CN106479984A - A kind of dual virus colloidal gold Rapid detection test strip of CMV PVY - Google Patents
A kind of dual virus colloidal gold Rapid detection test strip of CMV PVY Download PDFInfo
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- CN106479984A CN106479984A CN201610846043.0A CN201610846043A CN106479984A CN 106479984 A CN106479984 A CN 106479984A CN 201610846043 A CN201610846043 A CN 201610846043A CN 106479984 A CN106479984 A CN 106479984A
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- cmv
- pvy
- colloidal gold
- virus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of dual virus colloidal gold Rapid detection test strip of CMV PVY, is made up of sample pad, colloidal gold pad, NC film, absorbent filter and backing；CMV specific antibody and the PVY specific antibody of colloid gold label is coated with the colloidal gold pad, and two kinds of antibody are produced by hybridoma cell strain BALBc 15 27, BALBc 15 8 secretion respectively；Detection line and control line are provided with the NC film, in detection line, are coated with the specific antigen of two-strain；Be coated with colloid gold label on control line two resist.Test strips detection especially to CMV, PVY of Tobacco-growing areas in Guangdong, quick, sensitive, accurate, low cost, easy to operate, realize primary sample, two-strain is while diagnose, being especially suitable for live primary dcreening operation being carried out to batch samples, having very much using value in actual production, popularizing application prospect is good.
Description
Technical field
The invention belongs to pathogen detection technique field.Fast more particularly, to a kind of dual virus colloidal gold of CMV-PVY
Fast test strip.
Background technology
Cucumber mosaic virus (Cucumber mosaic virus, CMV), marmor upsilon (Potato virus Y,
PVY) it is the main virus that infects tobacco.The virosis that they cause causes the huge economic loss of tobacco industry every year.Quickly,
Qualitative detection correlated virus are the upper prevention and control diseases of production, reduce one of important means of loss exactly.
In scientific research, the detection method of plant virus mainly has biology detection, and (symptom type is distinguished, discriminating is posted
Main etc.), electron microscopy, serological detection method (Enzyme-linked Immunosorbent Assay react (Enzyme linked immunosorbent
Assay, ELISA) etc.) and molecular biological assay (including round pcr, Nucleic Acid Probe Technique etc.) etc..Isolated viral is according to electricity
Although mirror is the effective means of diagnosis virus, its high specificity, but wastes time and energy very much, needs technical professional, uncomfortable
Conjunction is promoted the use of.Serology test is relatively time-consuming, laborious.Although the methods such as immunofluorescence, ELLSA with micro, special,
Fast and accurately advantage, but the test apparatus that needs comparison complete and experienced technical staff are operating and judged result,
Detect that a batch sample whole flow process needs 4～8 hours, grass-roots work place is difficult to accomplish.PCR and the diagnosis of nucleic acid probe
With greater need for having special instrument and medicine, technology content is very high, is typically adapted only to laboratory diagnosis or research application, it is difficult in base
Layer is promoted.Therefore, in the urgent need to setting up viral diagnosis side that is a kind of simple, quick, sensitive, cheap and being suitable for basic unit's application
Method.
Collaurum starts for immunohistochemistry as label within 1971.Faulk etc. applies Electronic Speculum immune colloid gold
Decoration method observes salmonella.Many scholars are further characterized by collaurum and can stablize promptly adsorbed proteins, and protein
Biologically active nothing substantially changes.It can be carried out cell surface and intracellular polysaccharide, protein, polypeptide, antigen, swash as probe
Being accurately positioned of the large biological molecules such as element, nucleic acid, it is also possible to for daily immunodiagnosis, carry out immunohistochemical localization.
Colloidal gold solution refers to aurosol of the dispersed phase particles diameter between 1～150nm, belongs to multiphase heterogeneous system, and color is in
Salmon pink is to aubergine.Immune colloidal gold chromatography technology (Gold immunochromatography assay, GICA) is used as one
New immunological detection method is planted, is that the one kind that sets up in immuno-gold labeling technical foundation the eighties in 20th century is new
The unique diagnostic techniques of type.This technology is by colloidal gold-labeled method, immunoassay technology, Chromatographic techniques, list (many) gram
Multiple methods such as grand antibody technique and new material technology are organically combined, with simple, quick, accurate, pollution-free, operation
Simple and the advantages of without the need for special installation, obtain in medical science, the animal and plant quarantine, each field of food safety supervision increasingly extensive
Application.Early 1990s, Gold-immunochromatography assay diagnostic techniques initially entered commercial applications to mid-term.Using this
The colloidal gold fast detecting test paper strip of kind of technological development have easy to operate, quick, can single part of detection, be easy to preserve, be not required to spy
The advantages of different equipment, (clinic) primary dcreening operation can be carried out to target cause of disease at the scene, save substantial amounts of human and material resources.In medical domain,
This technology has also been developed into poison in addition to for hormone, the antigen of infectious disease pathogens and antibody, bacterium, the detection of parasite
The detection of the small-molecule substances such as product.The specimen types of detection also contemplated serum, blood plasma, whole blood, urine, excrement and saliva etc., show
Show wide prospect and huge using value.
At present, in terms of colloidal gold strip is mainly used in medical domain and animal and veterinary field, plant virus at home
Seldom see.In agriculture and animal husbandry field, rarer ripe product, more occur in the scientific research project of correlation.Sun Yan etc.
(2011) application colloidal gold technique has carried out cucumber bacterial angular leaf spot (Pseudomona.s.syringae
Pv.Lachrymans quick detection research).The country is related to the commercial colloidal gold strip of tobacco virus detection very
Few.External commercialization test strips are very expensive, and price is unsuitable for producing upper large quantities of tobacco seedlings at 50～60 yuans/
Detection is used.
Content of the invention
The technical problem to be solved in the present invention is defect and the deficiency for overcoming above-mentioned prior art, provides a kind of Tobacco-growing areas in Guangdong
The dual virus colloidal gold Rapid detection test strip of CMV-PVY.Core technology is to develop virus cucumber main for tobacco in seedling stage
Mosaic virus (Cucumber mosaic virus, CMV), the dual collaurum of marmor upsilon (Potato virus Y, PVY)
Test strip.Primary sample is realized, two-strain is diagnosed simultaneously, makes tobacco grower easy to operate, diagnosis is promptly and accurately.
It is an object of the invention to provide one plant of hybridoma cell strain BALBc-15-27 that can produce the special monoclonal antibody of CMV, with
And one plant of hybridoma cell strain BALBc-15-8 that can produce the special monoclonal antibody of PVY.
The present invention another object is that a kind of dual virus colloidal gold Rapid detection test strip of CMV-PVY of offer.
Another object of the present invention is to provide the CMV-PVY application of dual virus colloidal gold Rapid detection test strip.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The hybridoma cell strain BALBc-15-27 of the special monoclonal antibody of one plant of generation CMV, was preserved on June 30th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC No.12679, preservation address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Meanwhile, the CMV specific antibody for producing is secreted also in the guarantor of the present invention by above-mentioned hybridoma cell strain BALBc-15-27
Within the scope of shield.
The hybridoma cell strain BALBc-15-8 of the special monoclonal antibody of one plant of generation PVY, in being preserved on June 30th, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.12677, preservation address：North
The institute 3 of Jing Shi Chaoyang District North Star West Road 1.
Meanwhile, the PVY specific antibody for producing is secreted also in the guarantor of the present invention by above-mentioned hybridoma cell strain BALBc-15-8
Within the scope of shield.
In addition, the dual virus colloidal gold of CMV-PVY prepared by above-mentioned CMV specific antibody and PVY specific antibody is quickly examined
Test paper slip, also within protection scope of the present invention.
Preferably, the dual virus colloidal gold Rapid detection test strip of the CMV-PVY by sample pad, colloidal gold pad, NC film,
Absorbent filter and backing composition；The sample pad, colloidal gold pad and NC film are tied successively according to order from left to right, from top to bottom
Close on backing the same face, the end of colloidal gold pad and NC film overlaps；The absorbent filter combines the other end in NC film；Described
CMV specific antibody and the PVY specific antibody of colloid gold label is coated with colloidal gold pad, is provided with detection line (T on the NC film
Line) and control line (C line), and position of the detection line (T line) Wei Yu colloidal gold pad and control line (C line) between；Detection line (T line)
On be coated with the specific antigen of two-strain；Be coated with colloid gold label on control line (C line) two resist.
The dual virus colloidal gold Rapid detection test strip of above-mentioned CMV-PVY is in the context of detection of CMV and/or PVY virus, tool
There is good application prospect, in particular for the CMV and/or PVY virus of Tobacco-growing areas in Guangdong, detection sensitivity can reach 1g/
100mL.
The present invention utilizes A competitive inhibition method, successfully constructs the double check test strips of CMV-PVY.Its general principle is such as
Under：
CMV, PVY specific antibody of colloid gold label adsorbs on pad (i.e. colloidal gold pad), two-strain special
Antigen is fixed on ribbon in p-wire (T line) place of nitrocellulose membrane, and the two of colloid gold label anti-is fixed on cellulose nitrate
Control line (C line) place of film.After sample to be checked is added in the sample pad of test strips one end, moved forward by capillarity,
React to each other after colloid gold label specific reagent on dissolving pad, then when being moved to fixing antigenic domains, thing to be checked with
The conjugate of golden labelled antibody is specifically bound again therewith.
When one or two in the middle of sample to be tested contains CMV, PVY, it and the collaurum mark being dissolved in sample pad
The specific antibody of the corresponding virus of note reacts to each other；When being moved to fixing antigenic domains again, anti-without enough gold marks
Body and fixing antigen-reactive, do not have rufous lines to occur at T line, experimental result is the positive.Free gold labeling antibody or
Gold labeling antibody complex logistics are through, when at C, there is rufous quality control band with two anti-bindings at this.
When sample to be tested does not have any one in the middle of CMV, PVY viral, it and the collaurum being dissolved on pad
Labelled antibody does not react；When being moved to fixing antigenic domains again, there are enough gold labeling antibodies and fixing antigen-reactive,
And be trapped and be gathered on T line, rufous band can be observed by the naked eye, experimental result is feminine gender.
The invention has the advantages that：
The present invention constructs the test strip of dual virus first for plant virus, and novelty is very strong, exploitation
The dual virus Rapid detection test strip of CMV-PVY utilizes colloidal gold immunochromatographimethod technology, combines chromatography and immune response
Principle, it is achieved that the quickly and accurately purpose of qualitative detection cause of disease, can quick in field, large-scale detection sample, inspection
Survey result accurate, reliable.Compared with ELISA method, the test strips of the present invention have quick, sensitive, directly perceived, with low cost, behaviour
The features such as making detection that is easy, harmless, being easy to great amount of samples, realizes primary sample, and two-strain allows cigarette while diagnose
Agriculture is easy to operate, and diagnosis is promptly and accurately.
Importantly, the specificity of the virus of the ELISA test strip of the present invention is very strong, Tobacco-growing areas in Guangdong is specific to
The detection of CMV, PVY.The key antibody that the test strips are adopted is specific to the popular strain of Tobacco-growing areas in Guangdong CMV, PVY and builds
Arrive, prepared targetedly monoclonal antibody sensitivity is very high, can reach 1g/100mL, to Tobacco-growing areas in Guangdong CMV, PVY
The quick detection of two-strain has great importance.
In addition, the detection that can realize two-strain of the present invention simultaneously, whole small product size is little, easy to carry, is not required to
Instrument and equipment is wanted, simple to operate.Can Site Detection, result can be gone out in 3～10min；As a result can be by naked eyes according to T line color
The depth judged.Primary sample, two-strain are diagnosed simultaneously, make tobacco grower easy to operate, and diagnosis is promptly and accurately.This method
Be especially suitable for live primary dcreening operation being carried out to batch samples, have very much using value, with powerful popularization and application in actual production
Prospect.
Description of the drawings
Fig. 1 is connected to the recombinant vector schematic diagram of pET30a-GST carrier for CP-C-GD.
Fig. 2 is CP-C-GD protein mini-expression detection of expression electrophoretogram；M：Marker, 1：Target protein, 2：Control is not induced.
Fig. 3 is that CP-C-GD albumen great expression detects electrophoretogram；M：Marker, 1：Supernatant, 2：Precipitation.
Fig. 4 is CP-C-GD protein purification result；M：Marker, 1：100 times of Sample Dilution.
Fig. 5 is connected to the recombinant vector schematic diagram of pET30a-GST carrier for CP-P-GD.
Fig. 6 is CP-P-GD protein mini-expression detection of expression electrophoretogram；M：Marker, 1：Target protein, 2：Control is not induced.
Fig. 7 is that CP-P-GD albumen great expression detects electrophoretogram；M：Marker, 1：Supernatant, 2：Precipitation.
Fig. 8 is CP-P-GD protein purification result；M：Marker, 1：100 times of Sample Dilution.
Fig. 9 is the schematic diagram of colloidal gold fast detecting test paper strip.
Figure 10 is the dual virus colloid fast gold test strip Detection results of CMV-PVY；(left side is feminine gender sample,
The right is positive sample).
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent that the present invention is adopted, method and apparatus are that the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
The separation of 1 Tobacco-growing areas in Guangdong's CMV, PVY Strain of embodiment
1st, 2012～2014 years, Nanxiong from Shaoguan City of Guangdong Province, begin to flourish, newborn source, Lechang, the company continent of Qingyuan City, Meizhou
29, the 9 Ge Di county town such as the Wuhua in city, Jiangling, Dabu and Mei County, the tobacco for showing common mosaic in the vega in 33 villages are planted
It is sampled in strain.Sample point covers all cigarette districts in Guangdong Province.Totally 72 samples.It is carried out biology and ELISA respectively
The separation of (enzyme linked immunosorbent assay (ELISA)), identification.
Isolate and 24 CMV separators are obtained, 13 PVY separators, all obtain ELISA detection checking.Biology reflects
Determine result to show：Adopted CMV, PVY separator belongs to respective common strain.
2nd, respectively to two-strain full CP (coat protein)) gene expands.By after PCR primer glue reclaim with
PMD-18T carrier connects, and converts E. coli DH5 α.Picking positive colony expanding propagation culture, alkaline lysis are extracted
After plasmid, double digestion identification is carried out, obtain the positive transformant containing recombinant plasmid.Two kinds of positive transformants are sent Beijing AudioCodes
Bioisystech Co., Ltd is sequenced.
The CMV CP gene that sequencing is obtained is 654bp (sequence is as shown in SEQ ID NO.2), PVY CP gene is 796bp
(sequence is as shown in SEQ ID NO.3), it is respectively designated as CP-C-GD, CP-P-GD.
Enter NCBI database, with Blast instrument by two sequences of CP-C-GD, CP-P-GD respectively with lane database
Data compare.
3rd, analysis result shows：The similitude of the sequence of the CMV CP gene that CP-C-GD sequence is reported with other is
69.5%～97%.CP-P-GD sequence is 80.5%～95% with the similitude of the sequence of the PVY CP gene of other reports.This
Illustrate this research obtain account for CMV, PVY separator of main advantage status in Tobacco-growing areas in Guangdong and common CMV, PVY strain is deposited
In different.
Embodiment 2 prepares two kinds of CP gene prokaryotic differential proteins
CP-C-GD, CP-P-GD are building up to respectively on pET30a-GST carrier, BL21 bacterial strain are converted, carries out protokaryon table
Reach, purify expressing protein respectively.
1st, CP-C-GD prokaryotic expression protein is prepared, and relevant information is summarized as follows shown in table 1.
Table 1
Concrete grammar is as follows.
(1) objective gene sequence：According to e. coli codon optimize after sequence, the enzyme-added enzyme site EcoR I in two ends and
Xho I.Synthesis genes of interest is to pUC57 carrier.
(2) pass through digestion, connection, genes of interest is connected to pET30a-GST carrier, the recombinant vector after structure is illustrated
Figure is as shown in Figure 1.
Genetic fragment digestion：43 μ l recombinant plasmids, 1 μ l EcoR I, 1 μ l Xho I, 5 μ 10 × Buffer of l, 37 DEG C of mistakes
Night reacts.(Ago-Gel DNA QIAquick Gel Extraction Kit, BPI).
Carrier digestion：43 μ l carrier (pET30a-GST) plasmids, 1 μ l EcoR I, 1 μ l Xho I, 5 μ l10 × Buffer,
37 DEG C of reaction overnight.(Ago-Gel DNA QIAquick Gel Extraction Kit, BPI).
Connection：1 μ l carrier endonuclease bamhi, 3 μ l gene endonuclease bamhis, 1 μ l ligase (BPI), 5 μ l2 × Rapid
Buffer, mixes, room temperature reaction 30min.
(3) recombinant vector is converted BL21 competent cell：
1) 100 μ l competent cell (BL21) of -80 DEG C of preservations are taken out, is placed on slow on ice defrosting；
2) competent cell is added in the pipe of 1 μ l recombinant plasmid, mixes, place 30min on ice；
3) 42 DEG C of heat shock 90s；
4), after ice bath 2min, the nonresistant LB culture medium of 800 μ l is added；
5) 37 DEG C of culture 45min；
6) 5000rpm centrifugation 3min, abandons most of supernatant, stays about 100-150 μ l, and resuspended thalline, selection have corresponding resistant
LB flat board, coated plate；
7) dry, in 37 DEG C of incubators, be inverted overnight incubation.Then sequence verification is correct.
(4) a small amount of detection of expression：
1) flat board from conversion chooses monoclonal in 1.5ml LB fluid nutrient medium, 37 DEG C, and 200rpm is cultivated；
2) cultivate and induce to OD=0.6, IPTG (0.5mM), 37 DEG C, 200rpm cultivates 2h；
3) bacterium solution of 1ml induction taken, 12000rpm, 1min is centrifuged, abandons supernatant, precipitate with 50-100 μ l10mM Tris-
HCl (pH8.0) solution dispels (adding the amount of buffer solution depending on biomass)；
4) add and the isopyknic 2 × loading buffer of buffer solution, 100 DEG C are boiled 5min, electrophoresis detection (15%SDS-
PAGE).
As a result as shown in Figure 2, there is correct band of expression.
(5) great expression：
1) the correct bacterial strain of checking is selected, and the bacterium solution of 5～10 μ l activation is connect in 5ml LB fluid nutrient medium, 37 DEG C,
200rpm, culture；
2) bacterium solution of culture is transferred to 500mL LB fluid nutrient medium to mix, 37 DEG C, 200rpm, cultivates to OD=0.6,
IPTG (0.5mM) induces 4h；
3) bacterium is received in a large number：With the big concentrator bowl of 400ml, 6000rpm, 5min is centrifuged, abandons supernatant；
4) carrying out ultrasonic bacteria breaking：Precipitation is dispelled with 25ml 10mM Tris-HCl (pH 8.0) solution, ultrasound；
5) electrophoresis determines expression-form：Take 100 μ l ultrasound (500W, 90 times, each 3s, be spaced 6s) after bacteria suspension,
12000rpm, is centrifuged 10min, takes 50 μ l supernatants and manage to another EP, after supernatant is removed totally, precipitates with 50 μ l 10mM Tris-
HCl (pH 8.0) solution is dispelled, and adds 50 μ l 2 × loading buffer, and 100 DEG C are boiled 5min, electrophoresis detection (15%SDS-
PAGE).
As a result as shown in Figure 3, albumen has expression, mainly expresses in precipitation.
(6) protein purification (washing inclusion body)：
1) precipitation that the resuspended ultrasound centrifugation of 20～30ml 10mM Tris-HCl (pH8.0) solution is obtained, stands 10min；
2) 12000rpm, is centrifuged 10min, and supernatant is proceeded in another pipe and preserved；
3) the resuspended precipitation of 20～30ml 10mM Tris-HCl (pH8.0) solution, stands 10min；
4) 12000rpm, is centrifuged 10min, abandons supernatant；
5) repeat 3), 4) once；
6) the resuspended precipitation of a small amount of 10mM Tris-HCl (pH8.0) solution is initially charged, then plus 5～10ml is containing 8M urea
10mM Tris-HCl (pH8.0) solution soluble protein；
7) 12000rpm, is centrifuged 10min, collects supernatant, takes 50 μ l electrophoresis (15%SDS-PAGE).
As a result as shown in Figure 4, purifying obtains destination protein CP-T-GD.
2nd, CP-P-GD prokaryotic expression protein is prepared, and relevant information is summarized as follows shown in table 2.
Table 2
Concrete grammar is prepared with above-mentioned CP-T-GD albumen.
Recombinant vector schematic diagram after structure is as shown in Figure 5.
Protein mini-expression detection of expression as shown in Figure 6, has correct band of expression.
Albumen great expression detects that albumen has expression, mainly expresses in precipitation as shown in Figure 7.
As shown in Figure 8, purifying obtains destination protein CP-P-GD to protein purification result.
The hybridoma cell strain of the special monoclonal antibody of 3 construction expression CMV, PVY two-strain of embodiment
The two kinds of CP gene prokaryotic differential proteins obtained using embodiment 2, with corresponding expressing protein immune mouse；
Test for fusion is carried out, is produced the positive hybridoma cell strain of the special monoclonal antibody of two-strain respectively.
1st, the hybridoma cell strain of the special monoclonal antibody of construction expression CMV virus, method are as shown in table 3.
Table 3
Concrete grammar is as follows：
(1) immunity
1) " CMV " is used, by the amount of a 60ug albumen/mouse, 4 SPF BALB/c female mices of subcutaneous initial immunity, compiles
Number it is：1st, 2,3,4.
2), after just exempting from two weeks, subcutaneous first time booster immunization, immunity amount are 30ug albumen/only.
3) first time booster immunization is after two weeks, subcutaneous second booster immunization, and immunity amount is 30ug albumen/only.
4) second booster immunization be after two weeks, subcutaneous third time booster immunization, and immunity amount is 30ug albumen/only.
5) after one week, eye socket takes blood to second booster immunization, surveys serum titer.
(2) immunizing potency detection：
With " CMV ", 2ug/ml, 4 DEG C are coated overnight；2% milk, 37 DEG C of closing 2h；Serum starts 2 times of gradients from 200 times
Dilution, blank (blank) are PBS, and negative control (negative) is 200 times of dilutions of negative serum.
4 immunizing potency of table (potency be more than the dilution factor corresponding to the minimum OD reading of maximum OD/2)
No. 4 mouse of impact are chosen according to result and does cell fusion experiment.Before fusion, with immunogene " CMV " 50ug, abdominal cavity rushes
Hit immune No. 4 mouse.
(2) cell fusion
Mouse boosting cell and SP2/0 cell is taken, is merged using PEG method.Merge cell semisolid culturemedium (to contain
HAT) screening and culturing is carried out.
1) experiment equipment：The operating theater instruments of sterilizing is (including three scissors, three tweezers, cell sieve, a syringe
Inner core, a plate), wet box, 2 500ml beakers, 2 50ml centrifuge tubes, 3 15ml centrifuge tubes.
2) experiment reagent：IMDM culture medium；IMDM complete medium (containing 15% serum)；2.2% methylcellulose：Factory
Family：SIGMA, article No.：M0262-100G；NBCS 10ml；PEG1500：Producer：Roche, article No.：78364；HAT：Factory
Family：Sigma, article No.：H0262-10VL；HT：Producer：Sigma, article No.：H0137-10VL.
3) fusion experiment step
A. blow and beat soft for sp2/0 cell in good condition from culture bottle wall, be drawn in 50ml centrifuge tube.
B. mouse is plucked eyeball and takes blood, then draws neck to put to death, and is put into immersion 5min in 75% alcohol.
C. pour the IMDM of a small amount of serum-free in plate into, cell sieve and plunger are put in plate.Use scissors
The spleen of mouse is removed with tweezers, is put in cell sieve.Lightly spleen is fully pulverized with the inner core of syringe, thin by ground
Born of the same parents are drawn in the centrifuge tube of dress sp2/0, are centrifuged 1500rad/min, 5min.
D. the thymus gland of mouse is removed with scissors and tweezers, is pulverized.By the thymocyte for having ground in 15ml centrifuge tube, then plus
Enter the HAT of 1ml, be placed on standby in incubator.
E. by the cell being centrifuged, supernatant is outwelled, cell is carefully gently blown with the IMDM of serum-free even, centrifugation
(1500rad/min, 5min).
F. the cell conditioned medium being centrifuged is outwelled as far as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37
In DEG C warm water, the PEG of 1ml is slowly added in 1 minute, after adding, stand 1min in warm water.Then it is slowly added in 2min
The IMDM of the serum-free of 2ml, is then slowly added to the IMDM of 8ml serum-free in 2min.Centrifugation 1000rad/min, 5min.
G. supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours above ready thymocyte into.
The sterilized semisolid culturemedium of 25ml is added, is fully mixed.Then uniformly pour in 30 Tissue Culture Dish.Cell is trained
Foster ware is put in wet box, is then placed in cultivating in incubator.
(3) clone is chosen
10 plate × 93 cell monoclonals are chosen, is incubated at 96 porocyte culture plates and (uses thymocyte bed board, 100ul/ in advance
Hole).
(4) monoclonal cell 1 is sieved
With " CMV " wrapper sheet, the clone to selecting adopts ELISA method, does screening for the first time, obtains 48 plants of positive hybridomas
Cell line.
1) experiment reagent：
It is coated liquid：Sodium carbonate-bicarbonate buffer solution, pH9.6
PBS pH7.4
Confining liquid：2% milk in PBS
Washing lotion：PBS-T (0.05% tween, PBS)
Nitrite ion:1%A liquid+10%B liquid (A liquid：1%TMB in DMSO；B liquid：0.1%H2O2in citrate buffer solution)
Terminate liquid：2M sulfuric acid
Two resist：Goat anti-mouse IgG/HRP
2) experimental procedure
" CMV " is diluted with liquid is coated, final concentration of 2ug/ml, 100ul/ hole, 4 DEG C, overnight；Afterwards with wash liquid 3 times.
A.2% milk confining liquid closing, 200ul/ hole, 37 DEG C of incubators, 2h；Afterwards with wash liquid 3 times.
B. one is added to resist (cells and supernatant), negative control (SP2/0 culture supernatant), blank (PBS), the positive right
According to (1000 times of dilutions of positive serum PBS), 100ul/ hole, 37 DEG C of incubators, 1h is；Afterwards with wash liquid 3 times.
C. two anti-, the 100ul/ holes for adding PBS to dilute 20000 times, 37 DEG C of incubators, 1h；With wash liquid 3 times after taking-up.
D. develop the color, nitrite ion 100ul/ hole, developing time are 5min or so.
E. 50ul terminate liquid is added to terminate per hole.
F. dual wavelength (450,630) surveys light absorption value, and record preserves data.Including the hybridization is positive by immune protein screening
The data of tumor cell strain, positive control reading, blank reading and negative control reading.
Obtain screening immune protein the hybridoma cell strain being positive according to result screening, totally 48 plants.
(5) monoclonal cell 2 is sieved
By 48 plants of positive cell lines, " CMV " and label protein wrapper sheet again is used, using ELISA method, does second sieve
Choosing, obtains 24 plants of positive hybridoma cell strains.
(6) screen 24 plants of positive cell strains are carried out subgroup identification, the positive for finally obtaining 13 plants of IgG types is miscellaneous
Hand over tumor cell strain.
1) experiment reagent
Coated antibody：(Southern Biotech)
Confining liquid：2%BSA+3% sucrose in PBS；
Nitrite ion:0.2ml A liquid+10ul 30%H2O2In 10ml B liquid (A liquid：15mg/ml ABTS in H2O；B liquid：
Citrate buffer solution, pH4.0)
2) various subclass two resists：(Southern Biotech) experimental procedure：
A. with 100mM PBS (pH7.4) dilution coated antibody to 0.5ug/ml, add 0.1ml per hole, 4 DEG C, overnight.
B.PBS-T is washed 2 times, adds 200ul confining liquid per hole, and 370C is incubated 2h.
C.PBS-T washes 3 times；100ul hybridoma supematant is added per hole, and 370C is incubated 1h.
D.PBS-T washes 3 times；With confining liquid 1：10000 (κ, λ) or 1：The antibody of the HRP mark that 20000 (other) dilute
0.1ml is separately added in appropriate hole per hole, and 370C is incubated 1h.
E.PBS-T washes 3 times；Add 50ul substrate solution per hole, in 10-20min, light absorption value is surveyed in dual wavelength (450,630),
Record preserves data.
Table 5
The hybridoma cell strain of the special monoclonal antibody of 13 plants of generation CMV is finally given, is chosen best one plant and preservation is cultivated, and
Hybridoma cell strain BALBc-15-27 is named as, and Chinese microorganism strain preservation conservator is preserved on June 30th, 2016
Meeting common micro-organisms center, deposit number are CGMCC No.12679, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
2nd, the hybridoma cell strain of the special monoclonal antibody of construction expression PVY virus, method is ibid.
Table 6
The hybridoma cell strain of the special monoclonal antibody of 19 plants of generation PVY is finally given, is chosen best one plant and preservation is cultivated, and
BALBc-15-8 is named as, and China Committee for Culture Collection of Microorganisms's common micro-organisms is preserved on June 30th, 2016
Center, deposit number are CGMCC No.12677, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Embodiment 4 prepares the dual virus colloidal gold Rapid detection test strip of CMV-PVY
1st, experiment material
(1) colloid gold particle：Sodium citrate reducing process prepares (30nm)
(2) specific antibody of CMV, PVY two-strain
Two kinds of positive hybridoma cell strains of embodiment 3 are carried out culture expression, is carried out Protein A and post purifying is crossed, obtain
Specific monoclonal antibody to two-strain.
(3) two resist：Goat-anti-Rabbit Ig G
The pH of specific antibody colloid gold label is 8.2, and the anti-pH of colloid gold label two is 9.0.
Gold labeling antibody makees stabilizer with BSA.
2nd, instrument and equipment：
JY-EQ03 continous way draws film instrument, JY-EQ02 metal spraying machine, and JY-EQ01 cuts formula cutting knife, I JY-EQ05 case pressing machine.
3rd, consumptive material：
JY-D101DB-6 base plate, JY-X115H5072 blotting paper, JY-BX101 gold standard pad, 3 sample of JY-JZ112fusion
Product pad, JY-C111A-11 plastic clip.
4th, the assembling of test strips
(1) as shown in Figure 9, by backing (backing), sample pad, adsorptive pads (absorbent filter), nitrocellulose filter (NC
Film), gold standard pad (colloidal gold pad) stick together, be cut into the wide test strips of 4.5mm using cutting machine, in 4 DEG C of kept dry
Standby.
(2) test strips assembling condition：
The group reload request of test strips under the environment of the constant drying of room temperature, high humidity or temperature is too high can affect glass fibers
Dimension, the property of NC film and two are anti-, the activity of coated antibody, gold labeling antibody, and then affect the Tomography Velocity of test strips and develop the color anti-
Should.When each several part is assembled, to paste closely and NC film should not be drawn, can otherwise affect outlet effect.This test strips 25 DEG C of room temperature,
Humidity is assembled under the conditions of being less than 40%.
5th, the structure of the dual Viral diagnosis test strips of the CMV-PVY of the present invention is described as follows：
It is made up of sample pad, colloidal gold pad, NC film, absorbent filter and backing；The sample pad, colloidal gold pad and NC film are pressed
Combined successively on backing the same face according to order from left to right, from top to bottom, the end of colloidal gold pad and NC film overlaps；Described
Absorbent filter combines the other end in NC film；CMV, PVY specific antibody of colloid gold label, institute is coated with the colloidal gold pad
State and detection line (T line) and control line (C line) on NC film, is provided with, and detection line (T line) is located at colloidal gold pad and control line (C line)
Between position；The specific antigen of two-strain is coated with detection line (T line)；；Gold labeling antibody in the colloidal gold pad can
To react and develop the color with the antigen binding in detection line；Be coated with colloid gold label on control line (C line) two resist.
The detection general principle of the test strips is as follows：
CMV, PVY specific antibody of colloid gold label adsorbs on pad (i.e. colloidal gold pad), two-strain special
Antigen is fixed on ribbon in p-wire (T line) place of nitrocellulose membrane, and the two of colloid gold label anti-is fixed on cellulose nitrate
Control line (C line) place of film.After sample to be checked is added in the sample pad of test strips one end, moved forward by capillarity,
React to each other after colloid gold label specific reagent on dissolving pad, then when being moved to fixing antigenic domains, thing to be checked with
The conjugate of golden labelled antibody is specifically bound again therewith.
When one or two in the middle of sample to be tested contains CMV, PVY, it and the collaurum mark being dissolved in sample pad
The specific antibody of the corresponding virus of note reacts to each other；When being moved to fixing antigenic domains again, anti-without enough gold marks
Body and fixing antigen-reactive, do not have rufous lines to occur at T line, experimental result is the positive.Free gold labeling antibody or
Gold labeling antibody complex logistics are through, when at C, there is rufous quality control band with two anti-bindings at this.
When sample to be tested does not have any one in the middle of CMV, PVY viral, it and the collaurum being dissolved on pad
Labelled antibody does not react；When being moved to fixing antigenic domains again, there are enough gold labeling antibodies and fixing antigen-reactive,
And be trapped and be gathered on T line, rufous band can be observed by the naked eye, experimental result is feminine gender.
The sample detection of 5 colloidal gold fast detecting test paper strip of embodiment
1st, take the tobacco for having infected CMV, PVY two-strain, and the blade of health tobacco is used as experiment material, respectively
0.2g, plus PBS (pH8.0,0.01M), are ground, should not dilute leaf with running water.
Respectively take 50uL lapping liquid to be added in the test strips sample pad for preparing, sentence read result after five minutes.
2nd, result is as shown in Figure 10.At test strips C of susceptible tobacco sample, there is obvious rufous band in quality inspection band,
Experimental result is the positive.
At test strips C of health tobacco sample, at quality inspection and T, there are obvious 3 rufous bands in detection band band, real
Result is tested for feminine gender.
As a result show, the dual virus colloidal gold Rapid detection test strip of CMV-PVY of the present invention is to CMV, PVY two-strain
Detection results good.
The test of the sensitivity of 6 colloidal gold fast detecting test paper strip of embodiment
1st, 0.1g tobacco disease sample is diluted 6 series concentration with the PBS of 0.01mol/L, mixed with sample treatment liquid equal-volume
After conjunction, final concentration of 10,1,10-1、10-2、10-3、10-4Mg/mL, it is negative right to be mixed into sample treatment liquid equal-volume with PBS
According to measure test strips sensitivity.
2nd, result shows, when the concentration of tobacco disease sample is 10-3During mg/mL, the test strip of test strips is smudgy.Cause
This, the detection sensitivity of test strips of the present invention can reach 10-2mg/mL.
Claims (9)
1. one plant generation CMV special monoclonal antibody hybridoma cell strain BALBc-15-27, it is characterised in that in June 30 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.12679,
Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. the specific antibody of a kind of CMV virus, it is characterised in that secreted by claim 1 hybridoma cell strain BALBc-15-27
Produce.
3. one plant generation PVY special monoclonal antibody hybridoma cell strain BALBc-15-8, it is characterised in that in June 30 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.12677,
Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
4. the specific antibody of a kind of PVY virus, it is characterised in that secreted by claim 3 hybridoma cell strain BALBc-15-8
Produce.
5. the dual virus colloidal gold Rapid detection test strip of a kind of CMV-PVY, it is characterised in that by sample pad, colloidal gold pad, NC
Film, absorbent filter and backing composition；The sample pad, colloidal gold pad and NC film according to order from left to right, from top to bottom according to
On backing the same face, the end of colloidal gold pad and NC film overlaps for secondary combination；The absorbent filter combines the other end in NC film；
CMV specific antibody and the PVY specific antibody of colloid gold label is coated with the colloidal gold pad, is provided with detection on the NC film
Line and control line, and position of the detection line Wei Yu colloidal gold pad and control line between；The spy of two-strain is coated with detection line
Hapten；Be coated with colloid gold label on control line two resist.
6. the dual virus colloidal gold Rapid detection test strip of CMV-PVY according to claim 5, it is characterised in that described
CMV specific antibody is produced by hybridoma cell strain BALBc-15-27 secretion described in claim 2.
7. the dual virus colloidal gold Rapid detection test strip of CMV-PVY according to claim 5, it is characterised in that described
PVY specific antibody is produced by hybridoma cell strain BALBc-15-8 secretion described in claim 3.
8. the dual virus colloidal gold Rapid detection test strip of the arbitrary CMV-PVY of claim 5～7 is in CMV and/or PVY disease
The application of the context of detection of poison.
9. application according to claim 8, it is characterised in that CMV the and/or PVY virus refers to the CMV of Tobacco-growing areas in Guangdong
And/or PVY virus.
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