CN108957002A - Antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines - Google Patents
Antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines Download PDFInfo
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- CN108957002A CN108957002A CN201810720514.2A CN201810720514A CN108957002A CN 108957002 A CN108957002 A CN 108957002A CN 201810720514 A CN201810720514 A CN 201810720514A CN 108957002 A CN108957002 A CN 108957002A
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- gold
- swine fever
- fever virus
- rabbit igg
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
Abstract
The invention belongs to animal immune detection fields, and in particular to the antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines.The test card include sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, be coated with double detection lines of swine fever virus E2a albumen, goat anti-rabbit igg nature controlling line nitrocellulose filter and PVC offset plate；Colloidal gold is using double antigens sandwich and double detection lines；The swine fever virus gold mark E2 antigen and gold mark rabbit igg of e. coli bl21/pE2 secretion proteantigen building that deposit number is CCTCC NO:M2018060 are coated in swine fever virus albumen gold-labelled pad；It is coated with the swine fever virus E2a antigen for e. coli bl21/pE2a secretion proteantigen building that deposit number is CCTCC NO:M2018059 on the nitrocellulose filter, constitutes the nature controlling line area in a pair of detection line area and goat anti-rabbit igg composition.
Description
Technical field
The invention belongs to animal immune applied technical fields.More particularly to a kind of with double antigens sandwich and double detection lines
Antibody against swine fever virus quantitative testing test paper card.
Test card of the invention can be used for carrying out real-time, quick quantitative detection to antibody against swine fever virus, to swinery health
Antibody surveillance after horizontal, immune is of great significance.
Background technique
Swine fever (classical Swine fever, CSF) is by swine fever virus (classical Swine fever
Virus, CSFV) caused by acute, hot, the high contact of one kind pig infectious disease.Swine fever virus mainly passes through alimentary canal and exhales
Road transmission is inhaled, spread speed is fast, and morbidity and mortality are high, huge economic loss is brought to global pig breeding industry, by the world
Animal health tissue (OIE) is classified as one of the zoonosis of 16 kinds of legal notifications of A class.Swine fever also endangers China and supports for many years
The development of pig industry, be it is current occur at most, harm is maximum, one of popular most wide infectious disease, its legal is by the Ministry of Agriculture of China
A kind of infectious disease.
Swine fever is a kind of acute and severe infectious disease, and China depends on vaccine immunity to the successful control of swine fever.Therefore,
The superiority and inferiority of immune effect of vaccine plays decisive role to the prevention and control of swine fever.The detection of hog cholera antibody level is immune effect of vaccine
The main method of evaluation, while the detection of hog cholera antibody level is also of great significance for the formulation of hog cholera immune vaccine.
Serologic detection is horizontal commonly used in the herd immunity for understanding pig, evaluates immune effect of vaccine.Commonly
Detection method has the technologies such as the experiment of swine fever forward direction indirect hemagglutination, virus neutralization tests, enzyme-linked immunosorbent assay.Wherein indirect blood
Solidifying subjectivity is strong, detection efficiency is low, is not suitable for gross sample detection.Enzyme-linked immunosorbent assay sensibility is high, reproducible, but
It is easy to appear nonspecific reaction, and is only used for qualitative or half-quantitative detection.Traditional hog cholera antibody method for quantitatively determining is
Virus neutralization tests, but this experiment needs to cultivate the swine fever virus for having infection ability, does not have qualified laboratory to be difficult to carry out this
Experiment.And these methods have higher requirement to operator, equipment etc., take a long time mostly (such as hemagglutination-inhibition test need 1~
2 hours；ELISA test need 2~3 hours etc.), it is not easy to real-time, quick diagnosis, so can not push away on a large scale in base
It is wide to use.
Colloidal gold immunochromatographimethod (gold-immunochromatography assay GICA) is using colloidal gold as showing
Track object is applied to a kind of immunolabelling technique of antigen-antibody reaction based on colloidal gold-labeled method.Its core technology is with nitre
Acid cellulose film be solid phase carrier, promote sample solution swimming on chromatography strip because of capillarity, and make in sample to
In a short time high specific, high-affinity occur for the receptor (such as antigen or antibody) on inspection object and chromatographic film for object to be checked
Immune response.It has simplicity, quickly, high specificity, high sensitivity, the low advantage of expense.
Based on the technology, a kind of open examination of quickly detection hog cholera antibody of Chinese invention patent application 2008100004492
Paper slip, the test strips be judge by naked eyes detection line with or without carrying out negative or the positive the qualitative inspection to hog cholera antibody
It surveys, thus can not accurate quantification antibody content；Chinese invention patent application 201610454481.2 discloses a kind of fast quantification
The paper box and preparation method thereof of hog cholera antibody is detected, the kit is anti-respectively as label using swine fever E2 and E0 fused antigen
Former and capture antigen measures antibody against swine fever virus content, the linear model of the quantitative detection of sample concentration using single detection line technology
It encloses with certain limitation.In conventional single detection ray mode, antigen-antibody can only be reacted in finite region, while by
The sample liquid swimming caused by water absorption pad and nitrocellulose filter is rapid, causes the antigen-antibody reaction time of short duration, so that detection
Line colour developing is unobvious or not significant to high-content detectable substance quantitative detection color difference degree.
Summary of the invention
It is an object of the invention to overcome existing colloidal gold rapid detection method to qualitative detection or sxemiquantitative and quantitatively
Sensitivity in detection method is low and detects the inapparent disadvantage of color difference degree, provides a kind of with dual anti-former and pair detection lines
Antibody against swine fever virus quantitative testing test paper card and application.
Test card of the invention includes: sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, packet
By double detection lines of swine fever virus E2a albumen, the nitrocellulose filter of the nature controlling line of goat anti-rabbit igg, PVC offset plate.Of the invention
Colloidal gold is using double antigens sandwich and double detection ray modes, wherein being coated with deposit number in the gold-labelled pad is CCTCC
The swine fever virus gold mark E2 antigen of the e. coli bl21 of NO:M2018060/pE2 secretion proteantigen building and gold mark rabbit
IgG is coated with e. coli bl21/pE2a that deposit number is CCTCC NO:M2018059 on the nitrocellulose filter
The swine fever virus E2a antigen of the proteantigen building of secretion, the matter that the two constitutes a pair of detection line area and goat anti-rabbit igg is constituted
The area Kong Xian.When containing antibody against swine fever virus in measuring samples, antibody against swine fever virus swimming in sample to gold-labelled pad position with
Gold mark E2 antigen reacts, antigen antibody complex chromatography to E2a antigen when detecting line position, being coated in detection line
It reacts again with the antibody in antigen antibody complex, to form " gold mark Ag-Ab-coating in detection line position
Antigen " double antigens sandwich compound, and macroscopic red stripes are presented；The gold mark rabbit igg in gold-labelled pad will be with matter simultaneously
The goat anti-rabbit igg reaction for controlling line region forms red stripes in Quality Control line position.When antibody content is higher in sample, nitric acid is fine
The red stripes color in Wei Sumoshang test reaction area detection line region is deeper, on the contrary then more shallow.The intensity of detection line colour developing becomes
Change and be positively correlated with test antibodies content, by immune quantitative tacheometer to the automatic identification and interpretation of reaction band color density value
Post analysis goes out the contents level of determinand, to realize quantitative detection.
In the present invention, swine fever virus E2 and E2a recombinant antigen the preparation method is as follows: with final concentration 0.1mM IPTG points
It You Dao not recombinant bacterial strain e. coli bl21/pE2 (Escherichia coli BL21/pE2) and e. coli bl21/pE2a
(Escherichia coli BL21/pE2a), obtains swine fever virus E2 recombinant antigen and swine fever respectively after expression product is purified
Viral E2a recombinant antigen.
Recombinant bacterial strain e. coli bl21/pE2 preparation method:
The present invention searches CSFV E 2 Antigen proteinic genome sequence in ncbi database, chooses GenBank:
FJ598611.1(Classical swine fever virus strain C-strain-ZJ E2glycoprotein
Gene) it is used as target gene (nucleotide sequence is shown in SEQ ID NO:1).E2 protein gene is expanded, is connected with pET-28a (+) carrier
It connects, construction recombination plasmid pET-28a-E2 (map of recombinant plasmid pET-28a-E2 is shown in Fig. 1).
Above-mentioned recombinant plasmid pET-28a-E2 is converted into Escherichia coli (Escherichia coli) BL21 competent cell,
It obtains recombinant bacterial strain e. coli bl21/pET-28a-E2 (Escherichia coli BL21/pE2), applicant is by the recombination
Escherichia coli are named as e. coli bl21/pE2, Escherichia coli BL21/pE2, deliver on 01 25th, 2018
The China typical culture collection center preservation of the Chinese Wuhan Wuhan University, deposit number are CCTCC NO:M 2018060.
Recombinant bacterial strain e. coli bl21/pE2a's the preparation method comprises the following steps:
CSFV E 2 Antigen proteinic genome sequence is searched in ncbi database, chooses GenBank:FJ598611.1
(Classical swine fever virus strain C-strain-ZJ E2glycoprotein gene) portion gene
Sequence is as E2a target gene (nucleotide sequence is shown in SEQ ID NO:2).
E2a protein gene is expanded, is connect with pET-28a (+) carrier, building obtains recombinant plasmid pET-28a-E2a.
E. coli bl21 competent cell is converted with recombinant plasmid pET-28a-E2a, obtains recombinant bacterial strain Escherichia coli
The Strain Designation is large intestine by BL21/pET-28a-E2a (Escherichia coli BL21/pET-28a-E2), applicant
Bacillus BL21/pE2a, Escherichia coli BL21/pE2a delivered the Chinese Wuhan, the Wuhan on 01 25th, 2018
University's China typical culture collection center preservation, deposit number are CCTCC NO:M 2018059.
The preparation method of applicant provide a kind of antibody against swine fever virus quantitative testing test paper card with double detection lines, institute
The method stated includes: by sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, coating swine fever virus E2a
The matter of double detection lines (preparation method of swine fever virus E2a recombinant protein is shown in " specific embodiment ") and goat anti-rabbit igg of albumen
Nitrocellulose filter, the PVC offset plate of control line are successively assembled according to Fig. 3 sequence, obtain antibody against swine fever virus quantitative testing test paper card.
Wherein:
CSFV E 2 protein gold-labelled pad the preparation method comprises the following steps: colloidal gold solution is taken, with 300~700 μ L 0.1mM K2CO3
Adjust pH value；500~750 μ L 1mg/mL swine fever virus E2 recombinant protein (swine fever virus E2 are slowly added under stirring
The preparation method of recombinant protein is shown in " specific embodiment "), stir 30min；5%BSA solution is added to final concentration of 1%, after
Continuous stirring 30min；12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid；1500r/min, 4 DEG C from
Heart 20min abandons precipitating to get the CSFV E 2 protein of colloid gold label is arrived.The gold mark hog cholera that will be prepared with gold spraying instrument
For malicious E2 albumen even application to gold-labelled pad, discharge rate is 4.0~8.0 μ L/cm, is placed in the dry 12h~18h of 37 DEG C of incubators, is prepared into
It is spare comprising CSFV E 2 protein gold-labelled pad.
The preparation method of immune colloid gold solution: it measures 500mL ultrapure water and is added in 500mL round-bottomed flask, be put into magnetic
In power heating stirrer, 7.5ml 1%HAuCl is added4Solution is heated to boiling, and 6~9mL, 2% 2 water is added while stirring
Trisodium citrate is closed, heating stirring is continued, until colloid gold particle size is uniform, no irregular shape occurs.Stop heating, to
It is down to solution room temperature, is settled to 500mL, 4 DEG C save backup.
Rabbit igg gold-labelled pad the preparation method comprises the following steps: the colloidal gold solution of above-mentioned preparation is taken, with 300~700 μ L 0.1mM
K2CO3Adjust pH value；It is slowly added to 250~500 μ L 1mg/mL rabbit igg antibody (being purchased from ... company) under stirring, stirs
30min；5% bovine serum albumin(BSA) (BSA) solution is added to final concentration of 1%, continues to stir 30min；12000r/min, 4℃
It is centrifuged 30min, abandons supernatant, precipitating is resuspended with re-suspension liquid；1500r/min, 4 DEG C of centrifugation 20min abandon precipitating to get colloidal gold is arrived
The rabbit igg antibody of label.With gold spraying instrument by the gold mark rabbit igg albumen even application prepared to gold-labelled pad, discharge rate is 1.0~
8.0 μ L/cm are placed in the dry 12h~18h of 37 DEG C of incubators, are prepared into rabbit igg gold-labelled pad, spare.Specifically rabbit igg antibody is purchased from
Wuhan Boster Biological Technology Co., Ltd., article No. BA1045.
The preparation method of immune colloid gold:
Be coated with the detection line of swine fever virus E2a recombinant protein the preparation method comprises the following steps: with continuous point film instrument respectively by 0.2~
1.5mg/mL various concentration swine fever virus E2a recombinant protein is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm,
37 DEG C of incubators dry 18h, spare.
The nature controlling line of goat anti-rabbit igg is coated with the preparation method comprises the following steps: respectively that 0.2~0.8mg/mL difference is dense with continuous point film instrument
Degree goat anti-rabbit igg is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare.Specifically
Ground goat anti-rabbit igg, purchased from the outstanding Bioisystech Co., Ltd in Shanghai, article No. JY-QT04
Sample pad impregnates 30min with confining liquid, dries in 37 DEG C, spare
After test card assembling, detection line is away from gold-labelled pad end 5mm, and nature controlling line is away from water absorption pad end 3mm.
Compared with prior art, the present invention has the advantages that following prominent:
(1) test card of the present invention is made using dual-antigen sandwich method and double detection ray modes, in which: dual-antigen sandwich method is
Albumen is captured respectively as golden labelled protein and detection line using CSFV E 2 protein and swine fever virus E2a albumen；It is biggish
The space CSFV E 2 protein (molecular weight 33.3KD) binding site is more, easily in conjunction with tested antibody against swine fever virus；Hog cholera
Malicious E2a albumen (molecular weight 26KD) is smaller, is swine fever virus immunologic specificity albumen, can be with chromatography to the gold for detecting line position
The specific binding of CSFV antigen antibody complex is marked, the sensitivity of entire antibody test is improved.It is proposed by the present invention double
Ray mode is detected, insufficient deficiency of single detection line reaction time is overcome, antigen-antibody is made to can be carried out 2 association reactions, from
And the reaction time is increased, react antigen-antibody sufficiently, colleague also makes colour developing between high-content antibody samples that difference be presented.Pass through
What the color signal value and detected serum titer (i.e. antibody content) of double detection lines and nature controlling line in test zone were presented
Corresponding relationship realizes real-time, quantitative detection test serum potency.The present invention provides a kind of science for prevention and control swine fever
Effective technical support, while also a kind of reference frame is provided for quick, the Quantitative Monitoring of other diseases antibody.
(2) present invention is using colloidal gold immune chromatography test card as level-one sensor, i.e., gold mark antigen resists with to be measured first
Body, which combines, forms gold mark antigen antibody complex, makes test strip with capture antigen binding fixed in test strips again after swimming
Color change is generated, Strength Changes and the test antibodies content of colour developing are positively correlated, close to reaction band color by computer
The contents level of determinand can be analyzed after the automatic identification and interpretation of angle value quickly.
(3) antibody against swine fever virus detection reagent card is used in combination the present invention with immune quantitative tacheometer, forms hog cholera
Malicious antibody rapid quantitative detection test card, realizes monitoring real-time to antibody against swine fever virus, quick, quantitative.Of the invention answers
Caused error rate is determined with can be reduced to be seen by artificial eye, not only having improved the timeliness of detection, but also improve the objective of testing result
Property, accuracy.
(4) antibody against swine fever virus colloidal gold test paper card of the invention, has high specificity, and high sensitivity directly reads number
The advantages that value, detection time is short (15 minutes), and judgement is intuitive accurate.
(5) test card of the invention is easy to operate, and storage is convenient, of less demanding to storage temperature, guaranteeing the quality at room temperature
Phase is at least 1 year.
Detailed description of the invention
Sequence table SEQ ID NO:1 is the nucleotide sequence of CSFV E 2 protein.
Sequence table SEQ ID NO:1 is the amino acid sequence of CSFV E 2 protein.
Sequence table SEQ ID NO:1 is the nucleotide sequence of swine fever virus E2a albumen.
Sequence table SEQ ID NO:1 is the amino acid sequence of swine fever virus E2a albumen.
Fig. 1: the map for the recombinant plasmid pET-28a-E2 that the present invention constructs.
Fig. 2: the map for the recombinant plasmid pET-28a-E2a that the present invention constructs.
Fig. 3: the assembling schematic diagram of test strip of the present invention.Description of symbols:
1 is sample pad in Fig. 3, and 2 be CSFV E 2 protein gold-labelled pad in Fig. 3, and 3 be rabbit igg gold-labelled pad in Fig. 3, in Fig. 3
4 be nitrocellulose filter, and 5 be water absorption pad in Fig. 3, and 6 be detection line T1 in Fig. 3, and 7 be detection line T2 in Fig. 3, and 8 be matter in Fig. 3
Line is controlled, 9 be PVC backing in Fig. 3, and 10 be that test is got stuck in Fig. 3.
Fig. 4: test strip schematic illustration prepared by the present invention.
Fig. 5: the present invention examines test card result judgement schematic diagram.
Fig. 6: immune quantitative tacheometer schematic diagram.
1 indicates that detection CSFV is positive findings in Fig. 6；2 indicate that detection CSFV is negative findings in Fig. 6；3, Fig. 6 in Fig. 6
In 4 indicate test cards failure.
Specific embodiment
The preparation of 1 swine fever virus E2 and E2a recombinant antigen of embodiment
Present embodiments provide a kind of swine fever virus E2 and E2a recombinant antigen the preparation method is as follows: being lured respectively with IPTG
Artificial delivery E2 and E2a Protein reconstitution bacterial strain, obtains swine fever virus E2 recombinant antigen and swine fever virus respectively after expression product is purified
E2a recombinant antigen.
(1) swine fever virus E2 recombinant antigen preparation method is specific as follows:
CSFV E 2 Antigen proteinic genome sequence is searched in ncbi database, chooses GenBank:FJ598611.1
(Classical swine fever virus strain C-strain-ZJ E2glycoprotein gene) is used as target
Gene, specific gene order is as shown in SEQ ID NO:1.
Using the PCR primer of Primer premier5 software design amplification raq gene, the sequence of the primer is as follows:
P1:5'-CGGAATTCCGGCTAGCCTGCAAGGAAGATTAC-3'；
P2:5'-ACTCGAGAACTTCTGACTCAATTGTTCTCGC-3'.
Referring to E2 antigenic protein gene sequence and P1, P2 primer sequence, primer pair is limited by Nanjing Jin Sirui biotechnology
Company's synthesis.
Using the raq gene of synthesis as template, P1, P2 are primer, expand raq gene sequence.Reaction condition is 95 DEG C of 5min；
95 DEG C of 1min, 45 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min.E2 antigenic protein gene is obtained after reaction.
E2 protein gene and pET-28a (+) carrier are respectively after Ndel, BamHI restriction enzyme enzymatic treatment, by raq gene
It is attached with pET-28a (+), 16 DEG C of connections are overnight to get to recombinant plasmid pET-28a-E2 (see Fig. 1).
Above-mentioned recombinant plasmid pET-28a-E2 is converted into Escherichia coli (Escherichia coli) BL21 competent cell,
E. coli bl21/pE2 is obtained, Escherichia coli BL21/pE2 delivers Chinese Typical Representative culture on 01 25th, 2018
Object collection preservation, deposit number are CCTCC NO:M 2018060.
Recombination bacillus coli BL21/pE2 induces 16h through 0.1mMIPTG under the conditions of 16 DEG C；Recombination thallus is collected by centrifugation；
Thallus is crushed with sonicator at 4 DEG C；4 DEG C of 12000r/min are centrifuged 30min, collect supernatant；0.45 μm of filter membrane of supernatant
Filtering arrives swine fever virus E2 recombinant antigen protein with HisTrap FF crude affinity chromatography column purification.
(2) swine fever virus E2a recombinant antigen preparation method is specific as follows:
CSFV E 2 Antigen proteinic genome sequence is searched in ncbi database, chooses GenBank:FJ598611.1
(Classical swine fever virus strain C-strain-ZJ E2glycoprotein gene) portion gene
As E2a target gene, specific gene order is shown in SEQ ID NO:2.
Using the PCR primer of Primer premier5 software design amplification E2a gene, the sequence of the primer is as follows:
F1:5'-CATATGCGTCTGGCATGTAAAGAAGATTAT-3'；
F2:5'-GGATCCCGGATAATGCGGCAGGCCATCCG-3'.
Referring to E2a antigenic protein gene sequence and F1, F2 primer sequence, primer pair is limited by Nanjing Jin Sirui biotechnology
Company's synthesis.
Using the E2a gene of synthesis as template, F1, F2 are primer, expand E2a gene order.Reaction condition is 95 DEG C of 5min；
95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min.E2a antigen protein base is obtained after reaction
Cause.
E2a protein gene and pET-28a (+) carrier are respectively after Ndel, BamHI restriction enzyme enzymatic treatment, by E2 base
Because being attached with pET-28a (+), 16 DEG C of connections are overnight to get to recombinant plasmid pET-28a-E2a (see Fig. 2).
Above-mentioned recombinant plasmid pET-28a-E2a conversion Escherichia coli (Escherichia coli) BL21 competence is thin
Born of the same parents obtain e. coli bl21/pE2a, Escherichia coli BL21/pE2a, deliver China on 01 25th, 2018
Type Tissue Collection preservation, deposit number are CCTCC NO:M 2018059.
Recombination bacillus coli BL21/pE2a induces 16h through 0.1mMIPTG under the conditions of 16 DEG C；Recombinant bacterium is collected by centrifugation
Body；Thallus is crushed with sonicator at 4 DEG C；4 DEG C of 12000r/min are centrifuged 30min, collect supernatant；Supernatant is with 0.45 μm
Membrane filtration arrives swine fever virus E2a recombinant antigen protein with HisTrap FF crude affinity chromatography column purification.
The assembling of 2 antibody against swine fever virus quantitative testing test paper card of embodiment
The embodiment of the present invention provides a kind of system of antibody against swine fever virus quantitative testing test paper card based on double detection line technologies
Preparation Method, this method comprises: by sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, coating hog cholera
The nitrocellulose filter of the nature controlling line of double detection lines and goat anti-rabbit igg of malicious E2a albumen, PVC offset plate according to Fig. 3 sequence successively
Assembling, obtains antibody against swine fever virus quantitative testing test paper card.
Wherein, the preparation method of swine fever virus E2 and E2a recombinant antigen is referring to embodiment 1.
The preparation method of CSFV E 2 protein gold-labelled pad: taking colloidal gold solution, with 300 μ L 0.1mM K2CO3Adjust pH
Value；500 μ L 1mg/mL swine fever virus E2 recombinant proteins are slowly added under stirring, and (preparation of E2 recombinant protein is referring to reality
Apply example 1), stir 30min；5%BSA solution is added to final concentration of 1%, continues to stir 30min；12000r/min, 4 DEG C from
Heart 30min, abandons supernatant, and precipitating is resuspended with re-suspension liquid；1500r/min, 4 DEG C of centrifugation 20min abandon precipitating to get colloidal gold mark is arrived
The CSFV E 2 protein of note.The gold prepared is marked into CSFV E 2 protein even application to gold-labelled pad, discharge rate with gold spraying instrument
For 4.0~8.0 μ L/cm, it is placed in the dry 12h~18h of 37 DEG C of incubators, is prepared into CSFV E 2 protein gold-labelled pad, it is spare.
Further, the preparation method of colloidal gold solution includes: and is added to 500mL round bottom with measurement 500mL ultrapure water to burn
It in bottle, is put into magnetic heating stirrer, 7.5ml 1%HAuCl is added4Solution is heated to boiling, be added while stirring
2% two citric acid monohydrate trisodiums of 7.5mL continue heating stirring, until colloid gold particle size is uniform, no irregular shape goes out
It is existing.Stop heating, solution room temperature to be down to is settled to 500mL, and 4 DEG C save backup.
The preparation method of rabbit igg gold-labelled pad includes: to take colloidal gold solution, with 300 μ L 0.1mM K2CO3Adjust pH value；?
It is slowly added to 250 μ L 1mg/mL rabbit igg antibody under stirring, stirs 30min；5%BSA solution is added to final concentration of
1%, continue to stir 30min；12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid； 1500r/min,
4 DEG C of centrifugation 20min abandon precipitating to get the rabbit igg antibody of colloid gold label is arrived.The gold mark rabbit igg egg that will be prepared with gold spraying instrument
For white even application to gold-labelled pad, discharge rate is 1.0 μ L/cm, is placed in the dry 12h of 37 DEG C of incubators, is prepared into rabbit igg gold-labelled pad, spare.
Further, the preparation method of colloidal gold includes: same embodiment 2.
It is coated with swine fever virus E2a recombinant protein (the detection line preparation side of (preparation of E2a recombinant protein is referring to embodiment 1)
Method: uniformly being crossed 0.2mg/mL swine fever virus E2a recombinant protein to nitrocellulose filter with continuous point film instrument, is drawn film amount and is
1 μ L/cm, 37 DEG C of incubators dry 18h, spare.
It includes: with continuous point film instrument that 0.2mg/mL goat anti-rabbit igg is uniform for being coated with the nature controlling line preparation method of goat anti-rabbit igg
On scribing line to nitrocellulose filter, drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare.
Sample pad impregnates 30min with confining liquid, dries in 37 DEG C, spare
After test card assembling, detection line is away from gold-labelled pad end 5mm, and nature controlling line is away from water absorption pad end 3mm.
The preparation of 3 antibody against swine fever virus quantitative testing test paper card of embodiment
The embodiment of the present invention provides a kind of system of antibody against swine fever virus quantitative testing test paper card based on double detection line technologies
Preparation Method, this method comprises: by sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, coating hog cholera
The nitrocellulose filter of the nature controlling line of double detection lines and goat anti-rabbit igg of malicious E2a albumen, PVC offset plate according to Fig. 3 sequence successively
Assembling, obtains antibody against swine fever virus quantitative testing test paper card.
Wherein, the preparation method of swine fever virus E2 and E2a recombinant antigen is referring to embodiment 2.
The preparation method of CSFV E 2 protein gold-labelled pad: taking colloidal gold solution, with 700 μ L 0.1mM K2CO3Adjust pH
Value；It is slowly added to 750 μ L 1mg/mL swine fever virus E2 recombinant proteins under stirring, stirs 30min；It is molten that 5% BSA is added
Liquid continues to stir 30min to final concentration of 1%；12000r/min, 4 DEG C of centrifugation 30min abandon supernatant, precipitating re-suspension liquid weight
It is outstanding；1500r/min, 4 DEG C of centrifugation 20min abandon precipitating to get the CSFV E 2 protein of colloid gold label is arrived.It will with gold spraying instrument
For the gold mark CSFV E 2 protein even application prepared to gold-labelled pad, discharge rate is 8.0 μ L/cm, and it is dry to be placed in 37 DEG C of incubators
18h is prepared into CSFV E 2 protein gold-labelled pad, spare.
Further, the preparation method of colloidal gold includes: same embodiment 1.
The preparation method of rabbit igg gold-labelled pad: taking colloidal gold solution, with 700 μ L 0.1mM K2CO3Adjust pH value；It is stirring
It is slowly added to 250~500 μ L 1mg/mL rabbit igg antibody under state, stirs 30min；5%BSA solution is added to final concentration of
1%, continue to stir 30min；12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid； 1500r/min,
4 DEG C of centrifugation 20min abandon precipitating to get the rabbit igg antibody of colloid gold label is arrived.The gold mark rabbit igg egg that will be prepared with gold spraying instrument
For white even application to gold-labelled pad, discharge rate is 8.0 μ L/cm, is placed in the dry 12h~18h of 37 DEG C of incubators, is prepared into rabbit igg gold-labelled pad,
It is spare.
Further, the preparation method of colloidal gold includes: same embodiment 2.
It is coated with the detection line preparation method of swine fever virus E2a recombinant protein: with continuous point film instrument by 1.5mg/mL hog cholera
Malicious E2a recombinant protein is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare.
It includes: with continuous point film instrument that 0.8mg/mL goat anti-rabbit igg is uniform for being coated with the nature controlling line preparation method of goat anti-rabbit igg
On scribing line to nitrocellulose filter, drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare.
Sample pad impregnates 30min with confining liquid, dries in 37 DEG C, spare
After test card assembling, detection line is away from gold-labelled pad end 5mm, and nature controlling line is away from water absorption pad end 3mm.
The preparation of 4 antibody against swine fever virus quantitative testing test paper card of embodiment
The embodiment of the present invention provides a kind of system of antibody against swine fever virus quantitative testing test paper card based on double detection line technologies
Preparation Method, this method comprises: by sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, coating hog cholera
The nitrocellulose filter of the nature controlling line of double detection lines and goat anti-rabbit igg of malicious E2a albumen, PVC offset plate according to Fig. 3 sequence successively
Assembling, obtains antibody against swine fever virus quantitative testing test paper card.
Wherein, the preparation method of swine fever virus E2 and E2a recombinant antigen is referring to embodiment 1.
The preparation method of CSFV E 2 protein gold-labelled pad: taking colloidal gold solution, with 600 μ L 0.1mM K2CO3Adjust pH
Value；It is slowly added to 600 μ L 1mg/mL swine fever virus E2 recombinant proteins under stirring, stirs 30min；5% cow's serum is added
Albumin (BSA) solution continues to stir 30min to final concentration of 1%；12000r/min, 4 DEG C of centrifugation 30min abandon supernatant, sink
Shallow lake is resuspended with re-suspension liquid；1500r/min, 4 DEG C of centrifugation 20min abandon precipitating to get the swine fever virus E2 egg of colloid gold label is arrived
It is white.With gold spraying instrument by the gold mark CSFV E 2 protein even application prepared to gold-labelled pad, discharge rate is 5.0 μ L/cm, is placed in 37
The dry 18h of DEG C incubator, is prepared into CSFV E 2 protein gold-labelled pad, spare.
Further, the preparation method of colloidal gold includes: same embodiment 2.
The preparation method of rabbit igg gold-labelled pad includes: to take colloidal gold solution, with 500 μ L 0.1mM K2CO3Adjust pH value；?
It is slowly added to 300 μ L 1mg/mL rabbit igg antibody under stirring, stirs 30min；5%BSA solution is added to final concentration of
1%, continue to stir 30min；12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid； 1500r/min,
4 DEG C of centrifugation 20min abandon precipitating to get the rabbit igg antibody of colloid gold label is arrived.The gold mark rabbit igg egg that will be prepared with gold spraying instrument
For white even application to gold-labelled pad, discharge rate is 2.0 μ L/cm, is placed in the dry 8h of 37 DEG C of incubators, is prepared into rabbit igg gold-labelled pad, spare.
Further, the preparation method of colloidal gold includes: same embodiment 2.
The detection line preparation method of coating swine fever virus E2a recombinant protein includes: with continuous point film instrument by 1.2mg/mL pig
Pestivirus E2a recombinant protein is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, standby
With.
It includes: with continuous point film instrument that 0.35mg/mL goat anti-rabbit igg is equal for being coated with the nature controlling line preparation method of goat anti-rabbit igg
In even scribing line to nitrocellulose filter, drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare.
Sample pad impregnates 30min with confining liquid, dries in 37 DEG C, spare
After test card assembling, detection line is away from gold-labelled pad end 5mm, and nature controlling line is away from water absorption pad end 3mm.
Antibody against swine fever virus quantitative testing test paper card prepared by embodiment 4 is tested for the property and is tested, specifically such as
Under:
(1) foundation of quantitation curves
Swine fever positive serum National reference is subjected to doubling dilution, is prepared into 29、28…、23、22、21Series of positive blood
Clearly.The above-mentioned series of positive serum of 50 μ L and former serum are taken respectively, are detected with test card of the present invention, the positive of each dilution
Serum repeats detection 10 times, and test card is put into immune quantitative tacheometer after reaction by detection, reads double detection lines and Quality Control
The color signal value of line.Using the serum titer of swine fever positive serum National reference as ordinate, double detection lines and nature controlling line face
The ratio of chrominance signal value is abscissa, the linear relationship between serum titer and color signal value ratio is established, by linear relationship
In information input immune quantitative tacheometer software.
Swine fever positive serum National reference, purchased from Chinese veterinary microorganism culture presevation administrative center, antibody titer is
210, number is CVCC Z102, specification 1mL.
1 swine fever positive serum potency of table and color signal value ratio relation
| Swine fever positive serum potency | Color signal value ratio |
| 210 | 1.3429 |
| 29 | 1.3588 |
| 28 | 1.1204 |
| 27 | 0.9119 |
| 26 | 0.8105 |
| 25 | 0.6551 |
| 24 | 0.4716 |
| 23 | 0.2282 |
| 22 | 0.0784 |
| 21 | 0.0639 |
As shown in Table 1, when positive serum potency is 22With 23Between when, there is notable difference in color signal value ratio, shows
The Monitoring lower-cut of test card of the present invention is 23；Positive serum potency is 29With 210Between when color signal value it is almost the same, show
The upper limit of detection of test card of the present invention is 29.Thus infer, the quantitative detection potency range of test card of the present invention is 23-29。
(2) specificity experiments
Take porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, swine foot-and-mouth disease virus (FMDV) antibody positive blood
Clearly, Pseudorabies virus (PRV) positive serum reference material, pig circular ring virus (PCV) CAP protein antibody, recombination porcine parvovirus
(PPV) each 50 μ L of VP2 protein antibodies is detected with test card of the present invention respectively, reacts 15min, is distinguished with immune quantitative tacheometer
Read testing result.
Porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, purchased from Chinese veterinary microorganism culture presevation management
Center, number: CVCC Z230, specification: 5mL.
Swine foot-and-mouth disease virus (FMDV) Positive Sera, is derived from Schweineseuche antibody ELISA kit, visits purchased from Beijing
Er Di Bioisystech Co., Ltd, brand: Cusabio, article No.: CSB-EQ027745PI.
Pseudorabies virus (PRV) positive serum reference material is compiled purchased from Chinese veterinary microorganism culture presevation administrative center
Number: CVCC Z289, specification: 1mL.
Pig circular ring virus (PCV) CAP protein antibody is purchased from prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state, brand: rich
Gloomy, article No. difficult to understand: bs-20021R.
Pig recombination porcine parvovirus (PPV) VP2 protein antibodies are purchased from prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state, product
Board: Bo Aosen, article No.: bs-2309R.
Testing result: after reaction 15min, eye sees test card test reaction area, detects PRRSV, FMDV, PRV, PCV, PP
When antibody, there is apparent aubergine band in nature controlling line, double detection lines then not displaing amaranth band.It is immune fixed that test card is put into
It measures tacheometer and reads testing result, detect PRRSV, FMDV, PRV, PCV-2, PPV antibody, testing result is that serum titer is small
In 22；Detect antibody against swine fever virus standard positive serum, testing result with it is almost the same with Antibody serum titer in practice.
It follows that test card specificity of the present invention is high, the antibody no cross reaction with other important diseases virus of pig.
(3) stability test
Test card of the present invention uses vacuum sealed package, is individually positioned in 2~8 DEG C of refrigerators and 25 DEG C of environment.In test 0
A month, 3 months, 6 months, 9 months, 12 months and 15 months taking-up test card detects the swine fever positive blood of known serum titer
Clear National reference.Concrete outcome is as shown in table 2.
The test card storage life test result of the invention of table 2
As shown in Table 2, test card of the present invention saves 15 months in 2~8 DEG C of refrigerators and 25 DEG C of environment, in different time sections
Test card is taken to detect its performance, the accuracy of test card is unchanged, shows test card of the present invention in 2~8 DEG C of refrigerators or 25 DEG C of rings
Storage life under border is at least 1 year.
(4) with the comparative test of antibody against swine fever virus virus neutralisation
145 parts of pig anteserum samples are acquired altogether from Hubei herding Co., Ltd, Guangdong farm, Henan farm,
Serum hog cholera antibody potency is detected with test card of the present invention and viral neutralisation respectively.Concrete outcome is shown in Table 3, serum antibody titer
Greater than 24It is judged to the positive, otherwise is feminine gender.
The comparison result of the test card of the invention of table 3 and antibody against swine fever virus virus neutralisation
As shown in Table 3, test card of the present invention detects 39 parts of antibody against swine fever virus negative sample, 106 parts of positive sample；Disease
Malicious neutralisation detects 33 parts of antibody against swine fever virus negative sample, 112 parts of positive sample；The negative match-rate of two methods
96.97%, positive coincidence rate 93.75%, the total coincidence rate 94.48% of two methods.The result shows that test card of the present invention and disease
The testing result of malicious neutralisation is almost the same, and coincidence rate is preferable.
Sequence table
<110>Hubei Hua Da riel Science and Technology Ltd.
<120>the antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines
<141> 2018-06-27
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 831
<212> DNA
<213>swine fever virus (classical Swine fever virus)
<220>
<221> gene
<222> (1)..(831)
<400> 1
cgtctggcat gtaaagaaga ttatcgttat gccattagca gtaccgatga aattggtctg 60
ctgggtgcag gtggcctgac caccacctgg aaagaatata atcatgatct gcagctgaat 120
gatggtaccg ttaaagcaag ttgtgttgcc ggcagtttta aagtgaccgc cctgaatgtt 180
gttagccgtc gctatctggc aagtctgcat aaaaaagccc tgccgaccag cgttaccttt 240
gaactgctgt ttgatggtac caatccgagc accgaagaaa tgggcgatga ttttcgtagc 300
ggtctgtgtc cgtttgatac cagtccggtg gttaaaggca aatataatac caccctgctg 360
aatggtagtg cattttatct ggtgtgcccg attggttgga ccggtgttat tgaatgtacc 420
gcagtgagcc cgaccaccct gcgtaccgaa gttgtgaaaa cctttcgtcg cgataaaccg 480
tttccgcatc gtatggattg tgtgaccacc accgttgaaa atgaagatct gttttattgt 540
aagctgggcg gtaattggac ctgcgttaaa ggtgaaccgg tggtgtatac cggtggcgtt 600
gttaaacagt gccgttggtg tggctttgat tttgatggcc cggatggcct gccgcattat 660
ccgattggca aatgtattct ggccaatgaa accggttatc gcattgttga tagcaccgat 720
tgcaatcgtg atggtgtggt tattagcacc gaaggtagcc atgaatgcct gattggcaat 780
accaccgtta aagttcatgc cagtgatgaa cgcctgggcc cgatgccgca t 831
<210> 2
<211> 276
<212> PRT
<213>swine fever virus (classical Swine fever virus)
<220>
<221> BINDING
<222> (1)..(276)
<400> 2
Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp
1 5 10 15
Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu
20 25 30
Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ser Cys
35 40 45
Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg
50 55 60
Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe
65 70 75 80
Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp
85 90 95
Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys
100 105 110
Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val
115 120 125
Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro
130 135 140
Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro
145 150 155 160
Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu Asp
165 170 175
Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu
180 185 190
Pro Val Val Tyr Thr Gly Gly Val Val Lys Gln Cys Arg Trp Cys Gly
195 200 205
Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys
210 215 220
Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr Asp
225 230 235 240
Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys
245 250 255
Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg Leu
260 265 270
Gly Pro Met Pro
275
<210> 3
<211> 663
<212> DNA
<213>swine fever virus (classical Swine fever virus)
<220>
<221> gene
<222> (1)..(663)
<400> 3
cgtctggcat gtaaagaaga ttatcgttat gccattagca gtaccgatga aattggtctg 60
ctgggtgcag gtggcctgac caccacctgg aaagaatata atcatgatct gcagctgaat 120
gatggtaccg ttaaagcaag ttgtgttgcc ggcagtttta aagtgaccgc cctgaatgtt 180
gttagccgtc gctatctggc aagtctgcat aaaaaagccc tgccgaccag cgttaccttt 240
gaactgctgt ttgatggtac caatccgagc accgaagaaa tgggcgatga ttttcgtagc 300
ggtctgtgtc cgtttgatac cagtccggtg gttaaaggca aatataatac caccctgctg 360
aatggtagtg cattttatct ggtgtgcccg attggttgga ccggtgttat tgaatgtacc 420
gcagtgagcc cgaccaccct gcgtaccgaa gttgtgaaaa cctttcgtcg cgataaaccg 480
tttccgcatc gtatggattg tgtgaccacc accgttgaaa atgaagatct gttttattgt 540
aagctgggcg gtaattggac ctgcgttaaa ggtgaaccgg tggtgtatac cggtggcgtt 600
gttaaacagt gccgttggtg tggctttgat tttgatggcc cggatggcct gccgcattat 660
ccg 663
<210> 4
<211> 221
<212> PRT
<213>swine fever virus (classical Swine fever virus)
<220>
<221> BINDING
<222> (1)..(221)
<400> 4
Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp
1 5 10 15
Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu
20 25 30
Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ser Cys
35 40 45
Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg
50 55 60
Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe
65 70 75 80
Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp
85 90 95
Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys
100 105 110
Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val
115 120 125
Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro
130 135 140
Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro
145 150 155 160
Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu Asp
165 170 175
Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu
180 185 190
Pro Val Val Tyr Thr Gly Gly Val Val Lys Gln Cys Arg Trp Cys Gly
195 200 205
Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro
210 215 220
Claims (5)
1. a kind of antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines, which is characterized in that institute
Stating test card includes: sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, coating swine fever virus E2a egg
The nitrocellulose filter and PVC offset plate of the nature controlling line of white double detection lines, goat anti-rabbit igg；Colloidal gold using double antigens sandwich and
Double detection lines, in which: it is the big of CCTCC NO:M2018060 that deposit number is coated in the swine fever virus albumen gold-labelled pad
The swine fever virus gold mark E2 antigen and gold mark rabbit igg of the proteantigen building of enterobacteria BL21/pE2 secretion；The nitric acid is fine
Tie up the e. coli bl21/pE2a secretion proteantigen structure for being coated with that deposit number is CCTCC NO:M2018059 on plain film
The swine fever virus E2a antigen built, the nature controlling line area that the two constitutes a pair of detection line area and goat anti-rabbit igg is constituted；
Sample pad, CSFV E 2 protein gold-labelled pad, rabbit igg gold-labelled pad, water absorption pad, the double inspections for being coated with swine fever virus E2a albumen
The nitrocellulose filter of the nature controlling line of survey line and goat anti-rabbit igg, PVC offset plate sequence successively assemble.
2. the preparation method of swine fever virus E2 and E2a recombinant antigen, it is characterised in that:
Recombination bacillus coli BL21/pE2 and recombination bacillus coli BL21/pE2a are induced respectively with final concentration of 0.1mM IPTG,
Swine fever virus E2 recombinant antigen and swine fever virus E2a recombinant antigen are obtained after expression product is purified respectively.
3. a kind of e. coli bl21/pE2 preparation method of recombination, which comprises the following steps:
CSFV E 2 Antigen proteinic genome sequence is searched in ncbi database, the selection number of logging in is GenBank:
The gene order of FJ598611.1 is as target gene, and nucleotide sequence is as described in SEQ ID NO:1；
E2 protein gene is expanded, it is connect with pET-28a (+) carrier, building obtains recombinant plasmid pET-28a-E2；
With recombinant plasmid transformed e. coli bl21 competent cell, the weight that deposit number is CCTCC NO:M 2018060 is obtained
Group e. coli bl21/pE2.
4. a kind of e. coli bl21/pE2a preparation method of recombination, which comprises the following steps:
CSFV E 2 Antigen proteinic genome sequence is searched in ncbi database, the selection number of logging in is GenBank:
FJ598611.1 partial gene sequence is as E2a target gene, and nucleotide sequence is as described in SEQ ID NO:1；
E2a protein gene is expanded, it is connect with pET-28a (+) carrier, building obtains recombinant plasmid pET-28a-E2a；
With recombinant plasmid transformed e. coli bl21 competent cell, the weight that deposit number is CCTCC NO:M 2018059 is obtained
Group e. coli bl21/pE2a.
5. a kind of preparation method of antibody against swine fever virus quantitative testing test paper card with double detection lines described in claim 1,
It is characterized in that the following steps:
A. the preparation of CSFV E 2 protein gold-labelled pad:
Colloidal gold solution is taken, with 300~700 μ L 0.1mM K2CO3Adjust pH value；500~750 are slowly added under stirring
μ L 1mg/mL swine fever virus E2 recombinant protein stirs 30min；5%BSA solution is added to final concentration of 1%, continues to stir
30min；12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid；1500r/min, 4 DEG C of centrifugation 20min,
Precipitating is abandoned to get the CSFV E 2 protein of colloid gold label is arrived；The gold mark CSFV E 2 protein that will be prepared with gold spraying instrument
For even application to gold-labelled pad, discharge rate is 4.0~8.0 μ L/cm, is placed in the dry 12h~18h of 37 DEG C of incubators, obtains swine fever virus E2
Albumen gold-labelled pad；
B. the preparation of rabbit igg gold-labelled pad:
Colloidal gold solution is taken, with 300~700 μ L 0.1mM K2CO3Adjust pH value；250~500 are slowly added under stirring
μ L 1mg/mL rabbit igg antibody stirs 30min；5%BSA solution is added to final concentration of 1%, continues to stir 30min；
12000r/min, 4 DEG C of centrifugation 30min, abandon supernatant, and precipitating is resuspended with re-suspension liquid；1500r/min, 4 DEG C of centrifugation 20min, it is heavy to abandon
It forms sediment to get the rabbit igg antibody of colloid gold label is arrived；Gold mark rabbit igg albumen even application to the gold prepared is marked with gold spraying instrument
Pad, discharge rate are 1.0~8.0 μ L/cm, are placed in the dry 12h~18h of 37 DEG C of incubators, obtain rabbit igg gold-labelled pad；
C. it is coated with the preparation of swine fever virus E2a recombinant protein detection line: with continuous point film instrument respectively by 0.2~1.5mg/mL difference
Concentration swine fever virus E2a recombinant protein is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm, and 37 DEG C of incubators are dry
18h, it is spare；
D. it is coated with the preparation of the nature controlling line of goat anti-rabbit igg: with continuous point film instrument respectively by 0.2~0.8mg/mL various concentration goat-anti
Rabbit igg is uniformly crossed to nitrocellulose filter, and drawing film amount is 1 μ L/cm, and 37 DEG C of incubators dry 18h, spare；
E. sample pad confining liquid is impregnated into 30min, is dried at 37 DEG C, the formula of the confining liquid be 0.5g Tris,
2gNaCl, 2g PVP K30,2g casein, 0.2g NaN3, 5ml TritonX-100, be added to 900ml pure water dissolution, to complete
After fully dissolved, mix, with 2mol/L NaOH tune pH to 9.0, then plus pure water be settled to 1000ml, 0.22 μm of filtering with microporous membrane,
4 DEG C save backup.
F. the finished product detection line-spacing gold-labelled pad end for controlling test card is 5mm, and control nature controlling line is 3mm away from water absorption pad end.
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| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
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