CCEV-110 recombinant protein, application and indirect ELISA detection method of camel contagious pustular dermatitis virus antibody

Abstract

The invention relates to the technical field of virus detection, in particular to CCEV-110 recombinant protein, application of the CCEV-110 recombinant protein and an indirect ELISA detection method of a camel contagious pustular dermatitis virus antibody. In the kit used in the method, an ELISA plate is coated with CCEV-110 recombinant protein. The CCEV-110 recombinant protein can be used as a coating antigen of the camel contagious pustular dermatitis virus indirect ELISA detection kit, so that the rapid detection of the camel contagious pustular dermatitis virus antibody is realized; the detection method has the advantages of being good in specificity, sensitivity and repeatability, easy and convenient to operate, low in cost, capable of achieving large-scale detection and the like. The method is efficient, accurate and practical, and provides powerful technical support for follow-up research of camel contagious pustular disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus detection, in particular to a CCEV-110 recombinant protein, application and indirect ELISA detection method of camel contagious ecthyma virus antibody. BACKGROUND

[0002] Camel contagious ecthyma virus (CCEV) is a pathogen that is extremely harmful to the health of camels. After infection with the virus, camels will develop blisters and ulcers on the lips, nose mirrors, hooves and other parts. These lesions can cause severe pain to camels and affect their normal feeding, chewing and walking.

[0003] CCEV is contagious. Camels can transmit the virus through direct contact, such as licking and mutual scratching. In addition, contaminated environments, such as stalls, feed and water, can also become a medium for the spread of the virus. If sick camels cannot be detected and isolated in time, the virus will quickly spread among the camel population, leading to a large-scale outbreak of the epidemic.

[0004] Accurate and rapid detection methods are the key to effectively preventing and controlling CCEV. Currently, although there are some detection techniques for CCEV, such as polymerase chain reaction (PCR) technology, which can detect the presence of the virus at the nucleic acid level, such methods have high technical requirements for experimental conditions and operators, and cannot directly reflect the immune status of the animal body. In contrast, serological detection methods are based on the detection of specific antibodies in animal serum and have the advantages of simple operation, low cost and large-scale detection, and can be used to assess the infection status of animal populations, immune effect and monitor the epidemic situation. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a CCEV-110 recombinant protein, application and indirect ELISA detection method of camel contagious ecthyma virus antibody.

[0006] The technical solution of the present application to solve the above technical problem is as follows:

[0007] The present application provides a CCEV-110 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0008] The present application also provides an indirect ELISA detection kit for detecting camel contagious ecthyma virus antibody, comprising an ELISA plate coated with the CCEV-110 recombinant protein as described above.

[0009] Further, the coating concentration of the CCEV-110 recombinant protein is 7-9 μg / mL.

[0010] Furthermore, it also includes camel infectious pustular virus negative serum, camel infectious pustular virus positive serum, enzyme-labeled antibody working solution, diluent, washing solution, blocking solution, colorimetric reagent and stop solution.

[0011] The present invention also provides an indirect ELISA detection method for camel infectious pustular virus antibodies, using the kit described above for detection.

[0012] Furthermore, the following steps are included:

[0013] The ELISA plate coated with the CCEV-110 recombinant protein was blocked using a blocking solution;

[0014] The diluted serum sample to be tested was added to the ELISA plate and incubated for the first time.

[0015] Add diluted enzyme-labeled secondary antibody and perform a second incubation. The enzyme-labeled secondary antibody is HRP-labeled rabbit anti-camel IgG.

[0016] After washing the plate, add the color developer to develop the color.

[0017] Termination was performed by adding a stop solution, and the OD was measured. 450 Value, based on the OD 450 It is worth getting the test results.

[0018] Furthermore, the serum sample to be tested is diluted with blocking buffer at a volume ratio of 1:600-900; the enzyme-labeled secondary antibody is diluted with blocking buffer at a volume ratio of 1:15000.

[0019] Furthermore, the sealing time is 120 minutes, and the first incubation time is 60 minutes.

[0020] Furthermore, the second incubation time is 60 minutes and the color development time is 15 minutes.

[0021] Furthermore, when the OD of the serum sample to be tested... 450 When the OD value is greater than the positive threshold, the test result is judged as positive. 450 When the value is less than or equal to the negative threshold, the test result is judged as negative. 450 If the value is less than or equal to the positive threshold but greater than the negative threshold, the test result is deemed suspicious.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) The CCEV-110 recombinant protein of the present application can be used as a coating antigen of an indirect ELISA detection kit for camel infectious pustular disease virus antibody, so that the camel infectious pustular disease virus antibody can be rapidly detected;

[0024] (2) The indirect ELISA detection method for camel infectious pustular disease virus antibody of the present application can effectively detect the camel infectious pustular disease virus antibody, and has good specificity, sensitivity and repeatability;

[0025] (3) The indirect ELISA detection method for camel infectious pustular disease virus antibody of the present application is based on the detection of specific antibodies in Bactrian camel serum, and has the advantages of simple operation, low cost and large-scale detection;

[0026] (4) The indirect ELISA detection method for camel infectious pustular disease virus antibody of the present application is efficient, accurate and practical, and provides strong technical support for the follow-up research of camel infectious pustular disease. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a result graph of the CCEV-110 protein induced at 28 DEG C in Example 2 of the present application;

[0028] Figure 2 It is a result graph of the CCEV-110 protein induced at 37 DEG C in Example 2 of the present application;

[0029] Figure 3 It is a result graph of the CCEV-110 protein purified from the recombinant protein in Example 2 of the present application;

[0030] Figure 4 It is a result graph of Western blot in Example 2 of the present application;

[0031] Figure 5 It is a protein standard curve in Example 2 of the present application;

[0032] Figure 6 It is a result graph of the optimal antigen coating concentration and serum dilution in Example 4 of the present application;

[0033] Figure 7 It is a detection result graph of the positive and negative critical values in Example 8 of the present application;

[0034] Figure 8 It is an OD value of different dilution multiples in the sensitivity evaluation in Example 9 of the present application. 450 value. DETAILED DESCRIPTION

[0035] The principles and characteristics of the present application are described below in combination with the drawings, and the examples are only used to explain the present application and not to limit the scope of the present application.

[0036] The CCEV-110 recombinant protein of the present application has an amino acid sequence as shown in SEQ ID NO. 1.

[0037] The CCEV-110 recombinant protein of the present application is encoded by a CCEV-110 gene fragment optimized in the CCEV genome, and the CCEV-110 gene fragment is as shown in SEQ ID NO. 2.

[0038] The indirect ELISA detection method of the camel contagious ecthyma virus of the present application uses an indirect ELISA detection kit for detection, which includes an ELISA plate coated with a CCEV-110 recombinant protein; the method can effectively detect the antibodies produced by the camel contagious ecthyma virus and has good specificity, sensitivity and repeatability.

[0039] Preferably, the coating concentration of the CCEV-110 recombinant protein of the ELISA plate is 7-9 μg / mL.

[0040] Preferably, the kit further includes camel contagious ecthyma virus negative serum, camel contagious ecthyma virus positive serum, enzyme-labeled antibody working solution, diluent, washing solution, blocking solution, color developing reagent and termination solution.

[0041] Preferably, the method includes the following steps:

[0042] The ELISA plate coated with the recombinant protein is blocked with a blocking solution;

[0043] The diluted serum sample to be tested is added to the ELISA plate and incubated for the first time;

[0044] The diluted enzyme-labeled secondary antibody is added and incubated for the second time;

[0045] After washing the plate, the color developing solution is added for color development;

[0046] The termination solution is added for termination, and the OD value is determined. 450

[0047] Preferably, the serum sample to be tested is diluted with a blocking solution, and the dilution volume ratio is 1:600-900; the enzyme-labeled secondary antibody is diluted with a blocking solution, and the dilution volume ratio is 1:15000.

[0048] Preferably, the blocking time is 120 min, and the first incubation time is 60 min.

[0049] ​Preferably, the second incubation time is 60 min, and the color development time is 15 min.

[0050] Preferably, when the OD value of the serum sample to be tested is greater than the positive critical value, the detection result is determined to be positive, when the OD value of the serum sample to be tested is less than or equal to the negative critical value, the detection result is determined to be negative, and when the OD value of the serum sample to be tested is less than or equal to the positive critical value and greater than the negative critical value, the detection result is determined to be suspicious. 450 450 450

[0051] The detection method of the present application is based on detecting camel infectious pustular disease virus specific antibodies in Bactrian camel serum, and has the advantages of simple operation, low cost, and large-scale detection. The detection method is efficient, accurate and practical, and provides strong technical support for the follow-up study of camel infectious pustular disease.

[0052] The present application will be specifically described below through examples.

[0053] The standard positive serum used in each example is prepared from naturally occurring camel blood, which is positive by qPCR detection, and can specifically react with B2L antigen after 1:5000 dilution by Western blot test. The negative serum is collected from a healthy baby camel just after birth, which is negative by qPCR detection, and the undiluted negative serum cannot specifically react with B2L protein antigen by Western blot test. The gene encoding B2L protein is the only gene currently available on NCBI for CCEV.

[0054] The main reagents and main instruments used in each example are shown in Table 1 and Table 2.

[0055] Table 1 Main reagents

[0056]

[0057] Table 2 Main instruments

[0058]

[0059] Example 1 Construction of recombinant plasmid

[0060] The nucleotide sequence used to encode CCEV-110 protein is obtained, and the two are connected to the pET-32a empty plasmid by genetic engineering means, respectively, to obtain pET32a-CCEV-110 recombinant plasmid.

[0061] The above two recombinant plasmids are transformed into E. coli BL21(DE3) competent cells and cultured to obtain CCEV-110-BL21 bacterial liquid.​​​

[0062] The specific experimental procedures of the present embodiment can use conventional genetic engineering methods, and the nucleotide sequence encoding CCEV-110 protein is obtained by virus extraction amplification method. In the specific experimental procedures of the present embodiment, the sequence of the obtained product is verified to be correct by sequencing if necessary.

[0063] Example 2 Prokaryotic expression and purification of CCEV-110 recombinant protein

[0064] (1) Screening of optimal induction expression conditions: the CCEV-110-BL21 obtained in Example 1 was resuscitated and inoculated into LB liquid medium (working concentration of ampicillin was 100 μg / mL) at a volume ratio of 1:100. The absorbance was measured, and when OD 620nm = 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0, 0.1, 0.5, 1, 5 mM, respectively, the induction temperature was 28°C, 37°C, the induction time was 18 h, 10,000 rpm centrifugation was used for 10 min, the bacterial liquid precipitate after induction was resuspended with PBS buffer, the bacterial body was ultrasonically broken using a cell disrupter, the liquid after centrifugation was centrifuged at 10,000 rpm for 10 min at 4°C, 80 μL of supernatant was taken, the precipitate was resuspended and taken again, 20 μL of 5x protein loading buffer was added, respectively, and boiled in water for 10 min before SDS-PAGE electrophoresis.

[0065] The results of SDS-PAGE electrophoresis are shown in Figure 1 and Figure 2 ; Figure 1 and Figure 2 The specific bands in each lane are as follows: 1 indicates pET32a empty supernatant, 2 indicates pET32a empty precipitate, 3 indicates 0 mM IPTG supernatant, 4 indicates 0 mM IPTG precipitate, 5 indicates 0.1 mM IPTG supernatant, 6 indicates 0.1 mM IPTG precipitate, 7 indicates 0.5 mM IPTG supernatant, 8 indicates 0.5 mM IPTG precipitate, 9 indicates 1 mM IPTG supernatant, 10 indicates 1 mM IPTG precipitate, 11 indicates 5 mM IPTG supernatant, and 12 indicates 5 mM IPTG precipitate.

[0066] As can be seen from Figure 1 and Figure 2 , the optimal induction conditions for CCEV-110 recombinant protein are: IPTG final concentration of 1 mM, induction temperature of 28°C, and induction time of 24 h.

[0067] (2) Recombinant protein purification: After the recovery of PET-32a-CCEV-110-BL21 transformed strain, inoculate into LB liquid medium (Amp concentration is 100 μg / mL) at a volume ratio of 1:100.

[0068] Determine the optical density value, when OD 620nm When OD is 0.6-0.8, add IPTG to PET-28a-CCEV-110-BL21 at a final concentration of 1 mMOL / L, the induction temperature is 28°C, and the induction time is 18 h.

[0069] Collect the bacterial liquid precipitate after induction at 10,000 rpm for 10 min, resuspend the bacterial liquid precipitate with PBS buffer, and then centrifuge again, repeating 3 times.

[0070] Resuspend the bacterial precipitate of pET-32a-CCEV-110-BL21 with non-denaturing lysis solution, add lysozyme at a final concentration of 1 mg / ml, mix well, and stand on ice for 30 min. Ultrasonic lysis of bacteria on ice. Each ultrasonic treatment is 5 s, with an interval of 5 s, and a total of 15 min. Centrifuge the liquid after ultrasonic treatment at 4°C, 10,000 g for 20 min, filter the bacterial lysis liquid supernatant with a 0.45 μm filter, and place it in an ice water bath or on ice.

[0071] Take an appropriate amount of mixed 50% BeyoGold TM His-tag Purification Resin (denaturant type), centrifuge and discard the storage solution at 4°C, add one column volume of denaturing or non-denaturing lysis solution to the gel and mix well to equilibrate the gel, centrifuge and discard the liquid at 4°C, and repeat the equilibration 2 more times and discard the liquid.

[0072] According to the proportion of 4 mL of bacterial lysis liquid supernatant added to 1 mL of 50% gel (1:8), mix BeyoGold TM His-tag Purification Resin and bacterial lysis liquid supernatant. Slowly shake at 4°C on a side swing shaker or horizontal shaker for 60 min. The supernatant of pET-32a-CCEV-110-BL21 is combined with the gel equilibrated with non-denaturing lysis solution, and the protein is purified with non-denaturing washing solution and non-denaturing elution solution.

[0073] Mix the lysis solution and BeyoGold TMHis-tag Purification Resin (resistant to denaturing) was mixed into a suitable empty column tube. The lid at the bottom of the purification column was opened, and the liquid in the column was allowed to flow out under the action of gravity, and about 80 μL of the flow-through liquid was collected for subsequent analysis. The column was washed 5 times, and 1 mL of denaturing lysis solution or non-denaturing lysis solution was added each time. The column was washed again 10 times, and 1 mL of denaturing washing solution or non-denaturing washing solution was added each time. Finally, the column was washed 5 times, and 1 mL of denaturing elution solution or non-denaturing elution solution was added each time, and the liquid collected after the flow-through this time was the target protein.

[0074] The obtained target protein was subjected to SDS-PAGE electrophoresis, and the electrophoresis staining results are shown in Figure 3

[0075] It can be seen that CCEV-110 expressed and purified a 55 KDa protein, and the size of the target protein was correct, proving that the recombinant protein was successfully purified.

[0076] (3) Protein ultrafiltration concentration and Western blot identification: the obtained CCEV-110 protein solution was added to the treated 30 kDa protein ultrafiltration tube, and the protein was ultrafiltrated and concentrated. The ultrafiltration method is as follows:

[0077] Pure water, 20% ethanol, and pure water were used in sequence and added to the upper layer of the ultrafiltration tube, and centrifugal washing was performed at 4°C and 3,500 rpm for 5 min.

[0078] The protein solution was added to the upper layer of the ultrafiltration tube, and the protein was ultrafiltrated and concentrated by repeated centrifugation at 4°C, 3,500 rpm, and 5 min. After the end, the upper layer of the protein solution was stored, 80 μL of which was added to 20 μL of 5× protein loading buffer, and after boiling in water for 10 min, SDS-PAGE electrophoresis was performed.

[0079] Pure water and 20% ethanol were used in sequence to clean the ultrafiltration tube, and 20% ethanol was used for soaking and storage. Western blotting experiment was performed using the ultrafiltrated protein. SDS-PAGE electrophoresis was performed first, and then a PVDF membrane of appropriate size was cut according to the size of the gel. The transfer membrane device was assembled in the order of "negative electrode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode", ensuring that there were no air bubbles between the layers. Then, the membrane was transferred at 100 V for 1 hour. After the end, 5% skimmed milk powder was used for blocking, and then the membrane was blocked at room temperature on a shaker for 2 hours. The membrane was washed with TBST for 30 min. The primary antibody was diluted with 5% skimmed milk powder at a ratio of 1:8000, and the membrane was blocked again after being blocked at room temperature on a shaker for 2 hours. The secondary antibody was diluted at a ratio of 1:10000, and the membrane was blocked again after being blocked at room temperature on a shaker for 2 hours. Finally, the chemiluminescence instrument was used for color development detection.

[0080] ​SDS-PAGE electrophoresis was performed using the concentrated protein, and the protein gel was stained and identified by Western blot. The SDS-PAGE electrophoresis result staining result is shown in Figure 4 .

[0081] According to Figure 4 It can be seen that the size of the CCEV-110 recombinant protein is correct, proving that the concentration of the recombinant protein is successful. The Western blot result shows that the band size is consistent with the expected size, indicating that the obtained CCEV-110 recombinant protein has reactivity.

[0082] Establishment of protein standard curve: The protein concentration determination kit (BCA protein concentration determination kit, product number: PC0020) of Beijing Solabio Biotechnology Co., Ltd. was used, and the protein standard curve was established according to the instructions.

[0083] Specifically, the protein standard curve was plotted with the recombinant protein concentration as the abscissa and the corresponding microplate reader reading as the ordinate. As shown in Figure 5 , the slope of the protein standard curve is 0.9996, the intercept is -0.1454, the equation is y = 0.9996x - 0.1454, and the correlation coefficient R 2 = 0.9987, which satisfies R 2 > 0.99, 110% > E > 90%, indicating that there is a good linear relationship between the protein concentration and the microplate reader reading. Based on the standard curve, the concentration of CCEV-110 protein is 1.84 mg / mL.

[0084] Example 3 Serological detection method of camel contagious ecthyma virus

[0085] The specific steps of the camel contagious ecthyma virus indirect ELISA detection method of the present example are as follows:

[0086] (1) The expressed and purified recombinant protein CCEV-110 obtained in Example 2 was diluted to the required concentration with 0.1 moL / L carbonate buffer (pH = 9.6), and coated into an ELISA plate at a volume of 100 μL / well, sealed, and placed at 4°C overnight.

[0087] (2) Wash the plate: wash the plate 3 times with PBST, 5 min each time, and pat dry.

[0088] (3) Blocking: add blocking solution (0.5% skim milk powder) at a volume of 200 μL per well, and block at 37°C for 2 h.

[0089] (4) Wash the plate: wash the plate as in step (2) and pat dry.

[0090] (5) Incubate the primary antibody: add 100 μL of the serum sample to be tested diluted with the blocking solution to each well, and incubate at 37°C for 1 h.

[0091] (6) Washing the plate: Same as step (2) washing the plate and patting it dry.

[0092] (7) Incubate with enzyme-labeled secondary antibody; add 100 μL of horseradish peroxidase-labeled secondary antibody diluted with blocking buffer to each well, and incubate at 37°C for 45 min. The enzyme-labeled secondary antibody used in this example is HRP-labeled rabbit anti-camel IgG.

[0093] (8) Washing the plate: Same as step (2) washing the plate and patting it dry.

[0094] (9) Color development: Add 100 μL of TMB single-component color development solution to each well and develop the color at 37°C in the dark for 15 min.

[0095] (9) Termination: Add 50 μL of stop solution (3 mol / L concentrated sulfuric acid) to each well, shake the plate for 30 seconds to mix, and measure the OD using a microplate reader. 450 value.

[0096] Example 4: Optimal Coating Concentration and Serum Dilution

[0097] Using the detection method described in Example 3, multiple concentration gradients of CCEV-110 recombinant protein were set up, namely 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, with serum dilutions of 1:200, 1:400, 1:800, and 1:1600. The test was conducted using the checkerboard titration method, and the absorbance values ​​were converted into P / N values. The optimal coating concentration and serum dilution were determined using the P / N values.

[0098] The experimental results of this embodiment are as follows: Figure 6 As shown. According to Figure 6 It can be seen that the P / N value of the CCEV-110 recombinant protein is the largest at a coating concentration of 8 μg / mL and a serum dilution of 1:800, which is 9.84151.

[0099] Example 5: Optimal dilution of enzyme-labeled secondary antibody

[0100] Using the detection method of Example 3 and the optimal coating concentration and serum dilution obtained from the screening in Example 4, the enzyme-labeled secondary antibody dilution factors were set to 1:2500, 1:5000, 1:7500, 1:10000, 1:1500, and 1:2000. The absorbance values ​​were converted into P / N values, and the optimal dilution of the enzyme-labeled secondary antibody was determined by the P / N values.

[0101] The experimental results of this example are shown in Table 3. It can be seen that when the dilution factor of the enzyme-labeled secondary antibody is 1:15000, the P / N values of the CCEV-110 protein-coated enzyme-labeled plates are all maximum, and the P / N value of CCEV-110 is 17.5543.

[0102] Table 3 Determination results of optimal dilution of enzyme-labeled secondary antibody

[0103]

[0104] Example 6 Optimal blocking time and serum incubation time

[0105] Using the detection method of Example 3, the optimal antigen coating concentration and serum dilution obtained by screening of Example 4, and the optimal dilution of enzyme-labeled secondary antibody obtained by Example 5, the optimal blocking time is set to 60 min, 90 min, and 120 min, and the optimal serum incubation time is set to 60 min, 90 min, and 120 min. The chessboard titration method is used for testing, the absorbance value is converted into P / N value, and the optimal blocking time and serum incubation time are determined by P / N value.

[0106] The experimental results of this example are shown in Table 4. It can be seen that when the blocking time is 120 min and the serum incubation time is 60 min, the P / N values of the two enzyme-labeled plates are all maximum, and the P / N value of CCEV-110 is 9.6982.

[0107] Table 4 Determination results of optimal blocking time and serum incubation time

[0108]

[0109] Example 7 Optimal enzyme-labeled secondary antibody incubation time and color development time

[0110] Using the detection method of Example 3, the optimal antigen coating concentration and serum dilution obtained by screening of Example 4, the optimal dilution of enzyme-labeled secondary antibody obtained by Example 5, and the optimal blocking time and serum incubation time obtained by Example 6, the optimal enzyme-labeled secondary antibody incubation time is set to 30 min, 45 min, and 60 min, and the optimal color development time is set to 10 min, 15 min, and 20 min. The chessboard titration method is used for testing, the absorbance value is converted into P / N value, and the optimal enzyme-labeled secondary antibody incubation time and color development time are determined by P / N value.

[0111] The experimental results of this example are shown in Table 5. It can be seen that the P / N value of the CCEV-110 recombinant protein-coated enzyme-labeled plate is maximum, which is 9.69852, when the secondary antibody incubation time is 60 min and the color development time is 15 min.

[0112] Table 5 Determination results of optimal enzyme-labeled secondary antibody incubation time and color development time

[0113]

[0114]

[0115] Example 8: Determination of positive and negative threshold values ​​for the detection method

[0116] This embodiment determines the positive and negative threshold values ​​for the detection method based on the optimized method described in the above embodiments. Specifically, the main parameters of the optimized method are as follows:

[0117] The CCEV-110 recombinant protein was coated with antigen at a concentration of 8 μg / mL, and the serum dilution was 1:800. The enzyme-labeled secondary antibody was diluted 1:15000, the blocking time was 120 min, the serum incubation time was 60 min, and the coated ELISA plate was incubated with the secondary antibody for 60 min and the color development time was 15 min.

[0118] The experimental procedure in this embodiment involves testing 20 camel CCEV-negative serum samples to determine the positive / negative cutoff values. The OD values ​​of the 20 camel-negative serum samples were calculated using ELISA. 450 average and standard deviation (S). When the sample When, the result is judged as positive, when If the result is negative, the two are considered suspicious.

[0119] like Figure 7 As shown, the results of ELISA testing using CCEV-110 protein-coated microplates revealed that the OD values ​​of 20 camel-negative serum samples were significantly lower than those detected by ELISA. 450 average The value is 0.4565, and the standard deviation (S) is 0.1048. When the sample... The result was positive. If the result is negative, the two are considered suspicious.

[0120] Example 9 Sensitivity Evaluation

[0121] This embodiment evaluates the sensitivity of the method optimized from the above embodiments. Specifically, the main parameters of the optimized method are the same as those in Embodiment 8.

[0122] The experimental procedure in this embodiment involved diluting CCEV-positive serum at different concentration gradients: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:104800, 1:209600, and 1:4019200. Each dilution was repeated three times. OD was then observed. 450The sensitivity of this method is determined by the trend of change, with the highest fold of serum dilution resulting in a positive outcome.

[0123] Test results as follows Figure 8 As shown in the figure, the highest detectable dilution of the microplate coated with CCEV-110 protein is 1:6400.

[0124] Example 10 Repeatability Test

[0125] This embodiment demonstrates a repeatability test on the method optimized from the above embodiments. Specifically, the main parameters of the optimized method are the same as those in Embodiment 8.

[0126] The experimental procedure in this embodiment is as follows: using ELISA plates coated with the same batch and ELISA plates coated with three batches, five negative and five positive sera were tested, and each serum sample was replicated in three wells. The intra-batch and inter-batch repeatability of the method was determined based on the coefficient of variation (CV). The formula for calculating the coefficient of variation is as follows. The time results of this embodiment are shown in Table 6.

[0127] Coefficient of variation (CV) = standard deviation / mean.

[0128] As shown in Table 6, the ELISA plate coated with CCEV-110 recombinant protein had an intra-assay coefficient of variation of 0.69%–7.00% and an inter-assay coefficient of variation of 2.32%–8.98%. The low intra-assay and inter-assay coefficients of variation (less than 9%) indicate that the method has good reproducibility.

[0129] Table 6 Results of Repeatability Tests

[0130]

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. CCEV-110 recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. The application of the CCEV-110 recombinant protein as described in claim 1, characterized in that, The CCEV-110 recombinant protein can be used to prepare an indirect ELISA detection kit for detecting camel infectious pustular virus antibodies.

3. An indirect ELISA detection kit for detecting antibodies against camel infectious pustular virus, characterized in that, Includes an ELISA plate coated with the recombinant protein as described in claim 1.

4. The indirect ELISA detection kit for detecting camel infectious pustular virus antibodies according to claim 3, characterized in that, The coating concentration of the recombinant protein is 7-9 μg / mL.

5. The indirect ELISA detection kit for detecting camel infectious pustular virus antibodies according to claim 3, characterized in that, It also includes camel infectious pustular virus negative serum, camel infectious pustular virus positive serum, enzyme-labeled antibody working solution, diluent, washing solution, blocking solution, colorimetric reagent and stop solution.

6. An indirect ELISA detection method for camel infectious pustular virus antibodies, characterized in that, The test was performed using the kit described in any one of claims 3-5.

7. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 6, characterized in that, Includes the following steps: The ELISA plate coated with the CCEV-110 recombinant protein was blocked using a blocking solution; The diluted serum sample to be tested was added to the ELISA plate and incubated for the first time. Add diluted enzyme-labeled secondary antibody and perform a second incubation. The enzyme-labeled secondary antibody is HRP-labeled rabbit anti-camel IgG. After washing the plate, add the color developer to develop the color. Termination was performed by adding a stop solution, and the OD was measured. 450 Value, based on the OD 450 It is worth getting the test results.

8. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 7, characterized in that, The serum sample to be tested was diluted with blocking buffer at a volume ratio of 1:600-900; the enzyme-labeled secondary antibody was diluted with blocking buffer at a volume ratio of 1:15000; the blocking time was 120 min; and the first incubation time was 60 min.

9. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 7, characterized in that, The second incubation time was 60 minutes, and the color development time was 15 minutes.

10. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 7, characterized in that, When the OD of the serum sample to be tested 450 When the OD value is greater than the positive threshold, the test result is judged as positive. 450 When the value is less than or equal to the negative threshold, the test result is judged as negative. 450 If the value is less than or equal to the positive threshold but greater than the negative threshold, the test result is deemed suspicious.

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