CN103360472A - Acian Metapneumovirus antibody ELISA detection kit - Google Patents

Abstract

The invention provides an Acian Metapneumovirus antibody ELISA detection kit. The kit is the ELISA kit established for detecting the Acian Metapneumovirus antibody on the basis of treating the monomer protein of the A subgroup Acian Metapneumovirus F protein as a coating antigen. The invention also provides a preparation method of the monomer protein of the coating antigen F protein for making the kit. The F protein prepared through the method has a good reactionogenicity, and the established ELISA detection kit provides an effective method for the early-stage diagnosis and detection of the Acian Metapneumovirus infection, and plays a substantial promotion effect in the prevention researches of the Acian Metapneumovirus infected diseases.

Description

Fowl metapneumovirus antibody ELISA detection kit

Technical field

The present invention relates to the immunological technique field, be specifically related to a kind of monomeric protein of A subgroup fowl metapneumovirus F albumen and for detection of the ELISA test kit of above-mentioned A subgroup fowl metapneumovirus antibody.

Background technology

Fowl metapneumovirus (Avian Metapneumovirus, aMPV), have another name called Turkey Rhinotracheitis Virus (Turkey Rhinotracheitis Virus, TRTV), belonging to Paramyxoviridae Pneumovirinae Pneumovirus, is the bird important pathogen that distributes in a kind of world wide.This virus mainly causes the upper respiratory tract systemic disease of turkey and chicken, such as turkey rhinotracheitis (Turkey Rhinotracheitis, TRT), fowl rhinotracheitis (Avian Rhinotracheitis, ART), and in close relations with the swollen syndrome (Swollen Head Syndrome, SHS) of broiler chicken.Wherein, the turkey rhinotracheitis is a kind of acute height contagious disease of harm turkey aquaculture, this disease take have a running nose, sneeze, under hole swelling under the socket of the eye, head swelling and the laying rate degradation symptom as principal character, infectious rate, sickness rate can be up to 100%, mortality ratio changes with feeding and management and sanitary condition, if polyinfection is arranged, mortality ratio can be up to 25%～40%.This disease transmission is strong, propagates rapidly, can cause multiple bird infection morbidity and cause serious financial loss.At present China is the poultry big country that ranks the first in the world, and 9,600,000,000 of the numbers of animals raised of chicken, the annual mortality ratio of bringing because of all kinds of poultry dieases be up to 20%～25%, loss hundreds of hundred million Renminbi.

At present, serology is the most frequently used method of aMPV diagnosis, particularly vaccination chicken group not.Method commonly used has indirect enzyme-linked immunosorbent adsorption experiment, neutralization of virus, indirect immunofluorescence and immunodiffusion(ID).Wherein, ELISA is the most frequently used method, has quick, sensitive, few, the easy-operating characteristics of amount of serum.Commercialization aMPV ELISA detection kit is from Europe and the U.S. on the market, is mainly used in the detection of serum antibody, and its coated antigen is the totivirus that extracts behind the virus inoculation cell, brings potential harm usually for the propagation of this disease; In addition, these detection reagent are inadequate such as the susceptibility of the antibody test of A subgroup to specific subgroup; Particularly still do not use at present the recombinant protein of expressing to do the development that envelope antigen carries out the ELISA detection kit.AMPV contains multiple virus structural protein, and wherein F albumen is the major structural protein of virus, contains the neutrality epitope that can protect body opposing aMPV to infect, affects recombinant expressed in expression system of this albumen but this F albumen contains a plurality of cross-films district.How expressing the recombinant protein that contains the neutrality epitope sets up the detection that ELISA method exploitation antibody assay kit is used for this disease as envelope antigen and has realistic meaning.At present the plant that infects in the multiple poultry kind of China of fowl metapneumovirus occurs with popular, therefore, develops special, sensitive, easy diagnostic kit and be the active demand that the current prevention and control of China should disease.

Summary of the invention

In order to overcome the defective of prior art, an object of the present invention is for the preparation of the ELISA test kit that detects fowl metapneumovirus antibody, this test kit provides a cover effective instrument for the detection of fowl metapneumovirus antibody.

Another object of the present invention provides a kind of preparation and purification method of monomeric protein of A hypotype fowl metapneumovirus F albumen, for fowl metapneumovirus antibody assay kit provides envelope antigen.

A further object of the present invention is the application of above-mentioned ELISA test kit on rapid detection fowl metapneumovirus.

For realizing above purpose, the present invention at first provides a kind of A hypotype fowl metapneumovirus F albumen, and this albumen is on the basis with A hypotype fowl metapneumovirus F albumen wild-type, removes its part cross-film district and obtains, and its aminoacid sequence is shown in SEQ ID No.2.The present invention also comprises the gene of encoding sequence, and its nucleotide sequence is shown in SEQ ID No.1.The research discovery, this albumen has good immune response originality, can be used for the antibody test of aMPV.

Accordingly, the invention provides a kind of fowl metapneumovirus antibody ELISA detection kit, it comprises the coated elisa plate of monomeric protein of A type fowl metapneumovirus F albumen.

The ELISA test kit of above-mentioned detection fowl metapneumovirus also comprises ELIAS secondary antibody, positive control, negative control, washings, sample diluting liquid, substrate solution A, substrate solution B and stop buffer.

Wherein, described ELIAS secondary antibody is horseradish peroxidase mark goat-anti chicken monoclonal antibody; Described positive control is the chicken serum of natural infection A subgroup avian pneumovirus; Described negative control is normal chicken serum; Described coating buffer, washings, confining liquid, sample diluting liquid, substrate solution A, component and the proportioning of substrate solution B and stop buffer are as follows:

Coating buffer: anhydrous Na ₂CO ₃1.59g, NaHCO ₃2.93g adding distil water is to 1000mL;

Washings: NaCl8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O2.9g, KCl0.2g, Tween-200.5mL adds distilled water to 1000mL;

Confining liquid: 5% skim-milk;

Sample diluting liquid: the washings that contains 5% skim-milk;

Substrate solution A: urea peroxide 1.0g, Na ₂HPO ₄12H ₂O35.8g, citric acid H ₂O10.3g, Tween-20100 μ L adds 1000mL distilled water;

Substrate solution B:TMB700mg, DMSO40mL, fully dissolving, adding distil water 960mL then, citric acid H ₂O10.3g;

Stop buffer: 2mol/L sulphuric acid soln.

Above-mentioned for the monomeric protein preparation of ELISA test kit as the A type fowl metapneumovirus F albumen of enzyme plate envelope antigen, may further comprise the steps: synthetic A subgroup fowl metapneumovirus F protein gene sequence is that template amplification obtains the sequence shown in the SEQ ID No.1, the above-mentioned purpose gene is imported in the pBCX carrier, product after the connection transforms DH5 α competent cell, evaluation and screening goes out positive recombinant expression plasmid pBCX-aMPV-A-F after extracting the plasmid double digestion, with abduction delivering behind the above-mentioned positive recombinant expression plasmid conversion BL21 competent cell, SDS-PAGE electrophoretic analysis expressing protein, adopt again Ni post affinity chromatography method, target protein is carried out purifying, then detect the protein purification result with SDS-PAGE and Western blot respectively, target protein behind the purifying is processed with enteropeptidase, cut the result with SDS-PAGE check enzyme after processing, cutting glue with the KCl staining after electrophoresis finishes reclaims, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify, elisa assay is cut the antigenicity that glue reclaims albumen.

Particularly, the preparation of the monomeric protein of A type fowl metapneumovirus F albumen can may further comprise the steps:

1) amplifying target genes.Take synthetic A subgroup fowl metapneumovirus F protein gene sequence as template, by PCR method its F gene is increased, clones and obtain goal gene (F gene, its disappearance part cross-film district), upstream primer adds Kpn I restriction enzyme site, downstream primer adds Hind III restriction enzyme site, and primer sees Table 1:

The amplimer of table 1F gene

2) carry out double digestion with Kpn I, Hind III behind the recovery of F gene and the PBCX plasmid extraction, reclaim the purpose fragment, under 16 ° of C, connect 12-16h under the effect of T4DNA ligase enzyme goal gene being connected 5:1 with carrier, transformed competence colibacillus cell DH5 α, extract plasmid, after Kpn I and the evaluation correctly of Hind III double digestion, obtain positive recombinant expression plasmid pBCX-aMPV-A-F.

3) then get plasmid pBCX-aMPV-A-F and transform the BL21 competent cell, the positive plasmid bacterium that obtains is cultivated in 37 ° of C, treat that the A600 value reaches at 0.4～0.6 o'clock, adding IPTG is that 1.2mmol/L carries out abduction delivering to final concentration, the thalline of 4h behind the collection abduction delivering, ultrasonic disruption is got precipitation after centrifugal and is carried out the SDS-PAGE electrophoretic analysis.

4) then adopt Ni post affinity chromatography method, the purifying target protein, target protein behind the purifying carries out SDS-PAGE and Western blot identifies, process with enteropeptidase through the F albumen of identifying, then carry out the SDS-PAGE electrophoresis, take off gel after electrophoresis finishes, first with the distilled water washing, then immerse the 5min that develops the color in the KCl solution of 250mmol/L of 4 ° of C precoolings, the target protein band that is painted silvery white is downcut, PBS washing 3 times moves into adhesive tape in the one clean pouch and to pulverize, and broken end is scraped in the EP pipe, add PBS concussion mixing multigelation 3 times, the centrifugal 2min of 12000r/min draws supernatant, and the SDS-PAGE electrophoresis carries out recovering effect to be identified.

5) elisa assay is cut the antigenicity that glue reclaims albumen.

The present invention also provides the preparation method of mentioned reagent box, comprise the preparation of the elisa plate of A subgroup fowl metapneumovirus F albumen, its method is: with carbonate (pH9.6, concentration 0.5mol/L) damping fluid is made coating buffer, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use again.

Based on test kit of the present invention, can adopt following ELISA method, detect A subgroup fowl metapneumovirus antibody:

1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 30min;

2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 30min;

4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15～20min;

6) add stop buffer: add stop buffer 50 μ L in every hole;

7) measure: the absorption photometric value (OD450 value) of measuring every hole with microplate reader at the 450nm place.

The invention provides and use solubility pBCX carrier, expressed the F albumen in A subgroup fowl metapneumovirus disappearance part cross-film district, and utilize 6 * His label of pBCX carrier, the fusion rotein of expressing is carried out purifying, the albumen that Western-blot detects the proof purifying has reactionogenicity, cut with the albumen of enteropeptidase to purifying afterwards, obtain to remove the monomeric protein of carrier label, obtained the higher albumen of immunogenicity.This protein constructs provided by the invention is to have solved the problem of F protein monomer purification difficult.

The present invention is prepared from as the basis take the F albumen of gene engineering expression, and the F protein monomer is non-totivirus antigen, and security is good, does not contain irrelevant foreign protein, and has good antigenicity, and the detection kit of therefore inventing has very high specificity and susceptibility.

The present invention makes up is used for the test kit that A subgroup fowl metapneumovirus detects, easy to operation, detects responsively fast, is specially adapted to clinically the rapid detection to fowl metapneumovirus antibody, for the Prevention Research of this disease has played remarkable promoter action.

Description of drawings

Fig. 1 is the electrophorogram of F gene amplification, the purpose band of 1:F albumen wherein, 2: blank, M:MarkerDL2000.

Fig. 2 is the recombinant plasmid PCR evaluation figure that the F gene clone obtains to the pBCX carrier, M:DNA MarkerDL2000 wherein, 1～10: the recombinant plasmid pcr amplified fragment.

The enzyme of Fig. 3 recombinant plasmid pBCX-F that to be the F gene clone obtain to the pBCX carrier is cut evaluation figure, M:DNAMarkerDL2000 wherein, 1～3: the endonuclease bamhi of positive recombinant plasmid pBCX-F.

Fig. 4 is after pBCX-aMPV-A-F is transformed into the BL21 competent cell, extracts plasmid PCR and identifies figure, M:DNAMarkerDL2000 wherein, 1: the PCR product of recombinant plasmid, 3: blank.

Fig. 5 is the SDS-PAGE analysis chart of induction expression protein, M:175Marker wherein, and 1: the empty plasmid expression product, 2～8 are respectively the bacteria lysis product that 1.2mmol/LIPTG induces 0～6h.

Fig. 6 is the soluble analysis evaluation figure of fusion rotein, M:175Marker wherein, 1: bacteria lysis gross product, 2: the bacteria lysis precipitation; 3: the bacteria lysis supernatant.

Fig. 7 is the SDS-PAGE analysis chart of purifying protein, M:175Marker wherein, 1～4: the pipe of the 1st to 4 behind purifying wash-out product.

Fig. 8 is the Western-blot analysis chart of purifying protein, (a) middle M:175Marker, 1: be the fusion protein product of purifying; (b) M:175Marker in, 1,2,3: for being the fusion protein product of purifying.

Fig. 9 is that enzyme is cut rear KCl dyeing and cut the qualification result that glue reclaims target protein, M:175Marker wherein, 1,2: be respectively two pipe products after enzyme cuts back to close.

Embodiment

Following examples are used for further specifying of the present invention, but should not be construed limitation of the present invention.Under the prerequisite that does not deviate from the present invention's spirit and essence, modification or replacement to the present invention does all belong to category of the present invention.

Preparation and the purifying of the protein monomer of embodiment 1A subgroup fowl metapneumovirus F albumen

The gene order of the A subgroup fowl metapneumovirus F albumen shown in the synthetic SEQ ID NO.1, with a pair of Auele Specific Primer of Oligo6.0 software design, upstream primer adds Kpn I restriction enzyme site, and downstream primer adds Hind III restriction enzyme site, and upstream primer is: 5 '-GG GGTACCGAGGCAGTATCCACATTAGGG-3 ', downstream primer is: 5 '-CCC AAGCTTCTTGGCATCTGCACCTAG-3 '.(ddH increases in 20 μ L reaction systems take pcmy-myc-L plasmid (being purchased from Invitrogen company) as template ₂O11.5 μ L, 5 * Buffer4 μ L, Taq enzyme 0.2 μ L, dNTP2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, dna profiling 0.3 μ L). amplification condition is: 94 ° of C denaturation 3min; 94 ° of C sex change 30s, 59 ° of C annealing 45s, 72 ° of C extend 1.5min, totally 35 circulations; 72 ° of C extend 10min; 4 ° of C preserve (amplification is seen Fig. 1).

Get 100 μ L PCR products and carry out agarose gel electrophoresis, 100V electrophoresis 35～40min, ultraviolet detection product analysis result, and cut glue and reclaim.The purpose fragment through the Kpn I be connected with Hind III double digestion with the plasmid of being connected with Hind III double digestion through the Kpn I in molar ratio 5:1 under 16 ° of C, be connected 12～16h, transform DH5 α, 37 ° of C overnight incubation.Choosing colony extracts plasmid, uses Auele Specific Primer to carry out pcr amplification, and with above-mentioned restriction endonuclease digestion, electrophoresis is identified (the results are shown in Figure 2).Positive bacterium colony after identifying correctly checks order through plasmid and further verifies, and confirms that its gene order is correct, and the correct recombinant expression plasmid called after pBCX-aMPV-A-F(of order-checking be the results are shown in Figure 3).

From the correct bacterium colony that checks order, extract Plasmid Transformation BL21 competent cell, obtain recombinant bacterium (the results are shown in Figure 4), in the LB liquid nutrient medium, cultivate recombinant bacterium, treat that bacterium liquid is at 600nm optical density(OD) (optical density, OD) value under reaches at 0.4～0.6 o'clock, it is 1.2mmol/L that adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) makes its final concentration, abduction delivering.Collect the thalline that different induction times are expressed, ultrasonic disruption, centrifugally get respectively cleer and peaceful precipitation and carry out the SDS-PAGE electrophoresis afterwards, the result shows and induces the target protein overwhelming majority that reaches maximum (the results are shown in Figure 5) behind the 4h and merge to express (the results are shown in Figure 6) with soluble form.

After fusion rotein is defined as secretion type expression, directly the fusion rotein bacterial lysate is carried out purifying with the Ni-NTA purification column to target protein, with elutriant wash-out purification column, collect elutriant, contain the purpose fusion rotein in the elutriant, the F albumen that removes part cross-film district adds the label protein on the carrier, and the albumen size after the fusion is 74kDa, measure the concentration of albumen in the elutriant with ultraviolet spectrophotometer, the highest one is 27mg/mL (the results are shown in Figure 7).

Through the SDS-PAGE electrophoresis, utilize Western-blot that the fusion rotein of purifying is carried out the reactionogenicity analysis, primary antibodie is the monoclonal antibody of A hypotype aMPV F albumen, two anti-be the goat anti-mouse IgG of HRP mark, detected result shows: a darker purpose band is arranged at the 74kDa place, the fusion rotein of this explanation restructuring has reactionogenicity, and the albumen that the size of purpose band and SDS-PAGE express size position consistency, band is single, the WB detected result of albumen before the WB detected result of albumen behind the purifying and the purifying is compared, can prove fusion protein purification success (the results are shown in Figure 8).

F albumen is processed with enteropeptidase, then carry out the SDS-PAGE electrophoresis, electrophoresis takes off gel after finishing, wash with distilled water first, then immerse the 5min that develops the color in the KCL solution of 250mmol/L of 4 ° of C precoolings, the target protein band that is painted silvery white is downcut, PBS washing 3 times, adhesive tape moved in the clean pouch pulverize, broken end is scraped in the EP pipe, add PBS concussion mixing multigelation 3 times, the centrifugal 2min of 12000r/min, draw supernatant, the SDS-PAGE electrophoresis carries out recovering effect and identifies (the results are shown in Figure 9).

The Immunity identification of the F protein monomer that embodiment 2 the present invention make up

The F albumen that enzyme is cut back to close and the F albumen cut without enzyme are diluted to coated 96 orifice plates of 10 μ g/mL with the 0.5mol/L carbonate buffer solution of pH9.6, take fowl metapneumovirus infected chicken positive serum and negative serum as primary antibodie takes turns doing 3 gradient dilutions, be respectively 1:100,1:300,1:500.Take the anti-chicken IgG of the goat of HRP mark as two anti-, working concentration is 1:10000; Each hole OD after the TMB colour developing, is read in parallel 2 holes of doing of each sample _450nmValue, result show: without the enteropeptidase enzyme cut and with the albumen of carrier label as the elisa plate envelope antigen, when detecting serum, the OD value of negative serum is between 0.6-0.8, near the OD value of positive serum; And process enteropeptidase enzyme only keeps monomeric protein as the envelope antigen of elisa plate after cutting processing, when detecting serum, negative OD value is between 0.1-0.2, the P/N value of positive OD value and negative OD value is between 3.6-4.45, as seen ELISA result is good, the monomeric protein that enzyme cuts back to close has good reactionogenicity, and detected result sees Table 2:

Table 2ELISA detects the antigenicity that the KCl staining is cut glue purification albumen

The foundation of embodiment 3 fowl metapneumovirus ELISA detection methods

1.A determining of subgroup fowl metapneumovirus antibody ELISA detection kit optimum reaction condition

1) the best coated concentration of antigen and enzyme mark monoclonal antibody best effort concentration determines

Take the F monomeric protein of purifying as envelope antigen, the goat-anti chicken monoclonal antibody of HRP mark is second antibody, carries out the square formation burette test at enzyme plate.Make respectively 15 μ g/ml, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL with the F albumen that carbonate buffer solution cuts back to close enzyme, dilute 4 extent of dilution, each extent of dilution repeats 2 holes, 100 μ L/ holes, and 4 ℃ of coated elisa plates spend the night.Feminine gender and the positive serum of chicken are made respectively 1:100,1:300,1:500 doubling dilution, and each extent of dilution repeats 2 holes, and 100 μ L/ holes form the coated concentration of the best and primary antibodie thinning ratio row that square formation is determined recombinant protein.Behind the sealing 1h, each extent of dilution repeats 2 holes, 100 μ L/ holes, and each goes on foot 37 ℃ of reaction 30min, stops behind 37 ℃ of colour developing 5min, measures each hole OD in microplate reader _450nmValue.As can be seen from Table 3, antigen coated amount is the 1000ng/ hole, and the serum weaker concn is considered as best results when being 1:100, (as calculated, when the coated concentration of albumen be the 1000ng/ hole, when serum dilution is 1:100, OD _450nmRecord absorbancy P/N value maximum)

Determining of the antigen coated concentration of table 3 and serum dilution

2) selection of best coated condition

With the antigen coated concentration coated elisa plate of the best, each coated concentration repeats 2 holes, respectively with 4 ° of C spend the night, behind 37 ° of C effect 1h again 4 ° of C spend the night coatedly, measure each hole OD _450nmValue is calculated the P/N value and is selected best coated condition.As can be seen from Table 4, behind 37 ° of C1h 4 ° of C to spend the night be the coated condition of the best.

The selection of the coated condition of table 4

3) selection of confining liquid and off-period

With the suitableeest antigen coated concentration coated elisa plate, with the coated condition envelope antigen of the best, test with following three kinds of confining liquids respectively: 5% skimmed milk, 5% calf serum, 1%BSA, each confining liquid repeats 2 holes; With following 3 conditions sealing, test: 37 ° of C30min, 37 ° of C1h, 37 ° of C1.5h, 37 ° of C2h, each sealing condition repeats 2 holes respectively.Survey each hole OD ₄₅₀Value is calculated the P/N value.As can be seen from Table 5, the 2h5% skimmed milk is top condition.

Determining of table 5 sealing condition

4) two anti-optimum dilution degrees and the selection in reaction times

With the coated condition of the best, by the suitableeest antigen coated concentration coated elisa plate, use best confining liquid under best sealing condition, to seal enzyme plate, two anti-ly press respectively 1:10000,1:20000,1:30000,1:40000,1:50000 dilution, 37 ° of C are respectively 30min, 1h, 1.5h and 2h lower action time, each two anti-condition repeats 2 holes, records each hole OD _450nmValue is calculated the P/N value.As can be seen from Table 6,1:4000030min is best two anti-activity and the times.

Table 6 two anti-concentration and the selection of two anti-action times

5) selection of substrate developing time

With the coated condition of the best, by the suitableeest antigen coated concentration coated elisa plate, under best confining liquid and sealing condition, respectively at color development at room temperature 10min, 15min, 20min, 25min.Record each hole OD _450nmValue is calculated the P/N value, can find out according to table 7, and the P/N value was larger when developing time was 15min, and positive the value about 1.0, and negative value is top condition about 0.1, be best substrate developing time therefore select colour developing 15min.

Determining of table 7 substrate developing time

6) the Cut-off value determines

Select 20 parts of negative chicken serums, under the suitableeest antigen coated concentration and serum dilution, carry out the ELISA reaction under above definite optimum reaction condition, the result is as shown in table 8:

The measurement result of table 820 part negative chicken serum

Serum	1	2	3	4	5	6	7	8	9	10
											OD 450	0.157	0.172	0.177	0.18	0.202	0.207	0.179	0.157	0.172	0.18
Serum	11	12	13	14	15	16	17	18	19	20
											OD 450	0.177	0.173	0.158	0.182	0.108	0.118	0.179	0.142	0.121	0.137

The mean value X=0.1639 of 20 parts of serum, standard deviation SD=0.026052, according to formula, positive cut-off value is 0.2419(Cp=X+3SD), the cut-off value of feminine gender is 0.2159(Cn=X+2SD).With formula S/P=(S-N)/(P-N) come result of determination, if S/P 〉=Cp, serum to be checked is positive; If S/P＜Cn, serum to be checked is negative; If Cp＞S/P 〉=Cn is judged to suspiciously, need redeterminate.(S represents the OD450 value of sample to be checked; P represents the OD of positive control ₄₅₀Value; N represents the OD of negative control ₄₅₀Value).

7) repeated experiment between plate

Get the coated enzyme plate of 4 different batches, detect 4 parts of known positive serums, every block of plate of each sample arranges 2 repetitions, calculates interassay coefficient of variation CV=(SD/OD450nm mean value) * 100%, the results are shown in Table 9:

Determining of table 9 interassay coefficient of variation

OD450nm mean value=0.963969, SD=0.07474 calculates interassay coefficient of variation CV=(SD/OD450nm mean value according to formula) * 100%=7.75%, CV＜10%, illustrate that the degree of variation of same serum sample between different plates is very little, have good stability.

8) repeated experiment in the plate

Repeat in the same enzyme plate is criticized, detect 4 parts of known positive serums, each sample arranges 4 repetitions, calculates variation within batch coefficient CV=(SD/OD450nm mean value) * 100%, the results are shown in Table 10:

Determining of table 10 variation within batch coefficient

The chicken serum numbering	Positive value (P)	The chicken serum numbering	Positive value (P)	N
					A1	0.947	B1	0.947	0.13
A2	0.958	B2	0.998	0.167
					A3	0.944	B3	0.924	0.145
A4	0.950	B4	1.021	0.129
					C1	1.097	D1	0.896	0.121
C2	1.102	D2	0.898	0.161
					C3	1.052	D3	0.894	0.17
C4	1.087	D4	0.890	0.168

OD450nm mean value=0.975313, SD=0.072513 calculates interassay coefficient of variation CV=(SD/OD450nm mean value according to formula) * 100%=7.43%, CV＜10%, illustrate that the degree of variation of different serum samples in same plate is very little, have good repeatability.

9) specificity of indirect ELISA method experiment

Utilize the indirect ELISA method of setting up, detect positive serum, control group adopts AEV/ND/REO/IBD/IB serum and negative serum, positive serum OD _450nmNear 1.0, negative serum OD _450nmAt 0.1-0.2, control group serum OD _450nmAt 0.1-0.2, prove that the indirect ELISA method specificity of setting up with this albumen is better, the results are shown in Table 11:

The specificity of table 11 indirect ELISA method

10) sensitivity experiments of indirect ELISA method

One antiserum(antisera) is diluted in the ratio of 1:200,1:400,1:600,1:800,1:1000, to determine its susceptibility.Still can detect the positive when data results is presented at Dilution ratio and reaches 1:1000, prove that the susceptibility of this method is fine, the results are shown in Table 12:

The susceptibility of table 12 indirect ELISA method

Dilution ratio	Positive serum	Negative serum
			1:200	1.45	0.203
1:400	1.278	0.167
			1:600	0.969	0.142
1:800	0.464	0.107
			1:1000	0.33	0.096

11) comparative experiments of indirect ELISA method

With ELISA method and the American I DEXX aMPV of the company antibody assay kit (ELISA) set up 164 parts of clinical censorship serum are carried out Parallel testing, the result shows, the positive coincidence rate of the two is 96.77% (120/124), negative match-rate is 92.5% (37/40), total coincidence rate reaches 95.73% (157/164), the results are shown in Table 13 to table 16, the result shows that the indirect ELISA method of foundation has higher specificity and susceptibility.

The ELISA detected result of 40 parts of serum of table 13 Nanning sampling

The ELISA detected result of the 46 parts of chicken serums in Daxing, table 14 Beijing

The ELISA detected result of 40 parts of serum of table 15 Pinggu County, beijing

The ELISA detected result of the 38 parts of serum in Haidian, table 16 Beijing

The assembling of embodiment 4 fowl metapneumovirus antibody ELISA detection kits

1.ELISA test kit assembling

⑴ 96 hole enzyme plates: the monomeric protein that is coated with F albumen;

⑵ standard positive control: infect fowl metapneumovirus duck positive serum;

⑶ standard negative control: a normal health duck serum;

⑷ the goat-anti chicken monoclonal antibody of horseradish peroxidase-labeled;

⑸ coating buffer: anhydrous Na ₂CO ₃1.59g, NaHCO ₃2.93g adding distil water is to 1000ml;

⑹ washings: NaCl8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O2.9g, KCl0.2g, Tween200.5mL adds distilled water to 1000mL;

(7) substrate solution A: urea peroxide 1.0g, Na ₂HPO ₄12H ₂O35.8g, citric acid H ₂O10.3g, Tween-20100 μ L adds 1000mL distilled water;

(8) substrate solution B:TMB700mg, DMSO40mL, fully dissolving, adding distil water 960mL then, citric acid H ₂O10.3g;

(9) confining liquid: 5% skim-milk;

(10) diluent: the washings that contains 5% skim-milk; ⑼ stop buffer: 2mol/L sulphuric acid soln.

2. the preparation of enzyme plate

With carbonate (pH9.6, concentration 0.5mol/L) damping fluid is made coating buffer, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use again.

The mensuration program of embodiment 5A hypotype fowl metapneumovirus antibody ELISA detection kit

1. reagent is prepared and diluted sample

1) reagent need not preparation in the test kit of the present invention, can directly use;

2) dilution of sample to be checked and standard reference material: the standard positive, the standard negative reference substance that provide in serum sample to be detected and the test kit are used afterwards with 10 times of sample diluting liquid dilutions.

2. determination step

1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 60min;

2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 60min;

4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15min;

6) add stop buffer: add stop buffer 50 μ L in every hole;

7) measure: use microplate reader at OD _450nmThe place measures the absorption photometric value (OD in every hole _450nmValue).

3. the result judges

Calculate mean number (X) and the standard deviation (SD) of its absorbance, then positive Cut-off value Cp=X+3SD; Negative Cut-off value Cn=X+2SD.Using formula S/P=(S – N)/(P – N) comes result of determination (S: the OD of sample to be checked _450nmValue; P: the OD of positive control _450nmValue; N: the OD of negative control _450nmValue).If S/P 〉=Cp, serum to be checked is positive; If S/P＜Cn, serum to be checked is negative; If Cp＞S/P 〉=Cn is judged to suspiciously, need redeterminate.(S represents the OD of sample to be checked _450nmValue; P represents the OD of positive control _450nmValue; N represents the OD of negative control _450nmValue).

Claims (12)

1.A subgroup fowl metapneumovirus F albumen, its aminoacid sequence is shown in SEQ ID No.2.

2. the gene of coding claim 1 described F albumen.

3. gene according to claim 2, its nucleotide sequence is shown in SEQ ID No.1.

4. the ELISA detection reagent that contains the described A subgroup of claim 1 fowl metapneumovirus F albumen.

5. A subgroup fowl metapneumovirus antibody ELISA detection kit, it comprises the elisa plate of coated A subgroup fowl metapneumovirus F albumen claimed in claim 1.

6. test kit according to claim 5 is characterized in that, also comprises in ELIAS secondary antibody, sample diluting liquid, concentrated cleaning solution, substrate solution A, substrate solution B, stop buffer, positive control and the negative control one or more.

7. ELISA detection kit according to claim 6 is characterized in that, described ELIAS secondary antibody is horseradish peroxidase-labeled goat-anti chicken monoclonal antibody; Described positive control is the chicken serum of natural infection A subgroup avian pneumovirus; Described negative control is normal chicken serum; Contain NaCl8.0g, KH in every liter of the described washings ₂PO ₄0.2g, Na2HPO ₄12H ₂O2.9g, KCl0.2g, Tween-200.5mL; Described sample diluting liquid is the washings that contains 5% skim-milk; Contain urea peroxide 1.0g, Na among every liter of the described substrate solution A ₂HPO ₄12H ₂O35.8g, citric acid H ₂O10.3g, Tween-20100 μ L; Contain TMB700mg, DMSO40mL, citric acid H among every liter of the substrate solution B ₂O10.3g; Described stop buffer is the 2mol/L sulphuric acid soln.

8. prepare the method for the described A subgroup of claim 1 fowl metapneumovirus F albumen, it may further comprise the steps:

Synthetic A subgroup fowl metapneumovirus F protein gene sequence is that template amplification obtains the sequence shown in the SEQ ID No.1, the above-mentioned purpose gene is imported in the pBCX carrier, product after the connection transforms DH5 α competent cell, evaluation and screening goes out positive recombinant expression plasmid pBCX-aMPV-A-F after extracting the plasmid double digestion, with abduction delivering behind the above-mentioned positive recombinant expression plasmid conversion BL21 competent cell, SDS-PAGE electrophoretic analysis expressing protein, adopt again Ni post affinity chromatography method, target protein is carried out purifying, then detect the protein purification result with SDS-PAGE and Western blot respectively, target protein behind the purifying is processed with enteropeptidase, cut the result with SDS-PAGE check enzyme after processing, cutting glue with the KCl staining after electrophoresis finishes reclaims, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify, elisa assay is cut the antigenicity that glue reclaims albumen.

9. the preparation method of A subgroup fowl metapneumovirus F albumen according to claim 8, it may further comprise the steps:

1) take synthetic A subgroup fowl metapneumovirus F protein gene sequence as template, by PCR method its F gene is increased, clones and obtain goal gene;

The upstream and downstream primer of amplification F gene is respectively:

Upstream primer: 5 '-GG GGTACCGAGGCAGTATCCACATTAGGG-3 '

Downstream primer: 5 '-CCC AAGCTTCTTGGCATCTGCACCTAG-3 ';

2) simultaneously goal gene and pBCX carrier being carried out enzyme with Kpn I, Hind III cuts, reclaim the purpose fragment, 16 ° of C connections are spent the night, and transform DH5 α competent cell, extract plasmid, after Kpn I and the evaluation correctly of Hind III double digestion, obtain positive recombinant expression plasmid pBCX-aMPV-A-F;

3) then get plasmid pBCX-aMPV-A-F and transform the BL21 competent cell, the positive plasmid bacterium that obtains is cultivated in 37 ° of C, treat that the A600 value reaches at 0.4～0.6 o'clock, adding IPTG is that 1.2mmol/L carries out abduction delivering to final concentration, the thalline of 4h behind the collection abduction delivering, ultrasonic disruption is got precipitation after centrifugal and is carried out the SDS-PAGE electrophoretic analysis;

4) then adopt Ni post affinity chromatography method, the purifying target protein, target protein behind the purifying carries out SDS-PAGE and Westernblot identifies, process with enteropeptidase through the F albumen of identifying, then carry out the SDS-PAGE electrophoresis, cut glue with the KCl staining after electrophoresis finishes and reclaim, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify;

5) elisa assay is cut the antigenicity that glue reclaims albumen.

10. the preparation method of each described test kit of claim 5～7, it comprises the elisa plate that adopts the coated A subgroup fowl metapneumovirus F albumen claimed in claim 1 of following steps preparation: make coating buffer with carbonate buffer solution, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, again with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use.

11. the application of the described ELISA detection kit of claim 5～7 any one on rapid detection A subgroup fowl metapneumovirus antibody.

12. application according to claim 11 is characterized in that, may further comprise the steps:

1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 30min;

2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 30min;

4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;

5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15～20min;

6) add stop buffer: add stop buffer 50 μ L in every hole;

7) measure: the absorption photometric value of measuring every hole with microplate reader at the 450nm place.

Images