CN109557316A - A kind of detection method of duck Tan Busu Yolk antibody - Google Patents
A kind of detection method of duck Tan Busu Yolk antibody Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, disclose a kind of detection method of duck Tan Busu Yolk antibody, take 100ul that elisa plate, 37 DEG C of reaction 1h are added respectively duck egg yolk samples, positive control and negative control to be measured；It after washing 3-5 and pats dry, every minor tick 5min, goat-anti chicken IgY ELIAS secondary antibody, 37 DEG C of reaction 45min are added in every hole；It is separately added into tmb substrate developing solution, terminate liquid is added after 37 DEG C of reaction 10min；Microplate reader surveys the light absorption value at 450nm；The ratio of the OD450 value of the OD450 value and standard negative control duck egg yolk reaction hole of duck egg yolk samples reacting hole to be measured is confirmed.The present invention without blood sampling, only need to acquire be immunized the virus/E protein antigen chicken production egg, reduce because take a blood sample to laying hen caused by stress be with the requirement to technical staff；Yolk antibody yield is high, materials are simple.
Description
Technical field
The invention belongs to gene engineering technology field more particularly to a kind of detection methods of duck Tan Busu Yolk antibody.
Background technique
Currently, the prior art commonly used in the trade is such that
It is corresponding mainly to expand the detections such as experiment through PCR identification, indirect ELISA and fine jade for monitoring in relation to duck Tan Busu disease antibody
Antigen and antibody.And PCR is identified, is needed the samples such as take a blood sample and organize, is operated and take a long time, less suitable substrate farm
It needs.ELISA method high specificity, high sensitivity and detection speed are fast.The sensibility of AGP agar diffusion test not as good as ELISA method,
It is high to the purity requirement of antigen, it is not suitable for monitoring in large quantity and the detection of laboratory duck tembusu virus antibody.In conclusion
ELISA can be used for epidemiological survey, monitoring, anti-system of duck Tan Busu disease etc. and provide a kind of quickly and effectively detection method.
Duck tembusu virus is causing a disease for duck tembusu virus sick (duck Tembusu virus disease, DTVD)
Original is that duck is caused to be laid eggs sharply acute, deadly infectious disease a kind of flavivirus such as decline, hemorrhagic oaritis, and the disease was in 2010
April breaks out duck culturing dense region for the first time in south China, causes huge economic loss to China's duck culturing industry.
Chicken yolk immune globulin (IgY) is also known as Yolk antibody, is the specificity secreted after birds are stimulated by specific antigen
Antibody.Due to compared with serum IgG, having heat-resisting, acidproof containing the specific antibody for duck tembusu virus recombinant protein E
With the characteristics such as proteolytic degradation, the detection of sample can be completed by acquiring egg, for the research of day rear defence preparation with answer
With offer new approaches.Enzyme-linked immunosorbent assay (ELISA) is widely used in a kind of conventional method of disease diagnosis, in the short time
The interior large batch of egg yolk samples of processing, compared with fine jade expands experiment, latex agglutination experiment etc., specificity and sensitivity are higher.
In conclusion problem of the existing technology is:
(1) in the prior art, need to take a blood sample when the detection of duck Tan Busu Yolk antibody, cause to cause laying hen stress, and
It is required that technical staff's profession degree is high；
(2) serum antibody low output, materials are complicated；
(3) there are no the kits of detection Yolk antibody currently on the market, and the prevention and control monitoring of the smooth Soviet Union's virosis of duck is caused to arrange
It is poor to apply effect.
Solve the difficulty and meaning of above-mentioned technical problem:
The generation of Yolk antibody need to could obtain the high Yolk antibody exempted from after 2 immune duck tembusu virus, need to be into
Row sampling carries out SDS-PAGE, and whether qualitative evaluation Yolk antibody generates.
And the Yolk antibody detection method provided is provided, it can be used for the detection of sample when duck laying period, be that prevention and control duck is smooth
Egg production decline caused by cloth Soviet Union virus provides monitoring method, and the research for the anti-preparation of Yolk antibody provides new approaches.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of detection methods of duck Tan Busu Yolk antibody.
The invention is realized in this way a kind of detection method of duck Tan Busu Yolk antibody, the duck Tan Busu yolk is anti-
The detection method of body is detected using duck tembusu virus disease E-ELISA detection kit, is specifically included:
(1) take 100ul that elisa plate, 37 DEG C of reactions are added respectively duck egg yolk samples, positive control and negative control to be measured
1h；
(2) it washs after 3-5 and pats dry, every minor tick 5min, goat-anti chicken IgY ELIAS secondary antibody, 37 DEG C of reactions are added in every hole
45min；
(3) by after above-mentioned washing, it is separately added into tmb substrate developing solution, terminate liquid is added after 37 DEG C of reaction 10min；
(4) microplate reader surveys the light absorption value at 450nm；
(5) the OD450 value of duck egg yolk samples reacting hole to be measured and the OD450 value in standard negative control duck egg yolk reaction hole
Ratio is confirmed, when being more than or equal to 2.1, indicates duck egg yolk samples to be measured for the positive, duck infected duck tembusu virus to be measured；On
When stating ratio less than 2.1, duck egg yolk samples to be measured are indicated as feminine gender, duck to be measured does not have infected duck tembusu virus.
Further, the detection method of the duck Tan Busu Yolk antibody further comprises,
The preparation and identification of Yolk antibody:
The immune bird inlay for taking 9 120 age in days week old of laying hen is grouped, every group of 3 hens, sub-cage rearing, the
1,2 groups are test group, and the 3rd group is control group；The recombinant E protein that negative control group muscle multi-point injection 1ml was emulsified；After head exempts from
14d carries out two and exempts from；
Yolk slightly refers to identification:
7 days acquisition eggs beat easily yolk liquid after exempting from two, are put in -20 DEG C and freeze, and supernatant is taken to carry out the SDS- of Yolk antibody
PAGE, and identified by stripe size；IgY result should have 2 bands；Positive Yolk antibody is subjected to E-ELISA detection.
Further, the coated elisa plate of E protein is prepared by following procedure:
Purified E recombinant protein is diluted to 5 μ g/ml with 0.05mol/LPBS；PBS containing recombinant protein is added
Into 96 hole elisa Plates, 100 holes μ l/ are placed in 4 DEG C；10mmol/L PBST is added in liquid in 96 orifice plates, and 200 holes μ l/ wash 3
It is secondary, each 5min；The liquid in 96 orifice plates is confided all, confining liquid is added, is placed in 37 DEG C of 2.5h；The liquid in 96 orifice plates is confided all, is added
Enter 10mmol/LPBST (pH value 7.4), 200 holes μ l/ are washed 3 times, each 5min.The liquid in 96 orifice plates is thoroughly confided all, will be coated with
There are 96 orifice plates of His-E albumen to be placed in 37 DEG C, dry 2h has been put into desiccant plastic bag；Standard negative control yolk is SPF
Chicken with yolk, extracting yolk standard positive control yolk is to take a blood sample after SPF chicken is immunized with duck tembusu virus.
Further, other preparation of reagents methods for including in duck tembusu virus disease E-ELISA detection kit include:
1) preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of 9.6 coating buffer of pH value；
Solution A: Na2CO310.6g adds deionized water dissolving to 500ml；
Second liquid: NaHCO38.4g adds deionized water dissolving to 500ml；
Solution A 16ml, second liquid 34ml are taken, adds deionized water to 200ml, as 9.6 carbonate buffer of 0.05mol/LpH value
Liquid；
2) sample diluting liquid: BSA or skimmed milk power 2-5g, addition deionized water to 100mL；
3) sample cleaning solution: the process for preparation of 10 × concentration washing lotion: KH2PO42.6g, Na2HPO4-12H2O28.9g,
NaCl 87.1g, deionized water 800ml adjust pH value to 7.4, add deionized water to 1L, add 5ml in this solution
Tween-20；
4) sample confining liquid: BSA or skimmed milk power 2-5g, addition deionized water are added in the solution to 100mL
0.5ml Tween-20；
5) tmb substrate developing solution, each 1 bottle/box 50ml of kit loading amount；
6) terminate liquid: the preparation of terminate liquid: 2mol/L sulfuric acid solution, concentrated sulfuric acid solution concentration be 18mol/L, the concentrated sulfuric acid with
Deionized water is prepared by 1:9 becomes 2mol/L sulfuric acid solution.
Further, microplate reader surveys the light absorption value at 450nm, P/N=OD (positive-blank)/OD (feminine gender-blank) >=2.1
When be determined as the positive.
Another object of the present invention is to provide a kind of duck of detection method using the duck Tan Busu Yolk antibody is smooth
Cloth Soviet Union virosis E-ELISA detection kit, including standard positive control duck yolk, standard negative control duck yolk, the smooth cloth of duck
The coated elisa plate of virus E protein institute, Soviet Union, goat-anti duck ELIAS secondary antibody and other reagents.
Further, the kit, standard positive control duck yolk are to extract the immune SPF of personal duck tembusu virus
The yolk of chicken, the standard negative control duck yolk are the yolk for extracting from SPF chicken；
The duck tembusu virus E protein coated elisa plate recombinantly expressed by being used in prokaryotic expression system
Duck tembusu virus E protein is coated with elisa plate and is made.
Further, duck tembusu virus E protein pass through will be attached with shown in coding tembusu virus E protein nucleotides sequence
Column are cloned in procaryotic cell expression carrier and express in prokaryotic cell and are made.
Further, the goat-anti duck ELIAS secondary antibody is the goat-anti chicken IgY of HRP label.
Further, other reagents include sample diluting liquid, 10 × concentration washing lotion, tmb substrate developing solution, terminate liquid.
In conclusion advantages of the present invention and good effect are as follows:
Without blood sampling, the egg that the production of the virus/E protein antigen chicken is immunized need to be only acquired, reduces and is made because taking a blood sample to laying hen
At stress be with the requirement to technical staff；
Yolk antibody yield is high, materials are simple；
The high immunity yolk antibody of acquisition provides new selection for the anti-preparation of duck tembusu virus disease；
Prevention and control monitoring for the smooth Soviet Union's virosis of duck provides detection method.
ELISA testing result
Detailed description of the invention
Fig. 1 is the detection method flow chart of duck Tan Busu Yolk antibody provided in an embodiment of the present invention.
Fig. 2 is the SDS-PAGE detection figure one of IgY provided in an embodiment of the present invention.
In figure: M: Protein Marker；1, recombinant E protein group yolk is not immunized；2, recombinant E protein group yolk is immunized.
Fig. 3 is the SDS-PAGE detection figure two of IgY provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In the prior art, need to take a blood sample when the detection of duck Tan Busu Yolk antibody, cause to cause laying hen stress, and require
Technical staff's profession degree is high；Yolk antibody low output, materials are complicated.
Application of the invention is further described below with reference to embodiment.
Duck tembusu virus disease E-ELISA detection kit provided in an embodiment of the present invention, the kit include standard
Positive control duck yolk, standard negative control duck yolk, the coated elisa plate of duck tembusu virus E protein institute, goat-anti duck enzyme mark
Secondary antibody and other reagents.
The kit, wherein the standard positive control duck yolk is to extract personal duck tembusu virus to be immunized
The yolk of SPF chicken, the standard negative control duck yolk are the yolk for extracting from SPF chicken.
The kit, wherein the coated elisa plate of duck tembusu virus E protein institute is by being used in prokaryotic expression
The duck tembusu virus E protein coating elisa plate recombinantly expressed in system is made.
The kit, wherein the duck tembusu virus E protein is by encoding tembusu virus E shown in be attached with
The nucleotide sequence of albumen is cloned in procaryotic cell expression carrier and expresses in prokaryotic cell and is made.Tembusu virus E egg
White nucleotide sequence are as follows:
SEQ ID NO:1
FSCLGMQNRDFVEGVNGVGWIDVVLEGGSCVTITAKDRPTIDVKMMNMEATELAVVRSYCYEPKVSDV
TTESRCPTMGEAHNPKATYAEYICKKDFVDRGWGNGCGLFGKGSIQTCAKFDCTKKAEGRIVQKENVQFEVAVFIH
GSTEASTYHNYSAQQSLKHAARFVITPKSPVYTAEMEDYGTVTLECEPRSGVDMGQFYVFTMNTKSWLVNRDWFHD
LNLPWTGSSAGTWQNKESLIEFEEAHATKQSVVALASQEGALHAALAGAIPVKYSGSKLEMTSGHLKCRVKMQGLK
LKGMTYPMCSNTFSLVKNPTDTGHGTVMVELSYVGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTSSTGAKIM
VEVEPPFGDSFILVGSGKGQIRYQWHRSGSTIGKAFTSTLKGAQRMVALGDTAWDFGSVGGVLTSIGKGIHQVFGS
AFKSLFGGMSWITQGMLGALLLWMGLNARDRSISITFLAVGGILVFLAVNVNA。
The kit, wherein the goat-anti duck ELIAS secondary antibody is the goat-anti chicken IgY of HRP label.
The kit, wherein other described reagents include sample diluting liquid, 10 × concentration washing lotion, tmb substrate colour developing
Liquid, terminate liquid.
Such as Fig. 1, the detection method of duck Tan Busu Yolk antibody provided in an embodiment of the present invention, comprising:
S101: reaction step: duck egg yolk samples, positive control and negative control to be measured are taken respectively described in 100ul addition
Elisa plate, 37 DEG C of reaction 1h；
S102: with secondary antibody reaction step: after washing 3-5 and patting dry, every minor tick 5min, the goat-anti chicken is added in every hole
IgY ELIAS secondary antibody, 37 DEG C of reaction 45min；
S103: development step: by after above-mentioned washing, it is separately added into tmb substrate developing solution, is added after 37 DEG C of reaction 10min
Terminate liquid；
S104: reading step: microplate reader surveys the light absorption value at 450nm；
S105: judgment step: the OD450 value of duck egg yolk samples reacting hole to be measured and standard negative control duck egg yolk reaction hole
OD450 value ratio be more than or equal to 2.1 when, indicate duck egg yolk samples to be measured for the positive, illustrate that duck to be measured has infected the smooth cloth of duck
Soviet Union's virus；When above-mentioned ratio is less than 2.1, indicate that duck egg yolk samples to be measured for feminine gender, illustrate that duck to be measured does not have infected duck Tan Busu
Virus.
Application of the invention is further described below with reference to concrete analysis.
The diagnosis of the clinical symptoms such as the reduction of postpartum egg production is opened kind of a duck ovarian hypoplasia, laying duck in foundation of the invention
And the evaluation of tembusu virus immune effect of vaccine has a very important significance.
E-ELISA detection kit prepared by the present invention is used for the metainfective antibody test of duck tembusu virus.Institute as above
It states, duck tembusu virus recombinant protein E is laboratory preservation.
Application of the invention is further described below with reference to concrete analysis.
The preparation and identification of Yolk antibody provided in an embodiment of the present invention:
The immune bird inlay for taking 9 120 age in days week old of laying hen is grouped, every group of 3 hens, sub-cage rearing, the
1,2 groups are test group, and the 3rd group is control group.The recombinant E protein that negative control group muscle multi-point injection 1ml was emulsified.After head exempts from
14d carries out two in the same way and exempts from.
Yolk provided in an embodiment of the present invention slightly refers to identification:
7 days acquisition eggs beat easily yolk liquid after exempting from two, are put in -20 DEG C and freeze, and supernatant is taken to carry out the SDS- of Yolk antibody
PAGE, and identified by stripe size.IgY result should have 2 bands (heavy chain 60, light chain 20).By positive Yolk antibody into
Row E-ELISA detection (Yolk antibody 1:100 dilution, secondary antibody 1:4000 dilution).
It is provided in an embodiment of the present invention using duck tembusu virus E protein as the enzyme-linked immunosorbent assay (E- of envelope antigen
ELISA) detection kit, the kit include standard control positive duck yolk, standard control feminine gender duck yolk, duck Tan Busu
The coated elisa plate of virus E protein institute, goat-anti duck ELIAS secondary antibody and other reagents, by standard control positive chicken with yolk, standard pair
It is filled according to negative chicken with yolk, the coated elisa plate of duck tembusu virus E protein, goat-anti chicken IgY ELIAS secondary antibody and other reagent sets,
The kit is obtained, other compositions include sample diluting liquid, 10 × concentration washing lotion, tmb substrate developing solution, H2SO4 termination
Liquid, the kit are characterized in that the envelope antigen of enzyme mark elisa plate is the tembusu virus E protein of the prokaryotic expression of purifying,
It is obtained after being coated with elisa plate after purifying quantitatively；Positive criteria yolk is that duck tembusu virus E protein stoste is immune without specific
It is obtained after cause of disease (SPF) chicken, negative standards' yolk is SPF chicken with yolk.
For example, the coated elisa plate of the E protein can be prepared by following procedure:
Purified E recombinant protein is diluted to 5 μ g/ml with 0.05mol/LPBS (pH value 9.6)；Recombinant protein will be contained
PBS be added in 96 hole elisa Plates, 100 holes μ l/ are placed in 4 DEG C (15~18h)；10mmol/ is added in liquid in 96 orifice plates
LPBST (pH value 7.4), 200 holes μ l/, is washed 3 times, each 5min；The liquid in 96 orifice plates is confided all, confining liquid is added, is placed in 37 DEG C
2.5h；The liquid in 96 orifice plates is confided all, is added 10mmol/LPBST (pH value 7.4), 200 holes μ l/ are washed 3 times, each 5min.It is thorough
The liquid in 96 orifice plates is confided all at bottom, 96 orifice plates for being coated with His-E albumen is placed in 37 DEG C, dry 2h has been put into desiccant modeling
In material bag.Standard negative control yolk is SPF chicken with yolk, conventionally extracts yolk.Standard positive control yolk is to use
Duck tembusu virus (for example, JL2011 plants of duck tembusu virus) is taken a blood sample after SPF chicken is immunized, and conventionally extracts yolk.
Can conventionally to distinguish a large amount of ovum extracted after SPF chicken with yolk and duck tembusu virus immune duck for kit
Huang, and as standard items deposit, it is extracted again without detecting every time.
Goat-anti chicken ELIAS secondary antibody can be bought from proteintech company.
Application of the invention is further described combined with specific embodiments below
Other reagents in the kit include: sample diluting liquid, 10 × concentration washing lotion, tmb substrate developing solution, termination
Liquid.Its ingredient and preparation method are as follows:
(1) preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of 9.6 coating buffer of pH value；
Solution A: Na2CO310.6g adds deionized water dissolving to 500ml；
Second liquid: NaHCO38.4g adds deionized water dissolving to 500ml；
Solution A 16ml, second liquid 34ml are taken, adds deionized water to 200ml, as 9.6 carbonate buffer of 0.05mol/LpH value
Liquid.
(2) sample diluting liquid: BSA or skimmed milk power 2-5g, addition deionized water to 100mL.
(3) sample cleaning solution: the process for preparation of 10 × concentration washing lotion (100mmol/LPBST, pH7.4) is as follows:
KH2PO42.6g, Na2HPO4-12H2O 28.9g, NaCl 87.1g, deionized water 800ml adjust pH value to 7.4, addition go from
Sub- water adds 5ml Tween-20 to 1L in this solution.
(4) sample confining liquid: BSA or skimmed milk power 2-5g adds deionized water to 100mL, adds in this solution
0.5ml Tween-20。
(5) tmb substrate developing solution is purchased from Wuhan doctor moral company, each 1 bottle/box 50ml of kit loading amount.
(6) terminate liquid: 1 bottle of bottle of each kit loading amount, 250ml/ bottles；The preparation of terminate liquid: 2mol/L sulfuric acid solution
(2mol/LH2SO4), concentrated sulfuric acid solution concentration is 18mol/L, and the concentrated sulfuric acid and deionized water, which press 1:9 preparation, can become 2mol/L
Sulfuric acid solution.
Result judgement: the light absorption value at microplate reader survey 450nm, P/N=OD (positive-blank)/OD (feminine gender-blank) >=
It is determined as the positive when 2.1.
It specifically includes:
Sample coating buffer (0.05mol/LPBS, pH 9.6): Na2CO31.59g NaHCO32.93g plus deionized water dissolving
To 1000ml；
Sample diluting liquid: BSA or skimmed milk power 2-5g, addition deionized water to 100mL.Sample cleaning solution: 10 × concentration
The process for preparation of washing lotion (100mmol/LPBST, pH7.4) is as follows: KH2PO42.6g, Na2HPO4-12H2O 28.9g, NaCl
87.1g, deionized water 800ml adjust pH value to 7.4, and addition deionized water to 1L adds 5ml Tween-20 in this solution.
Sample confining liquid: BSA or skimmed milk power 2-5g adds deionized water to 100mL, adds 0.5ml in this solution
Tween-20。
Tmb substrate developing solution is purchased from Wuhan doctor moral company, each 1 bottle/box 50ml of kit loading amount.
Terminate liquid: 1 bottle of bottle of each kit loading amount, 250ml/ bottles；The preparation of terminate liquid: 2mol/L sulfuric acid solution (2mol/
LH2SO4), concentrated sulfuric acid solution concentration is that 18mol/L, the concentrated sulfuric acid and deionized water can be molten as 2mol/L sulfuric acid by 1: 9 preparation
Liquid.
(5) it assembles: by the ingredient of the coated elisa plate of above-mentioned duck tembusu virus recombinant protein E and suitable (2)-(4)
It is assembled into kit.
Fig. 3 is the SDS-PAGE detection figure of IgY provided in an embodiment of the present invention.
Fig. 2 is the SDS-PAGE detection figure one of IgY provided in an embodiment of the present invention.
In figure: M: Protein Marker；1, recombinant E protein group yolk is not immunized；2, recombinant E protein group yolk is immunized.
Fig. 3 is the SDS-PAGE detection figure two of IgY provided in an embodiment of the present invention.
Application of the invention is further described below with reference to specific experiment.
Yolk antibody identification
SDS-PAGE runs the Yolk antibody Western- of dying method with coomassie brilliant blue Detection and Extraction after purification after glue
Blotting identifies Yolk antibody and E protein antigen-binding activity, and successively 10ul E protein is added to the glue hole that glue is concentrated in every hole
In, albumen is transferred on NC film after SDS-PAGE is separated, and the Yolk antibody that extraction is added is incubated overnight as 4 DEG C of primary antibody shaking table
It educates, carries out ECL development after adding the rabbit-anti chicken IgY secondary antibody incubation at room temperature 1h of HRP label, result is saved.
7 days acquisition eggs beat easily yolk liquid after exempting from two, are put in -20 DEG C and freeze, and supernatant is taken to carry out the SDS- of Yolk antibody
PAGE, and identified by stripe size.IgY result should have 2 bands (heavy chain 60, light chain 20).By positive Yolk antibody into
Row E-ELISA detection (Yolk antibody 1:100 dilution, secondary antibody 1:4000 dilution).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences
<120>a kind of detection method of duck Tan Busu Yolk antibody
<130> 2018S1389IWH
<141> 2018-12-03
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 501
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Phe Ser Cys Leu Gly Met Gln Asn Arg Asp Phe Val Glu Gly Val Asn
1 5 10 15
Gly Val Gly Trp Ile Asp Val Val Leu Glu Gly Gly Ser Cys Val Thr
20 25 30
Ile Thr Ala Lys Asp Arg Pro Thr Ile Asp Val Lys Met Met Asn Met
35 40 45
Glu Ala Thr Glu Leu Ala Val Val Arg Ser Tyr Cys Tyr Glu Pro Lys
50 55 60
Val Ser Asp Val Thr Thr Glu Ser Arg Cys Pro Thr Met Gly Glu Ala
65 70 75 80
His Asn Pro Lys Ala Thr Tyr Ala Glu Tyr Ile Cys Lys Lys Asp Phe
85 90 95
Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser
100 105 110
Ile Gln Thr Cys Ala Lys Phe Asp Cys Thr Lys Lys Ala Glu Gly Arg
115 120 125
Ile Val Gln Lys Glu Asn Val Gln Phe Glu Val Ala Val Phe Ile His
130 135 140
Gly Ser Thr Glu Ala Ser Thr Tyr His Asn Tyr Ser Ala Gln Gln Ser
145 150 155 160
Leu Lys His Ala Ala Arg Phe Val Ile Thr Pro Lys Ser Pro Val Tyr
165 170 175
Thr Ala Glu Met Glu Asp Tyr Gly Thr Val Thr Leu Glu Cys Glu Pro
180 185 190
Arg Ser Gly Val Asp Met Gly Gln Phe Tyr Val Phe Thr Met Asn Thr
195 200 205
Lys Ser Trp Leu Val Asn Arg Asp Trp Phe His Asp Leu Asn Leu Pro
210 215 220
Trp Thr Gly Ser Ser Ala Gly Thr Trp Gln Asn Lys Glu Ser Leu Ile
225 230 235 240
Glu Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala Leu Ala
245 250 255
Ser Gln Glu Gly Ala Leu His Ala Ala Leu Ala Gly Ala Ile Pro Val
260 265 270
Lys Tyr Ser Gly Ser Lys Leu Glu Met Thr Ser Gly His Leu Lys Cys
275 280 285
Arg Val Lys Met Gln Gly Leu Lys Leu Lys Gly Met Thr Tyr Pro Met
290 295 300
Cys Ser Asn Thr Phe Ser Leu Val Lys Asn Pro Thr Asp Thr Gly His
305 310 315 320
Gly Thr Val Met Val Glu Leu Ser Tyr Val Gly Thr Asp Gly Pro Cys
325 330 335
Arg Val Pro Ile Ser Met Ser Ala Asp Leu Asn Asp Met Thr Pro Val
340 345 350
Gly Arg Leu Ile Thr Val Asn Pro Tyr Val Ser Thr Ser Ser Thr Gly
355 360 365
Ala Lys Ile Met Val Glu Val Glu Pro Pro Phe Gly Asp Ser Phe Ile
370 375 380
Leu Val Gly Ser Gly Lys Gly Gln Ile Arg Tyr Gln Trp His Arg Ser
385 390 395 400
Gly Ser Thr Ile Gly Lys Ala Phe Thr Ser Thr Leu Lys Gly Ala Gln
405 410 415
Arg Met Val Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly
420 425 430
Gly Val Leu Thr Ser Ile Gly Lys Gly Ile His Gln Val Phe Gly Ser
435 440 445
Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Met
450 455 460
Leu Gly Ala Leu Leu Leu Trp Met Gly Leu Asn Ala Arg Asp Arg Ser
465 470 475 480
Ile Ser Ile Thr Phe Leu Ala Val Gly Gly Ile Leu Val Phe Leu Ala
485 490 495
Val Asn Val Asn Ala
500
Claims (10)
1. a kind of detection method of duck Tan Busu Yolk antibody, which is characterized in that the detection side of the duck Tan Busu Yolk antibody
Method is detected using duck tembusu virus disease E-ELISA detection kit, is specifically included:
(1) take 100ul that elisa plate, 37 DEG C of reaction 1h are added respectively duck egg yolk samples, positive control and negative control to be measured；
(2) it washs after 3-5 and pats dry, every minor tick 5min, goat-anti chicken IgY ELIAS secondary antibody, 37 DEG C of reaction 45min are added in every hole；
(3) by after above-mentioned washing, it is separately added into tmb substrate developing solution, terminate liquid is added after 37 DEG C of reaction 10min；
(4) microplate reader surveys the light absorption value at 450nm；
(5) ratio of the OD450 value of duck egg yolk samples reacting hole to be measured and the OD450 value in standard negative control duck egg yolk reaction hole
Confirmed, when being more than or equal to 2.1, indicates duck egg yolk samples to be measured for the positive, duck infected duck tembusu virus to be measured；Above-mentioned ratio
When value is less than 2.1, duck egg yolk samples to be measured are indicated as feminine gender, duck to be measured does not have infected duck tembusu virus.
2. the detection method of duck Tan Busu Yolk antibody as described in claim 1, which is characterized in that the duck Tan Busu yolk
The detection method of antibody further comprises,
The preparation and identification of Yolk antibody:
The immune bird inlay for taking 9 120 age in days week old of laying hen is grouped, every group of 3 hens, sub-cage rearing, and the 1st, 2 group
For test group, the 3rd group is control group；The recombinant E protein that negative control group muscle multi-point injection 1ml was emulsified；Head exempt from rear 14d into
Row two is exempted from；
Yolk slightly refers to identification:
7 days acquisition eggs beat easily yolk liquid after exempting from two, are put in -20 DEG C and freeze, and supernatant is taken to carry out the SDS-PAGE of Yolk antibody,
And it is identified by stripe size；IgY result should have 2 bands；Positive Yolk antibody is subjected to E-ELISA detection.
3. the detection method of duck Tan Busu Yolk antibody as claimed in claim 2, which is characterized in that the coated ELISA of E protein
Plate is prepared by following procedure:
Purified E recombinant protein is diluted to 5 μ g/ml with 0.05mol/L PBS；PBS containing recombinant protein is added to
In 96 hole elisa Plates, 100 holes μ l/ are placed in 4 DEG C；10mmol/L PBST is added in liquid in 96 orifice plates, and 200 holes μ l/ wash 3
It is secondary, each 5min；The liquid in 96 orifice plates is confided all, confining liquid is added, is placed in 37 DEG C of 2.5h；The liquid in 96 orifice plates is confided all, is added
Enter 10mmol/L PBST (pH value 7.4), 200 holes μ l/ are washed 3 times, each 5min.The liquid in 96 orifice plates is thoroughly confided all, will be wrapped
There are 96 orifice plates of His-E albumen to be placed in 37 DEG C, dry 2h has been put into desiccant plastic bag；Standard negative control yolk is
SPF chicken with yolk, extracting yolk standard positive control yolk is to take a blood sample after SPF chicken is immunized with duck tembusu virus.
4. the detection method of duck Tan Busu Yolk antibody as described in claim 1, which is characterized in that duck tembusu virus disease E-
Other preparation of reagents methods for including in ELISA detection kit include:
1) preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of 9.6 coating buffer of pH value；
Solution A: Na2CO3 10.6g adds deionized water dissolving to 500ml；
Second liquid: NaHCO3 8.4g adds deionized water dissolving to 500ml；
Solution A 16ml, second liquid 34ml are taken, adds deionized water to 200ml, as 9.6 carbonate buffer solution of 0.05mol/LpH value；
2) sample diluting liquid: BSA or skimmed milk power 2-5g, addition deionized water to 100mL；
3) sample cleaning solution: the process for preparation of 10 × concentration washing lotion: KH2PO42.6g, Na2HPO4-12H2O 28.9g, NaCl
87.1g, deionized water 800ml adjust pH value to 7.4, and addition deionized water to 1L adds 5ml Tween-20 in this solution；
4) sample confining liquid: BSA or skimmed milk power 2-5g, addition deionized water add 0.5ml in the solution to 100mL
Tween-20；
5) tmb substrate developing solution, each 1 bottle/box 50ml of kit loading amount；
6) terminate liquid: the preparation of terminate liquid: 2mol/L sulfuric acid solution, concentrated sulfuric acid solution concentration be 18mol/L, the concentrated sulfuric acid and go from
Sub- water is prepared by 1:9 becomes 2mol/L sulfuric acid solution.
5. the detection method of duck Tan Busu Yolk antibody as claimed in claim 4, which is characterized in that
Microplate reader surveys the light absorption value at 450nm, and when P/N=OD (positive-blank)/OD (feminine gender-blank) >=2.1 is determined as sun
Property.
6. a kind of duck tembusu virus disease E-ELISA using the detection method of duck Tan Busu Yolk antibody described in claim 1
Detection kit, which is characterized in that the duck tembusu virus disease E-ELISA detection kit includes standard positive control duck ovum
Huang, standard negative control duck yolk, the coated elisa plate of duck tembusu virus E protein institute, goat-anti duck ELIAS secondary antibody and other examinations
Agent.
7. as claim 6 duck tembusu virus disease E-ELISA detection kit, which is characterized in that the kit,
Standard positive control duck yolk is the yolk for extracting the immune SPF chicken of personal duck tembusu virus, the standard negative control duck
Yolk is the yolk for extracting from SPF chicken；
The duck tembusu virus E protein coated elisa plate it is smooth by being used in the duck that recombinantly expresses in prokaryotic expression system
Cloth Soviet Union virus E protein coating elisa plate is made.
8. as claim 6 duck tembusu virus disease E-ELISA detection kit, which is characterized in that duck tembusu virus E
Albumen is by the way that the nucleotide sequence of coding tembusu virus E protein shown in note to be cloned in procaryotic cell expression carrier simultaneously
It expresses and is made in prokaryotic cell.
9. as claim 6 duck tembusu virus disease E-ELISA detection kit, which is characterized in that
The goat-anti duck ELIAS secondary antibody is the goat-anti chicken IgY of HRP label.
10. as claim 6 duck tembusu virus disease E-ELISA detection kit, which is characterized in that other reagents include
Sample diluting liquid, 10 × concentration washing lotion, tmb substrate developing solution, terminate liquid.
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| CN112300275A (en) * | 2020-02-19 | 2021-02-02 | 南京蛋球球生物医学技术合伙企业(有限合伙) | Yolk antibody for inhibiting new coronavirus SARS-CoV-2 and its preparation method and application |
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