Preparation and Application of Recombinant Porcine C-Reactive Protein and Monoclonal Antibody

Abstract

The present invention discloses a coding gene sequence and application of porcine C-reactive protein and monoclonal antibody. By optimizing the sequence, the porcine C-reactive protein gene is artificially synthesized, and the synthesized gene is inserted into the pET-28a vector, and then co-transformed into the BL21(DE3) host bacterium together with the chaperone plasmid pTF16 to co-express porcine C-reactive protein and the chaperone protein tig to promote the soluble expression of porcine C-reactive protein; a hybridoma cell is obtained by immunizing with this recombinant protein, and the secreted monoclonal antibody contains the heavy chain variable region named VH and the light chain variable region VL, and is composed of the complementarity-determining regions CDR1, CDR2, CDR3 and the framework region. Experiments prove that the recombinant expressed porcine C-reactive protein has high specificity, and the monoclonal antibody produced by preparation and screening through the recombinant protein can be used in a protein detection kit. The porcine C-reactive protein ELISA detection kit of the present invention is used to detect porcine C-reactive protein, can effectively detect the concentration of porcine C-reactive protein in the serum of pig herds, detect the group inflammation level, and evaluate the health level of pig herds in pig farms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a porcine C-reactive protein, a monoclonal antibody against porcine C-reactive protein, a detection kit and applications thereof. Background Art

[0002] C-reactive protein (CRP) is an acute-phase protein mainly synthesized and secreted by hepatocytes in the liver. The production of CRP is mainly a response of hepatocytes to inflammatory factors such as IL-6 in the body. When the body is damaged in terms of immune homeostasis due to infections, tumor growth or tissue trauma, etc., inflammatory stimuli will prompt the liver to increase the synthesis of CRP, resulting in an increase in the CRP level in the blood. The CRP level is not specific to any particular type of inflammation. Currently, the detection of human CRP levels is widely applied, including the diagnosis and detection of inflammation and infections, the assessment of cardiovascular disease risks, the monitoring of autoimmune diseases, cancer management, and the monitoring of postoperative infections, etc. CRP is one of the commonly used tools for assisting in the diagnosis and management of various diseases. By considering the CRP level together with other clinical information and test results, it can help make accurate diagnostic and treatment decisions.

[0003] Currently, swine diseases in China can be divided into viral diseases (African swine fever, foot-and-mouth disease, classical swine fever, porcine epidemic diarrhea, pseudorabies, etc.) and bacterial diseases (swine streptococcosis, porcine pleuropneumonia, porcine pasteurellosis multocida, etc.). When pigs are infected with bacteria and cause an inflammatory response, the serum CRP content increases significantly, and different infectious diseases have different levels of increase in serum CRP. Usually, the CRP concentration of healthy pigs is about 1 - 2 μg / mL, the CRP concentration in pigs infected with Streptococcus suis is about 15 - 20 μg / mL, and the CRP concentration caused by Pasteurella multocida infection can reach about 200 μg / mL. The CRP concentration is a powerful indicator of the systemic inflammatory response. While assessing the inflammatory level of the pig herd, it can assist in diagnosing the general pathogens infecting the pig herd, providing a strong guiding role for early diagnosis, isolation, and treatment. In addition, the application of this detection kit is also helpful for researchers in the fields of inflammation, immunology, and infectious diseases to conduct research.

[0004] A large number of small and medium-sized pig farms have cases of pig infections, among which bacterial infections are particularly serious. However, the current detection methods mainly include bacterial classification and PCR identification, which have cumbersome processes and take too long, seriously affecting the early diagnosis and treatment of pig farms. Currently, the main methods for detecting porcine serum CRP are latex agglutination detection and routine blood CRP detection. Latex agglutination detection cannot accurately detect the CRP concentration. Routine blood CRP detection is difficult to popularize in small and medium-sized pig farms due to machine costs and sample requirements.

[0005] In summary, according to the technical defects existing in the current detection methods on the market, there is an urgent need to find an effective means with high sensitivity, strong specificity, and capable of rapidly detecting porcine serum CRP in pig farms. Summary of the Invention

[0006] To solve the above technical problems existing in the prior art, the present invention provides an optimized porcine C-reactive protein gene sequence for soluble expression, obtains a porcine C-reactive protein monoclonal antibody through screening, and establishes a sandwich ELISA detection method for porcine serum CRP to achieve quantitative detection of porcine C-reactive protein.

[0007] To achieve the above object, the embodiments of the present invention provide the following technical solutions:

[0008] The first object of the present invention is to provide a gene encoding porcine C-reactive protein, and the nucleotide sequence is as shown in SEQ ID NO.1.

[0009] The second object of the present invention is to provide a recombinant vector, and the recombinant vector contains the aforementioned gene sequence encoding porcine C-reactive protein.

[0010] In a specific embodiment, the basic vector of the recombinant vector is pET-28a.

[0011] The third object of the present invention is to provide a genetically engineered bacterium, and the genetically engineered bacterium contains the aforementioned recombinant vector and a chaperone protein expression vector co-expressed with the recombinant vector.

[0012] In a specific embodiment, the chaperone protein expression vector is the molecular chaperone plasmid pTF16. pTF16 can promote the soluble expression of C-reactive protein in the engineered bacterium; the theoretical function is that protein misfolding is the reason for the loss of protein activity and insolubility; we co-express the molecular chaperone protein and C-reactive protein in the host, and the molecular chaperone protein promotes the correct folding of C-reactive protein to achieve the expression of soluble C-reactive protein.

[0013] Furthermore, the genetically engineered bacterium can express soluble porcine C-reactive protein.

[0014] The fourth object of the present invention is to provide a recombinant porcine C-reactive protein, and the amino acid sequence of the recombinant porcine C-reactive protein is as shown in SEQ ID NO.2, and the amino acid sequence is encoded by SEQ ID NO.1.

[0015] In a specific embodiment, the recombinant porcine C-reactive protein is expressed by the aforementioned genetically engineered bacterium.

[0016] The fifth object of the present invention is to provide the application of the aforementioned recombinant porcine C-reactive protein in the preparation of monoclonal antibodies or polyclonal antibodies against porcine C-reactive protein.

[0017] The sixth object of the present invention is to provide a monoclonal antibody against porcine C-reactive protein, which is prepared by using the above-mentioned recombinant porcine C-reactive protein as an antigen, and the monoclonal antibody contains V H heavy chain variable region and V L light chain variable region;

[0018] The amino acid sequence of the V H of the monoclonal antibody is shown in SEQ ID NO.4;

[0019] The amino acid sequence of the V L of the monoclonal antibody is shown in SEQ ID NO.6;

[0020] The V H and V L include complementary determining regions, and the complementary determining regions are composed of CDR1, CDR2, and CDR3;

[0021] The amino acid sequence of CDR-H1 of the V H of the monoclonal antibody is shown in positions 1-30 of SEQ ID NO.4;

[0022] The amino acid sequence of CDR-H2 of the VH of the monoclonal antibody is shown in positions 36-49 of SEQ ID NO.4;

[0023] The amino acid sequence of CDR-H3 of the VH of the monoclonal antibody is shown in positions 99-111 of SEQ ID NO.4;

[0024] The amino acid sequence of CDR-L1 of the V L of the monoclonal antibody is shown in positions 24-38 of SEQ ID NO.6;

[0025] The amino acid sequence of CDR-L2 of the V L of the monoclonal antibody is shown in positions 54-60 of SEQ ID NO.6;

[0026] The amino acid sequence of CDR-L1 of the V L of the monoclonal antibody is shown in positions 93-96 of SEQ ID NO.6.

[0027] Furthermore, the nucleotide sequence of the V H heavy chain variable region encoding the monoclonal antibody is shown in SEQ ID NO.3; the nucleotide sequence of the V L heavy chain variable region encoding the monoclonal antibody is shown in SEQ ID NO.5.

[0028] The seventh object of the present invention is to provide the application of the aforementioned gene encoding porcine C-reactive protein, or the aforementioned recombinant vector, or the aforementioned genetically engineered bacterium, or the aforementioned recombinant porcine C-reactive protein, or the aforementioned monoclonal antibody against porcine C-reactive protein in the preparation of an ELISA detection kit for porcine C-reactive protein.

[0029] The eighth object of the present invention is to provide an ELISA detection kit for porcine C-reactive protein, and the kit includes the aforementioned monoclonal antibody against porcine C-reactive protein.

[0030] The ninth object of the present invention is to provide an ELISA method for detecting porcine C-reactive protein. In the ELISA method, the aforementioned monoclonal antibody against porcine C-reactive protein is used for coating as the capture antibody, and the aforementioned porcine C-reactive protein is used as the antigen to establish a standard curve for detecting porcine C-reactive protein.

[0031] The conditions of the ELISA method include: the linear range of the standard curve is 31.25 - 1000 ng / mL, the concentration of the capture antibody is 4 μg / mL, the coating condition is 4°C for 12 h, the blocking solution is 5% skim milk, the blocking condition is 37°C for 2 h, the dilution ratio of the binding antibody is 1:2000, the action condition of the binding antibody is 37°C for 1 h, the dilution ratio of the enzyme-labeled antibody is 1:4000, the action condition of the binding antibody is 37°C for 45 min, and the color development time is 37°C for 30 min.

[0032] In a specific embodiment, the ELISA method is used to detect C-reactive protein in porcine serum.

[0033] Advantages of the present invention:

[0034] The present invention provides a codon-optimized gene sequence of porcine C-reactive protein, a soluble porcine C-reactive protein and a monoclonal antibody against porcine C-reactive protein. The sandwich ELISA detection kit for porcine C-reactive protein prepared by using the protein and the antibody optimizes the concentration of the capture antibody, the concentration of the antigen and the binding time, the concentration of the binding antibody and the binding time, the concentration of the HRP antibody and the binding time. Finally, a sandwich ELISA detection method for effectively detecting the concentration of C-reactive protein in porcine serum is established. The method has high sensitivity and strong specificity, can realize the rapid detection of C-reactive protein in the serum of pig herds in pig farms, and has high practical value. Description of the drawings

[0035] Figure 1 It is a schematic diagram of SDS-PAGE analysis of the induced expression of porcine C-reactive protein in the embodiment of the present invention. In the figure, each lane is: M. 180 kDa protein Marker; 1. Bacterial whole cell before induction; 2. Bacterial whole cell after induction; 3. Supernatant after ultrasonic disruption; 4. Precipitate after ultrasonic disruption.

[0036] Figure 2 Schematic diagram of the identification of the His column-purified porcine CRP recombinant protein by Western-blot in the embodiment of the present invention. In the figure, each lane is as follows: M. 180 kDa protein Marker; 1. Ultrasonic supernatant protein sample; 2. Flow-through sample; 3. 25 mM imidazole elution sample; 4. 100 mM imidazole elution sample; 5. Dialyzed and concentrated recombinant protein.

[0037] Figure 3 For the four-quadrant fitting curve and standard curve of the CRP sandwich ELISA detection method in the embodiment of the present invention, the linear range of the standard curve is 31.25 - 1000 ng / mL, and the fitting curve equation is: Y = 2361X - 323.8, R 2 = 0.996.

[0038] Figure 4 For the sera collected from healthy pigs at 60 days old and pigs infected with Streptococcus zooepidemicus detected by the CRP sandwich ELISA in the embodiment of the present invention, the median CRP concentration in the sera of healthy pigs is 1.127 μg / mL (0.404 - 3.318 μg / mL), and the median CRP concentration in the sera of pigs infected with Streptococcus zooepidemicus is 14.467 μg / mL (10.081 - 29.112 μg / mL). Detailed implementation manners

[0039] The following embodiments are used to further explain the present invention, but the embodiments do not limit the present invention in any form.

[0040] Example 1 Preparation of porcine C-reactive protein

[0041] 1. Optimization and synthesis of porcine C-reactive protein gene

[0042] According to the porcine C-reactive protein gene sequence (accession number NM_213844.2) published on NCBI, the codon preference of Escherichia coli was optimized. After optimization, the codon adaptation index was 0.65 and the GC content was 51.85%. The sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. After double digestion with BamHⅠ and XhoⅠ, the synthesized sequence was ligated to the pET-28a vector to obtain the recombinant plasmid pET-28a-CRP.

[0043] The nucleotide sequence results are as follows:

[0044] Optimized porcine C-reactive protein nucleotide sequence (SEQ ID NO.1)

[0045] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCCAAACAGATATGATAGGAAAAGCTTTTGTATTCCCGAAAGAGTCCGAGAACAGCTATGTGAGCTTGACGGCGCGTCTGACGAAGCCGCTGACTGCGTTCACCGTGTGCCTGCGTGTTTACACCGACCTGAATCGTGACTACTCTTTGTTCTCCTACGCCACCAAAACCCAGTATAACGAGATCTTGCTCTTCCGCGGTAAGACGGCTGTGTATTCTATTAGCGTTGGTGGCGCTGATGTCGTGTTTAAACCGCATCAGAGCTCGGAGCCGATGCACTTTTGTATGACCTGGGAATCGACCAGCGGTATTACCGAAC TGTGGGTTGATGGTAAGCCGATGGTTCGTCGTAGCCTTAAGCGCGGTTATTCCTTAGGCACCCAAGCGAGCATTATCCTGGGACAAGAGCAAGATGCATTCGCCGGTGGTTTTGAAAAAAATCAGTGCTTGGTTGGCGACATCGGCGACGTAAATATGTGGGATTACGTGCTGAGCCCGGAAGAGATCAACACCGTTTACGCGGGCGGCACTTTTTCTCCCAACGTGCTGAACTGGCGTGCACTGCGCTATGAAATGAGCGGTGAAGTTTACGTCAAACCACAGCTGTGGCCGCTCGAGCACCACCACCACCACCACTGA

[0046] Optimized amino acid sequence of porcine C-reactive protein (SEQ ID NO.2):

[0047] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSQTDMIGKAFVFPKESENSYVSLTARLTKPLTAFTVCLRVYTDLNRDYSLFSYATKTQYNEILLFRGKTAVYSISVGGADVVFKPHQSSEPMHFCMTWESTSGITELWVDGKPMVRRSLKRGYSLGTQASIILGQEQDAFAGGFEKNQCLVGDIGDVNMWDYVLSPEEINTVYAGGTFSPNVLNWRALRYEMSGEVYVKPQLWPLEHHHHHH

[0048] 2. Expression and purification of porcine C-reactive protein

[0049] The recombinant plasmid pET-28a-CRP and the chaperone plasmid pTF16 were co-transformed into the competent BL21(DE3) cells. The host bacteria identified as positive by double resistance screening with kanamycin and chloramphenicol were preserved. A single colony was picked and inoculated into 8 mL of LB liquid medium with double resistance to kanamycin and chloramphenicol, and cultured overnight at 37 °C with 180 rpm / min. The next day, the bacterial solution was inoculated into 500 mL of LB liquid medium with double resistance to kanamycin and chloramphenicol at a ratio of 1:100, and L-arabinose with a final concentration of 1 g / L was added. The culture was carried out at 37 °C with 180 rpm / min for 3 - 4 h. When the OD 600nm value reached 0.6 - 0.8, isopropyl β-D-thiogalactopyranoside with a final concentration of 0.2 mmol / L was added, and induction was carried out at 16 °C with 90 rpm / min for 16 h. The correct folding of C-reactive protein was promoted by co-expressing the chaperone protein to achieve the expression of soluble C-reactive protein.

[0050] The bacteria were collected by centrifugation (5000 rpm / min, 10 min), the supernatant was discarded, and the bacteria were resuspended at a ratio of the mass of bacteria (g) to the volume (mL) of buffer A (50 mM Tris Base, 500 mM NaCl, 0.05% Tween-20, pH 7.4) of 1:10. After ultrasonic disruption (power 130 W, ultrasonic treatment for 40 min), SDS-PAGE identification was carried out. As Figure 1 shown, an obvious band was found at about 28 kDa, which was consistent with the expected protein size (27.49 kDa), proving that the target protein was successfully expressed.

[0051] After determining the expression of the target protein, centrifuge (10000 rpm / min, 20 min), collect the expression supernatant, filter it through a 0.22 μm filter, and perform column chromatography. Install the His chromatography column on the chromatograph, equilibrate it with 5 column volumes of buffer A, add the sample to the His chromatography column, wash it with 5 column volumes of buffer A, elute it with 5 - 10 column volumes of buffer B (50 mM TrisBase, 500 mM NaCl, 250 mM imidazole, pH 7.4), collect the eluted target protein, concentrate the target protein using a 10 kDa ultrafiltration concentrator tube, and replace the medium with PBS. Detect by Western-blot. As Figure 2 shown, after determining it as the target protein, measure the protein concentration and store it at -40 °C.

[0052] Example 2 Preparation of Porcine C-Reactive Protein Mouse Monoclonal Antibody and Rabbit Polyclonal Antibody

[0053] 1. Preparation and Identification of Porcine C-Reactive Protein Mouse Monoclonal Antibody

[0054] Mix the purified porcine CRP recombinant protein with ISA 206 adjuvant at a volume ratio of 1:1, and immunize three 5 - 6-week-old female BALB / c mice by multiple subcutaneous injections on the back. The immunization dose of the recombinant protein is 200 μg for the first immunization, 100 μg for the second immunization 2 weeks later, 100 μg for the third immunization 1 week later, and 50 μg for the booster immunization by intraperitoneal injection of the recombinant protein without adjuvant 1 week later. Collect blood from the tail vein 3 days after each immunization and detect the serum titer by indirect ELISA.

[0055] Decapitate and sacrifice the mice 4 days after the booster immunization, take the spleen, rinse it with DMEM medium and grind it through a filter screen to collect splenocytes. Mix the splenocytes with SP2 / 0 myeloma cells at a ratio of 10:1, centrifuge the mixed cells (800 rpm / min, 10 min), discard the supernatant, add 1 mL of PEG fusogen in a 37 °C water bath environment, slowly add 10 mL of pre-warmed DMEM medium at 37 °C within 5 min, centrifuge after making up the medium to 30 mL (800 rpm / min, 10 min), discard the supernatant, add 40 mL of pre-warmed HAT medium at 37 °C, inoculate 150 μL per well into a 96-well cell culture plate, and culture it in a 37 °C, 5% CO2 incubator. Select wells with single cell clusters for subcloning by the limited dilution method, perform subcloning three times in total, gradually reduce the concentration of HAT medium, and finally replace it with HT medium for culture. Detect the culture supernatant by indirect ELISA, finally expand the culture of the hybridoma cell line stably secreting porcine CRP protein antibody, detect the titer and cryopreserve the cells.

[0056] Five 5-6-week-old female BALB / c mice were taken, and the body was sensitized by intraperitoneal injection of 1 mL of liquid paraffin oil. After about two weeks, 1×10 6 hybridoma cells were intraperitoneally injected. When the abdomen of the mice was significantly distended, ascites was collected by inserting a 20 mL syringe needle into the side of the abdominal nipple. After centrifugation (2000 rpm / min, 10 min), the cell precipitate was separated. After centrifugation (8000 rpm / min, 10 min), the antibody was collected and the monoclonal antibody was purified using Protein A+G agarose. The purified antibody was aliquoted and stored frozen. The antibody titer was detected by indirect ELISA, and the monoclonal antibody titer was 1:409600.

[0057] Using the mouse monoclonal antibody subtype detection kit from Beijing Boaolong Company, the above antibody was detected for its subtype. The results showed that the obtained monoclonal antibody against porcine C-reactive protein was an IgG1 antibody with κ light chain.

[0058] 2. Preparation of rabbit polyclonal antibody against porcine C-reactive protein

[0059] The purified recombinant porcine CRP protein was mixed with ISA 206 adjuvant at a volume ratio of 1:1. Three 12-week-old male New Zealand white rabbits (weighing 2.2 kg - 2.3 kg) were selected and immunized by subcutaneous multi-point injection on the back in different batches. Each immunization was 0.5 mg of porcine CRP protein, and the immunization was carried out once every two weeks for a total of four times. After 4 immunizations, blood was collected and the serum titer was detected. The serum protein concentration was 40 mg / mL, the titer was 1:102400, and there was no significant difference in the batch-to-batch serum titer.

[0060] Example 3 Sequencing of the heavy and light chain variable region genes of the mouse monoclonal antibody against porcine C-reactive protein

[0061] 1. RNA extraction and reverse transcription of hybridoma cells

[0062] The hybridoma cells were cultured until the cell number reached 5×10 6 , and the total cell RNA was extracted according to the instructions of the Omega RNA extraction kit. According to the instructions of the Novizan reverse transcription kit, the extracted total RNA was reverse transcribed into cDNA.

[0063] 2. PCR amplification and cloning of the target fragment

[0064] The heavy and light chain variable region genes were amplified using the universal degenerate primers for mouse-derived IgG1 antibody with κ light chain. The PCR products were subjected to 2% agarose gel electrophoresis and the gel was cut. The target gene was extracted according to the instructions of the Omega agarose gel recovery kit. The target gene was TA cloned into the pMD19-T vector, then transformed into DH-5α competent cells, and spread on an LB solid plate containing ampicillin resistance. Positive monoclonal colonies were picked and sequenced for identification.

[0065] The sequences of the heavy chain and light chain are as follows:

[0066] Nucleotide sequence of the heavy chain (SEQ ID NO.3)

[0067] CAAGTGAAGCTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGAAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTTATGACAGGACTGCCATCTACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTCTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGCATAACTGTGCAAGATGGGCCTACGGTAGTAGCAACCACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG

[0068] Amino acid sequence of the heavy chain (SEQ ID NO.4)

[0069] QVKLQESGGGLVQPGGSRKLSCEASGFTFSSFGMHWVRQAPEKGLEWVAYISYDRTAIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMHNCARWAYGSSNHWYFDVWGAGTTVTVSS

[0070] Nucleotide sequence of the light chain (SEQ ID NO.5)

[0071] GACATTGTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGG

[0072] Light chain amino acid sequence (SEQ ID NO.6)

[0073] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIR

[0074] 3. Variable region (CDR) sequence analysis

[0075] The CDR region analysis of the variable region amino acid sequences of the Heavy chain and Light chain was performed through the Kabat definition scheme at https: / / www.novopro.cn / tools / cdr.html

[0076] Antibody Heavy chain CDR region:

[0077] CDR-H1: QVKLQESGGGLVQPGGSRKLSCEASGFTFS, as shown in positions 1-30 of SEQ ID NO.4;

[0078] CDR-H2: YISYDRTAIYYADTVKG, as shown in positions 36-49 of SEQ ID NO.4;

[0079] CDR-H3: WAYGSSNHWYFDV, as shown in positions 99-111 of SEQ ID NO.4;

[0080] Antibody Light chain CDR region:

[0081] CDR-L1: RASKSVSTSGYSYMH, as shown in positions 24-38 of SEQ ID NO.6;

[0082] CDR-L2: LVSNLES, as shown at positions 54-60 of SEQ ID NO.6;

[0083] CDR-L3: QHIR, as shown at positions 93-96 of SEQ ID NO.6.

[0084] Establishment of a sandwich ELISA for porcine C-reactive protein and optimization of reaction conditions

[0085] 1. Establishment of the sandwich ELISA

[0086] According to the checkerboard titration method, mouse monoclonal antibodies at 0 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL were used as capture antibodies, and coated at 100 μL / well at 4 °C for 12 h; washed 3 times with PBST, 5 min each time, blocked with 200 μL of 5% skim milk at 37 °C for 2 h; washed 3 times with PBST, added 4 μg / mL of recombinant porcine C-reactive protein, incubated at 37 °C for 1 h; washed 3 times with PBST, and rabbit polyclonal antibodies at 0 μg / mL, 2.5 μg / mL (1:16000), 5 μg / mL (1:8000), 10 μg / mL (1:4000), 20 μg / mL (1:2000), 40 μg / mL (1:1000), 80 μg / mL (1:500), 160 μg / mL (1:250), and 320 μg / mL (1:125) were used as binding antibodies, incubated at 100 μL / well at 37 °C for 1 h; washed 3 times with PBST, 100 μL of goat anti-rabbit HRP-labeled antibody (1:5000) was added to each well, incubated at 37 °C for 1 h; washed 3 times with PBST, 100 μL of TMB was added to each well, after incubating at 37 °C for 15 min, 100 μL of 2M H2SO4 was added to terminate the color development, and the OD 450 value was detected by an enzyme-linked immunosorbent assay reader. P / N value = OD of the test sample 450 value / OD of the negative sample 450 value. Based on the P / N value and the principle of the least amount of antibody used, the optimal concentration of the capture antibody was determined to be 4 μg / mL, and the working concentration of the binding antibody (1:2000, 20 μg / mL). The specific results are shown in Table 1.

[0087] Table 1 Determination of the working concentration of antibodies for porcine C-reactive protein sandwich ELISA (P / N value)

[0088]

[0089]

[0090] 2. Determination of the working concentration and time of the enzyme-labeled antibody

[0091] According to the above antibody optimization conditions, use 4 μg / mL mouse monoclonal antibody as the capture antibody, coat at 4 °C with 100 μL per well for 12 h; wash 3 times with PBST, 5 min each time, add 200 μL of 5% skim milk at 37 °C and block for 2 h; wash 3 times with PBST, add 4 μg / mL recombinant porcine C-reactive protein, incubate at 37 °C for 1 h; wash 3 times with PBST, use 20 μg / mL (1:2000) rabbit polyclonal antibody as the binding antibody, incubate at 37 °C with 100 μL per well for 1 h; wash 3 times with PBST, add goat anti-rabbit HRP enzyme-labeled antibody diluted at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 to each well respectively, incubate at 37 °C for 1 h; wash 3 times with PBST, add 100 μL of TMB to each well, after incubating at 37 °C for 15 min, add 100 μL of 2 M H2SO4 to terminate the color development, and detect the OD 450 value. Determine the working concentration of the goat anti-rabbit HRP enzyme-labeled antibody as 1:4000 through the P / N value. The specific results are shown in Table 2. By adding the goat anti-rabbit HRP enzyme-labeled antibody diluted at 1:4000, incubate at 37 °C for 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min respectively, and determine the working time of the goat anti-rabbit HRP enzyme-labeled antibody as 45 min through the P / N value. The specific results are shown in Table 3.

[0092] Table 2 Determination of the working concentration of the enzyme-labeled antibody in the porcine C-reactive protein sandwich ELISA (P / N value)

[0093]

[0094] Table 3 Determination of the working time of the enzyme-labeled antibody in the porcine C-reactive protein sandwich ELISA (P / N value)

[0095]

[0096] 3. Establishment of the standard curve of the porcine C-reactive protein sandwich ELISA method

[0097] According to the above antibody optimization conditions, use 4 μg / mL murine monoclonal antibody as the capture antibody, coat at 100 μL / well at 4 °C for 12 h; wash 3 times with PBST, 5 min each time, add 200 μL of 5% skim milk at 37 °C for blocking for 2 h; wash 3 times with PBST, and add 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.62 ng / mL, 7.81 ng / mL respectively, incubate at 37 °C for 1 h; wash 3 times with PBST, use 20 μg / mL (1:2000) rabbit polyclonal antibody as the binding antibody, incubate at 100 μL / well at 37 °C for 1 h; wash 3 times with PBST, add 100 μL of goat anti-rabbit HRP-labeled antibody (1:4000) to each well, incubate at 37 °C for 45 min; wash 3 times with PBST, add 100 μL of TMB to each well, after incubating at 37 °C for 15 min, add 100 μL of 2M H2SO4 to terminate the color development, and detect the OD value by an enzyme-linked immunosorbent assay instrument. 450 The specific results are as Figure 3 shown. The results show that they are the four-parameter fitting curve and the standard curve respectively, both of which can achieve high precision. The linear range of the standard curve detection result is 31.25 - 1000 ng / mL, and the fitting curve equation is Y = 2361X - 323.8, with R 2 reaching 0.996; for more accurate detection of C-reactive protein concentration, the standard curve is used as the standard for subsequent detections.

[0098] Example 5 Evaluation and Application of Porcine C-Reactive Protein Sandwich ELISA Detection Method

[0099] 1. Detection of coefficient of variation and coincidence rate of porcine C-reactive protein sandwich ELISA method

[0100] According to the above optimized conditions, perform coating, antigen binding, antibody binding and color development, and detect the addition of low, medium and high concentration recombinant CRP protein samples as antigens. Each group of samples contains 6 replicates, and this experiment is carried out 3 times. The within-batch coefficient of variation is less than 10%, and the between-batch coefficient of variation is less than 15%, indicating that this method has good repeatability and determines that the ELISA detection kit meets the in-line use standards; the sample coincidence rate is 96% - 106%, and the average coincidence rate is 101%, determining that the ELISA detection method has high accuracy. The specific results are shown in Table 4.

[0101] Table 4 Detection of coefficient of variation and coincidence rate of porcine C-reactive protein sandwich ELISA

[0102]

[0103]

[0104] 2. Detection of porcine C-reactive protein sandwich ELISA method in porcine serum samples

[0105] Blood was collected from the anterior vena cava of 6 healthy pigs at 60 days of age. The blood samples were placed at 4°C for 12 hours, centrifuged (4000 rpm / min, 20 minutes) and the upper serum samples were collected and stored at -40°C. 6 CFU Streptococcus zooepidemicus, 36-48h after injection, blood was collected and serum samples were collected according to the above method. The above samples were diluted tenfold three times and tested by the porcine C-reactive protein sandwich ELISA method. The results showed that the median serum CRP concentration of healthy pigs was 1.127201μg / mL, and the median serum CRP concentration of pigs infected with Streptococcus zooepidemicus was 14.4667μg / mL. This shows that the porcine C-reactive protein sandwich ELISA method can detect clinical pig serum samples, effectively identify the C-reactive protein concentration of healthy pigs and the high concentration of C-reactive protein concentration after Streptococcus zooepidemicus infection, and provide protection for the health of the pig herd. The specific results are as follows Figure 4 shown.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against porcine C-reactive protein, characterized in that, The monoclonal antibody contains a V H heavy chain variable region and a V L light chain variable region; The monoclonal antibody V H has the amino acid sequence shown in SEQ ID NO.4; The monoclonal antibody V L has the amino acid sequence shown in SEQ ID NO.

6.

2. Use of the porcine C-reactive protein monoclonal antibody according to claim 1 in the preparation of a porcine C-reactive protein ELISA detection kit.

3. A porcine C-reactive protein ELISA detection kit, characterized in that, The kit includes the porcine C-reactive protein monoclonal antibody according to claim 1.

4. An ELISA method for detecting porcine C-reactive protein, characterized in that, In the ELISA method, the capture antibody coating is carried out using the porcine C-reactive protein monoclonal antibody according to claim 1, and a standard curve is established using the porcine C-reactive protein described in SEQ ID NO.

2.

5. The ELISA method according to claim 4, wherein The conditions of the ELISA method include: the linear range of the standard curve is 31.25 - 1000 ng / mL, the concentration of the capture antibody is 4 µg / mL, the coating condition is 4°C for 12 h, the blocking solution is 5% skim milk, the blocking condition is 37°C for 2 h, the dilution ratio of the binding antibody is 1:2000, the dilution ratio of the enzyme-labeled antibody is 1:4000, and the color development time is 30 min at 37°C.

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