Detailed Description
For a detailed description of the above objects and features of the present invention, the following description will further explain the embodiments of the present invention by referring to the figures of the specification, but the present invention is not limited to the following examples.
Example 1
1. Acquisition of p27 Gene and construction of expression vector
Clinical cases of goat local intranasal tumor are found from a goat farm, tumor tissues are obtained after dissection, proviral DNA is extracted, PCR amplification is carried out by comparing published ENTV-2 sequences, specific primers are designed in a conservation region, the amplified products are connected with a T carrier and then sequenced, and the goat local intranasal tumor proviral sequence is obtained through BLAST verification. On the basis, a specific primer is designed to amplify the p27 gene sequence, and the primers are as follows:
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The amplified product is cloned into a pET-30a vector after double enzyme digestion of EcoRI and XholI enzyme digestion sites, and the recombinant plasmid pET-30a-p27 is obtained after the plasmid is extracted after the sequencing is correct.
2. Expression and purification of p27 proteins
Recombinant plasmid pET-30a-p27 was transformed into BL21 (DE 3) competence, 0.5mM IPTG induced expression, nickel column purification, SDS-PAGE identified the protein. The method comprises the following specific steps:
1) Protein expression by inoculating recombinant expression bacteria into Carna-resistant LB culture medium at a volume ratio of 1:100, culturing at 37deg.C (or 25deg.C) at 160rpm until OD value is 0.6-0.8, and adding IPTG with final concentration of 0.5mM at 37deg.C (or 25deg.C) at 130 rpm for induction culture for 12 hr.
2) And (3) thallus breaking, namely collecting the induced bacterial liquid, centrifuging at 60000rpm for 5min, discarding the supernatant, re-suspending, washing and precipitating with 10mL of 1 XPBS, centrifuging again, and placing 40mL Binding buffer re-suspended thallus in an ice-water mixture for ultrasonic breaking. The ultrasonic treatment is stopped for 5s, and the ice bath ultrasonic treatment is performed for 10min. After the bacterial liquid is broken, collecting the whole bacterial liquid, and simultaneously centrifuging at 10000rpm and 4 ℃ for 10min, and respectively collecting the supernatant and the sediment.
3) The whole bacteria, supernatant and pellet were subjected to SDS-PAGE and Wester Blot identification, respectively, and the results are shown in FIGS. 1 and 2. From the results, it can be seen that the supernatant of the 25 ℃ induced recombinant expression bacterium was selected for the following protein purification work, since the content of p27 protein was high and the amount of foreign proteins was small.
4) And (3) protein purification, namely adding the supernatant collected in the step (2) into a balanced nickel column, suspending and combining for 2 hours at the temperature of 4 ℃, eluting the impurity protein by using 30mL of washing solution, eluting the target protein by using the eluting solution, and collecting the eluting solutions of the first, fourth, sixth and eighth times respectively.
5) The purified recombinant p27 protein was subjected to SDS-PAGE, and the results are shown in FIG. 3, wherein lanes 1, 2, 3 and 4 are the SDS-PAGE results of the first, fourth, sixth and eighth eluents, respectively.
3. Preparation of anti-p 27 protein monoclonal antibody
Animal immunization 8 week old Balb/c mice were immunized 100 μg each with purified p27 protein as immunogen (antigen) with 14d booster immunization. The antigen was emulsified with an equal volume of Freund's complete adjuvant at the time of the first immunization and with an equal volume of Freund's incomplete adjuvant at the time of the booster immunization. And after finishing the three-immunity for one week, the tail vein blood sampling is performed to detect the serum antibody titer, and the non-emulsified antigen is used for enhancing the immunity after the serum antibody titer meets the immunity requirement by intraperitoneal injection, and the fusion is performed after 72 hours.
Preparation of spleen cells of mice, namely, selecting a Balb/c mouse after booster immunization, removing eyeballs, taking blood, separating serum, soaking the mouse after death by 75% alcohol, fixing the mouse on a dissecting plate, cutting the abdomen of the mouse by using high-pressure scissors and tweezers, separating spleen and transferring the spleen to a plate, repeatedly blowing spleen by using a 10mL syringe to extract about 10mL of preheated RPMI-1640 culture medium, and enabling the spleen of the mouse to be in an off-white state after multiple times of spleen blowing, thus finishing spleen blowing. The culture solution containing the spleen cells was filtered through a cell sieve into a 50mL centrifuge tube, supplemented to 25mL with the remaining RPMI-1640 medium, and counted after mixing.
Cell fusion, namely resuscitating SP2/0 cells one week before fusion, selecting cells with uniform size and neat edges for fusion, and counting after resuspension of 25mL of RPMI-1640 medium of the SP2/0 cells. Mixing spleen cells and SP2/0 cells at a ratio of 8:1, centrifuging at 1000rpm for 5min, discarding supernatant, and beating the bottom of the centrifuge tube with palm to uniformly spread the cells on the bottom of the tube. Placing the bottom of the centrifuge tube into a large beaker containing 37 ℃ water, slowly adding the fusion agent PEG preheated at 37 ℃ into the centrifuge tube within 1min, rotating the centrifuge tube while adding, standing for 1min at 37 ℃ after adding, and slowly adding 1mL of RPMI-1640 medium along the wall of the centrifuge tube. And slowly adding 6mL of RPMI-1640 culture medium into the cell mass at the bottom of the centrifuge tube, blowing large cell mass to scatter the cell mass in the adding process, gently inverting the centrifuge tube to mix the cells uniformly after the adding, centrifuging at 800rpm for 5min, and discarding the supernatant. HAT medium containing 10% Clone Easy is added to the pellet, the pellet is resuspended, and the pellet is split into 96-well cell culture plates, 200 mu L of each well is placed in a 37 ℃ 5% CO2 cell incubator for culture, the culture solution is replaced after one week of culture, and the supernatant is taken after 24 hours for preliminary screening of hybridoma cells.
Positive hybridoma cell selection recombinant p27 protein was diluted to 1. Mu.g/ml with PBS and coated at 4℃for 12h at 100. Mu.L per well. After three PBST washes 200 μl 5% skim milk was added for 2h at 37 ℃, and after three PBST washes cell supernatants were added for 1h incubation at 37 ℃ at 100 μl per well. Adding 100 mu L of HRP-labeled goat anti-mouse IgG secondary antibody after PBST is washed three times, incubating for 1h at 37 ℃, adding 100 mu L of TMB color development liquid after PBST is washed three times, adding 50 mu L of sulfuric acid stop solution after light shielding for 10min for stopping, placing into an enzyme-labeling instrument, reading an absorbance value of 450nm, and judging positive when the P/N value is larger than 2.
Subcloning, namely selecting positive hybridoma cell holes of single cell groups under a microscope, re-suspending the positive hybridoma cell holes by using 100 mu L of HT culture medium, counting 20 mu L, taking cell suspension containing 150-180 hybridoma cells into 20mL of HT culture medium after calculation, adding the mixture into 96-well cell culture plates after fully mixing, and culturing 200 mu L of the mixture in a 37 ℃ 5% CO2 cell culture box. The monoclonal Hybridoma cell line which stably secretes ENTV-2 p27 protein antibody is selected after three subclones, and is named as Hybridoma cell line ENTV-2C3 (hybrid oma CELL LINE ENTV-2C 3), classified as Hybridoma cell line ENTV-2C3 (hybrid oma CELL LINE ENTV-2C 3) and preserved in China center for type culture collection (CHINA CENTER for Type Culture Collection, CCTCC for short), with the preservation number of CCTCC NO: C2024412 and the preservation time of 2024 for 12 months and 30 days at university of Wuhan and Wuhan in China. Meanwhile, the secreted anti-p 27 protein monoclonal antibody was designated as 2C3.
Ascites preparation and purification, namely, taking 150-200-day-old Balb/C mice, injecting 0.5mL of Freund's incomplete adjuvant into each mouse intraperitoneally, and injecting 1X 106-1×107 hybridoma cell strains ENTV-2C3 into each mouse intraperitoneally after three days. Collecting ascites after the abdomen of the mouse is obviously enlarged, centrifuging the ascites at 10000rpm for 10min, taking supernatant, purifying the ascites by using ProteinG purification columns, wherein the concentration of the purified monoclonal antibody 2C3 is 1.75mg/mL. The SDS-PAGE identification of monoclonal antibody 2C3 before and after purification is shown in FIG. 4.
4. Preparation of polyclonal antibody against p27 protein
Animal immunization New Zealand white rabbits were immunized by subcutaneous multipoint injection on the back, 1mg each time, with 14d intervals as the immunogen to boost. The antigen was emulsified with an equal volume of Freund's complete adjuvant at the time of the first immunization and with an equal volume of Freund's incomplete adjuvant at the time of the booster immunization. And after four-immunity is completed for one week, the serum antibody titer is detected by blood sampling of the auricular margin, and after the serum antibody titer meets the immune requirement, the polyclonal antibody of the anti-p 27 protein is obtained by separating serum by heart blood sampling.
Example 2 establishment and optimization of double antibody sandwich ELISA detection method
1. Basic experimental steps of the double-antibody sandwich ELISA detection method include:
1) Coating antibody, namely adding diluted anti-p 27 protein monoclonal antibody 2C3 into a 96-well ELISA plate, placing the ELISA plate at 100 mu L at 4 ℃ for 12h per well, discarding coating liquid after 12h, washing with 200 mu L of PBST for 3 times, oscillating for 2min each time, beating the ELISA plate on absorbent paper each time after washing, and beating liquid in the wells.
2) And (3) sealing, namely adding 200 mu L of sealing liquid into each hole, sealing for 2 hours at 37 ℃, and washing the ELISA plate 3 times after sealing is finished, wherein the method is the same as that described above.
3) P27 antigen. Gradient diluted p27 antigen was added to the ELISA plate, incubated at 37℃for 2h at 100. Mu.L per well, and after incubation, the liquid in the wells was removed and the ELISA plate was washed 3 times.
4) Detection antibody 100. Mu.L of diluted anti-p 27 protein polyclonal antibody is added into each hole, the mixture is incubated for 2 hours at 37 ℃, liquid in the holes is thrown out after the incubation is completed, and the ELISA plate is washed for 3 times.
5) HRP goat anti-rabbit three antibodies 100 μl of diluted HRP-labeled goat anti-rabbit IgG was added to each well and incubated at 37deg.C for 1h, after incubation was completed, the liquid in the wells was removed, and the ELISA plate was washed 3 times.
6) Color development, adding 100 mu L TMB color development liquid into each hole, and reacting for 10min at room temperature.
7) Stop the color development by adding 50. Mu.L of 2M sulfuric acid stop solution per well.
8) And (3) measuring absorbance values, namely placing the ELISA plate into an ELISA instrument, reading absorbance values of 450nm, and recording results.
2. Optimization of detection conditions
2.1 Determination of optimal concentration of Capture antibody and detection antibody
The capture antibody (anti-P27 protein monoclonal antibody 2C 3) was diluted transversely to 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL according to the checkerboard method, and the detection antibody (anti-P27 protein polyclonal antibody) was diluted longitudinally according to 1:250000, 1:500000, 1:1000000, 1:2000000, and the maximum concentration of P/N value was determined as the optimal working concentration. The results are shown in Table 1 and show an optimal coating concentration of capture antibody of 1. Mu.g/mL and an optimal dilution of detection antibody of 1:250000.
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2.2 Determination of the sealing fluid and the sealing time
2% W/v skim milk, 5% w/v skim milk, 2% w/v NH4Cl、5%w/v NH4 Cl, 2% w/v gelatin, 5% w/v gelatin, 2% w/v BSA, 5% w/v BSA, 2% v/v fetal bovine serum, 2% v/v horse serum, commercial protein stabilizer I (from Huzhou Ind. Biotechnology Co., ltd.), commercial blocking buffer I (from Huzhou Ind. Biotechnology Co., ltd.) were each selected as blocking solution and blocked at 37℃for 1h, 1.5h, 2h, 2.5h, respectively, with the P/N values being maximum to the optimal blocking solution, the P/N values being not significantly different and the blocking time being shorter to the optimal blocking time by detection analysis. The results are shown in tables 2 and 3, and show that the optimal blocking solution is 5% w/v skim milk and the optimal blocking time is 1h.
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2.3 Determination of antigen incubation conditions
The antigen incubation conditions were set to incubation times of 0.5h, 1h, 1.5h, 2h, respectively, with maximum p/N values at 37 ℃ as optimal incubation times, and the results are shown in table 4, which shows that the antigen optimal incubation conditions were 37 ℃ for 1.5h.
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2.4 Determination of incubation conditions for detection antibodies
The antigen incubation conditions were set to the incubation times of maximum p/N values at 25 ℃, 37 ℃ for 0.5h, 1h, 1.5h, 2h, respectively, as the optimal incubation times, and the results are shown in table 5, which shows that the optimal incubation conditions for the detection antibody were 37 ℃ for 2h.
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2.5 Determination of incubation time of HRP goat anti-rabbit three antibodies
The HRP-labeled goat anti-rabbit IgG secondary antibody was added, incubated at 37℃for 30min, 45min, 60min, and the incubation time with the maximum P/N value was used as the optimal incubation time, and the results are shown in Table 6, and the results show that the optimal incubation time for the HRP goat anti-rabbit three antibody was 45min.
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2.6 Determination of the color development time
TMB color development liquid is added, and the mixture is respectively incubated for 10min, 15min and 20min at 25 ℃, the P/N value is larger, the background value is lower, the color development time is taken as the optimal color development time, the result is shown in Table 7, and the optimal color development time is shown as 10min.
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3. Experiment verification
3.1 Specificity verification
The specificity of the diagnostic method was determined using established ELISA methods using virus antigens as antigens to be tested in the section assay ENTV-2 positive goat nasal swab, JSRV Gag protein, foot and mouth disease inactivated vaccine (FMDV, purchased from Jin Yubao Programme biological vaccine Co., ltd.), ovine streptococcus septicemia inactivated vaccine (Caprine Streptococci, purchased from Hayata group biological vaccine Co., ltd.), ovine infectious pleuropneumonia vaccine (Mycoplasma mycidessub sp. Capri, purchased from Hayata group biological vaccine Co., ltd.), ovine colibacillosis vaccine (Caprine colibacillosis, purchased from Hayata group biological vaccine Co., ltd.), ovine infectious pustular dermatitis live vaccine (OrfV, purchased from Shandong Hua Hong biological engineering Co., ltd.), capripox live vaccine (GTPV, purchased from Hayata group biological vaccine Co., ltd.).
The specificity of JSRV is demonstrated by synthesizing its main antigen protein Gag protein, which is specifically obtained by downloading sheep lung adenoma Gag gene sequence from NCBI, synthesizing it after codon optimization by biological company (GenBank: AF 105220.1), and connecting pcDNA3.1 vector to construct pcDNA3.1-JSRV-Gag expression plasmid. The pcDNA3.1-JSRV-Gag expression plasmid is transfected into 293T cells with 2 mug, cell lysate and supernatant are collected after 48 hours, and secreted JSRV Gag protein is obtained through purification and used for the specificity test of the invention.
The results of the specificity test are shown in Table 8, and only ENTV-2 and JSRV are positive in the detected antigen, which shows that the method has good specificity.
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+ Means containing the virus, -means a negative control without the virus.
3.2 Sensitivity verification
The p27 antigen was diluted to different concentrations at a double ratio of 5.00ng/mL, 2.50ng/mL, 1.25ng/mL, 0.63ng/mL, 0.31ng/mL, 0.16ng/mL, 0.08ng/mL, 0.04ng/mL in this order, and the sensitivity of the ELISA method established by the present invention was tested.
The sensitivity test results are shown in Table 9, and the method can detect 0.16ng/mL of p27 antigen, which indicates that the method has good sensitivity.
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3.3 Repeatability verification
In order to prove the repeatability of the ELISA method established by the invention, p27 antigen is diluted into 5 different concentrations, the ELISA plates coated in the same batch are used for testing, each concentration is repeated for 3 times, the detection result is used for calculating the variation coefficient of the experiment in the batch, the 5 ELISA plates in different batches are used for testing according to the same method, and the detection result is used for calculating the variation coefficient of the experiment in the batch. The coefficient of variation was calculated as CV (%) =sd/x×100%. Wherein SD is standard deviation, and X is average number.
The repeatability test results are shown in tables 10 and 11, and the intra-batch difference and the inter-batch difference are less than 10%, which shows that the method has good repeatability.
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