing North Star West Road 1.
Brief description of the drawings
Fig. 1 be purifying recombinant protein p29 polyacrylamide gel electrophoresises result (wherein swimming lane 1 be protein molecular quality
Standard, swimming lane 2 is the recombinant protein p29 of purifying)；
Fig. 2 is that (wherein swimming lane 1 is protein molecular quality standard to Western blotting result figures, and swimming lane 2 is restructuring egg
White p29)；
Fig. 3 is immune colloid gold test paper assembling schematic diagram.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Biological material source：
Scylla paramamosain reovirus SsRV：Applicant laboratory separates from clear water disease mud crab is suffered from and obtains；
PET28a (+), coli expression carrier is introduced from Novogen companies of the U.S.；
Escherichia coli (E.coli) BL21 (DE3)：Introduced from Novogen companies of the U.S.；
PMD18-S12, (Molecular characterization of eight are obtained by laboratory where applicant
segments of Scylla serrata reovirus(SsRV)provides the complete genome
sequence；Jigang Chen,Juan Xiong,Bojing Cui,Jifang Yang,Wenchen Li,Zhijuan
Mao；Arch Virol.2012,157(8):1551-1557.〕.
Embodiment 1
Such as accompanying drawing, the immune colloid gold test paper that Scylla paramamosain reovirus can be detected of the invention, the test strips be by
Sample pad 1, pad 2, nitrocellulose membrane 3, absorption pad 4, RPVC liner plate 5 are combined into one and structure by bonding
Into, wherein, detection line 6 and nature controlling line 7 are coated with the middle part of nitrocellulose membrane 3, gold conjugation pad 2 is by glass fibre element film
It is made, the anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies of colloid gold label is sprayed on the gold conjugation pad 2
1M9 (G-1M9), monoclonal antibody is with purifying His-p29 protein immunization Balb/c mouse, through cell fusion, what screening was obtained
Hybridoma cell strain 1M9 secretions；Nitrocellulose NC films are the detection film of test strips, and the side that keeps left above is detection line 6, matter
Control line 7 is located at right side, the spraying rabbit-anti Scylla paramamosain reovirus SsRV p29 protein polyclone antibody PcAb1 of detection line 6, matter
The control spraying rabbit anti-mouse igg antibody of line 7 PcAb2.When Scylla paramamosain reovirus is contained in test sample, immune colloid gold mark
Remember anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies G-1M9 and the Scylla paramamosain reovirus SsRV surfaces of thing
Albumen p29 is combined, and forms immune complex matter G-1M9-SsRV as chromatography is moved, can be by the PcAb1 in spraying detection line 6
Capture, forms G-1M9-SsRV-PcAb1 compounds, and enrichment forms macroscopic red, and the G-1M9-SsRV being not associated with is answered
Compound continues to move on nature controlling line 7, is captured by PcAb2, forms G-1M9-PcAb2-SsRV compounds, shows that naked eyes are visible
Red；It is feminine gender if only there is nature controlling line 7；Two lines all occur being then the positive；If nature controlling line 7 and detection line 6 are not
Occur, then illustrate test strips failure.
The preparation of the anti-Scylla paramamosain reovirus p29 Protein reconstitution antigens of embodiment 2
Using specific primer, by polymerase chain reaction method to Scylla paramamosain reovirus p29 albumen correspondence
Viral genome Section 12 section (S12) expanded, Successful amplification obtain SsRV S12 gene opens reading frame (ORF), will
S12 genes ORF is cloned into coli expression carrier pET28a (+), builds RT-PCR expression vector pET28-S12:
Forward primer：5’-CGGGATCCATGAACCTGGAAATTAACAAC-3’(SEQ ID NO.1)
Reverse primer：5’-CCCAAGCTTAGTAATCGAGAACCCACTT-3’(SEQ ID NO.2)
Enter performing PCR amplification, 25 μ L by template of the recombinant vector PMD18-S12 containing reovirus S12 sections total lengths
Reaction system contains：Plasmid PMD18-S12 (100ng/ μ L) 1 μ L, upstream and downstream primer (20 μM) each 1 μ L, the μ of dNTP mix (10mM) 1
L, the μ L of 10 × PCR Buffer, 2.5 μ L, Taq archaeal dna polymerases 0.5, deionized water are mended to 25 μ L.
30 times cycle P CR programs are：95 DEG C of predegenerations 3 minutes, 95 DEG C are denatured 1 minute, 47 DEG C of annealing 45sec, and 72 DEG C are prolonged
Stretch 2 minutes, totally 30 circulate into performing PCR, finally extend 10 minutes.
After amplified production restriction enzyme BamH I, Hind III digestion, linearize pET-28a (+) with same enzyme and be connected, obtain
Recombinant vector pET-S12；With recombinant vector Transformed E .coli BL21 (DE3) competent cell, containing 50 μ g/mL kanamycins
37 DEG C of overnight incubations on agar plate, obtain pET-S12 recombinant bacteriums；LB of the picking single bacterium colony inoculation containing 50 μ g/mL kanamycins
Culture medium, 37 DEG C of overnight incubations are used as seed liquor；By 1:100 ratios inoculation 1000mL contains 50 μ g/mL kanamycins LB culture mediums,
37 DEG C of (200 revs/min) of concussions are cultivated to OD600When reaching 0.8, IPTG to final concentration 1mmol/L, 37 DEG C of vibrations (200 are added
Rev/min) Fiber differentiation 5h；4 DEG C, 5000 revs/min of centrifugations, 2 minutes collects thallines.Purpose product is purified under Denaturing to press
The QIAexpressionist of QIAGEN companiesTMProtein purification system operating guidance is carried out, and is improved slightly.Concretely comprise the following steps：
Thalline is suspended in buffer B (100mM NaH in the ratio of 5mL/g2PO4, 10mM TrisCl, 8M urea, pH8.0), in room
The lower slight oscillatory 45~60 minutes (avoiding bubble from producing) of temperature, 30 times are impacted with Ultrasonic Cell Disruptor 400W ultrasonic waves, 12000 turns/
Minute, 4 DEG C are centrifuged 15 minutes, take supernatant and add 2mL Ni-NTA, and 200 revs/min, 37 DEG C vibrate 30 minutes, make recombinant protein
And Ni2+After-NTA solid phases are fully combined, with 4mL eluents C (100mM NaH2PO4, 10mM TrisCl, 8M urea, 20mM
Imidazol, pH6.3) after wash-out 4 times, then with 0.5mL eluents D (100mM NaH2PO4, 10mM TrisCl, 8M urea,
) and 0.5mL eluents E (100mM NaH pH5.92PO4, 10mM TrisCl, 8M urea, pH 4.5) and each wash-out 4 washes, collects
Eluent E had both been the albumen His-p29 of purifying.Take 0.5 μ L purified products carries out polyacrylamide with protein molecular weight standard
Gel electrophoresis separate (5% concentration glue, 12% separation gel), through Coomassie blue stain after, with gel imaging system observe weight
Histone purification effect, as a result shows that eluent E can obtain single recombinant protein (Fig. 1)；According to albumen transfer instrument, (BioRad is public
Department) specification, gel separation albumen is gone into print nitric acid fibre tunica fibrosa (Whatman companies), resisted with the anti-6 × His monoclonals of mouse
Body is primary antibody (Invitrogen companies), and the rabbit anti-mouse igg (Invitrogen companies) of horseradish peroxidase-labeled is second
Antibody, conventionally carries out Western blotting analyses, as a result shows that the anti-6 × His monoclonal antibodies of mouse can be special
The His-p29 recombinant antigens (Fig. 2) of purifying are recognized, as a result proves successfully to realize expression of the SsRV p29 albumen in Escherichia coli
And high-purity purifying.
The preparation of the monoclonal antibody 1M9 of the anti-Scylla paramamosain reovirus p29 albumen of embodiment 3
Using the QIAexpressionist of Qiagen companiesTMProtein purification system purifies the group of Recombinant protein expression
(recombinant protein is referred to as the p29 albumen of His tag (His)：His-p29)；With the His-p29 albumen and equivalent Freund that purify
Freund's complete adjuvant mixes, and hypodermic injection is immunized BALB/c mouse.After 3 weeks, intraperitoneal injection respectively is mixed with equivalent incomplete Freund's adjuvant
The His-p29 albumen of the expression and purification of conjunction.His-p29 albumen booster immunization is used after 3 weeks 1 time.3rd immune rear 3d, anesthesia is caused
It is dead to harvest spleen, splenocyte is merged with nonsecreting type myeloma cell (SP2/0), RPMI of the fused cell containing HAT and HT
1640 solution are continuously cultivated.Screened through the culture medium containing HAT and the culture medium of HT, detected in culture by indirect ELISA
Antibody in clear, the strong positive clones of screening specific anti-SsRV p29 protein monoclonal antibodies respectively, through 3 time clonings after, obtain
Obtain the cell line 1M9 of the monoclonal antibody of the energy anti-mud crab reovirus p29 albumen of stably excreting.Indirect elisa method detection is aobvious
Show the identification His-p29 albumen that 1M9 hybridoma supernatants can be special.Hybridoma cell strain 1M9 include numerous inoculation small
Mouse prepares ascites, then determines potency using indirect ELISA, and the titer of ascites for finding hybridoma cell strain 1M9 is 107.To list
Anti- subclass analysis show that monoclonal antibody 1M9 belongs to IgG1 subclass, and light chain is k chains.Western blotting are detected
Result display monoclonal antibody can specific recognition purifying His-p29 and SsRV 29ku virus structural proteins, instruction sheet gram
Grand antibody 1M9 has compared with high specific；Monoclonal antibody mouse ascites caprylic acid-ammonium initial purification, and G- albumen parent
After purification, the anti-SsRV p29 protein monoclonal antibodies of high-purity are obtained.
The preparation of the polyclonal antibody of the anti-Scylla paramamosain reovirus p29 albumen of the Bacillus coli expression of embodiment 4
Antigen immune new zealand white rabbit is made with the purifying His-p29 albumen prepared in embodiment 2：Initial immunity takes antigen
With the fully emulsified mixing of equivalent complete Freund's adjuvant, multiple spot hypodermic injection on the inside of the neck, back, back leg in rabbit.After 14 days
Mixed with incomplete Freund's adjuvant with antigen, subcutaneous multi-point injection；1 time is carried out after 7 days again to be immunized, immunizing dose and immunization route
It is immune with the 2nd time identical, gather a small amount of serum and be used as ELISA detection antibody potency, measure the ELISA antibody titers of restructuring p29
It is 1:512000；Carry out the 4th after 14 days to be immunized, immunizing dose and immunization route are immunized identical, laggard action in 14 days with the 3rd time
Thing is anaesthetized, by surgically separating arteria carotis, sterile collection blood, and 4 DEG C of the blood being collected into placement 2h, then 4 DEG C,
3000 revs/min are centrifuged 15 minutes, collect the anti-Scylla paramamosain reovirus polyvalent antibody that supernatant is preparation, polyvalent antibody
After using caprylic acid-ammonium initial purification, and G- albumen affinity purifications, the anti-Scylla paramamosain reovirus of high-purity is obtained
P29 protein polyclone antibodies.
The preparation of the collaurum of embodiment 5
Collaurum is prepared using trisodium citrate reduction method.Measure 99 mL tri- and boil off ionized water in clean flat burning
In bottle, magnetic stir bar is heated to adding the gold chloride (HAuC1 of 1 mL 1% at 65 DEG C or so while stirring4) solution, continue to add
Thermal agitation is rapidly added 1% trisodium citrate (37 DEG C of pre-temperatures) solution that 2 mL are newly prepared to after seething with excitement, and continues to heat.It is pale yellow
The aqueous solution of chloraurate of color trisodium citrate addition after in 2 minutes, its color present light yellow-black-purple-aubergine-
The change of claret.After solution be changed into claret or it is orange red after, be further continued for heating stirring 15 minutes；The collaurum outward appearance of preparation
In limpid transparent orange red, it scanned by spectrophotometer wavelength X corresponding to absorption maximum is produced in absorption spectrum
Max is 520 nm, and by electron microscopic observation, the colloid gold particle of preparation is uniform in size, and average diameter is 20 nm.
The Scylla paramamosain reovirus p29 protein monoclonal antibodies colloid gold label of embodiment 6 and purifying
7 small test tubes are taken, 5mL collaurums are separately added into, with 0.2 mol/L potassium carbonate (K2CO3) regulation collaurum pH value
6,6.5,7,7.5,8,8.5,9 are adjusted to respectively；Then 100.0 μ L concentration are separately added into the collaurum of every kind of different pH value is
The monoclonal antibody 1M9 of 1.0 mg/mL, with sealed membrane closed test tube mouthful, stands 15 minutes, 4 DEG C of l0 points of 2000 revs/min of centrifugations
Zhong Hou, takes supernatant ultra-violet and visible spectrophotometer (400~600nm) in visible-range and is scanned, and obtains colloid
Golden visible absorption spectrum, determines maximum absorption wavelength, light absorption value.It is to there is corresponding collaurum pH value during maximum absorption band
Optimum mark pH value.The optimal pH of the monoclonal antibody 1M9 colloid gold labels of survey is 8, when monoclonal resists in 1 mL collaurums
When body 1M9 additions are 10 μ g, corresponding absworption peak is maximum, i.e., most suitable labelled amount is 10 μ g/mL collaurums.20 mL are prepared
Colloidal gold solution be placed in the beaker of 50 mL, use 0.2 mol/LK2CO3The pH of solution is adjusted to optimum mark pH, is then pressed
Optimum antibody label concentration is slowly added to 0.5mL monoclonal antibodies 1M9.Stir and evenly mix antibody colloidal gold mixture 30 minutes, then
After standing 20 minutes, final concentration of 1% BSA solution is added, after continuing to stir 20 minutes, solution is transferred to 50mL centrifuge tubes
In, 4 DEG C, 3000 revs/min are centrifuged 30 minutes, collect supernatant.By supernatant in 4 DEG C, 13500 revs/min, it is centrifuged 30 minutes,
Supernatant is abandoned, precipitation is collected.To addition colloid cleaning solution (PBS of 1%BSA) resuspended precipitation in precipitation so that volume is after addition
The 10% of original volume.By re-suspension liquid in 4 DEG C, 13500 revs/min, it is centrifuged 30 minutes, this operation 2-3 times is repeated, fully to wash
Precipitation.The precipitation that will be finally obtained is resuspended (1%BSA, 1% sucrose, 0.1%Tween-20, PBS) with gold labeling antibody preservation liquid, and 4
DEG C save backup.
The nitrocellulose membrane of embodiment 7 and gold conjugation pad are coated with
Coating buffer solution is the 0.01mol/L phosphate buffers (PBS, pH7.2) containing 6% methyl alcohol of .22um membrane filtrations,
Confining liquid is to adjoin pyrrolidone K30 (PVPK30), 2.5% containing 2% bovine serum albumin(BSA) (BSA), 1% polysorbas20,0.5% polyethylene
Sucrose and 0.02% nitrine receive (NaN3) 0.01mol/L PBS (pH7.4) buffer solution, 0.22 μm of filtering with microporous membrane, 4 DEG C standby
With；Detection film is the (model of nitrocellulose membrane 3：Millipore HF135, aperture is 0.45 μm), first general, rabbit-anti SsRV p29 are more
After clonal antibody PcAb1, rabbit anti-mouse igg antibody PcAb2 are diluted with coating buffer solution, it is sprayed at respectively on nitrocellulose membrane 3, makees
It is detection line 6 and nature controlling line 7, is sprayed on the concentration of PcAb1 in the detection line 6 of nitrocellulose membrane 3 for 1mg/mL, quantity for spray is 3 μ L/
cm；The PcAb2 concentration on the nature controlling line 7 of nitrocellulose membrane 3 is sprayed on for 0.75mg/mL, quantity for spray is 2 μ L/cm, in 37 DEG C of incubators
Dry for standby, the material of gold conjugation pad 2 is glass fibre membrane, after colloidal gold labeled monoclonal antibody G-1M9 is diluted, is sprayed at glue
On body gold pad 2,37 DEG C of dry for standby.It is sprayed on the colloidal gold labeled monoclonal antibody G-1M9 (1.5mg/mL) of gold conjugation pad 2
Make 5 times of dilutions, quantity for spray is divided into 50 μ L/cm.
The assembling of the test paper of embodiment 8
The sample pad 1 that will be handled well, the collaurum for being coated with anti-SsRV p29 protein monoclonal antibodies-colloid gold label thing
Pad 2, anti-SsRV p29 polyclonal antibodies are coated with as detection line 6 and rabbit anti-mouse igg antibody is coated with as nature controlling line 7
Nitrocellulose membrane 3, adsorptive pads stick to successively on RPVC liner plate 5 in order.Its package assembly is：Cellulose nitrate
Film 3 is placed in above RPVC liner plate 5, and sample pad 1 is smooth in the left side of nitrocellulose membrane 3, and adsorptive pads are smooth in nitric acid
The right side of tunica fibrosa 3, gold conjugation pad 2 is smooth between sample pad 1 and nitrocellulose membrane 3, make its one end be pressed on sample pad 1 it
Under, the other end is overlying on nitrocellulose membrane 3.The test strips being assembled into, Vacuum Package, normal temperature is preserved, the term of validity 6 months.
The application of the immunochromatography colloid gold test paper of the detection Scylla paramamosain reovirus of embodiment 9
(1) pretreatment of mud crab sample：Take appropriate mud crab cheek tissue sample to be placed in lapping apparatus, add the TEN of equivalent
Grinding buffer solution is Tris-EDTA-NaCl, is fully ground homogenised tissue and is made suspension, and suspension stands 5 minutes or centrifugation
Afterwards, supernatant is taken as trial-production bar sample solution；
(2) detect：The μ L of sample solution 100 for taking step (1) are dropped in test paper sample pad 1 of the present invention, and knot is observed after 10 minutes
Really, while taking appropriate mud crab reovirus liquid and TEN grinding buffer solutions respectively respectively as positive and negative control；
(3) result judgement：It is blue or green when the detection line 6 is presented red line when detection sample is added in sample pad 1
Crab reovirus is positive, i.e., contain mud crab reovirus in sample；It is that mud crab exhales if detection line 6 occurs without red line
The lonely virus of intestines is negative, i.e., mud crab reovirus is not contained in sample；If red line does not occur in nature controlling line 7, test paper is invalid.
The application effect of the invention of embodiment 10 is illustrated
1st, specific test：The mud crab reovirus known oneself, WSSV, prawn Taura syndrome enter
Row cross matching.When mud crab reovirus is contained in sample, detection line 6 is positive (red), and works as and contain in sample
When having WSSV or shrimp Taura syndrome, detection line 6 is negative (colourless), TEN buffer solution negative controls, detection
Line 6 is also negative.Result is identical after above-mentioned experiment is repeated several times 5 times, illustrates that the method has the specificity of height.
2nd, stability test：By 5 batches of test paper of the invention (batch number 150303,150309,150315,150321,
150327) after being placed 7 days in 37 DEG C of constant incubators, detect that 10 parts of mud crabs exhale intestines lonely simultaneously with same batch of test paper for preserving 4 DEG C
Virus-positive sample and 10 parts of mud crab reovirus negative samples, as a result show：37 DEG C effect 7d to test paper of the invention without
Destruction, it was demonstrated that test paper is stablized at high temperature.
3rd, storage life experiment：By with the test paper of the invention of a collection of preparation, respectively in 4 DEG C and room temperature preservation 1 to 6 month,
Taken out in 0 month, 3 months, 6 months, detecting the mud crab reovirus of 1 part of doubling dilution carries out storage life sensitivity tests,
Mud crab reovirus, WSSV, prawn Taura syndrome positive are preserved known to 10 parts of detection
Phase specific test, observation period sensitiveness, specificity and stability, to determine the storage life of test paper.Result shows：It is of the invention
Test paper does not change through 6 months phases of room temperature preservation specificity, sensitiveness.
4 compare with one-step RT-PCR method：30 parts of cheek silks of tested mud crab reovirus suspected case, use RT-PCR
The positive is 20 parts, and feminine gender is 2 parts, and positive with detection paper of the present invention is 16 parts, and feminine gender is 14 parts, is as a result shown：Mud crab exhales intestines
Lonely virus test paper method Positive rate 53.3% of the present invention, the Positive rate compared with RT-PCR is low, and its reason is immune glue
Body gold detection sensitivity is low compared with RT-PCR detection sensitivities.
Claims (4)
1. a kind of Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip, the reagent strip is by sample pad (1), pad
(2), nitrocellulose membrane (3), absorption pad (4), RPVC liner plate (5) composition, it is characterised in that：The pad (2)
On be coated with the anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies of immuno-gold labeling thing, the cellulose nitrate
Film (3) is including being coated with the detection line (6) of anti-Scylla paramamosain reovirus p29 protein polyclone antibodies and being coated with rabbit-anti mouse
The nature controlling line (7) of IgG antibody；Wherein, the nitrocellulose membrane (3) is placed in the middle of RPVC liner plate (5), the sample
Pad (1) is arranged on the side of the RPVC liner plate (5), and the sample pad (1) is arranged on the hard polychlorostyrene second
The opposite side of alkene liner plate (5), the pad (2) is smooth between sample pad (1) and nitrocellulose membrane (3), the Scylla paramamosain
The preparation method of reovirus colloidal gold immunochromatographimethod reagent strip is comprised the following steps：
(1) acquisition of antigen：, as primer, expanded with PCR with sequence shown in SEQ ID NO.1 and SEQ ID NO.2
Increase the corresponding genetic fragments of anti-Scylla paramamosain reovirus p29, be cloned into prokaryotic expression carrier pET28a (+), lured with IPTG
Expression of recombinant e. coli is led, affinity chromatography purified recombinant antigens are used, to obtain the band of the Recombinant protein expression of purifying
Histidine-tagged p29 gene proteins His-p29；
(2) monoclonal antibody of anti-Scylla paramamosain reovirus p29 albumen is prepared：The His-p29 albumen purified with step (1)
Immune Balb/c mouse, through cell fusion, screening is by preserving number for the hybridoma cell strain 1M9 of CGMCC No.10888 is produced
To the monoclonal antibody for secreting anti-Scylla paramamosain reovirus p29 albumen；
(3) preparation of colloid gold particle：Colloidal gold solution is prepared as reducing agent using trisodium citrate, and it is molten to adjust collaurum
The pH of liquid is standby to 8；
(4) the colloid gold label thing of anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies is prepared：Step (2) is obtained
The monoclonal antibody of anti-Scylla paramamosain reovirus p29 albumen add standby colloidal gold solution, anti-Scylla paramamosain is exhaled
The quality of monoclonal antibody and the volume ratio of colloidal gold solution of intestines orphan's virus p29 albumen are 1：100, shape after purified and concentration
Into the anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies of collaurum binding marker；
(5) preparation of pad：The anti-Scylla paramamosain reovirus of the collaurum binding marker sprayed on the pad (2)
The concentration of p29 protein monoclonal antibodies is 1.5mg/ml, and quantity for spray is 40-50ul/cm；
(6) anti-Scylla paramamosain reovirus p29 protein polyclone antibodies and rabbit anti-mouse igg antibody are prepared；
(7) preparation of antibody solid phase nitrocellulose membrane：Anti- Scylla paramamosain reovirus p29 protein polyclone antibodies coating is arrived
Detection line (6) position on nitrocellulose membrane (3) captures antigen as detection line (6), and concentration is 1mg/ml, and quantity for spray is 2-
3ul/cm；The rabbit anti-mouse igg secondary antibody of anti-binding antibody sprays to nature controlling line (7) position on nitrocellulose membrane (3) as Quality Control
Line (7) captures antibody, and concentration is 0.75mg/ml, and coating parameter is 1-3ul/cm；
(8) by absorption pad (4), the nitrocellulose membrane (3) with detection line (6) and nature controlling line (7), pad (2), sample pad
(1), RPVC liner plate (5) is assembled into the colloidal gold immunochromatographimethod inspection that can detect anti-Scylla paramamosain reovirus p29
Test paper slip.
2. Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip according to claim 1, it is characterised in that：Institute
The preparation method for stating colloid gold particle in step (3) is as follows：By gold chloride and trisodium citrate according to 1：2 ratio mixes and adds
Heat is made colloidal gold solution, and the particle size diameter that collaurum is obtained is 10-20nm.
3. Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip according to claim 2, it is characterised in that：Institute
The preparation method for stating the colloid gold label thing of anti-Scylla paramamosain reovirus p29 protein monoclonal antibodies in step (4) is as follows:
Monoclonal antibody is added in the colloidal gold solution in step (3), even, addition bovine serum albumin is mixed slowly, it is heavy after centrifugation
Form sediment with the resuspended precipitation of the phosphate buffer containing 1% bovine serum albumin, add and overlap nitrogen, be made the monoclonal of colloid gold label
Antibody.
4. Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip according to claim 1, it is characterised in that：Institute
State the middle method for preparing anti-Scylla paramamosain reovirus p29 protein polyclone antibodies of step (6) as follows：By the step (1)
The purifying His-p29 albumen of preparation makees antigen immune new zealand white rabbit：Initial immunity takes antigen and equivalent complete Freund's adjuvant
Antigen and incomplete Freund are used in fully emulsified mixing, multiple spot hypodermic injection on the inside of the neck, back, back leg in rabbit after 14 days
Adjuvant mixes, subcutaneous multi-point injection；1 time is carried out after 7 days again to be immunized, immunizing dose and immunization route are immune with the 2nd time identical, adopt
Collect a small amount of serum and be used as ELISA detection antibody potency；Carry out the 4th after 14 days to be immunized, immunizing dose and immunization route and the 3rd time
Identical, the collection of laggard promoting circulation of blood liquid in 14 days, 4 DEG C of placement 2h of the blood being collected into, then 4 DEG C, 3000 revs/min of centrifugations 15 is immunized
Minute, collect the anti-mud crab reovirus polyvalent antibody that supernatant is preparation；Polyvalent antibody is pure for the first time with caprylic acid-ammonium
Change, and after G- albumen affinity purifications, obtain the anti-mud crab reovirus p29 protein polyclone antibodies of high-purity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102109526A (en) * | 2011-02-12 | 2011-06-29 | 鲁东大学 | Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof |
| CN202002934U (en) * | 2011-02-12 | 2011-10-05 | 鲁东大学 | Rapid detecting test paper for grass carp reovirus |
| CN204495838U (en) * | 2015-02-11 | 2015-07-22 | 河南科技学院 | A kind of test strips detecting Avianreovirus |
-
2015
- 2015-11-05 CN CN201510515279.1A patent/CN105319361B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102109526A (en) * | 2011-02-12 | 2011-06-29 | 鲁东大学 | Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof |
| CN202002934U (en) * | 2011-02-12 | 2011-10-05 | 鲁东大学 | Rapid detecting test paper for grass carp reovirus |
| CN204495838U (en) * | 2015-02-11 | 2015-07-22 | 河南科技学院 | A kind of test strips detecting Avianreovirus |
Non-Patent Citations (3)
| Title |
|---|
| Molecular characterization of eight segments of Scylla serrata reovirus （SsRV) provides the complete genome sequence;Jigang Chen et al;《Arch Virol》;20120426;第157卷;表1，第1555页右栏第2段，图1 * |
| 锯缘青蟹呼肠孤病毒p40蛋白的体外表达与单克隆抗体制备;张昭 等;《上海海洋大学学报》;20140731;第23卷(第4期);第487-491页 * |
| 青蟹呼肠孤病毒对拟穴青蟹的致病性研究;蔡小辉 等;《水产科学》;20130331;第32卷(第3期);第130-135页 * |
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