CN103360472B - Acian Metapneumovirus antibody ELISA detection kit - Google Patents

Abstract

The invention provides an ELISA detection kit for avian metapneumovirus antibody, which is an ELISA kit for detecting avian metapneumovirus antibody based on the monomer protein of A subgroup avian metapneumovirus F protein as a coating antigen. The present invention also provides a method for preparing the monomeric protein of F protein, which is the coating antigen of the above kit, the F protein prepared by the method provided by the present invention has good reactogenicity, and the ELISA established by the present invention The detection kit provides an effective method for the early diagnosis and detection of avian metapneumovirus infection, and has played a significant role in promoting the prevention research of the disease.

Description

Avian metapneumovirus antibody ELISA detection kit

technical field

The invention relates to the technical field of immunology, in particular to a monomeric protein of F protein of subgroup A avian metapneumovirus and an ELISA kit for detecting the antibody of subgroup A avian metapneumovirus.

Background technique

Avian Metapneumovirus (aMPV), also known as Turkey Rhinotracheitis Virus (TRTV), belongs to the Pneumovirus genus of the Paramyxoviridae Pneumoviridae subfamily, and is an important species of poultry distributed worldwide. pathogen. This virus mainly causes upper respiratory diseases of turkeys and chickens, such as turkey rhinotracheitis (TRT), avian rhinotracheitis (Avian Rhinotracheitis, ART), and is associated with broiler chicken swollen head syndrome (Swollen Head Syndrome, SHS) ) are closely related. Among them, turkey rhinotracheitis is an acute and highly contagious infectious disease that endangers the turkey farming industry. The main symptoms of the disease are runny nose, sneezing, swelling of the infraorbital sinus, swelling of the head, and decreased egg production rate. , The infection rate and morbidity rate can be as high as 100%, and the mortality rate varies with feeding management and sanitary conditions. If there is mixed infection, the mortality rate can be as high as 25% to 40%. The disease is highly contagious and spreads rapidly, which can cause a variety of poultry infections and cause severe economic losses. At present, my country is the largest chicken-raising country in the world, with 9.6 billion chickens. The mortality rate due to various poultry diseases is as high as 20% to 25% every year, and tens of billions of yuan are lost.

Currently, serology is the most commonly used method for aMPV diagnosis, especially in unvaccinated flocks. Commonly used methods are indirect enzyme-linked immunosorbent assay, virus neutralization reaction, indirect immunofluorescence and immunodiffusion. Among them, ELISA is the most commonly used method, which has the characteristics of rapidity, sensitivity, less serum consumption and easy operation. The commercial aMPV ELISA detection kits on the market are from Europe and the United States, and are mainly used for the detection of serum antibodies. The antigen coated is the whole virus extracted after the virus is inoculated into cells, which often brings potential harm to the spread of the disease; In addition, these detection reagents are not sensitive enough to detect antibodies of specific subgroups such as subgroup A; especially, there is still no development of ELISA detection kits using expressed recombinant proteins as coating antigens. aMPV contains a variety of viral structural proteins, among which F protein is the main structural protein of the virus, which contains neutralizing epitopes that can protect the body against aMPV infection, but the F protein contains multiple transmembrane regions that affect the expression of the protein in the system recombinant expression. How to express the recombinant protein containing neutralizing antigenic epitope as the coating antigen and establish the ELISA method to develop the antibody detection kit for the detection of the disease has practical significance. At present, avian metapneumovirus infection occurs and is prevalent in various poultry farms in my country. Therefore, the development of specific, sensitive and simple diagnostic kits is an urgent need for the prevention and control of this disease in my country.

Contents of the invention

In order to overcome the defects of the prior art, an object of the present invention is to prepare an ELISA kit for detecting the avian metapneumovirus antibody, which provides a set of effective tools for the detection of the avian metapneumovirus antibody.

Another object of the present invention is to provide a method for preparing and purifying monomeric protein of A-subtype avian metapneumovirus F protein, so as to provide coating antigen for the avian metapneumovirus antibody detection kit.

Another object of the present invention is the application of the above ELISA kit in rapid detection of avian metapneumovirus.

To achieve the above object, the present invention firstly provides a kind of A subtype avian metapneumovirus F protein, which is obtained by removing part of the transmembrane region of the A subtype avian metapneumovirus F protein wild type, Its amino acid sequence is shown in SEQ ID No.2. The present invention also includes the gene of coding sequence, its nucleotide sequence is as shown in SEQ ID No.1. The study found that the protein has good immunogenicity and can be used for antibody detection of aMPV.

Accordingly, the present invention provides an ELISA detection kit for avian metapneumovirus antibody, which comprises an ELISA plate coated with a type A avian metapneumovirus F protein monomer protein.

The above ELISA kit for detecting avian metapneumovirus also includes an enzyme-labeled secondary antibody, a positive control, a negative control, a washing solution, a sample diluent, a substrate solution A, a substrate solution B and a stop solution.

Wherein, the enzyme-labeled secondary antibody is horseradish peroxide-labeled goat anti-chicken monoclonal antibody; the positive control is chicken serum naturally infected with subgroup A avian pneumovirus; the negative control is normal chicken serum; The components and proportions of coating solution, washing solution, blocking solution, sample diluent, substrate solution A, substrate solution B and stop solution are as follows:

Coating solution: anhydrous Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, add distilled water to 1000mL;

Washing liquid: NaCl8.0g, KH ₂ PO ₄ 0.2g, Na ₂ HPO ₄ 12H ₂ O2.9g, KCl0.2g, Tween-200.5mL, add double distilled water to 1000mL;

Blocking solution: 5% skimmed milk powder;

Sample diluent: washing solution containing 5% skimmed milk powder;

Substrate solution A: carbamide peroxide 1.0g, Na ₂ HPO ₄ 12H ₂ O 35.8g, citric acid H ₂ O 10.3g, Tween-20 100μL, add 1000mL distilled water;

Substrate solution B: TMB700mg, DMSO40mL, fully dissolved, then add distilled water 960mL, citric acid· _H2O10.3g ;

Stop solution: 2mol/L sulfuric acid solution.

The above-mentioned monomeric protein preparation of the A-type avian metapneumovirus F protein used in the ELISA kit as the coating antigen of the enzyme plate comprises the following steps: artificially synthesizing the A subgroup avian metapneumovirus F protein gene sequence as a template to amplify The sequence shown in SEQ ID No.1, the above-mentioned target gene was introduced into the pBCX vector, the product after ligation was transformed into DH5α competent cells, the plasmid was extracted and double-digested, and the positive recombinant expression plasmid pBCX-aMPV-A-F was identified and screened. The above positive recombinant expression plasmids were transformed into BL21 competent cells to induce expression, analyzed the expressed protein by SDS-PAGE electrophoresis, and then purified the target protein by Ni column affinity chromatography, and then detected the protein purification by SDS-PAGE and Western blot respectively As a result, the purified target protein was treated with enterokinase, and after the treatment, the digestion result was checked by SDS-PAGE. After electrophoresis, the gel was cut and recovered by KCl staining, and the recovery effect of the recovered F protein was identified by SDS-PAGE electrophoresis. The antigenicity of the recovered protein was analyzed by ELISA.

Specifically, the monomeric protein preparation of type A avian metapneumovirus F protein may comprise the following steps:

1) Amplify the target gene. Using artificially synthesized subgroup A avian metapneumovirus F protein gene sequence as a template, its F gene was amplified and cloned by PCR to obtain the target gene (F gene, which lacked part of the transmembrane region), and the upstream primer plus Kpn Ⅰ Restriction sites, downstream primers plus Hind III restriction sites, primers are listed in Table 1:

Table 1F Gene Amplification Primers

2) After the recovery of the F gene and the extraction of the PBCX plasmid, perform double enzyme digestion with Kpn Ⅰ and Hind Ⅲ to recover the target fragment, and under the action of T4 DNA ligase, the target gene and the vector were ligated at a molar ratio of 5:1 at 16°C for 12- At 16 hours, the competent cell DH5α was transformed, and the plasmid was extracted, and the positive recombinant expression plasmid pBCX-aMPV-A-F was obtained after Kpn Ⅰ and Hind Ⅲ double enzyme digestion and identification.

3) Then take the plasmid pBCX-aMPV-A-F to transform BL21 competent cells, and culture the obtained positive plasmid bacteria at 37°C. When the A600 value reaches 0.4-0.6, add IPTG to a final concentration of 1.2mmol/L to induce expression. The bacterial cells 4 hours after the induced expression were collected, ultrasonically disrupted, and the precipitate was collected after centrifugation for SDS-PAGE electrophoresis analysis.

4) Then use Ni column affinity chromatography to purify the target protein. The purified target protein is identified by SDS-PAGE and Western blot. The identified F protein is treated with enterokinase, and then SDS-PAGE electrophoresis is performed, and the electrophoresis ends Finally, the gel was removed, washed with double distilled water, and then immersed in 4°C pre-cooled 250mmol/L KCl solution for 5min to develop color, and the band of the target protein stained silvery white was cut off, washed 3 times with PBS, Move the strip into a clean pouch and crush it, scrape the powder into an EP tube, add PBS to shake and mix, freeze and thaw 3 times, centrifuge at 12000r/min for 2min, absorb the supernatant, and identify the recovery effect by SDS-PAGE electrophoresis .

5) ELISA was used to analyze the antigenicity of the protein recovered from gel cutting.

The present invention also provides the preparation method of the above kit, including the preparation of the ELISA plate of A subgroup avian metapneumovirus F protein. The method is: use carbonate (pH9.6, concentration 0.5mol/L) buffer solution as Coating solution, use the above coating solution to dilute the F protein monomer to 10 μg/mL, add 100 μL/well into the ELISA reaction plate, block at 37°C for 1 hour, coat at 4°C overnight, pat dry, and then use 5 % skimmed milk was sealed at 37°C for 2 hours, washed with PBST containing 0.05% Tween, patted dry, sealed with aluminum film and stored in vacuum, and used for later use.

Based on the kit of the present invention, the following ELISA method can be used to detect the A subgroup avian metapneumovirus antibody:

1) Adding samples: Add 100 μL of the serum sample to be tested diluted 1:100 times with the sample diluent to the microwell of the microplate, and incubate at 37°C for 30 minutes;

2) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, wash 3 times and pat dry;

3) Add enzyme-labeled antibody: add 100 μL of enzyme-labeled antibody working solution to each well, and incubate at 37°C for 30 minutes;

4) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, wash 3 times and pat dry;

5) Add substrate solution: first add 50 μL of substrate solution A to each well, then add 50 μL of substrate solution B, and develop color in the dark for 15-20 minutes;

6) Add stop solution: add 50 μL of stop solution to each well;

7) Determination: Measure the absorbance value (OD450 value) of each well at 450nm with a microplate reader.

The present invention provides the use of soluble pBCX vectors to express the F protein of the transmembrane region missing from subgroup A avian metapneumovirus, and use the 6×His tag of the pBCX vector to purify the expressed fusion protein, which is proved by Western-blot detection The purified protein has reactogenicity, and then the purified protein is cut with enterokinase to obtain a monomeric protein with the carrier tag removed, and a protein with higher immunogenicity is obtained. The protein construction system provided by the present invention solves the problem of difficult purification of F protein monomers.

The present invention is prepared on the basis of the F protein expressed by genetic engineering. The F protein monomer is a non-whole virus antigen, which has good safety, does not contain irrelevant miscellaneous proteins, and has good antigenicity. Therefore, the invented detection kit has great High specificity and sensitivity.

The kit for the detection of subgroup A avian metapneumovirus constructed by the present invention is simple and easy to operate, fast and sensitive in detection, and is especially suitable for the rapid detection of antibodies to avian metapneumovirus clinically, and has played a role in the prevention and research of this disease. significant promotion.

Description of drawings

Figure 1 is the electropherogram of F gene amplification, in which 1: the target band of F protein, 2: blank control, M: MarkerDL2000.

Fig. 2 is the PCR identification diagram of the recombinant plasmid obtained by cloning the F gene into the pBCX vector, wherein M: DNAMarkerDL2000, 1-10: PCR amplified fragments of the recombinant plasmid.

Fig. 3 is the enzyme digestion identification diagram of the recombinant plasmid pBCX-F obtained by cloning the F gene into the pBCX vector, wherein M: DNAMarkerDL2000, 1-3: restriction enzyme fragments of the positive recombinant plasmid pBCX-F.

Figure 4 is a PCR identification map of the extracted plasmid after pBCX-aMPV-A-F was transformed into BL21 competent cells, where M: DNAMarkerDL2000, 1: PCR product of recombinant plasmid, 3: blank control.

Fig. 5 is the SDS-PAGE analysis diagram of the induced expression protein, wherein M: 175 Marker, 1: empty plasmid expression product, 2-8 are bacterial lysates induced by 1.2mmol/LIPTG for 0-6h respectively.

Figure 6 is a diagram of the soluble analysis and identification of the fusion protein, where M: 175Marker, 1: total bacterial lysate product, 2: bacterial lysate precipitate; 3: bacterial lysate supernatant.

Figure 7 is the SDS-PAGE analysis diagram of the purified protein, where M: 175 Marker, 1-4: the 1st to 4th tube products after purification and elution.

Figure 8 is the Western-blot analysis diagram of the purified protein, (a) M: 175Marker, 1: the purified fusion protein product; (b) M: 175Marker, 1, 2, 3: the purified fusion protein product .

Figure 9 is the identification result of the target protein recovered by KCl staining and gel cutting after enzyme digestion, where M: 175Marker, 1, 2: are the two tube products after enzyme digestion and recovery.

Detailed ways

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. On the premise of not departing from the spirit and essence of the present invention, all modifications or replacements made to the present invention belong to the scope of the present invention.

Preparation and purification of the protein monomer of embodiment 1A subgroup avian metapneumovirus F protein

Artificially synthesize the gene sequence of F protein of subgroup A avian metapneumovirus shown in SEQ ID NO.1, design a pair of specific primers with Oligo6.0 software, add KpnⅠ restriction site to the upstream primer, and add HindⅢ enzyme to the downstream primer Cutting site, the upstream primer is: 5'-GG GGTACC GAGGCAGTATCCACATTAGGG-3', the downstream primer is: 5'-CCC AAGCTT CTTGGCATCTGCACCTAG-3'. Using the pcmy-myc-L plasmid (purchased from Invitrogen) as a template, amplify in a 20 μL reaction system (ddH ₂ O 11.5 μL, 5×Buffer 4 μL, Taq enzyme 0.2 μL, dNTP 2 μL, upstream primer 1 μL, downstream primer 1 μL, DNA Template 0.3 μL). The amplification conditions are: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 59°C annealing for 45 seconds, 72°C extension for 1.5 minutes, a total of 35 cycles; 72°C extension for 10 minutes; 4°C Save (see Figure 1 for the amplification results).

Take 100 μL of PCR products for agarose gel electrophoresis, 100V electrophoresis for 35 to 40 minutes, detect the product analysis results with ultraviolet light, and perform gel cutting and recovery. After double digestion with Kpn Ⅰ and Hind Ⅲ, the target fragment was ligated with the plasmid after double digestion with Kpn Ⅰ and Hind Ⅲ at a molar ratio of 5:1 at 16°C for 12-16 hours, transformed into DH5α, and cultured at 37°C overnight. Select colonies to extract plasmids, use specific primers for PCR amplification, digest with the above-mentioned endonucleases, and identify by electrophoresis (results shown in Figure 2). The correctly identified positive colonies were further verified by plasmid sequencing, and their gene sequences were confirmed to be correct, and the recombinant expression plasmids with correct sequencing were named pBCX-aMPV-A-F (results shown in Figure 3).

Extract plasmids from colonies with correct sequencing to transform BL21 competent cells to obtain recombinant bacteria (results shown in Figure 4), culture recombinant bacteria in LB liquid medium, and wait for the value of the bacterial solution at 600nm optical density (OD) When it reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside (IPTG) to make the final concentration 1.2mmol/L to induce expression. The bacteria expressed at different induction times were collected, ultrasonically disrupted, and after centrifugation, the supernatant and precipitate were taken for SDS-PAGE electrophoresis. The results showed that the expression reached the maximum after 4 hours of induction (results shown in Figure 5) and most of the fused target protein was derived from expressed in soluble form (results shown in Figure 6).

After the fusion protein is confirmed to be secreted expression, the bacterial lysate of the fusion protein is directly purified with a Ni-NTA purification column to purify the target protein, and the purification column is eluted with an eluent, and the eluate is collected, which contains the target fusion protein , removing part of the F protein in the transmembrane region plus the tagged protein on the carrier, the size of the fused protein is 74kDa, and the concentration of the protein in the eluate is measured with a UV spectrophotometer, the highest one is 27mg/mL (results shown in Fig. 7).

After SDS-PAGE electrophoresis, Western-blot was used to analyze the reactogenicity of the purified fusion protein. The primary antibody was a monoclonal antibody to subtype A aMPV F protein, and the secondary antibody was HRP-labeled goat anti-mouse IgG. The test results showed: There is a darker target band at 74kDa, which indicates that the recombinant fusion protein is reactogenic, and the size of the target band is consistent with the size and position of the protein expressed by SDS-PAGE, and the band is single. WB of the purified protein Comparing the detection results with the WB detection results of the protein before purification can prove that the fusion protein was successfully purified (results shown in Figure 8).

The F protein was treated with enterokinase, and then SDS-PAGE electrophoresis was performed. After the electrophoresis, the gel was removed, washed with double distilled water, and then immersed in 4°C pre-cooled 250mmol/L KCL solution for 5min to develop color. Cut off the band of the target protein stained silvery white, wash with PBS 3 times, transfer the strip into a clean pouch and grind it, scrape the minced powder into an EP tube, add PBS to shake and mix, freeze and thaw 3 times repeatedly, 12000r Centrifuge for 2 min at 1/min, draw the supernatant, and conduct SDS-PAGE electrophoresis to identify the recovery effect (results shown in Figure 9).

Example 2 Identification of the immunogenicity of the F protein monomer constructed by the present invention

Dilute the recovered F protein and undigested F protein with pH9.6 0.5mol/L carbonate buffer solution to 10 μg/mL and coat a 96-well plate, and infect chicken positive serum and Negative sera were primary antibodies that were serially diluted in 3 gradients, 1:100, 1:300, and 1:500, respectively. HRP-labeled goat anti-chicken IgG was used as the secondary antibody, and the working concentration was 1:10000; two wells were prepared in parallel for each sample, and after color development by TMB, the OD _450nm value of each well was read, and the results showed that: without enterokinase digestion And the protein with the carrier tag is used as the antigen coated on the ELISA plate. When the serum is detected, the OD value of the negative serum is between 0.6-0.8, which is close to the OD value of the positive serum; after the enterokinase enzyme digestion treatment, only the monomer remains The protein is used as the coating antigen of the ELISA plate. When testing the serum, the negative OD value is between 0.1-0.2, and the P/N value between the positive OD value and the negative OD value is between 3.6-4.45. It can be seen that the ELISA result is good, and the enzyme digestion recovery The monomeric protein has good reactogenicity, and the test results are shown in Table 2:

Table 2 ELISA detection of antigenicity of KCl staining gel cutting purified protein

The establishment of embodiment 3 avian metapneumovirus ELISA detection method

1. Determination of the optimal reaction conditions of ELISA detection kit for subgroup A avian metapneumovirus antibody

1) Determination of the optimal coating concentration of the antigen and the optimal working concentration of the enzyme-labeled monoclonal antibody

The purified F monomeric protein was used as the coating antigen, and the HRP-labeled goat anti-chicken monoclonal antibody was used as the second antibody, and the square array titration test was carried out on the enzyme plate. Dilute the F protein recovered by enzyme digestion with carbonate buffer to 15 μg/ml, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, respectively, and dilute 4 dilutions, each dilution is repeated for 2 wells, 100 μL/well, Coat the plate overnight at 4°C. The negative and positive sera of the chicken were diluted 1:100, 1:300, 1:500 respectively, and each dilution was repeated for 2 wells, 100 μL/well, to form a square matrix to determine the optimal coating concentration of the recombinant protein and a Antidilution ratio column. After blocking for 1 hour, repeat 2 wells for each dilution, 100 μL/well, react at 37°C for 30 minutes in each step, stop after 5 minutes of color development at 37°C, and measure the OD _450nm value of each well with a microplate reader. As can be seen from Table 3, the antigen coating amount is 1000ng/well, and the effect is considered to be the best when the serum dilution concentration is 1:100. At 100, the absorbance P/N value measured at OD _450nm is the largest)

Table 3 Determination of antigen coating concentration and serum dilution

2) Selection of the best coating conditions

Coat the ELISA plate with the optimal antigen coating concentration, repeat 2 wells for each coating concentration, treat at 4°C overnight, 37°C for 1 hour, and then coat at 4°C overnight, measure the OD _450nm value of each well, Calculate the P/N value to select the best coating condition. As can be seen from Table 4, overnight at 4°C after 1h at 37°C is the best coating condition.

Table 4 Selection of Coating Conditions

3) Selection of blocking solution and blocking time

Coat the ELISA plate with the optimal antigen coating concentration, and coat the antigen with the optimal coating condition, and conduct the test with the following three blocking solutions: 5% skimmed milk, 5% calf serum, 1% BSA, each Repeat the blocking solution for 2 wells; respectively seal with the following 3 conditions for testing: 37°C for 30min, 37°C for 1h, 37°C for 1.5h, and 37°C for 2h, repeat 2 wells for each blocking condition. Measure the OD ₄₅₀ value of each well and calculate the P/N value. As can be seen from Table 5, 2h5% skimmed milk is the best condition.

Table 5 Determination of closing conditions

4) Selection of the optimal dilution and reaction time of the secondary antibody

Coat the microtiter plate with the optimum coating condition and the optimum antigen coating concentration, use the optimum blocking solution to block the microtiter plate under the optimum blocking condition, and use the secondary antibodies at 1:10000, 1:20000, and 1:20000 respectively. 30000, 1:40000, 1:50000 dilution, the action time at 37°C is 30min, 1h, 1.5h and 2h respectively, each secondary antibody condition is repeated for 2 wells, the OD _450nm value of each well is measured, and the P/N value is calculated . It can be seen from Table 6 that 1:4000030min is the optimal concentration and time of secondary antibody action.

Table 6 Selection of secondary antibody concentration and secondary antibody action time

5) Selection of substrate color development time

Coat the microtiter plate with the optimal coating conditions and the optimal antigen coating concentration, and develop color at room temperature for 10 min, 15 min, 20 min, and 25 min under the optimal blocking solution and blocking conditions, respectively. Measure the OD _450nm value of each well and calculate the P/N value. According to Table 7, it can be seen that the P/N value is larger when the color development time is 15 minutes, and the positive value is about 1.0, and the negative value is about 0.1, which is the best. Conditions, so 15 minutes for color development was selected as the best substrate color development time.

Determination of the color development time of the substrate in table 7

6) Determination of Cut-off value

Select 20 parts of negative chicken serum, under the optimal antigen coating concentration and serum dilution, carry out ELISA reaction under the optimal reaction condition determined above, the result is as shown in table 8:

The assay result of table 820 negative chicken sera

serum 1 2 3 4 5 6 7 8 9 10 _OD450 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 _OD450 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 sera, standard deviation SD=0.026052, according to the formula, the positive cut-off value is 0.2419 (Cp=X+3SD), the negative cut-off value is 0.2159 (Cn=X+2SD) . Use the formula S/P=(SN)/(PN) to judge the result. If S/P≥Cp, the serum to be tested is positive; if S/P<Cn, the serum to be tested is negative; if Cp>S/P≥ Cn is judged as suspicious and needs to be re-measured. (S represents the OD450 value of the sample to be tested; P represents the _OD450 value of the positive control; N represents the _OD450 value of the negative control).

7) Inter-plate repeatability experiment

Take 4 coated ELISA plates from different batches, detect 4 copies of known positive sera, set 2 replicates per plate for each sample, and calculate the inter-assay coefficient of variation CV=(SD/OD450nm average value)×100%, The results are shown in Table 9:

Determination of coefficient of variation between batches of table 9

The average value of OD450nm=0.963969, SD=0.07474, and the coefficient of variation between batches was calculated according to the formula CV=(SD/OD450nm average value)×100%=7.75%, CV<10%, indicating the degree of variation of the same serum sample among different plates Small and has great stability.

8) In-plate repeatability experiment

Intra-batch repetitions were carried out on the same ELISA plate, 4 copies of known positive sera were detected, 4 replicates were set for each sample, and the intra-assay coefficient of variation CV=(SD/OD450nm average value)×100% was calculated. The results are shown in Table 10 :

Determination of coefficient of variation within table 10 batches

chicken serum number Positive value (P) chicken serum number 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

The average value of OD450nm=0.975313, SD=0.072513, according to the formula to calculate the coefficient of variation between batches CV=(SD/OD450nm average value)×100%=7.43%, CV<10%, indicating the degree of variation of different serum samples in the same plate Small and very repeatable.

9) Specificity experiment of indirect ELISA method

Using the established indirect ELISA method to detect positive serum, the control group uses AEV/ND/REO/IBD/IB serum and negative serum, the positive serum OD _450nm is close to 1.0, the negative serum OD _450nm is at 0.1-0.2, and the control group serum OD _450nm is at 0.1-0.2, it proves that the specificity of the indirect ELISA method established with this protein is better, the results are shown in Table 11:

The specificity of table 11 indirect ELISA method

10) Sensitivity experiment of indirect ELISA method

The primary antiserum was diluted at ratios of 1:200, 1:400, 1:600, 1:800, and 1:1000 to determine its sensitivity. The results of the data show that positive results can still be detected when the dilution ratio reaches 1:1000, which proves that the sensitivity of this method is very good. The results are shown in Table 12:

Table 12 Sensitivity of 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 experiment of indirect ELISA method

Using the established ELISA method and the American IDEXX company aMPV antibody detection kit (ELISA) to conduct parallel detection on 164 clinically submitted sera, the results showed that the positive coincidence rate of the two was 96.77% (120/124), and the negative coincidence rate was 96.77%. 92.5% (37/40), the total coincidence rate reached 95.73% (157/164), the results are shown in Table 13 to Table 16, the results show that the established indirect ELISA method has high specificity and sensitivity.

Table 13 ELISA detection results of 40 samples of serum in Nanning, Guangxi

Table 14 ELISA detection results of 46 chicken sera in Beijing Daxing

Table 15 ELISA detection results of 40 sera in Pinggu, Beijing

Table 16 ELISA detection results of 38 sera in Haidian, Beijing

The assembly of embodiment 4 avian metapneumovirus antibody ELISA detection kit

1. ELISA kit assembly

(1) 96-well ELISA plate: monomeric protein coated with F protein;

(2) Standard positive control: Positive serum of ducks infected with avian metapneumovirus;

(3) Standard negative control: a normal healthy duck serum;

(4) horseradish peroxidase-labeled goat anti-chicken monoclonal antibody;

(5) Coating solution: anhydrous Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, add distilled water to 1000ml;

(6) Washing liquid: NaCl8.0g, _KH2PO40.2g , _Na2HPO4 · _12H2O2.9g , _KCl0.2g , Tween200.5mL, add double distilled water to 1000mL _;

(7) Substrate solution A: carbamide peroxide 1.0g, Na ₂ HPO ₄ 12H ₂ O 35.8g, citric acid H ₂ O 10.3g, Tween-20 100μL, add 1000mL distilled water;

(8) Substrate solution B: TMB700mg, DMSO40mL, fully dissolved, then add distilled water 960mL, citric acid H ₂ O10.3g;

(9) Blocking solution: 5% skimmed milk powder;

(10) Diluent: Washing solution containing 5% skimmed milk powder; ⑼Terminator solution: 2mol/L sulfuric acid solution.

2. Preparation of ELISA plate

Use carbonate (pH9.6, concentration 0.5mol/L) buffer as the coating solution, dilute the F protein monomer to 10μg/mL with the above coating solution, add 100μL/well into the ELISA reaction plate, and keep at 37° C to block for 1 hour, 4°C to coat overnight, pat dry, then block with 5% skimmed milk at 37°C for 2 hours, wash with PBST containing 0.05% Tween, pat dry and store in vacuum with aluminum film for future use.

The assay procedure of embodiment 5A subtype avian metapneumovirus antibody ELISA detection kit

1. Reagent preparation and sample dilution

1) The reagents in the kit of the present invention do not need to be prepared and can be used directly;

2) Dilution of samples to be tested and standard control substances: Dilute the serum samples to be tested and the standard positive and standard negative control substances provided in the kit by 10 times with the sample diluent before use.

2. Measurement steps

1) Adding samples: Add 100 μL of the serum sample to be tested diluted 1:100 times with the sample diluent to the microwell of the microplate, and incubate at 37°C for 60 minutes;

2) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, wash 3 times and pat dry;

3) Add enzyme-labeled antibody: add 100 μL of enzyme-labeled antibody working solution to each well, and incubate at 37°C for 60 minutes;

4) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, wash 3 times and pat dry;

5) Add substrate solution: first add 50 μL of substrate solution A to each well, then add 50 μL of substrate solution B, and develop color in the dark for 15 minutes;

6) Add stop solution: add 50 μL of stop solution to each well;

7) Determination: Use a microplate reader to measure the absorbance value (OD _450nm value) of each well at OD _450nm .

3. Result judgment

Calculate the mean (X) and standard deviation (SD) of the absorbance values, then the positive Cut-off value Cp=X+3SD; the negative Cut-off value Cn=X+2SD. Use the formula S/P=(S–N)/(P–N) to determine the result (S: OD _450nm value of the sample to be tested; P: OD _450nm value of the positive control; N: OD _450nm value of the negative control). If S/P≥Cp, the serum to be tested is positive; if S/P<Cn, the serum to be tested is negative; if Cp>S/P≥Cn, it is suspicious and needs to be re-tested. (S represents the OD _450nm value of the sample to be tested; P represents the OD _450nm value of the positive control; N represents the OD _450nm value of the negative control).

Claims (10)

1. A subgroup avian metapneumovirus F protein, its amino acid sequence is as shown in SEQ ID No.2. 2. The gene encoding the F protein of claim 1. 3. the gene according to claim 2, its nucleotide sequence is as shown in SEQ ID No.1. 4. contain the ELISA detection reagent of A subgroup avian metapneumovirus F protein described in claim 1. 5. A subgroup A avian metapneumovirus antibody ELISA detection kit, which comprises an ELISA plate coated with the A subgroup avian metapneumovirus F protein according to claim 1. 6. The kit according to claim 5, further comprising enzyme-labeled secondary antibody, sample diluent, concentrated washing solution, substrate solution A, substrate solution B, stop solution, positive control and negative control one or more of. 7. The ELISA detection kit according to claim 6, wherein the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-chicken monoclonal antibody; the positive control is a natural infection of A subgroup poultry Chicken serum of lung virus; the negative control is normal chicken serum; the washing liquid contains 8.0g of NaCl, KH ₂ PO ₄ 0.2g, Na ₂ HPO ₄ 12H ₂ O 2.9g, KCl 0.2g, Tween20 per liter 0.5mL; the sample diluent is a washing solution containing 5% skimmed milk powder; each liter of the substrate solution A contains carbamide peroxide 1.0g, Na ₂ HPO ₄ 12H ₂ O 35.8g, citric acid H ₂ O 10.3g, Tween-20 100μL; each liter of substrate solution B contains TMB 700mg, DMSO 40mL, citric acid·H ₂ O 10.3g; the stop solution is 2mol/L sulfuric acid solution. 8. prepare the method for A subgroup avian metapneumovirus F protein described in claim 1, it may further comprise the steps: Artificially synthesized subgroup A avian metapneumovirus F protein gene sequence was amplified as a template to obtain the sequence shown in SEQ ID No.1, the above target gene was introduced into the pBCX vector, the ligated product was transformed into DH5α competent cells, and the plasmid was extracted After double enzyme digestion, the positive recombinant expression plasmid pBCX-aMPV-A-F was identified and screened out, and the above positive recombinant expression plasmid was transformed into BL21 competent cells to induce expression, and the expressed protein was analyzed by SDS-PAGE electrophoresis, and Ni column affinity chromatography was used. Purify the target protein, and then use SDS-PAGE and Western blot to detect the protein purification results respectively. The purified target protein is treated with enterokinase, and the digestion result is checked by SDS-PAGE after treatment. After electrophoresis, the gel is cut with KCl staining method For recovery, the recovery effect of the recovered F protein was identified by SDS-PAGE electrophoresis, and the antigenicity of the recovered protein was analyzed by ELISA. 9. the preparation method of A subgroup avian metapneumovirus F protein according to claim 8, it comprises the following steps 1) Taking the artificially synthesized A subgroup A avian metapneumovirus F protein gene sequence as a template, amplifying and cloning its F gene by PCR method to obtain the target gene; The upstream and downstream primers for amplifying the F gene are: Upstream primer: 5'-GG GGTACC GAGGCAGTATCCACATTAGGG-3' Downstream primer: 5'-CCC AAGCTT CTTGGCATCTGCACCTAG-3'; 2) Simultaneously digest the target gene and pBCX vector with Kpn Ⅰ and Hind Ⅲ, recover the target fragment, connect overnight at 16°C, transform DH5α competent cells, extract the plasmid, and obtain it after identification by Kpn Ⅰ and Hind Ⅲ double enzyme digestion Positive recombinant expression plasmid pBCX-aMPV-A-F; 3) Then take the plasmid pBCX-aMPV-A-F to transform BL21 competent cells, and cultivate the positive plasmid bacteria obtained at 37°C. When the A600 value reaches 0.4-0.6, add IPTG to a final concentration of 1.2mmol/L to induce expression, collect The bacterial cells 4h after induction of expression were disrupted by ultrasonic waves, and the precipitate was collected after centrifugation for SDS-PAGE electrophoresis analysis; 4) Then use the Ni column affinity chromatography method to purify the target protein, the purified target protein is identified by SDS-PAGE and Western blot, the identified F protein is treated with enterokinase, and then SDS-PAGE electrophoresis is performed, and the electrophoresis ends Afterwards, the KCl staining method was used to cut the gel for recovery, and the recovered F protein was subjected to SDS-PAGE electrophoresis to identify the recovery effect; 5) ELISA analysis of the antigenicity of the protein recovered from gel cutting. 10. The preparation method of the kit according to any one of claims 5 to 7, which comprises the following steps to prepare the ELISA plate coated with the A subgroup avian metapneumovirus F protein according to claim 1: using carbonate buffer As the coating solution, F protein monomer was diluted to 10 μg/mL with the above coating solution, added to the ELISA reaction plate at 100 μL/well, blocked for 1 hour at 37°C, coated overnight at 4°C, patted dry, and then used for 5 % skimmed milk was sealed at 37°C for 2 hours, washed with PBST containing 0.05% Tween, patted dry, sealed with aluminum film in vacuum and stored for later use.