CN108828223A - A kind of indirect ELISA detection method and application based on DHAV-1 3A albumen - Google Patents
A kind of indirect ELISA detection method and application based on DHAV-1 3A albumen Download PDFInfo
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Abstract
The invention discloses a kind of indirect ELISA detection method based on DHAV-1 3A albumen and applications.A kind of kit involved in the detection method, for the kit using 3A-198 recombinant protein as envelope antigen, the 3A-198 recombinant protein is the recombination fusion protein for removing the 3A truncated gene coding of DHAV-1 3A gene transmembrane region.The advantages of detection method of the invention has repeatability and specificity good, the high sensitivity of detection, it is high with the indirect ELISA method coincidence rate based on DHAV-1 virus, it can be used for the detection of DHAV-1 antibody.
Description
Technical field
The invention belongs to gene engineering technology fields, specifically, being related to a kind of based on the indirect of DHAV-1 3A albumen
ELISA detection method and application.
Background technique
Duck virus hepatitis (Duck viral hepatitis, DVH) is by duck hepatitis virus (Duck hepatitis
Virus, DHV) caused by mainly for duckling a kind of height lethal virus infectious disease.It is maximum to be wherein distributed most wide harm
For 1 type duck hepatitis A virus (DHAV-1).
1 type duck hepatitis A virus category Picornaviridae (Picornaviridae), the virus nucleocapsid are symmetrical 20 face
Body, no cyst membrane, core are single-stranded positive RNA, size 20-40nm.1 type duck hepatitis A virus is resistant to the environment of pH3.0, to height
Temperature also has stronger tolerance.Virus can survive 37d in the natural environment, can then survive the several years under the conditions of -20 DEG C.The disease
Poison can be proliferated in the chorioallantoic cavity of duck embryos and goose embryo.I type duck hepatitis occurs only at duckling, adult kind duck in its natural state
It does not fall ill, and does not influence its laying rate.Other birds also have certain reaction after artificial challenge DHAV-1, wherein with young pheasant, goose and
The death rate is higher after the infection such as guinea fowl.Reuss is studies have shown that the disease can not be broadcast to rabbit, cavy, small white mouse and dog.It should
Disease can contact propagation, and propagation efficiency is high, can be by mouse as reservoir host.And it can be passed by the way that birds band poison is long-range
It broadcasts.The disease occurs occur throughout the year without apparent seasonality, and the disease incidence of duckling is 100%, the disease of 1 week old duckling
Dead rate is up to 95%, and disease incidence and case fatality rate that 1-3 week old duckling case fatality rate is 50% or lower, 4-5 week old duckling are all very
It is low.
Being directed to current China of detection of DHAV, there are no approved commercialization detection kits.Traditional ELISA inspection
Survey method is needed using a large amount of DHAV virion, and the purifying of virion is needed by high speed centrifuge, and gained
Viral purity it is difficult to ensure that, this just gives the research and development process of commercial kit greatly to limit.Although having at present for 1 type duck first
The research report of hepatovirus, but for its each protein protomer, it is especially very few to non-structural protein research, the research of 3A is had no
Report.
Summary of the invention
In view of this, the present invention is directed to above-mentioned problem, a kind of indirect ELISA based on DHAV-1 3A albumen is provided
Detection method and application.
In order to solve the above-mentioned technical problem, the indirect ELISA detection based on DHAV-1 3A albumen that the invention discloses a kind of
Kit, for the kit using 3A-198 recombinant protein as envelope antigen, the 3A-198 recombinant protein is to remove DHAV-1 3A
The recombination fusion protein of the 3A truncated gene coding of gene transmembrane region.
Further, the recombination fusion protein that the 3A truncated gene for removing DHAV-1 3A gene transmembrane region encodes
Amino acid sequence is as shown in SEQIDNO.4.
Further, the 3A-198 recombinant protein is prepared by the following method to obtain：Remove 3A gene by PCR acquisition
Target fragment is subcloned to pET-32a (+), construction recombination plasmid pET-32-3A- by the 3A truncated gene segment of transmembrane region
198；Recombinant plasmid transformed is entered to express bacterium BL21 (DE3) 3A that inducing expression is 31kD afterwards and truncates recombination fusion protein, name
For 3A-198；Maximum expression quantity is obtained with 0.1mmol/LIPTG, 37 DEG C of induction 6h, albumen is expressed in supernatant, affine using Ni
Chromatographic column method purifies 3A-198, and 3A-198 recombinant protein is prepared.
Further, the peridium concentration of envelope antigen is 6.185 μ g/mL in the kit.
Further, the kit includes confining liquid, and the confining liquid is the PBS containing 5%BSA.
Further, the kit further includes ELIAS secondary antibody, and the ELIAS secondary antibody is the anti-duck IgG of HRP label, enzyme mark
The dilution of secondary antibody is 1:2000.
The present invention also provides a kind of indirect ELISA detection methods based on DHAV-1 3A albumen, include the following steps：
(1) envelope antigen：The 3A-198 recombinant protein that claim 3 is prepared is used into 0.05mol/L as antigen
ELISA Plate is added after the dilution of pH9.6 carbonate buffer solution, 100 μ L are added in every hole, and 4 DEG C of coatings are overnight；The peridium concentration of antigen is
6.185μg/mL；
(2) ELISA Plate is washed：Coating buffer is discarded, is washed 3 times using the PBS containing 0.05% polysorbas20, pH7.4,5min/
It is secondary, dry residual liquid；
(3) it closes：Confining liquid, every 100 μ L of hole, 37 DEG C of incubations are added into ELISA Plate；Wherein, confining liquid is containing 5%BSA
PBS；
(4) ELISA Plate is washed：As step (2) method is washed；
(5) it is incubated for primary antibody：Duck blood after dilution is added is clear, every 100 μ L of hole, 37 DEG C of incubations；The clear dilution of duck blood is 1:
20；
(6) ELISA Plate is washed：As step (2) method is washed；
(7) ELIAS secondary antibody：The anti-duck IgG of HRP label after dilution is added, every 100 μ L of hole, 37 DEG C of incubations；HRP label
Anti- duck IgG is rabbit-anti duck IgG, dilution 1:2000；
(8) ELISA Plate is washed：As step (2) method is washed；
(9) it develops the color：TMB developing solution is added, every 100 μ L of hole, 37 DEG C are protected from light 10min；
(10) it terminates：2mol/L H is added2SO4Terminate reaction, every 100 μ L of hole；
(11) it reads：OD value is measured under 450nm and 630nm double wave using microplate reader.
Further, the incubation time in step (3) is 90min.
Further, the incubation time in step (5) and step (7) is 30min.
The invention also discloses a kind of application of above-mentioned kit in detection DHAV serum antibody.
Compared with prior art, the present invention can be obtained including following technical effect：
1) present invention truncates recombination fusion protein 3A-198 using prokaryotic expression system successful expression DHAV-1 3A, with
The 3A-198 recombinant protein of purifying successfully prepares the anti-3A-198 of mouse and resists more, and 3A-198 mostly anti-can identify in virus infection produces
Raw 3A albumen.
2) the research of the invention finds that the duck embryo fibroblasts of DHAV-1 infection and pCMV-myc-3A transfection, 3A albumen are equal
It is primarily located in endoplasmic reticulum.
3) truncating the indirect ELISA method that recombination fusion protein is established based on 3A has repeatability and specificity good, detection
High sensitivity the advantages of, it is high with the indirect ELISA method coincidence rate based on DHAV-1 virus, can be used for the inspection of DHAV-1 antibody
It surveys.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings：
Fig. 1 is 3A-198 gene PCR product electrophoretogram of the present invention；Wherein, M：Standard molecular weight DL500；1：DHAV-1
3A-198PCR product；
Fig. 2 is pET-32a (+) -3A-198 recombinant plasmid PCR identification electrophoretogram of the present invention；Wherein, M：Standard molecular weight
DL500；1：Negative control 2：DHAV-1 3A-198PCR product；
Fig. 3 is recombinant plasmid pET-32-3A-198 digestion qualification result of the present invention；Wherein, M1.DNA molecular weight standard
DL500；1. recombinant plasmid EcoRI single endonuclease digestion is identified；2. recombinant plasmid EcoRI, HindIII double digestion is identified；3.pET-32-
3A-198PCR identifies reference；M2.DNA molecular weight standard DL15000；
Fig. 4 is the expression of 3A-198 recombinant protein of the present invention；Wherein, M:Protein standards；A：3A full length recombinant albumen and
3A-198 recombinant protein expression analysis, 1 is the unloaded full bacterium of pET-32a, and 2-3 is respectively the total length expressed supernatant packet of pET-32-3A
Containing body, 4-5 is respectively pET-32-3A-198 expression supernatant occlusion body, and 6-7 is pET-32- zero load supernatant occlusion body；B：3A
Full length recombinant albumen is analyzed with 3A-198 recombinant protein difference inducing temperature expression, and 1-4 is that pET-32-3A overall length exists respectively
16 DEG C, 25 DEG C, 30 DEG C and 37 DEG C inductions, 5-8 are that pET-32-3A-198 is induced at 16 DEG C, 25 DEG C, 30 DEG C and 37 DEG C respectively, and 9 are
PET-32 is unloaded；C：PET32-3A-198 inducing expression under the conditions of the IPTG of various concentration is analyzed, and 1 is unloaded for pET-32,2-9
Respectively 0,0.1,0.3,0.5,0.7,1,1.5 and 2mmol/L of IPTG concentration；D:The optimization of pET-32-3A-198 induction time,
1-7 is respectively 0h, 2h, 4h, 6h, 8h, 10h and 12h, and 8 is unloaded for pET-32；
Fig. 5 is the purifying of 3A-198 recombinant protein of the present invention；Wherein, M：Protein standards；1 is unloaded for pET32；2 are
pET32-3A-198；3 cross albumen after column for pET32-3A-198；4 be the 3A-198 recombinant protein of purifying；
Fig. 6 is the Westernblot analysis of expression product of the present invention；Wherein, M：Albumen pre-dyed Marker；1：pET-32a-
After 3A-198 induction；2：After pET-32a zero load induction；
Fig. 7 is that fine jade of the present invention expands the how anti-potency of analysis；Wherein, 1-5 is respectively 1:2,1:4,1:8,1:16,1:32 is diluted
It is mostly anti-；6 negative mice serum controls；The 3A-198 recombinant protein of 7 purifying；
Fig. 8 is expression analysis of the pCMV-myc-3A of the present invention in duck embryo fibroblasts, wherein 1：PCMV-myc matter
Grain；2：PCMV-myc-3A recombinant plasmid；M：Colored ultralow protein standard molecular weight；
Fig. 9 is the subcellular localization of 3A albumen golgiosome in DEF of the present invention；Wherein, A-D:Control group；E-H transfection
Recombinant plasmid group 3A subcellular localization situation；I-L connects the 3A subcellular localization situation of golgiosome after malicious DHAV-1；
Figure 10 is the Subcellular Localization of 3A albumen endoplasmic reticulum in DEF of the present invention；Wherein, A-D:Control group；E-H turns
Contaminate recombinant plasmid group 3A subcellular localization situation；I-L connects the 3A subcellular localization situation of endoplasmic reticulum after malicious DHAV-1；
Figure 11 is figure sensitivity analysis of the present invention；
Figure 12 is cross reactivity analysis of the present invention；
Figure 13 is that DHAV-1 positive serum of the present invention blocks analysis.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
DHAV-1H plants (JQ301467), escherichia coli DH5a, BL21 (DE3) bacterial strain, prokaryotic expression carrier pET-32a
(+), carrier for expression of eukaryon pCMV-myc is saved by Sichuan Agricultural University's poultry disease prevention and control research center；PMDl9-T (Simple) is carried
Body is purchased from Takara.
DHAV-1, DHAV-3, duck plague virus, duck swell head septicaemia virus, duck source Escherichia coli, duck source avian influenza virus,
The positive serums such as Salmonella anatis, riemerella anatipestifer and DHAV-1 negative serum and clinical serum sample, by Sichuan
Agriculture university's poultry disease prevention and control research center provides.15 Kunming mouses test Company of Animals Ltd. up to rich fruit purchased from Chengdu；9 age in days ducks
Embryo, purchased from Yaan duck.
Remaining reagent is commercially available with consumptive material.
Prokaryotic expression, purifying and the identification of 1 DHAV-1 3A-198 recombinant protein of embodiment
1, the PCR amplification of 3A-198 gene
Using DHAV-1 viral RNA in the allantoic fluid of extracting as template, by reverse transcription of viral RNA at cDNA.With acquisition
CDNA is added the upstream and downstream 3A-198 primer and carries out PCR amplification as template.Using 2% agarose gel electrophoresis, find about
(there is specific band for 207bp) and is consistent with expected results (see Fig. 1) in amplified fragments size at 200bp.
Upstream primer：5'-CCGGAATTCTCTAAGGTGAGGCGTTTCTCT-3 ' (EcoRI), as shown in SEQIDNO.1；
Downstream primer：5'-CCCAAGCTTCCGATTCCGCTCCAGAAAACC-3 ' (HindIII), such as SEQIDNO.2 institute
Show；
It wherein, is restriction enzyme site at underscore.
PCR amplification PCR amplification system and program are as follows：
Table 1PCR amplification reaction system
PCR amplification program 95 DEG C of 5min, 1 time；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 50s, 30 times；72 DEG C of 10min, 1 time.
2, the building and identification of expression vector
By digestion recycling 3A-198 gene cloning into prokaryotic expression carrier pET-32a (+), using specific primer into
Row PCR identifies recombinant plasmid, and carries out double digestion identification using primer both ends restriction enzyme site EcoRI and HindIII.As a result table
Bright, PCR qualification result and digestion qualification result can obtain the segment (see Fig. 2 and Fig. 3) of purpose band size, send Invitrogen
(Shanghai) sequencing.The result shows that the 3A-198 gene expanded and the sequence of reference (GenBank accession number JQ301467.1)
100% meets.
3, the expression and purifying of 3A-198 recombinant protein
3A-198 gene coded protein is about 8kD, by its with after label amalgamation and expression on pET-32a, recombination fusion protein
Molecular size range is about 31kD, nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence is as shown in SEQ ID NO.4.
By the recombinant plasmid transformed BL21 competence of building, grope respectively in different temperatures (16 DEG C, 25 DEG C, 30 DEG C, 37 DEG C),
Under different IPTG concentration levels (0.1mmol/L-2mmol/L) and on the different inducing expression time, be collected by centrifugation each thallus into
Row SDS-PAGE analysis.By the thallus ultrasonication after induction, takes supernatant precipitating to carry out SDS-PAGE analysis respectively, as a result show
Show, 3A-198 recombination fusion protein mainly has (Fig. 4 A) in the form of supernatant.The result shows that the IPTG in 0.1mmol/L is dense
Degree, 37 DEG C of induction 6h have maximum expression quantity (Fig. 4 B, C, D).
Using Ni2+Affinity chromatography column purification 3A-198 recombinant protein, and carry out SDS-PAGE analysis.Obtain the 3A- of purifying
198 recombinant proteins (Fig. 5).
4, recombination fusion protein Westernblot is analyzed
The immune response of specificity can occur with the anti-DHAV-1 positive serum of duck for the recombinant protein of expression, show that expression produces
Object has preferable reactionogenicity.As a result see Fig. 6.
The subcellular localization of the preparation of 2 recombinant protein antiserum of embodiment and 3A albumen
1, recombinant protein polyclonal antibody titration
Small white mouse is immunized with the 3A-198 recombinant protein of purifying and prepares antiserum, how anti-3A-198 is prepares immune process such as table
Shown in 2, the serum titer of rear 7d acquisition is exempted from the detection of Ago-Gel diffusion experiment eventually, and discovery is diluted to 1 in serum:Have when 4 bright
Aobvious precipitation line, 1:Also there is faint precipitation line when 8 as it can be seen that negative control is then without precipitation line (Fig. 7).
How anti-2 3A-198 of table is prepares immune process
FCA:Freund's complete adjuvant, FICA:Incomplete Freund's adjuvant
2, the subcellular localization of 3A albumen
(1) building and expression of recombinant plasmid pCMV-myc-3A
Correct pCMV-myc-3A plasmid transfection will be sequenced to duck in construction recombination plasmid pCMV-myc-3A according to a conventional method
On embryo fibroblast, cell sample is harvested after 48h, is obtained the 3A albumen expressed in duck embryo fibroblasts, is passed through
The correctness of Westernblot experimental verification expression albumen.Since the recombinant protein is smaller, normal protein adhesive cannot be fine
Separation expression destination protein, therefore it is separated using Tricine-SDS-PAGE, the results showed that (Fig. 8), the party
Method can be good at being separated to destination protein myc-3A (3A about 11kD, fusion protein about 14kD), and illustrate pCMV-myc-
The anti-3A-198 polyclonal antibody identification that albumen expressed by 3A can be produced, the indirect immunofluorescence that can be used for next step are real
It tests.Also there is a band in 28kD, 3A albumen can exist in the form of polymer in many cases really, therefore the band is simultaneously
Non- non-specificity band, but the dimer of 3A albumen.
(2) common location of 3A albumen and golgiosome
Carry out immunofluorescence dyeing, by under laser confocal microscope it has been observed that the duck embryos of control group are at fiber finer
It in born of the same parents, can be combined by rabbit-anti β COP, and occur the fluorogen in side aggregation around nucleus, and in mammalian cell
Middle coloration result is identical, it is thus determined that the rabbit-anti β COP antibody based on the design of mouse β COP496-513 amino acids, can act on
Duck source cell.In control group, β COP can be colored, but after using the anti-3A serum of mouse to be incubated for, colour developing there are no red glimmering
Light occurs, and sees Fig. 9 (A-D).Duck embryo fibroblasts transfect pCMV-myc-3A, are copolymerized burnt picture clearance software analysis, red green glimmering
Light registration is 36.5%, shows that albumen can be detected normal expression, but do not have common location signal to be detected with β COP
(Fig. 9 E-H).Show red green fluorescence weight by infecting DHAV-1 virus coloration result obtained in duck embryo fibroblasts
Right is 34.1%, therefore under Virus Infection, and the common location signal (Fig. 9 I-L) of 3A albumen and β COP is also not present.
(3) common location of 3A albumen and endoplasmic reticulum
Immunofluorescence dyeing is carried out, is observed under laser confocal microscope, the duck embryos of control group and experimental group are at fiber finer
It in born of the same parents, can be identified by anti-ERp57 antibody, the segment of antibody identification is 202-453 ammonia of ERp57 corresponding sequence of the mankind
Base acid, therefore can be used as endoplasmic reticulum marker use.Coloration result in duck embryo fibroblasts in mammalian cells
Coloration result is similar, determines that it can act on duck source cell, can identify duck embryo fibroblasts endoplasmic reticulum.In control group
In, after endoplasmic reticulum can be colored, but the anti-3A serum of mouse is incubated for, colour developing there are no red fluorescence appearance, see Figure 10 A-D.Duck
Embryo fibroblast transfects pCMV-myc-3A, is copolymerized burnt picture clearance software analysis, and red green fluorescence registration is 87.6%, table
Bright albumen can be detected normal expression, and high (Figure 10 E-H) with the endoplasmic reticulum fluorescence registration of ERp57 label.By
DHAV-1 virus coloration result obtained is infected in duck embryo fibroblasts shows that red green fluorescence registration is 52.2%, because
Similarly there is the coincidence (Figure 10 I-L) of the endoplasmic reticulum fluorescence of 3A albumen and ERp57 label under Virus Infection in this.
The foundation of indirect ELISA method of the embodiment 3 based on 3A-198 recombinant protein
1, the optimization of 3A-198 recombinant protein indirect ELISA method
(1) determination of recombinant protein peridium concentration and serum dilution
BCA measurement result shows that recombinant protein original concentration is 1.237mg/mL.Best antigen packet is determined by chessboard method
By concentration and optimal serum dilution.The result shows that (table 3), when antigen diluent degree is 1:When 200, i.e. envelope antigen concentration is
6.185 μ g/mL, serum dilution 1:20, the OD of positive serum450nm/OD630nmValue reachable 0.972, and negative serum
OD450nm/OD630nmValue is 0.275, positive and negative serum OD450nm/OD630nmIt is maximum (P/N=3.540) to be worth ratio.Therefore,
6.185 μ g/mL be the best peridium concentration of antigen, 1:20 be best serum dilution.
The best antigen coat concentration of table 3 and serum dilution determine
Note：Runic is that selected optimal conditions corresponds to numerical value
(2) the most preferably determination of coating condition
Antigen is coated with best peridium concentration, respectively at 37 DEG C of 2h, 37 DEG C 2h+4 DEG C overnight, 37 DEG C 1h+4 DEG C overnight and
Under the conditions of 4 DEG C several overnight, indirect ELISA is carried out according to above-mentioned steps.It the results are shown in Table 4 and determine that the best coating condition is 4 DEG C
Overnight.
The selection of the coating condition of table 4
Note：Runic is that selected optimal conditions corresponds to numerical value
(3) determination of secondary antibody best effort concentration
Antigen is coated with best peridium concentration, 4 DEG C overnight.Serum makees 1:20 dilutions, ELIAS secondary antibody is done 500,1000,
2000 and 4000 times of dilutions, 100 holes μ L/ carry out indirect ELISA according to above-mentioned steps, record result (table 5).Test determines most
Good ELIAS secondary antibody dilution ratio is 1:2000.
The selection of 5 secondary antibody best effort concentration of table
Note：Runic is that selected optimal conditions corresponds to numerical value.
2, the indirect ELISA detection method based on DHAV-1 3A albumen
(1) envelope antigen：Antigen (3A truncates recombinant protein, is named as 3A-198) is used into 0.05mol/LpH9.6 carbonic acid
ELISA Plate is added in salt buffer dilution (6.185 μ g/mL protein concentration) afterwards, and 100 μ L are added in every hole, and 4 DEG C of coatings are overnight；
(2) ELISA Plate is washed：Coating buffer is discarded, is washed 3 times using the PBS (pH7.4) containing 0.05% polysorbas20,5min/
It is secondary, dry residual liquid；
(3) it closes：Confining liquid (PBS containing 5%BSA) is added into ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 90min；
(4) ELISA Plate is washed：As step (2) method is washed；
(5) it is incubated for primary antibody：Duck blood clear (1 after dilution is added:20 dilutions), every hole 100 μ L, 37 DEG C of incubation 30min；
(6) ELISA Plate is washed：As step (2) method is washed；
(7) ELIAS secondary antibody：The rabbit-anti duck IgG (1 of HRP label after dilution is added:2000 dilutions), 100 μ L of every hole, 37 DEG C
It is incubated for 30min；
(8) ELISA Plate is washed：As step (2) method is washed；
(9) it develops the color：TMB developing solution is added, every 100 μ L of hole, 37 DEG C are protected from light 10min；
(10) it terminates：2mol/L H is added2SO4Terminate reaction, every 100 μ L of hole；
(11) it reads：OD value is measured under 450nm and 630nm double wave using microplate reader.
The evaluation of embodiment 43A-198 recombinant protein indirect ELISA method
1, the determination of yin and yang attribute critical value
48 parts of DHAV-1 negative serums are detected, obtaining result (table 6) maximum value is 0.254, and minimum value is
0.080,It is 0.0389 for 0.157, SD (standard deviation), can obtain its critical value according to formula is：
2, sensitivity tests
Take 8 parts of DHAV-1 positive serums from 1：40 start to carry out doubling dilution, can detect positive maximum dilution times
Number is used as sensitivity.It is respectively set 1:40,1：80,1：160,1：320,1：640,1：1280,1：2560,1：5120, do 3 weights
It is multiple.As a result (Figure 11) shows because of 1：The OD value that 2560 times of diluted positive serums obtain is slightly less than critical value, so maximum 1:
1280 times of diluted positive serums can be detected.
6 48 parts of negative serum OD450nm/630nm values of table
Note：Runic is labeled as OD450nm/630nm peak and minimum
3, specific test
The OD value of specific cross test result (Figure 12) discovery detection DHAV-1 positive serum is much larger than critical value, average
It is 0.295 that the OD value that value reaches 1.024, DHAV-3 positive serum, which is slightly larger than critical value, other common cause of disease serum detect OD
Value is respectively less than critical value (0.274).Show that the detection method established in this research can detecte DHAV-1 positive serum；Inspection
The OD value for surveying DHAV-3 positive serum is slightly larger than critical value, illustrates that there are slight cross reactions for 1 type and 3 type duck hepatitis A virus；
Cross reaction does not occur clearly with the duck blood of other encountered pathogenics infection.
By the positive serum of DHAV-1, neutralization reaction, testing result (figure are carried out with DHAV-1 antigen and DPV antigen respectively
13) show the OD of the serum after being neutralized by DHAV-1 antigen under the same conditions450nm/OD630nmValue decline becomes apparent, and
Its detection of the serum OD handled using DPV450nm/OD630nmValue decline is unobvious.Therefore DHAV-1 positive serum can be by DHAV-
The blocking of 1 antigentic specificity, blocking rate are 31%.And DHAV-1 positive serum cannot then be blocked using DPV antigen.
4, repetitive test
Test is repeated in batch using 3 batch coatings of albumen obtained by same Batch purification point, by 3 parts of DHAV-1 positive serums
With 3 parts of DHAV-1 negative serums, 4 repetitions are arranged in every part of serum, according to the indirect ELISA method of foundation, carry out batch interior repetition
Property test, calculating average value simultaneously calculates standard deviation, and it is flat to obtain coefficient of variation CV (%)=SD/ by standard deviation and mean value calculation
Mean value.As a result (table 7) shows that variation within batch coefficient is between 1.57%-5.29%, average coefficient of variation 3.79%.
7 batches, table interior repetitive test results
Test is repeated between batch using 4 different batches purifying gained albumen, is coated with according to optimal antigen coat amount,
It is incubated for 3 parts of DHAV-1 positives and negative serum respectively, 4 repetitions of every part of setting are carried out according to the indirect ELISA method of foundation
Repetitive test between batch, obtains coefficient of variation CV (%)=SD/ average value by standard deviation and mean value calculation.As the result is shown
(table 8) interassay coefficient of variation is between 1.25%-7.73%, average coefficient of variation 5.09%.
8 batches of repetitive test results of table
In testing in batch and between criticizing, the coefficient of variation CV% of 6 parts of detected serum is not above 10%, shows the party
Method has preferable repeatability.
5 3A-198 recombinant protein indirect ELISA of embodiment and the assessment of DHAV-1 indirect ELISA coincidence rate
Use DHAV-1 virus as indirect ELlSA detection method (ShenY, ChengA, WangM, the et of antigen coat
al.Development of an indirect ELISA method based on the VP3protein of duck
hepatitis A virus type 1(DHAV-1)for dual detection of DHAV-1and DHAV-
3antibodies[J].Journal of Virological Methods,2015,225:30-34), with newly-established 3A-
198 recombinant protein indirect ELISA methods detect one group (96 parts) clinical duck blood final proofs simultaneously respectively, as a result (table 9) positive rate point
It Wei not 51% (49/96) and 56% (54/96).The wherein serum positive comprising the detection of portion DHAV-1 indirect ELISA, 3A-198
The detection of recombinant protein indirect ELISA is negative.Between the serum 3A-198 recombinant protein for thering are 6 parts of DHAV-1 indirect ELISAs to be negative
It meets ELISA and detected and be positive.Using DHAV-1 indirect ELISA as reference, 3A-198 recombinant protein indirect ELISA is calculated
The diagnostic sensitivity of the detection method is 88.9% (48/54), and specificity is 97.6% (48/49), and coincidence rate is
92.7% (89/96).
The coincidence rate of table 9 3A-198 recombinant protein indirect ELISA and DHAV-1 indirect ELISA method
Note：Runic is that two kinds of detection method yin-yang are consistent number
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Sequence table
<110>Sichuan Agricultural University
<120>A kind of indirect ELISA detection method and application based on DHAV-1 3A albumen
<130> 2017
<141> 2018-04-13
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
ccggaattct ctaaggtgag gcgtttctct 30
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
cccaagcttc cgattccgct ccagaaaacc 30
<210> 3
<211> 741
<212> DNA
<213>Duck hepatitis A virus (duck hepatitis A virus)
<400> 3
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120
ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300
aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360
catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420
ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480
gctgatatcg gatccgaatt ctctaaggtg aggcgtttct ctgacccgga gactcttttt 540
agtgatttgg aagatctaaa attggaattt gattttgatc aactcgagca acaagctaaa 600
ctttttgcaa aaccaaaaga aggaaaaatc tctaaattta gggcttgggt tagagactgc 660
acagggaaga ttaagggttt tctggagcgg aatcgggcta agcttgcggc cgcactcgag 720
caccaccacc accaccactg a 741
<210> 4
<211> 246
<212> PRT
<213>Duck hepatitis A virus (duck hepatitis A virus)
<400> 4
Met Ser Ala Leu Ile Ile His Leu Thr Ala Ala Ser Pro Ala Thr Ala
1 5 10 15
Val Leu Leu Ala Ala Gly Ala Ile Leu Val Ala Pro Thr Ala Gly Thr
20 25 30
Cys Gly Pro Cys Leu Met Ile Ala Pro Ile Leu Ala Gly Ile Ala Ala
35 40 45
Gly Thr Gly Gly Leu Leu Thr Val Ala Leu Leu Ala Ile Ala Gly Ala
50 55 60
Pro Gly Thr Ala Pro Leu Thr Gly Ile Ala Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Pro Leu Ala Gly Gly Val Ala Ala Thr Leu Val Gly Ala Leu Ser
85 90 95
Leu Gly Gly Leu Leu Gly Pro Leu Ala Ala Ala Leu Ala Gly Ser Gly
100 105 110
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
115 120 125
Ala Gly Ser Gly Met Leu Gly Thr Ala Ala Ala Leu Pro Gly Ala Gly
130 135 140
His Met Ala Ser Pro Ala Leu Gly Thr Ala Ala Ala Ala Leu Ala Met
145 150 155 160
Ala Ala Ile Gly Ser Gly Pro Ser Leu Val Ala Ala Pro Ser Ala Pro
165 170 175
Gly Thr Leu Pro Ser Ala Leu Gly Ala Leu Leu Leu Gly Pro Ala Pro
180 185 190
Ala Gly Leu Gly Gly Gly Ala Leu Leu Pro Ala Leu Pro Leu Gly Gly
195 200 205
Leu Ile Ser Leu Pro Ala Ala Thr Val Ala Ala Cys Thr Gly Leu Ile
210 215 220
Leu Gly Pro Leu Gly Ala Ala Ala Ala Leu Leu Ala Ala Ala Leu Gly
225 230 235 240
His His His His His His
245
Claims (10)
1. a kind of indirect ELISA testing kit based on DHAV-1 3A albumen, which is characterized in that the kit is with 3A-
198 recombinant proteins are envelope antigen, and the 3A-198 recombinant protein is the 3A truncated gene for removing DHAV-1 3A gene transmembrane region
The recombination fusion protein of coding.
2. kit according to claim 1, it is characterised in that：The 3A for removing DHAV-1 3A gene transmembrane region is cut
The amino acid sequence of the recombination fusion protein of short gene coding is as shown in SEQIDNO.4.
3. kit according to claim 1, which is characterized in that the 3A-198 recombinant protein is prepared by the following method
It obtains：The 3A truncated gene segment for removing 3A gene transmembrane region is obtained by PCR, and target fragment is subcloned to pET-32a
(+), construction recombination plasmid pET-32-3A-198；Entering to express bacterium BL21 (DE3) for recombinant plasmid transformed, inducing expression is 31kD afterwards
3A truncate recombination fusion protein, be named as 3A-198；Maximum expression quantity is obtained with 0.1mmol/L IPTG, 37 DEG C of induction 6h,
Albumen is expressed in supernatant, purifies 3A-198 using Ni affinity column method, 3A-198 recombinant protein is prepared.
4. kit according to claim 1, which is characterized in that the peridium concentration of envelope antigen is in the kit
6.185μg/mL。
5. kit according to claim 1, which is characterized in that the kit includes confining liquid, and the confining liquid is
PBS containing 5%BSA.
6. kit according to claim 1, which is characterized in that the kit further includes ELIAS secondary antibody, the enzyme mark
Secondary antibody is the anti-duck IgG of HRP label, and the dilution of ELIAS secondary antibody is 1:2000.
7. a kind of indirect ELISA detection method based on DHAV-1 3A albumen, which is characterized in that include the following steps：
(1) envelope antigen：The 3A-198 recombinant protein that claim 3 is prepared is used into 0.05mol/L as antigen
ELISA Plate is added after the dilution of pH9.6 carbonate buffer solution, 100 μ L are added in every hole, and 4 DEG C of coatings are overnight；The peridium concentration of antigen is
6.185μg/mL；
(2) ELISA Plate is washed：Coating buffer is discarded, is washed 3 times, 5min/ times, is got rid of using the PBS containing 0.05% polysorbas20, pH7.4
Dry residual liquid；
(3) it closes：Confining liquid, every 100 μ L of hole, 37 DEG C of incubations are added into ELISA Plate；Wherein, confining liquid is containing 5%BSA's
PBS；
(4) ELISA Plate is washed：As step (2) method is washed；
(5) it is incubated for primary antibody：Duck blood after dilution is added is clear, every 100 μ L of hole, 37 DEG C of incubations；The clear dilution of duck blood is 1:20；
(6) ELISA Plate is washed：As step (2) method is washed；
(7) ELIAS secondary antibody：The anti-duck IgG of HRP label after dilution is added, every 100 μ L of hole, 37 DEG C of incubations；The anti-duck of HRP label
IgG is rabbit-anti duck IgG, dilution 1:2000；
(8) ELISA Plate is washed：As step (2) method is washed；
(9) it develops the color：TMB developing solution is added, every 100 μ L of hole, 37 DEG C are protected from light 10min；
(10) it terminates：2mol/L H is added2SO4Terminate reaction, every 100 μ L of hole；
(11) it reads：OD value is measured under 450nm and 630nm double wave using microplate reader.
8. the indirect ELISA detection method according to claim 7 based on 3A-198 recombinant protein, which is characterized in that step
Suddenly the incubation time in (3) is 90min.
9. the indirect ELISA detection method according to claim 7 based on 3A-198 recombinant protein, which is characterized in that step
Suddenly the incubation time in (5) and step (7) is 30min.
10. application of any kit of claim 1-5 in detection DHAV serum antibody.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008016594A1 (en) * | 2006-07-31 | 2008-02-07 | The Trustees Of Columbia University In The City Of New York | Picornavirus and uses thereof |
| CN104297493A (en) * | 2014-10-31 | 2015-01-21 | 四川农业大学 | Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit |
| CN105330727A (en) * | 2015-11-02 | 2016-02-17 | 四川农业大学 | 1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein |
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2018
- 2018-04-13 CN CN201810331738.4A patent/CN108828223A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008016594A1 (en) * | 2006-07-31 | 2008-02-07 | The Trustees Of Columbia University In The City Of New York | Picornavirus and uses thereof |
| CN104297493A (en) * | 2014-10-31 | 2015-01-21 | 四川农业大学 | Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit |
| CN105330727A (en) * | 2015-11-02 | 2016-02-17 | 四川农业大学 | 1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein |
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| Title |
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| JITENDRA K BISWAL ET AL.: "Diagnostic application of recombinant non-structural protein 3A to detect antibodies induced by foot-and-mouth disease virus infection", 《BIOLOGICALS》 * |
| 刘伟 等: "检测鸭1型甲肝病毒抗体间接ELISA方法的建立", 《中国兽医杂志》 * |
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