Indirect ELISA kit for detecting bovine nodular skin disease virus antibody

Abstract

The invention provides an indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting a bovine nodular skin disease virus antibody, the kit comprises a coating antigen, and the coating antigen is bovine nodular skin disease virus 095 protein with an amino acid sequence as shown in SEQ ID NO.1. The kit disclosed by the invention has the characteristics of high sensitivity, strong specificity and good stability, can be used for evaluating LSDV infection and vaccine immune conditions, and has important significance on effective prevention and control of epidemic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an indirect ELISA kit for detecting antibodies against lumpy skin disease virus of cattle. Background Art

[0002] Lumpy Skin Disease (LSD) of cattle is an acute, subacute or chronic infectious disease caused by lumpy skin disease virus, and its main characteristics are fever, skin nodules, lymph node enlargement and decreased milk production. Since it was first reported in Zambia in 1929, the disease has spread to many regions such as Africa, the Middle East, Europe and Asia, causing serious economic losses to the global cattle industry. LSD is listed as a notifiable animal disease by the World Organization for Animal Health, and its prevention, control and diagnosis have attracted much attention. In 2019, the epidemic of lumpy skin disease in cattle was first confirmed in China, which has attracted wide attention. It is of great practical significance to strengthen the research, prevention and control of this disease.

[0003] Lumpy Skin Disease Virus (LSDV) is a double-stranded DNA virus. The virus particles are oval-shaped and have an envelope. Its genome size is about 151 kb and encodes 156 open reading frames. It has a homology of more than 98% with Sheeppox virus (SPPV) and a homology of up to 96% with Goatpox virus (GTPV). It has a high homology in antigenicity and gene sequence, but there are certain differences in aspects such as host range and pathogenicity.

[0004] In China, GTPV live vaccine and LSDV inactivated vaccine are currently used for immunization. Therefore, developing a good method with cross-immunity is an important means to evaluate the immunization effect of current vaccines. At present, there is still no relatively mature national standard detection kit. As the gold standard for LSD diagnosis, VNT takes a long time and requires a high-level biosafety laboratory. Moreover, the false positive rate of the kits on the market is high, and the evaluation of the detection effect of LSDV antibodies is not good.

[0005] Fu Cun, Screening of Targets for Detecting Antibodies Against Lumpy Skin Disease Virus of Cattle and Establishment of an ELISA Antibody Detection Method [D], Master's Thesis of Inner Mongolia University in 2023. By analyzing the functions of the 156 ORFs encoded by LSDV, 7 proteins encoded by the virus were selected for evaluation. Finally, the L5 protein with the best immunogenicity was selected as the coating antigen to establish an indirect ELISA detection method for detecting LSD antibodies, but the sensitivity of this method is not ideal.

[0006] The LSDV 095 protein is a core structural protein of virus particles encoded by ORF 095 in the LSDV genome. Its amino acid sequence has a high similarity with the homologous genes of GTPV and SPPV, and its sequence is highly conserved in the family Poxviridae. Currently, there is no report on the detection of LSDV antibodies using the 095 protein. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the object of the present invention is to provide an indirect ELISA kit for detecting antibodies against bovine nodular dermatitis virus.

[0008] The present invention provides an indirect ELISA kit for detecting antibodies against bovine nodular dermatitis virus. The kit contains a coated antigen, and the coated antigen is the 095 protein of bovine nodular dermatitis virus with the amino acid sequence shown in SEQ ID NO.1.

[0009] Among them, the coding nucleotide sequence of the 095 protein of bovine nodular dermatitis virus is shown in SEQ ID NO.2.

[0010] Among them, the preparation method of the 095 protein of bovine nodular dermatitis virus is as follows:

[0011] Clone the 095 sequence with the nucleotide sequence shown in SEQ ID NO.2 into the expression vector pET-28a, transform it into BL21(DE3) competent cells, add IPTG for induction expression, and after denaturation and renaturation of the expression product, obtain purified 095 recombinant protein.

[0012] Among them, the coating concentration of the 095 protein of bovine nodular dermatitis virus in the kit is 0.5 μg / ml.

[0013] Among them, the kit also optionally includes at least one of enzyme-labeled antibody, positive control serum, negative control serum, serum sample diluent, washing solution, chromogenic solution, and termination solution.

[0014] Among them, the enzyme-labeled antibody is a secondary antibody labeled with HRP.

[0015] Among them, the method includes the step of coating the 095 protein of bovine nodular dermatitis virus on an ELISA microtiter plate.

[0016] The present invention also provides the application of the above kit in the detection of antibodies against bovine nodular dermatitis virus.

[0017] The present invention also provides an immunogen, which includes the 095 protein with the amino acid sequence shown in SEQ ID NO.1, or the 095 gene with the nucleotide sequence shown in SEQ ID NO.2.

[0018] The present invention successfully expressed the LSDV 095 protein and obtained the corresponding polyclonal antibody. An indirect ELISA detection method based on the LSDV 095 protein antibody was successfully established and applied, which has a higher accuracy than commercial kits. It can only react with positive sera of LSDV and GPTV, has good cross-reactivity with viruses in the genus Capripoxvirus, and does not cross-react with positive sera of other viruses.

[0019] The indirect ELISA kit prepared with the 095 recombinant protein as an antigen in the present invention can effectively detect antibodies against lumpy skin disease virus. It has a low antigen coating amount and high sensitivity, and is still positive when the positive serum is diluted to 1:6400. It has the characteristics of high sensitivity, strong specificity and good stability, and can be used for large-scale detection of LSDV antibodies and epidemiological investigations, which is of great significance for effective epidemic disease prevention and control.

[0020] SEQ ID NO.1 LSDV 095 protein

[0021] MDFMKKYTKDLETTVKNKKDEEIASTSNLINNTSVTLTDVDTMLKSKEHLYQQMMMNQLEEKKTLKIKNIEIKNNSNKLNDQCSEKKQNDPLKKIKSISHDELVKELKDIKDKTKSLQDDSDSLIKDISVAKDTTFDAINSIMNDLKKRFNIDKLDDNNSK

[0022] SEQ ID NO.2 Coding nucleotide sequence of LSDV 095 protein

[0023] ATGGACTTCATGAAAAAATATACTAAAGATTTAGAAACAACGGTAAAGAATAAAAAAGATGAGGAAATAGCATCTACTTCAAATTTAATTAACAACACATCTGTTACATTAACTGATGTAGATACTATGTTAAAAAGTAAAGAACATTTATATCAACAAATGATGATGAATCAATTGGAAGAAAAAAAAACATTAAAAATCAAAAATATAGAAATCAAAAACAACAGTAATAAACTCAACGATCAATGTAGTGAAAAAAAACAAAATGATCCGTTAAAAAAAATAAAATCTATTAGCCATGATGAACTAGTAAAGGAACTGAAAGATATAAAAGATAAAACTAAATCACTTCAAGATGATTCTGATTCACTTATTAAAGATATTTCAGTTGCTAAAGATACAACTTTTGATGCTATAAACTCAATTATGAATGACTTAAAAAAGAGATTTAATATAGACAAACTGGATGATAATAACAGCAAATAA Description of the Drawings

[0024] Figure 1 For the restriction enzyme digestion identification of the recombinant plasmid; M: DNA molecular weight standard; 1: pET-28a-LSDV095.

[0025] Figure 2 For the effect of IPTG concentration gradient on the expression of LSDV 095 recombinant protein; M: Prestained protein molecular weight standard; 1: Empty vector control; 2: Uninduced recombinant bacteria control; 3 - 7: IPTG final concentration gradient induction groups (0.2, 0.4, 0.6, 0.8, 1.0 mM).

[0026] Figure 3 For the effect of time gradient on the expression of LSDV 095 recombinant protein; M: Prestained protein molecular weight standard; 1: Empty vector control; 2: Uninduced recombinant bacteria control; 3 - 7: Different time gradient induction groups (2, 4, 6, 8, 10 h).

[0027] Figure 4 For the solubility analysis of LSDV 095 recombinant protein; M: Prestained protein molecular weight standard; 1: Supernatant of the first sonication (12000 rpm, 15 min); 2: Precipitate of the first sonication (resuspended in PBS); 3: Supernatant of the second sonication (12000 rpm, 15 min).

[0028] Figure 5 For the purification verification of LSDV 095 protein; M: Protein Marker; 1: Penetrating solution; 2: Washing solution; 3 - 8: Elution solution.

[0029] Figure 6 For the Western blot verification of LSDV 095; M: Protein Marker; 1: LSDV 095 protein. Specific implementation manners

[0030] The present invention will be further described in detail below in conjunction with embodiments, but the present invention is not limited thereto.

[0031] For those not specifying specific technologies or conditions in the embodiments, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. The culture media, reagents and solutions used are all commercially available products, or can be prepared by methods known in this field.

[0032] Example 1

[0033] 1 Materials and methods

[0034] 1.1 Viruses and sera

[0035] Sheeppox virus, positive and negative sera of bovine nodular dermatitis, positive serum of bovine Newbury virus, positive serum of bovine norovirus, positive serum of bovine parainfluenza virus, positive serum of bovine infectious rhinotracheitis virus, positive serum of bovine coronavirus, positive serum of bovine viral diarrhea virus, etc. are all provided by this laboratory.

[0036] 1.2 Main reagents and consumables

[0037] BL21(DE3) competent cells, E.coli (DH5α) competent cells, restriction endonucleases EcoRⅠ and HindⅢ, T4 ligase, pMD19 - T vector are purchased from TaKaRa Biological Company; agar is purchased from Sangon Biotech (Shanghai) Co., Ltd.; GoldView nucleic acid dye is purchased from Zhonghui Hechai Biomedicine Technology Co., Ltd., DNA Marker is purchased from Rongwei Gene Biotechnology Co., Ltd., QuickTaq TMThe HSDyeMix was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; LB agar and LB broth were purchased from Hangzhou Microbial Reagent Co., Ltd.; the agarose gel recovery kit and plasmid miniprep kit were purchased from OMEGA Bio-Tek; the One-Step PAGE Gel Fast Preparation Kit was purchased from Nanjing Novozymes Biotech Co., Ltd., and fetal bovine serum was purchased from Beijing Quanshijin Co., Ltd.; antibiotics (kanamycin, ampicillin), skimmed milk powder, imidazole, sodium dodecyl sarcosinate, PEG6000, PEG20000 were purchased from Solarbio; nickel columns were purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; Tween 20, urea, arginine, bovine serum albumin were purchased from Saiguo Biotechnology Co., Ltd.; tris(hydroxymethyl)aminomethane was purchased from Beijing Bio-Top Technology Co., Ltd.; sodium chloride, potassium chloride, sodium hydroxide, methanol, etc. were purchased from Chengdu Kelong Chemical Co., Ltd.; IPTG and protease inhibitor mixture (general type) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the enhanced chemiluminescence (ECL) substrate was purchased from Semeibio Co., Ltd.; rabbit anti-bovine IgG antibody (IgG-HRP) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd.; protein Marker was purchased from Thermo Fisher Scientific; protein loading buffer (5×) and BCA protein concentration assay kit were purchased from Boster Biological Technology Co., Ltd.; PVDF membranes were purchased from Millipore; Tris-HCl buffer and carbonate buffer were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; PBS and TBS were purchased from Chengdu Duyou Biotechnology Co., Ltd.;

[0038] The sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the sequences are as follows:

[0039] 095F EcoRⅠ: CCGGAATTCATGGACTTCATGAAAAAA

[0040] 095R HindⅢ: CCCAAGCTTTTATTTGCTGTTATTATC

[0041] 1.3 Amplification of target gene, construction and identification of recombinant expression plasmid

[0042] The viral genomic DNA was extracted from the live goatpox vaccine by the phenol-chloroform extraction method, and a 50 μL PCR amplification system was established. After the amplification products were verified by 1% agarose gel electrophoresis, they were sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. The successfully sequenced PCR amplification products were purified by the OMEGA gel extraction kit and ligated to the pMD-19T vector. The recombinant plasmids confirmed by sequence alignment were named pMD-19T-LSDV 095 respectively. The recombinant plasmids were digested with EcoR I / Hind III double enzymes, and at the same time, the pET-28a(+) vector was digested with the same restriction enzymes. After separation by 1% agarose gel electrophoresis, the target fragments and the linearized vectors were recovered by cutting the gel, and ligated overnight at 16 °C with T4 DNA ligase to construct the pET-28a(+)-LSDV 095 expression vector. According to the standard molecular cloning procedure, the ligation products were transformed into Escherichia coli BL21(DE3) competent cells, and the recombinant plasmids were obtained by the plasmid extraction kit. The pET-28a-LSDV 095 was verified by double digestion with the restriction enzymes EcoR I / Hind III. After confirming the correct insertion of the target gene by the nucleic acid electrophoresis detection system, the positive clone plasmids were sent to Shanghai Sangon Biological Engineering Co., Ltd. for bidirectional sequencing verification.

[0043] 1.4 Expression and purification of recombinant LSDV 095 protein

[0044] A systematic optimization protocol for recombinant protein induction expression was adopted: single colonies were picked and inoculated into LB medium containing Kan for three-level amplification culture. When the OD 600 of the culture broth reached 0.6, IPTG was added for induction, and at the same time, a triple control system was set up: an uninduced recombinant bacteria control group, an empty vector pET-28a(+) induction group, and an expression control group. The bacterial solutions of each group were placed under the conditions of 37 °C and 220 rpm for induction culture.

[0045] 1.5 Optimization of the expression conditions of recombinant LSDV 095 protein

[0046] First, an IPTG concentration gradient experiment was carried out. At OD 600In the BL21(DE3) / pET-28a-LSDV recombinant bacterial culture system with a value of 0.6, IPTG with final concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mM was added respectively, and continuous induction was carried out at 37 °C for 10 h. After collecting the bacterial cells by centrifugation at 12000 rpm, SDS-PAGE detection was performed, and the gray value of the target band was quantitatively analyzed through a gel imaging system to determine the optimal induction concentration. Based on the concentration optimization results, a time gradient experiment was then carried out: in the same induction system, the optimal induction concentration of IPTG was added, and bacterial liquid samples (100 μL) were collected at 2, 4, 6, 8, and 10 h of induction respectively. Samples at each time point were subjected to SDS-PAGE detection, and Coomassie brilliant blue staining was used to determine the optimal induction duration.

[0047] 1.6 Solubility analysis of recombinant LSDV 095 protein expression

[0048] After culturing 100 mL of recombinant plasmid bacterial liquid according to the optimized induction conditions, the following protein extraction and detection operations were carried out: First, the bacterial cells were collected by centrifugation (12000 rpm, 10 min). After the bacterial cells were ultrasonically disrupted, centrifugation was carried out again to remove cell debris (8000 rpm, 10 min), and 100 μL of the supernatant and the precipitate component resuspended in PBS were collected respectively for standby. When preparing the protein sample, 100 μL of the sample to be tested was mixed with 20 μL of 5× protein loading buffer in equal volume, and after boiling water bath treatment for 7 min, it was cooled. 10 μL of the denatured sample was taken for 15% separating gel SDS-PAGE electrophoresis analysis, and Coomassie brilliant blue staining method was used to detect the recombinant protein expression level.

[0049] 1.7 Purification of recombinant LSDV 095 protein

[0050] According to the optimized induction conditions, the recombinant bacteria were inoculated into 600 mL of LB liquid medium containing Kan for large-scale induction expression. The Ni-NTA affinity chromatography system was used for recombinant protein purification, and each purified component was analyzed by SDS-PAGE electrophoresis. The component with a purity of the target band > 95% was selected for renaturation treatment. The target eluate was placed into a dialysis bag with a molecular weight cut-off of 10 kDa and gradient dialysis was carried out at 4 °C. After the renatured protein was ultrafiltered and concentrated by PEG20000, the protein concentration was measured by the BCA method to calculate the final concentration of the recombinant protein.

[0051] 1.8 Identification of recombinant LSDV 095 protein

[0052] For the Western-Blotting analysis of the recombinant protein, the sheep high-titer immune serum was diluted 1:100 with TBST, and the HRP-labeled rabbit anti-sheep IgG antibody (dilution ratio was 1:5000) was used. Gently oscillate and incubate at 37 °C for 2 h. The PVDF membrane was scanned using a scanner.

[0053] 1.11 Animal immunization and determination of polyclonal antibody titer

[0054] To obtain the positive serum for establishing the indirect ELISA antibody detection method, the purified LSDV 095 protein was mixed with an equal volume of 201 adjuvant and used to immunize beef cattle at a ratio of 1 mg / head. Seven to ten days after the third immunization, blood was collected from the tail vein to isolate serum, and the polyclonal antibody titer of the highly immunized serum against LSDV 095 was determined by the indirect ELISA detection method. Combining the principles of statistics and the instructions of some commercial ELISA kits, if the OD value of the negative control is low and the standard deviation is small, 2.1 times is used as the critical value S / N (sample OD value / negative control mean) to balance sensitivity and specificity.

[0055] 1.9 Determination of the optimal protein coating concentration and serum dilution

[0056] The purified 095 protein was diluted with coating buffer using a BCA protein concentration assay kit, and the final concentrations were 2.0, 1.0, and 0.5 μg / mL respectively. To determine the optimal antigen coating concentration and primary antibody dilution, the checkerboard method was used. Three replicate wells were coated with each concentration, 100 μL per well. The coated ELISA plate was placed in a 4°C refrigerator overnight to promote antigen immobilization. The remaining steps were as described in 2.1. 100 μL of diluted LSDV positive and negative sera were added longitudinally. The sera were diluted at 1:50, 1:100, 1:200, and 1:400 respectively. Three replicate controls were set for the positive and negative sera at each concentration. Finally, the OD450nm value was immediately read on an ELISA reader, and the positive / negative OD450nm value (P / N value) was calculated. The condition with the largest P / N value was taken as the optimal condition.

[0057] 1.10 Determination of the optimal antigen coating solution type

[0058] According to the determined antigen coating concentration and serum dilution, three types of antigen coating solutions were set, namely phosphate buffer (PBS), Tris-HCL, and carbonate buffer (CBS). Three replicate controls were set for the positive and negative sera in each group. By comparing the P / N values, the condition with the largest P / N value was taken as the optimal condition to determine the best antigen coating solution.

[0059] 1.11 Determination of the optimal antigen coating time

[0060] According to the determined optimal antigen coating concentration and coating solution type, the ELISA plate was coated in this study, and two different coating conditions were set, namely: overnight coating at 4°C and coating at 37°C for 2 h. Three replicate controls were set for the positive and negative sera at each coating time. By comparing the P / N values, the condition with the largest P / N value was taken as the optimal condition to determine the best antigen coating time.

[0061] 1.12 Determine the type of the optimal primary antibody diluent

[0062] According to the determined coating antigen concentration and coating conditions, three types of primary antibody diluents were set, namely PEG6000, 1% BSA, and PBST. Positive and negative sera were set in each group to make three duplicate controls respectively. By comparing the P / N values, the result with the largest P / N value was taken as the optimal condition to determine the optimal primary antibody diluent.

[0063] 1.13 Determine the type of the optimal blocking solution

[0064] According to the determined coating antigen concentration, coating conditions and the type of primary antibody diluent, five types of blocking solutions were set, namely 4% PEG6000, 1% and 2% BSA (diluted with PBST), PBS, 5% skim milk powder (diluted with PBST). Positive and negative sera were set in each group to make five duplicate controls respectively. By comparing the P / N values, the result with the largest P / N value was taken as the optimal condition to determine the optimal blocking solution.

[0065] 1.14 Determine the dilution ratio of the optimal secondary antibody

[0066] Under the above optimized conditions, four different dilution ratios of the secondary antibody were set, which were 1:4000, 1:6000, 1:8000, and 1:10000 in sequence. Positive and negative sera were set to make three duplicate controls respectively. By comparing the P / N values, the result with the largest P / N value was taken as the optimal condition to determine the dilution ratio of the optimal secondary antibody.

[0067] 1.15 Determine the optimal incubation time

[0068] Under the above optimized conditions, the incubation time was optimized (1.0 h, 1.5 h, 2.0 h). Positive and negative sera were set to make three duplicate controls respectively. By comparing the P / N values, the result with the largest P / N value was taken as the optimal condition to determine the optimal incubation time.

[0069] 1.16 Determine the color development time

[0070] Under the above optimized conditions, three different color development times (10 min, 20 min, 30 min) were set. Positive and negative sera were set to make three duplicate controls respectively. The remaining operations were carried out according to 2.1. By comparing the P / N values, the result with the largest P / N value was taken as the optimal condition to determine the optimal color development time.

[0071] 1.17 Specificity test

[0072] The cross-reactivity was verified by the indirect ELISA method established in this research: the positive sera of GTPV, NoV, NeV, BPIV, BCoV, IBRV, and BVDV were detected respectively, and a negative control (healthy bovine serum) and a positive control of LSDV were established simultaneously. The OD450nm value was measured by an enzyme-labeled instrument. Samples with an OD450nm value greater than 2.1 times that of the negative serum were judged as positive to verify the specificity of the detection method.

[0073] 1.18 Repeatability test

[0074] Within-batch repeatability test

[0075] The same batch of purified recombinant protein was used as the coating antigen for solid-phase coating on a 96-well enzyme-labeled plate (overnight at 4°C). Strictly following the optimized indirect ELISA detection parameters, the OD450 absorbance of 8 LSDV positive sera (including 3 immune sera) and 2 healthy bovine negative sera was detected, and all samples were set with 3 replicate controls.

[0076] Between-batch repeatability test

[0077] Three batches of independently purified recombinant antigens were used to coat 96-well enzyme-labeled plates. Strictly referring to the optimized indirect ELISA detection parameters, 8 LSDV positive sera (including 3 immune sera) and 2 healthy bovine negative control sera were detected, and 3 replicate controls were set for each batch of experiments.

[0078] 1.19 Sensitivity experiment

[0079] The sensitivity of the detection system was evaluated by the two-fold serial dilution method: the LSDV positive / negative control sera were serially diluted at a starting concentration of 1:100 (PBS diluent) for 7 gradients (1:100 - 1:12800). Referring to the optimized indirect ELISA parameters, the OD 450 value of each dilution was measured using an enzyme-labeled instrument, and 3 replicate controls were set. With an S / N value ≥ 2.1 as the positive determination threshold, the lowest detection limit was calculated.

[0080] 1.20 Detection of non-immunized negative sera in 2020 by a commercial kit and the indirect ELISA antibody detection method for LSDV 095 protein

[0081] According to the instructions of the commercial kit and the indirect ELISA antibody detection method based on the LSDV 095 protein established in this study, negative sera from Ganzi Tibetan Autonomous Prefecture, Sichuan Province were detected respectively, and the detection rates of LSDV by the two methods were compared. Ten serum samples randomly selected as positive by the commercial kit were subjected to neutralization tests. The serum samples were diluted from 1:2 to 1:256 and incubated with GTPV, then inoculated onto Vero cells and maintained in a constant temperature incubator at 37°C. The 96-well cell culture plates were taken out every day, and the infection status of cells in each well was observed under a microscope. According to previous experience, cytopathic effect (CPE) of Vero cells appeared at 72 h, and the CPE was stable after 96 h, and the neutralizing antibody could be determined. The CPE was observed and recorded day by day until the results were stable.

[0082] 1.21 Detection of LSDV non-immunized serum samples by the indirect ELISA antibody detection method for LSDV 095 protein

[0083] The indirect ELISA method established in the present invention was used for serological detection of 1863 healthy non-immunized yak serum samples from Ganzi Tibetan Autonomous Prefecture, Sichuan Province (542 serum samples from Ganzi County from 2023 to 2024, 724 serum samples from Dege County from 2023 to 2024, 297 serum samples from Luhuo County in 2024, and 300 serum samples from Litang County in 2024).

[0084] 2 Results

[0085] 2.1 Amplification of the target gene, construction and identification of the recombinant expression plasmid

[0086] To obtain ORF095, viral DNA was extracted from the live goatpox vaccine in this study for PCR amplification of the gene. To ligate ORF095 to the cloning vector, the successfully sequenced PCR product was recovered and purified by a kit and ligated to the cloning vector pMD-19T, and then transformed into the competent cell DH5α. The successfully transformed recombinant plasmid was named pMD-19T-LSDV095. After the recombinant plasmid was amplified in culture, the plasmid was extracted by a kit and double digested with the pET-28a(+) vector stored in the laboratory and then ligated and transformed into DH5α. To verify the successful ligation of the recombinant expression plasmid, the recombinant plasmid was double digested with restriction endonucleases in this study, and corresponding target gene bands and plasmid bands could be observed in each lane. The results were as Figure 1 shown, fragments of about 5400 bp and 486 bp were obtained respectively, which were consistent with the expected sizes. The above results indicate that the LSDV095 gene has been correctly inserted into the prokaryotic expression vector pET-28a(+).

[0087] 2.2 Optimization of the optimal IPTG induction concentration

[0088] To optimize the optimal IPTG induction concentration of LSDV recombinant protein, in this study, the pET-28a-LSDV 095 recombinant plasmid was transformed into BL21(DE3) competent cells for induced protein expression. For LSDV 095 protein, when the OD600nm was 0.6, different final concentrations of inducer were added at the same induction time, and the results were as Figure 2 As analyzed by SDS-PAGE, there were differences in the expression levels of the target protein. By comparison, it can be seen that under the condition of the same induction time, when inducing LSDV 095 protein with 0.8 mmol / L IPTG, the expression content was the highest.

[0089] 2.3 Optimization of the optimal induction time

[0090] To optimize the optimal induction time of LSDV recombinant protein, when the OD of the recombinant bacterial culture 600 reached 0.6, IPTG with a final concentration of 0.8 mM was added for induction (37 °C, 220 rpm). The results were as Figure 3 shown. SDS-PAGE analysis showed that the expression level of LSDV 095 recombinant protein reached the peak at 10 h. The above results indicate that the optimal induction time for LSDV 095 recombinant bacteria is 10 h to obtain the highest expression level of protein.

[0091] 2.4 Solubility analysis and identification of induced expression of recombinant protein

[0092] To identify the expression form of the recombinant protein, in this study, after inducing the expression of 100 mL of the culture of LSDV 095 recombinant bacteria by using the combined technology of ultrasonic disruption and centrifugation, ultrasonic disruption was carried out under ice bath conditions to separate the supernatant and precipitate components. The results were as Figure 4 SDS-PAGE analysis showed that significant bands of LSDV 095 protein appeared in the precipitate phase, and the molecular weight was 23 kDa. The above results indicate that through solubility analysis, it is confirmed that the LSDV 095 recombinant protein is mainly expressed in the form of inclusion bodies.

[0093] 2.5 Large-scale expression and purification of recombinant LSDV 095 protein

[0094] To obtain a large amount of LSDV recombinant protein, in this study, according to the optimal induction conditions, after inducing the large-scale expression of the recombinant protein, the induced sample was processed and filtered, and the recombinant protein was purified using the Ni-NTA affinity chromatography system, and all the collected components were verified by SDS-PAGE. The results were as Figure 5 shown. The SDS-PAGE results showed that the recombinant protein was all located in the eluate, and the band size was about 23 kDa, which was consistent with the size of the target band. The above results indicate that the purification of LSDV recombinant protein was successful.

[0095] To obtain high-purity recombinant protein for animal immunization and establish an indirect ELISA antibody detection method using it as an antigen. In this study, the fraction with the highest purity was dialyzed and refolded using a dialysis bag, and the refolded protein was concentrated with PEG20000. According to the BCA method, the concentration of the purified and concentrated LSDV 095 recombinant protein was measured, and a standard curve was established. The standard curve equation was y = 0.0005x + 0.0116, R 2 = 0.9984 > 0.995, which proved that the established standard curve was reliable. The results showed that the concentration of the 095 protein after purification was 619.6 μg / mL. The purified protein was aliquoted and stored frozen for animal immunization to prepare hyperimmune serum and provide antigen for establishing the LSDV indirect ELISA method. The above results indicated that through purification, we could obtain the recombinant protein required for animal immunization and establishing an indirect ELISA antibody detection method.

[0096] 2.6 Identification of recombinant LSDV 095 protein

[0097] To identify the antigenicity of the recombinant protein, this study used the Western-Blot method to verify the protein expression of the recombinant bacteria. Sheep hyperimmune serum was used as the primary antibody at a dilution ratio of 1:100, and rabbit anti-sheep enzyme-labeled secondary antibody was diluted at a ratio of 1:5000. As Figure 6 the WB results showed that the band was consistent with the expectation, and LSDV 095 was around 23 kDa. The above results indicated that the purification of recombinant LSDV 095 protein was successful and it had good antigenicity.

[0098] 2.7 Determination of the titer of animal polyclonal antibodies

[0099] To obtain hyperimmune serum against LSDV 095, this study immunized beef cattle. 7 - 10 days after the third immunization, the indirect ELISA detection method was used to measure the titer of LSDV 095 polyclonal antibodies in the immunized dairy cow serum. The OD greater than 2.1 times that of the negative serum was determined as positive. The results are shown in Table 1. When the hyperimmune serum against LSDV 095 protein was diluted to 1:51200, the OD 450nm was greater than the OD of the negative serum 450nm and 450nm (the OD of the negative serum against LSDV 095 protein 450nm was 0.16 ± 0.04). The above results indicated that the antibody titer of LSDV 095 protein was 1:51200, and immunizing beef cattle with LSDV 095 protein could induce a relatively high antibody level in beef cattle. The immunized bovine serum was aliquoted and used to establish an indirect ELISA antibody detection method.

[0100] Table 1 Detection of the titer of hyperimmune serum of cattle immunized with LSDV 095 protein

[0101]

[0102] 2.8 Determination of the optimal protein coating concentration and serum dilution factor

[0103] To determine the optimal coating concentration of LSDV 095 protein and serum dilution factor, in this study, the purified recombinant protein was serially diluted at three gradients of 2.0, 1.0, and 0.5 μg / mL with ELISA coating buffer. The positive and negative sera were serially diluted 2-fold from 1:50 to 1:400 with 1% BSA. After dilution, the experiment was carried out by the checkerboard titration method with 3 replicates within each group. The results are shown in Table 2. In the experiment, the optimal coating concentration of LSDV 095 protein was 0.5 μg / mL, the serum dilution factor was 1:100, and the highest P / N value was 23.26. The above results indicate that the optimal protein coating concentration of LSDV 095 protein is 0.5 μg / mL, and the serum dilution factor is 1:100.

[0104] Table 2 Establishment of the optimal coating concentration of LSDV 095 protein and serum dilution factor

[0105]

[0106] 2.9 Determination of the optimal type of antigen coating buffer

[0107] To determine the optimal type of antigen coating buffer for LSDV recombinant protein, in this study, the optimal coating concentration of 0.5 μg / mL of LSDV 095 protein was used to coat the ELISA plates, 100 μL per well, with a serum dilution ratio of 1:100, and other conditions remained unchanged. The antigen coating buffer was optimized. Each group was set with 3 replicate controls. The results are shown in Table 3. The P / N value of the PBS group for the LSDV 095 protein coating buffer was the highest, at 43.05. The above results indicate that for LSDV 095 protein, PBS is the best antigen coating buffer.

[0108] Table 3 Establishment of the optimal coating buffer for LSDV095 recombinant protein

[0109]

[0110] 2.10 Determination of the optimal antigen coating time

[0111] To determine the optimal antigen coating time for LSDV recombinant protein, in this study, the optimal coating concentration of 0.5 μg / mL of LSDV 095 protein was used to coat the ELISA plates, 100 μL per well, with a serum dilution ratio of 1:100, and other conditions remained unchanged. The antigen coating time was optimized. Each group was set with 3 replicate controls. As shown in Table 4, the results showed that the highest P / N value of 54.47 was obtained when the optimal antigen coating time of LSDV 095 was overnight at 4°C, indicating that the antigen coating effect of LSDV 095 protein is the best when coated overnight at 4°C.

[0112] Establishment of the optimal coating time for LSDV protein

[0113]

[0114] 2.11 Determine the type of the optimal primary antibody diluent

[0115] To determine the type of the optimal primary antibody diluent for LSDV 095 protein, in this study, the ELISA plates were coated with the optimal coating concentration of 0.5 μg / mL of LSDV 095 protein, 100 μL per well. The serum dilution ratio was 1:100, and other conditions remained unchanged. The antigen coating solution was optimized, and 3 replicates were set in each group. The results are shown in Table 5. The P / N value of the 1% BSA group, the optimal primary antibody diluent for LSDV 095 protein, was the highest at 13.45. The above results indicate that the effect is the best when the optimal primary antibody diluent for LSDV 095 protein is 1% BSA.

[0116] Table 5 Establishment of the type of the optimal primary antibody diluent for LSDV protein

[0117]

[0118] 2.12 Determine the type of the optimal blocking solution

[0119] To determine the type of the optimal blocking solution, 3 groups of blocking solutions with different types and concentrations were set in this study, and 3 replicates were set in each group. As shown in Table 6, the results showed that the P / N value of the 1% BSA group for LSDV 095 protein was the highest at 24.95. The above results indicate that the effect is the best when the blocking solution for LSDV 095 protein is 1% BSA.

[0120] Table 6 Establishment of the type of the optimal blocking solution for LSDV protein

[0121]

[0122] 2.13 Determine the optimal secondary antibody dilution ratio

[0123] To determine the optimal secondary antibody dilution ratio, 4 different secondary antibody dilution ratios were set in this study, and 3 replicates were set in each group. As shown in Table 7, for LSDV 095, the secondary antibody dilution ratio was 1:8000, and the average P / N value was the highest at 40.27. The above results indicate that the effect is the best when the secondary antibody dilution ratio for LSDV 095 protein is 1:8000.

[0124] Table 7 Establishment of the optimal secondary antibody dilution ratio for LSDV 095 protein

[0125]

[0126] 2.14 Determine the optimal incubation time

[0127] To determine the optimal incubation time, indirect ELISA assays were performed according to the optimal experimental conditions determined above, with other conditions remaining unchanged. To optimize the optimal incubation time, three control groups with reaction durations of 1.0 h, 1.5 h, and 2.0 h were set up, and each group had three replicate controls. The results are shown in Table 8. The P / N value of the incubation LSDV 0952 h group was the highest, at 21.05. The above results indicate that the best effect was obtained with an incubation time of 2 h.

[0128] Table 8 Establishment of the optimal incubation time for LSDV protein

[0129]

[0130] 2.15 Determination of the color development time

[0131] To determine the optimal color development time, according to the above optimal experimental conditions with other conditions unchanged, to optimize the optimal color development time, three different color development times (10 min, 20 min, 30 min) were set up, and each group had three replicate controls. The results are shown in Table 9. The P / N value of the LSDV 095 protein with an optimal color development time of 20 min was the highest, at 51.38, indicating that the optimal color development time for the LSDV 095 protein was 20 min.

[0132] Table 9 Establishment of the optimal color development time for LSDV protein

[0133]

[0134] 2.16 Specificity test

[0135] To verify the specificity of the established indirect ELISA antibody detection assay, the established indirect ELISA method was used in this study to detect bovine positive sera immunized with NeV, NoV, BPIV, etc. As shown in Table 10, the results showed that the OD 450nm values of the test samples were all less than 2.1 times that of the negative serum (the OD of the negative serum 450nm was 0.067), showing no significant difference from the negative serum, but significant differences from the LSDV positive serum and the GTPV positive serum. The above results indicate that this method has good specificity and good cross-immunity to capripoxviruses.

[0136] Table 10 Results of the specificity test of the LSDV recombinant protein

[0137]

[0138] 2.17 Repeatability test

[0139] 2.17.1 Intra-batch repeatability test

[0140] To verify the within - batch repeatability of the established indirect ELISA antibody detection assay, in this study, the same batch of purified LSDV 095 recombinant antigen was used to perform three repeated detections on 8 positive sera, immune sera, and 2 negative sera (1 - 8 are positive sera and immune sera, 9 - 10 are negative sera). The results are shown in Table 11. The coefficient of variation (CV) analysis between batches showed that the CV values of the LSDV 095 antigen group ranged from 0% to 6%. The above results indicate that the variation degree of the LSDV095 protein in the same batch of samples in the same batch of tests is small, proving that this method has good repeatability.

[0141] Table 11 Detection results of within - batch repeatability test of LSDV recombinant protein

[0142]

[0143] 2.17.2 Between - batch repeatability test

[0144] To verify the between - batch repeatability of the established indirect ELISA antibody detection assay, in this study, purified LSDV 095 recombinant antigens from different production batches were used to perform three repeated detections on 8 positive sera, immune sera, and 2 negative sera (1 - 8 are positive sera and immune sera, 9 - 10 are negative sera). The results are shown in Table 12. CV analysis showed that the CV values of the LSDV 095 antigen group ranged from 0% to 4%. The above results indicate that the variation degree of the LSDV095 protein in purified antigens from different batches in the same batch of tests is small, and the purification process has good repeatability.

[0145] Table 12 Detection results of between - batch repeatability test of LSDV recombinant protein

[0146]

[0147] 2.18 Sensitivity experiment

[0148] To verify the sensitivity of the established indirect ELISA antibody detection assay, in this study, the LSDV immune - positive sera and negative sera were serially diluted (1:100, 1:200,... 1:12800) and reacted under the optimal conditions. The results are shown in Table 13. When the positive sera were diluted to 1:6400, the critical S / N value ≥ 2.1 (OD of negative sera against LSDV 095 protein 450nm was 0.05 ± 0.00). The above results indicate that this method has good sensitivity.

[0149] Table 13 Results of sensitivity test of LSDV positive sera

[0150]

[0151] 2.19 Commercial kits and indirect ELISA antibody detection method for LSDV 095 protein to detect negative sera

[0152] To verify the differences between the indirect ELISA antibody detection kit for LSDV (Tianjin Baiwot, production batch number: 20240822) and the indirect ELISA antibody detection method established in this study, 46 negative sera from Sichuan Province were detected by two ELISA methods in this study.

[0153] The specific operation of the indirect ELISA method established in this study is as follows

[0154] (1) Dilute the recombinant protein LSDV 095 with coating buffer at 0.5 μg / mL, add 100 μL / well to the enzyme-linked immunosorbent assay (ELISA) plate, and coat overnight at 4°C.

[0155] (2) Discard the coating buffer, wash 3 times with 5% PBST, and pat the plate to remove residual liquid. Add 200 μL / well of 1% BSA and incubate at 37°C for 2 h.

[0156] (3) After blocking, wash with PBST (same as before), dilute the serum with 1% BSA primary antibody diluent, with a serum dilution ratio of 1:100, add 100 μL / well to the ELISA plate, place at 37°C for 2 h, discard the liquid, wash the plate 3 times and pat dry.

[0157] (4) After washing with PBST, add 100 μL / well of HRP-labeled rabbit anti-bovine IgG antibody working solution (1:8000) and incubate at 37°C in the dark for 2 h;

[0158] (5) After washing, add 100 μL / well of TMB chromogenic solution, react at room temperature in the dark for 20 min. Add 50 μL / well of 2 M H2SO4 to terminate the reaction. Immediately read the absorbance OD 450nm value.

[0159] The established indirect ELISA method was used to detect a large number of clinical samples and compared with the existing gold standard detection method. Through statistical analysis, it was found that when the S / P value ≥ 0.2 was used as the positive judgment standard, the coincidence rate with the gold standard method was relatively high, and it could accurately detect positive antibodies in clinical samples, with good clinical application value.

[0160] As shown in Table 14, the detection rate of the kit reached 82.61%, much higher than the domestic total prevalence rate. When using the indirect ELISA antibody detection method based on LSDV 095 protein established in this study to detect the same batch of negative sera, only 1 S / P value (sample OD value - negative control OD value / positive control OD value - negative control OD value) ≥ 0.2.

[0161] The above results indicate that the test results of the kit are unreliable and the false positive rate is high. Therefore, we need to use the neutralization test to determine the LSDV antibody titer of the serum samples that are positive in the kit test and compare the accuracy of the two methods.

[0162] To detect the LSDV antibody titer of the positive samples detected by the kit, 10 positive serum samples were randomly selected in this study for the neutralization test. The dilution ratio ranged from 1:2 to 1:256, and 3 replicates were made for each dilution ratio. The results showed that lesions occurred in all 10 samples at the dilution ratio of 1:2. The above results indicate that these 10 serum samples have no neutralization titer and are negative sera. The results of the neutralization test indicate that the indirect ELISA antibody test results of the commercial kit are inaccurate, and the indirect ELISA method established in this test has high accuracy and is suitable for the serological detection of LSDV antibodies.

[0163] Table 14 Detection results of LSDV negative sera in 2020

[0164]

[0165] LSD mainly occurs in the relatively hot regions in the south of China, and is more prevalent from August to October every year. The incidence of the disease in the epidemic area is 5% - 30%, while the detection rate of the kit is much higher than the detection rate in the domestic epidemic area. Moreover, LSD did not spread to Sichuan in 2020. Therefore, the sera collected in 2020 should be true negative sera, but the positive rate of the kit test results reached 82.61%. Therefore, it is considered that the kit test results are unreliable and the false positive rate is high.

[0166] 2.21 Detection of LSDV unimmunized serum samples by the indirect ELISA antibody detection method for LSDV 095 protein

[0167] To detect the positive rate of LSDV in 1863 yak serum samples from Sichuan Province from 2023 to 2024, the indirect ELISA method established in this study was used to detect 1863 healthy yak sera that had not been immunized with sheeppox vaccine.

[0168] The results are shown in Table 15. The total detection rate was 2.42%. Among them, the detection rate in County A was 1.85%, the detection rate in County B was 0.83%, the detection rate in County C was 9.76%, and the detection rate in County D was 0%. The above results indicate that the different detection results in different regions may be due to different infection situations. The detection results of the indirect ELISA antibody detection method based on LSDV 095 protein established in this study are in line with the domestic LSDV prevalence rate and are accurate.

[0169] Table 15 Detection results of clinically healthy yak serum samples in Sichuan Province from 2023 to 2024

[0170]

[0171] In summary, the present invention successfully expressed the LSDV 095 protein and obtained the corresponding polyclonal antibody. An indirect ELISA detection method based on the LSDV 095 protein antibody was successfully established and applied. The indirect ELISA kit prepared with the 095 recombinant protein as the antigen can effectively detect the antibodies against bovine nodular dermatitis virus, with a higher accuracy than the commercial kit. It can only react with the positive sera of LSDV and GPTV, and does not cross-react with the positive sera of other viruses.

Claims

1. An indirect ELISA kit for detecting antibodies against lumpy skin disease virus of cattle, characterized in that, The kit contains a coated antigen, and the coated antigen is the Lumpy skin disease virus 095 protein with the amino acid sequence shown in SEQ ID NO.

1.

2. The kit according to claim 1, wherein The coding nucleotide sequence of the Lumpy skin disease virus 095 protein is shown in SEQ ID NO.

2.

3. The kit according to claim 1, wherein The preparation method of the Lumpy skin disease virus 095 protein is as follows: Clone the 095 sequence with the nucleotide sequence shown in SEQ ID NO.2 into the expression vector pET-28a, transform it into BL21(DE3) competent cells, add IPTG for induction expression, and after denaturation and renaturation of the expression product, obtain the purified 095 recombinant protein.

4. The kit according to claim 1, characterized in that, The coating concentration of the Lumpy skin disease virus 095 protein in the kit is 0.5 μg / ml.

5. The kit according to any one of claims 1-4, characterized in that The kit also optionally includes at least one of an enzyme-labeled antibody, a positive control serum, a negative control serum, a serum sample diluent, a washing solution, a chromogenic solution, and a termination solution.

6. The kit according to claim 5, characterized in that, The enzyme-labeled antibody is a secondary antibody labeled with HRP.

7. A method for preparing the kit according to any one of claims 1-6, characterized in that: The method includes the step of coating the Lumpy skin disease virus 095 protein on an ELISA microtiter plate.

8. Use of the kit according to any one of claims 1-6 in the detection of antibodies against Lumpy skin disease virus.

9. An immunogen, characterized in that, It includes the 095 protein with the amino acid sequence shown in SEQ ID NO.1, or the 095 gene with the nucleotide sequence shown in SEQ ID NO.2.

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