CCEV-99 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-99 recombinant protein, application of the CCEV-99 recombinant protein and an indirect ELISA detection method of a camel contagious pustular dermatitis virus antibody. In a kit used in the method, an ELISA plate is coated with CCEV-99 recombinant protein. The CCEV-99 recombinant protein can be used as a coating antigen of the camel contagious pustular dermatitis virus indirect ELISA detection kit, and rapid detection of a camel contagious pustular dermatitis virus specific 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-99 recombinant protein, application and indirect ELISA detection method of camel contagious ecthyma virus antibody. BACKGROUND

[0002] Camel contagious ecthyma virus (CCEV) belongs to Parapoxvirus of Poxviridae, which seriously endangers the healthy development of camel breeding industry. The disease is widely prevalent in North Africa and West Asia. After camel is infected with the virus, typical pustule-like lesions appear on the face, mainly on the lips, mouth and nose, and around the eyes, and a few cases are also found on the cheeks, affecting the normal feeding of camels.

[0003] CCEV has strong infectivity, and its main transmission routes are direct contact, such as licking behavior; it can also be transmitted through indirect contact, such as contact with contaminated feed, water, utensils, etc. If the sick camels cannot be detected and isolated in time, the virus will spread rapidly in the camel population, leading to large-scale outbreak of the epidemic.

[0004] Accurate and rapid detection method is the key to effectively prevent and control CCEV. At present, there is no detection method for the virus worldwide, and the b2l gene of the virus can only be amplified by polymerase chain reaction (PCR) technology, and the presence of CCEV virus nucleic acid can be determined after sequencing comparison, but this method is time-consuming and laborious, and has poor specificity, and has high requirements for experimental conditions and technical requirements of operators. In contrast, the serological detection method is based on the detection of specific antibodies in Bactrian camel serum, which has the advantages of simple operation, low cost, large-scale detection, etc., and can be used to evaluate the infection status, immune effect and monitor the epidemic situation of Bactrian camel population. SUMMARY

[0005] The technical problem to be solved by the present application is the CCEV-99 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 1. CCEV-99 recombinant protein, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1.

[0008] The present application also provides an application of CCEV-99 recombinant protein, which can be used for detecting camel contagious ecthyma virus antibody.

[0009] The application also provides an indirect ELISA detection kit for detecting camel contagious ecthyma virus antibody, comprising an ELISA plate coated with the CCEV-99 recombinant protein as described above.

[0010] Further, the coating concentration of the CCEV-99 recombinant protein is 1-2 μg / mL.

[0011] Further, the kit further comprises camel contagious ecthyma virus negative serum, camel contagious ecthyma virus positive serum, enzyme-labeled antibody, diluent, washing solution, blocking solution, color developing reagent and termination solution.

[0012] The application also provides an indirect ELISA detection method for camel contagious ecthyma virus antibody, which uses the kit as described above for detection, and the method comprises the following steps:

[0013] The ELISA plate coated with the CCEV-99 recombinant protein is blocked with a blocking solution;

[0014] The diluted serum sample to be detected is added to the ELISA plate and subjected to first incubation;

[0015] The diluted enzyme-labeled secondary antibody, which is HRP-labeled rabbit anti-camel IgG, is added and subjected to second incubation;

[0016] After washing the plate, color developing solution is added for color development;

[0017] Termination solution is added for termination, and OD 450 value is determined, and the detection result is obtained according to the OD 450 value.

[0018] Further, the serum sample to be detected is diluted with a blocking solution, and the dilution volume ratio is 1:400-800; the enzyme-labeled secondary antibody is diluted with a blocking solution, and the dilution volume ratio is 1:15000.

[0019] Further, the blocking time is 120 min, and the first incubation time is 60 min.

[0020] Further, the second incubation time is 30 min, and the color development time is 15 min.

[0021] Further, when the OD 450 value of the serum sample to be detected is greater than the positive critical value, the detection result is determined as positive; when the OD 450 value of the serum sample to be detected is less than or equal to the negative critical value, the detection result is determined as negative; and when the OD 450 value of the serum sample to be detected is less than or equal to the positive critical value and greater than the negative critical value, the detection result is determined as suspicious.

[0022] The beneficial effects of the present application are:

[0023] (1) The CCEV-99 recombinant protein of the present application can be used as a coating antigen of an indirect ELISA detection kit of camel infectious pustular disease virus antibody, so as to realize rapid detection of camel infectious pustular disease virus;

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

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

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

[0027] Figure 1 It is a result graph of CCEV-99 protein induced test at 37 DEG C in Example 2 of the present application;

[0028] Figure 2 It is a Western blot result graph in Example 2 of the present application;

[0029] Figure 3 It is a protein standard curve in Example 2 of the present application;

[0030] Figure 4 It is a result graph of optimal antigen coating concentration and serum dilution degree in Example 4 of the present application;

[0031] Figure 5 It is a detection result graph of positive and negative critical values in Example 8 of the present application;

[0032] Figure 6 It is an OD value of different dilution multiples in sensitivity evaluation in Example 9 of the present application; 450

[0033] Figure 7 It is an average serum positive rate of camel infectious pustular disease virus in a surveyed area in Example 12 of the present application;

[0034] Figure 8 It is a comparison graph of serum positive rates of adult camels and young camels in a surveyed area in Example 12 of the present application. DETAILED DESCRIPTION​

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

[0036] The CCEV-99 recombinant protein for detecting camel contagious pustular disease virus antibody of the present application has an amino acid sequence as shown in SEQ ID NO. 1.

[0037] The CCEV-99 recombinant protein of the present application is encoded by an optimized CCEV-99 gene fragment in the CCEV genome, and the CCEV-99 gene fragment is as shown in SEQ ID NO. 2.

[0038] The indirect ELISA detection method for camel contagious pustular disease virus antibody of the present application uses an indirect ELISA detection kit for detection, which includes an ELISA plate coated with CCEV-99 recombinant protein; the method can effectively detect camel contagious pustular disease virus antibody and has good specificity, sensitivity and repeatability.

[0039] Preferably, the coating concentration of CCEV-99 recombinant protein of the ELISA plate is 1-2 μg / mL.

[0040] Preferably, the kit further includes camel contagious pustular disease virus negative serum, camel contagious pustular disease 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 1:400-800; 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 30 min, and the color development time is 15 min.

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

[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 through the following 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]

[0058] Table 2 Main instruments

[0059]

[0060] Example 1 Construction of recombinant plasmid

[0061] The nucleotide sequence for encoding CCEV-99 protein is obtained, and the two are connected to the pET-32a empty plasmid by genetic engineering means, to obtain the pET32a-CCEV-99 recombinant plasmid.

[0062] The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured to obtain CCEV-99-BL21 bacterial liquid.

[0063] The specific experimental steps of the embodiment can use conventional genetic engineering methods, and the nucleotide sequence encoding the CCEV-99 protein is obtained by the method of virus extraction and amplification. In the specific experimental steps of the embodiment, the sequence of the obtained product is verified to be correct by sequencing when necessary.

[0064] Example 2 Prokaryotic expression and purification of CCEV-99 recombinant protein

[0065] (1) Screening of optimal induction expression conditions: the CCEV-99-BL21 obtained in Example 1 was resuscitated and inoculated into LB liquid medium (the 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-thiogalactopyranoside (IPTG) was added at a final concentration of 0, 0.1, 0.5, 1 and 5 mM, respectively, the induction temperature was 28°C and 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 by 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, and 80 μL of resuspended precipitate was taken again, 20 μL of 5× protein loading buffer was added, respectively, and after boiling for 10 min, SDS-PAGE electrophoresis was performed.

[0066] The results of SDS-PAGE electrophoresis are shown in Figure 1 ; Figure 1 The specific bands in each lane are as follows: 1 represents pET32a empty supernatant, 2 represents pET32a empty precipitate, 3 represents 0 mM IPTG supernatant, 4 represents 0 mM IPTG precipitate, 5 represents 0.1 mM IPTG supernatant, 6 represents 0.1 mM IPTG precipitate, 7 represents 0.5 mM IPTG supernatant, 8 represents 0.5 mM IPTG precipitate, 9 represents 1 mM IPTG supernatant, 10 represents 1 mM IPTG precipitate, 11 represents 5 mM IPTG supernatant, and 12 represents 5 mM IPTG precipitate.

[0067] According to Figure 1 , it can be seen that the optimal induction conditions for the CCEV-99 recombinant protein are as follows: the final concentration of IPTG is 0.5 mM, the induction temperature is 37°C, and the induction time is 18 h.

[0068] (2) Purification of recombinant protein: After the recovery of the PET-32a-CCEV-99-BL21 transformed strain obtained in Example 1, inoculate into LB liquid medium (Amp concentration is 100 μg / mL) at a volume ratio of 1:100.

[0069] Determine the optical density value, and when OD 620nm When the optical density value is 0.6-0.8, add IPTG to the PET-28a-CCEV-99-BL21 at a final concentration of 0.5 mMol / L, and the induction temperature is 37°C.

[0070] 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.

[0071] Resuspend the bacterial precipitate of pET-32a-CCEV-99-BL21 with denaturing lysis solution.

[0072] Ultrasonically lyse the bacteria on ice. Each ultrasonic treatment is 5 s, with an interval of 5 s, and the total ultrasonic treatment is 15 min. Centrifuge the liquid after ultrasonic treatment at 4°C and 10,000 g for 20 min, filter the bacterial lysis solution supernatant using a 0.45 μm filter, and then place in an ice water bath or on ice.

[0073] Take an appropriate amount of mixed and uniform 50% BeyoGold TM His-tag Purification Resin (denaturant-resistant 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 to equilibrate the gel, centrifuge and discard the liquid at 4°C, and repeat the equilibration 2 more times and discard the liquid.

[0074] According to the ratio of 4 mL of bacterial lysis solution supernatant to 1 mL of 50% gel (1:8), mix the BeyoGold TM His-tag Purification Resin and the bacterial lysis solution supernatant, and slowly shake at 4°C on a side-to-side shaker or a horizontal shaker for 60 min. The supernatant of pET-32a-CCEV-99-BL21 is combined with the gel equilibrated with denaturing lysis solution, and the protein is purified using denaturing washing solution and denaturing elution solution subsequently.

[0075] Mix the lysis solution and BeyoGold TMHis-tag Purification Resin (resistant to denaturant) was packed into a column. The cap at the bottom of the column was opened and the liquid in the column was allowed to flow out under the force of gravity. About 80 μL of the flow-through was collected for subsequent analysis. The column was washed 5 times with 1 mL of denaturing lysis buffer or non-denaturing lysis buffer. The column was washed again 10 times with 1 mL of denaturing wash buffer or non-denaturing wash buffer. Finally, the column was washed 5 times with 1 mL of denaturing elution buffer or non-denaturing elution buffer. The liquid collected after the final flow-through was the target protein.

[0076] The target protein was subjected to SDS-PAGE electrophoresis. The results of the electrophoresis staining are shown in Figure 2. As can be seen, CCEV-99 expressed and purified a 70 KDa protein. The size of the target protein was correct, indicating that the recombinant protein was successfully purified. Figure 3

[0077] (3) Protein ultrafiltration and concentration and Western blot identification: The CCEV-99 protein solution obtained was added to a 30 kDa protein ultrafiltration tube that had been prepared. The protein was subjected to ultrafiltration and concentration. The ultrafiltration method was as follows:

[0078] The ultrafiltration tube was washed with pure water, 20% ethanol, and pure water in that order. The protein solution was added to the upper layer of the ultrafiltration tube. The protein was subjected to repeated centrifugal ultrafiltration and concentration at 4°C and 3,500 rpm for 5 min. After the end of the ultrafiltration and concentration, the upper layer of the protein solution was divided and stored. 80 μL of the protein solution was added to 20 μL of 5x protein loading buffer. After boiling for 10 min, the solution was subjected to SDS-PAGE electrophoresis.

[0079] The ultrafiltration tube was washed with pure water and 20% ethanol in that order and was stored by soaking in 20% ethanol. The protein after ultrafiltration was subjected to Western blotting. SDS-PAGE electrophoresis was performed first. After the end of the electrophoresis, a PVDF membrane of an 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. Subsequently, the membrane was transferred at 100 V for 1 hour. After the end of the transfer, the membrane was blocked with 5% skim milk. Subsequently, the membrane was blocked on a shaker at room temperature for 2 hours. The membrane was washed with TBST for 30 min. The primary antibody was diluted with 5% skim milk at a ratio of 1:8000. After blocking on a shaker at room temperature for 2 hours, the membrane was washed again. The secondary antibody was diluted at a ratio of 1:10000. After blocking on a shaker at room temperature for 2 hours, the membrane was washed again. Finally, the membrane was subjected to color development detection using a chemiluminescence instrument.

[0080] The concentrated protein was subjected to SDS-PAGE electrophoresis. The protein gel was stained and subjected to Western blotting. The CCEV-99 recombinant protein was of the correct size, indicating that the recombinant protein was successfully concentrated. ​

[0081] According to Figure 2 As can be seen, the Western blot results show that the band size is consistent with the expected size, indicating that the obtained CCEV-99 recombinant protein has reactivity.

[0082] Establishment of protein standard curve: The protein quantitative reagent 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 3 , the slope of the protein standard curve is 0.9996, the intercept is -0.1454, the equation is y = 0.9996x - 0.1454, the correlation coefficient R 2 = 0.9987, 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 the CCEV-99 recombinant protein obtained in this example is 1.84 mg / mL.

[0084] Example 3 Serological detection method for camel infectious pustular virus

[0085] The specific steps of the camel infectious pustular virus indirect ELISA detection method of this example are as follows:

[0086] (1) Dilute the expressed and purified recombinant protein CCEV-99 obtained in Example 2 to the desired concentration with 0.1 moL / L carbonate buffer (pH = 9.6), and coat it into the ELISA plate at a volume of 100 μL / well, seal the hole, and place it 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) Wash the plate: wash the plate as in step (2) and pat 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-99 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 4 As shown. According to Figure 4 It can be seen that the P / N value of the CCEV-99 recombinant protein is the largest, which is 11.86257, when the coating concentration is 1 μg / mL and the serum dilution is 1:800.

[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 embodiment 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 value of the enzyme-labeled plate coated with CCEV-99 protein is at its maximum value, and the CCEV-99 value is 10.7751.

[0102] Table 3. Results of Determination of Optimal Dilution of Enzyme-Labeled Secondary Antibodies

[0103]

[0104]

[0105] Optimal blocking time and serum incubation time of Example 6

[0106] Using the detection method of Example 3, the optimal antigen coating concentration and serum dilution obtained by screening of Example 4, and the optimal enzyme-labeled secondary antibody dilution obtained by Example 5, the optimal blocking time was set to 60 min, 90 min, and 120 min, and the optimal serum incubation time was set to 60 min, 90 min, and 120 min. The chessboard titration method was used for testing, and the absorbance value was converted to P / N value to determine the optimal blocking time and serum incubation time.

[0107] 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 value of the enzyme-labeled plate is the maximum value, and the CCEV-99 value is 12.7388.

[0108] Table 4 Determination results of optimal blocking time and serum incubation time

[0109]

[0110] Optimal enzyme-labeled secondary antibody incubation time and color development time of Example 7

[0111] Using the detection method of Example 3, the optimal antigen coating concentration and serum dilution obtained by screening of Example 4, the optimal enzyme-labeled secondary antibody dilution 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 was set to 30 min, 45 min, and 60 min, and the optimal color development time was set to 10 min, 15 min, and 20 min. The chessboard titration method was used for testing, and the absorbance value was converted to P / N value to determine the optimal enzyme-labeled secondary antibody incubation time and color development time.

[0112] The experimental results of this example are shown in Table 5. It can be seen that the P / N value of the enzyme-labeled plate coated with CCEV-99 recombinant protein is the maximum value of 7.93300 when the secondary antibody incubation time is 30 min and the color development time is 15 min.

[0113] Table 5 Determination results of optimal enzyme-labeled secondary antibody incubation time and color development time

[0114]

[0115] Determination of positive and negative critical values of the detection method of Example 8

[0116] This embodiment determines the positive and negative threshold values ​​for the method optimized from the above embodiments.

[0117] Specifically, the main parameters of the optimized method are as follows:

[0118] The CCEV-99 recombinant protein was coated with antigen at a concentration of 1 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 30 min and the color development time was 15 min.

[0119] 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 The result is judged as positive. If the result is negative, the two are considered suspicious.

[0120] like Figure 5 As shown, the OD values ​​of 20 camel-negative serum samples detected by ELISA were... 450 average The value is 0.3811, and the standard deviation (S) is 0.0427. When the sample... The result was positive. If the result is negative, the two are considered suspicious.

[0121] Example 9 Sensitivity Evaluation

[0122] 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.

[0123] 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. 450 The sensitivity of this method is determined by the trend of change, with the highest fold of serum dilution resulting in a positive outcome.

[0124] Test results as follows Figure 6 As shown in the figure, the highest detectable dilution of the microplate coated with CCEV-99 protein is 1:25600.

[0125] Example 10 Reproducibility test

[0126] This example is a reproducibility test of the method optimized in the above examples. Specifically, the main parameters of the optimized method are the same as in Example 8.

[0127] The experimental process of this example is to detect 5 negative and 5 positive sera on the same batch of coated ELISA plates and three batches of coated ELISA plates, with each serum repeated in three wells. The reproducibility of the method within and between batches is judged according to the coefficient of variation (CV), and the calculation formula of the coefficient of variation is as follows. The time results of this example are shown in Table 6.

[0128] Coefficient of variation (CV) = standard deviation / average value.

[0129] As can be seen from Table 6, using CCEV-99 recombinant protein coated ELISA plates, the within-batch coefficient of variation is 1.31%ˉ4.89%, and the between-batch coefficient of variation is 2.09%ˉ4.54%. The within-batch and between-batch test coefficients of variation are both less than 5%, indicating that the method has good reproducibility.

[0130] Table 6 Reproducibility test results

[0131]

[0132] Example 12 Detection of clinical samples

[0133] 100 camel sera from Altai region were detected using the established ELISA detection method.

[0134] As Figure 7 shown, the flow test results showed that the average serum positive rate of camel infectious pustular disease virus in the surveyed area was 16.69% (311 / 1863), among which Fuhai County was 15.83% (104 / 657), Jimunai County was 17.44% (158 / 906), and Habaxi County was 16.33% (49 / 300). The average serum positive rates of the three counties were not significantly different (P>0.05).

[0135] As Figure 8As shown, further analysis of serum positive condition of adult camels and young camels showed that the average serum positive rate of adult camels was 18.07% (281 / 1,555), which was significantly higher than the average serum positive rate of young camels, i.e. 9.74% (30 / 308) (P<0.05). Among them, the average serum positive rate of adult camels in Fukhai County was 17.20% (96 / 558), which was significantly higher than the average serum positive rate of young camels, i.e. 8.08% (8 / 99) (P<0.05). The average serum positive rate of adult camels in Jimenai County was 18.36% (141 / 768), and the average serum positive rate of young camels was 12.32% (17 / 138), and the difference was not significant (P>0.05). The average serum positive rate of adult camels in Habahai County was 19.21% (44 / 229), which was significantly higher than the average serum positive rate of young camels, i.e. 7.04% (5 / 71) (P<0.05).

[0136] Among the collected 80 camel blood samples, the clinical observation positive rate was 32.50% (24 / 80), the fluorescent quantitative PCR identification positive rate was 47.50% (38 / 80), 38 positive camels completely contained 24 positive camels observed clinically, and 14 camels without clinical symptoms were detected. The ELISA identification positive rate was 53.75% (43 / 80).

[0137] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. CCEV-99 recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. An application of a CCEV-99 recombinant protein, characterized in that, It can be used to detect antibodies against camel infectious pustular virus.

3. An indirect ELISA detection kit for detecting antibodies against camel infectious pustular virus, characterized in that, Includes an ELISA plate coated with the CCEV-99 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 CCEV-99 recombinant protein is 1-2 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, 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 detection is performed using the kit described in any one of claims 3-5, and the detection method includes the following steps: The ELISA plate coated with the CCEV-99 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.

7. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 6, characterized in that, The serum sample to be tested was diluted with blocking buffer at a volume ratio of 1:400-800; the enzyme-labeled secondary antibody was diluted with blocking buffer at a volume ratio of 1:15000.

8. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 6, characterized in that, The sealing time is 120 minutes, and the first incubation time is 60 minutes.

9. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 6, characterized in that, The second incubation time was 30 minutes, and the color development time was 15 minutes.

10. The indirect ELISA detection method for camel infectious pustular virus antibodies according to claim 6, 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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