CN113376380B

CN113376380B - ELISA kit for detecting dog IL-6 and application thereof

Info

Publication number: CN113376380B
Application number: CN202110618231.9A
Authority: CN
Inventors: 郭霄峰; 孙荣航; 罗钧; 褚颖
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-06-24
Anticipated expiration: 2041-05-31
Also published as: CN113376380A

Abstract

The invention discloses an ELISA kit for detecting dog IL-6 and application thereof. According to the invention, through primer design, protein induction and animal immunity of IL-6, a monoclonal antibody and a polyclonal antibody of canine IL-6 are prepared, and the two antibodies are respectively used as a detection antibody and a capture carrier in an ELISA double-antibody sandwich kit, so that the canine IL-6 detection kit and the use method thereof are established. The canine IL-6 detection kit established by the invention can quantitatively detect IL-6 in a sample, has the characteristics of high sensitivity and wide detection range, and has the lowest detection value of 1.17ng/mL and the detection range of 1.17 ng/mL-300 ng/mL.

Description

ELISA kit for detecting dog IL-6 and application thereof

Technical Field

The invention relates to the technical field of biotechnology, and relates to an ELISA kit for detecting dog IL-6 and application thereof.

Background

Interleukin-6 (IL-6), originally described as a B cell stimulating factor, now known as a pleiotropic cytokine, has a number of key biological functions, including stimulating inflammation and immune responses, promoting hematopoiesis, and tumorigenesis. It is transiently produced by immune cells such as monocytes and macrophages, but also by other cell lineages under various stimuli (e.g., infection or tissue damage). In addition, interleukin-6 is associated with hematopoiesis as a cofactor for stem cell expansion and differentiation.

The physiological properties of interleukin-6 are complex, with pro-inflammatory and anti-inflammatory effects in the immune system. Interleukin-6 regulates the transcription of several liver-specific genes, particularly C-reactive protein (CRP), under acute inflammatory conditions and controls the survival of normal plasma cells. In addition, interleukin-6 is an activator or inhibitor of the T cell response, and this interplay of proinflammatory and anti-inflammatory activities suggests that interleukin-6 may play a role in regulating the physiological response to disease. In view of the role of interleukin-6 in the immune response, enhanced susceptibility is a reasonable consideration in any therapeutic strategy directed to interleukin-6 signaling, such as interleukin-6 receptor (rituximab, sariluzumab) or interleukin-6 (steuximab), increasingly used to treat rheumatoid arthritis, juvenile idiopathic arthritis.

Increased expression of interleukin-6 has been implicated in a variety of disease processes, including alzheimer's disease, autoimmunity (e.g., rheumatoid arthritis), inflammation, myocardial infarction, paget's disease, osteoporosis, solid tumors, prostate and bladder cancer, certain neurological cancers, B cell malignancies. In some cases, interleukin-6 is associated with a proliferative pathway because it acts in conjunction with other factors, such as heparin binding epithelial growth factor and hepatocyte growth factor. Therefore, blocking interleukin-6 may be beneficial in many pathological situations.

Interleukin-6 plays a role not only in the pathogenesis of systemic inflammation, but also in the pathogenesis of local inflammation, and is involved in the growth and differentiation of many types of cells. In humans, interleukin-6 is produced and released rapidly after full-thickness skin injury, even for 24 hours after injury. Interleukin-1 β is produced primarily by macrophages and monocytes, as well as non-immune cells including activated fibroblasts and keratinocytes, and contributes to wound healing. In dogs, interleukin-1 β stimulates the production and release of interleukin-6 through a mitogen-activated protein kinase (MAPK) signaling pathway in a variety of cells, a key process in canine skin wound healing.

Although the detection of the IL-6 level can not be directly used as a basis for judging whether the animal has a certain disease or not, the IL-6 level in the animal body is associated with various diseases, so that an efficient detection method is established for detecting the IL-6 level in the animal body, is used for monitoring the health condition of the animal, the disease prognosis condition and the immune state of the animal, and has great significance for dogs. The level of cytokine in serum or cells of an immunized animal is one of the important indexes for measuring the immune effect of the vaccine. Therefore, the establishment of the detection method of the canine IL-6 has important significance for evaluating the immune effect of the vaccine.

Currently, Radioimmunoassay (RIA) and RT-PCR are commonly used as methods for detecting cytokines. The sensitivity of the radioimmunoassay is limited by the working principle of the method, certain substances with particularly low content in the body cannot be measured, and the problems of radioactive radiation, pollution and the like exist. The RT-PCR has the advantages of high specificity, high sensitivity and high detection speed. However, since the sensitivity is too high, the probability of false positive results is too high, and a negative control must be set at the same time. RT-PCR requires multiple touch conditions and requires skill in operation.

At present, although ELISA kits for detecting canine IL-6 are available on the market, the kits with high sensitivity have a smaller detection range; the sensitivity of the kit with a larger detection range is not high.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an ELISA kit for detecting canine IL-6 and application thereof, wherein the monoclonal antibody and the polyclonal antibody of the canine IL-6 are prepared by primer design, protein induction and animal immunity of the IL-6 and are respectively used as a detection antibody and a capture carrier in the ELISA double-antibody sandwich kit, and the detection kit for the canine IL-6 and the use method thereof are established.

The first purpose of the invention is to provide an ELISA kit for detecting canine IL-6.

The second purpose of the invention is to provide a using method of the ELISA kit.

In order to achieve the purpose, the invention is realized by the following scheme:

according to the invention, through primer design, protein induction and animal immunity of IL-6, a monoclonal antibody and a polyclonal antibody of canine IL-6 are prepared, and are respectively used as a detection antibody and a capture carrier for an ELISA double-antibody sandwich kit. The kit can be used for quantitatively detecting the canine IL-6, has good linear relation of a standard curve, and has higher sensitivity and wider detection range.

Accordingly, the invention claims the following:

an ELISA kit for detecting canine IL-6 comprising: the kit comprises a pET-IL6 monoclonal antibody, a pET-IL6 polyclonal antibody, an enzyme label plate, a confining liquid, a goat anti-rabbit enzyme labeled secondary antibody, a developing liquid and a stop solution;

wherein the pET-IL6 monoclonal antibody and the pET-IL6 polyclonal antibody are antibodies prepared by immunizing animals by pET-IL6 protein, and the amino acid sequence of the pET-IL6 protein is shown in SEQ ID NO. 1;

the pET-IL6 monoclonal antibody is used as a capture antibody, and the pET-IL6 polyclonal antibody is used as a detection antibody.

Preferably, the blocking liquid is one or more of 2-5% BSA and 2-5% skimmed milk powder, wherein the percentage is mass percent.

More preferably, the sealing liquid is 5% of skimmed milk powder by mass.

Preferably, the pET-IL6 protein is prepared by the following method:

amplifying an IL-6 gene, and connecting the IL-6 gene with a vector pET-32a (+) to obtain a pET-IL6 recombinant plasmid;

transforming the plasmid into BL21 escherichia coli to obtain pET-IL6 recombinant bacteria; inducing the pET-IL6 recombinant bacteria by using 0.1-1.5 mmol/L IPTG, carrying out induced culture at 22-42 ℃ for 2-6 h, collecting bacterial liquid, centrifuging, removing supernatant, carrying out ultrasonic crushing on the precipitate, taking the supernatant, and purifying the obtained protein to obtain pET-IL6 protein;

the nucleotide sequence of the IL-6 gene is shown in SEQ ID NO. 2.

More preferably, the specific primer group used for amplifying the IL-6 has a sequence shown in SEQ ID NO.3 and SEQ ID NO. 4.

More preferably, the concentration of IPTG is 0.1-0.8 mmol/L.

Most preferably, the IPTG concentration is 0.1 mmol/L.

More preferably, the temperature of the induction culture is 28-37 ℃.

Most preferably, the temperature of the induction culture is 37 ℃.

More preferably, the time of the induction culture is 3-5 h.

Most preferably, the time of the induction culture is 4 h.

Preferably, the concentration of the pET-IL6 monoclonal antibody is 24-48 mu g/mL, and the concentration of the pET-IL6 polyclonal antibody is determined according to the ratio of 1: 500-1: 100 dilution.

More preferably, the concentration of the pET-IL6 monoclonal antibody is 36-48 mu g/mL, and the concentration of the pET-IL6 polyclonal antibody is determined according to the ratio of 1: 100 dilution.

Preferably, the dilution of the goat anti-rabbit enzyme-labeled secondary antibody is 1: 1000-1: 500.

more preferably, the dilution of the goat anti-rabbit enzyme-labeled secondary antibody is 1: 500.

preferably, the color development liquid is tetramethyl benzidine.

Preferably, the stop solution is H₂SO₄。

The use method of the ELISA kit comprises the following steps:

s1, coating: adding 100 mu L of pET-IL6 monoclonal antibody into an ELISA plate, and keeping the mixture in a refrigerator overnight;

s2, washing a plate: discarding the liquid in the ELISA plate, and oscillating and rinsing for 4 times for 60s once according to 200 mu L/hole of PBST;

s3, sealing: adding 100 μ L of sealing solution into each well, sealing in a thermostat at 37 deg.C, and washing the plate in the same manner as S2;

s4, sample adding: incubating 100. mu.L of sample per well at 37 ℃, and washing the plate as in S2;

s5, adding a detection antibody: diluting pET-IL6 polyclonal antibody with blocking solution, adding 100 μ L of diluted pET-IL6 polyclonal antibody into each well, acting at 37 deg.C for 60min, and washing the plate with S2;

s6, adding a secondary antibody: adding 100 mu L of diluted goat anti-rabbit enzyme-labeled secondary antibody into each hole, incubating for 60min at 37 ℃, and washing the plate with S2;

s7, color development: adding color development solution in a dark environment, performing reaction at a temperature of 37 ℃ for 10min at a concentration of 100 mu L/hole;

s8, terminating: adding 50 mu L of stop solution into each hole to stop the reaction;

s9, reading: OD450 values were read for each well using a microplate reader.

Preferably, in the step S3, the sealing time is 1-2 h.

More preferably, in the step S3, the sealing time is 1 to 1.5 hours.

Most preferably, in step S3, the sealing time is 1 h.

Preferably, in the step S4, the incubation time of the sample is 1-2 h.

More preferably, in the step S4, the incubation time of the sample is 1.5-2 h.

Most preferably, in step S4, the incubation time of the sample is 1.5 h.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention adopts the monoclonal antibody as the capture antibody and the polyclonal antibody as the detection antibody, and compared with two same or different monoclonal antibodies, the established double-antibody sandwich ELISA method is respectively used as the capture antibody, has higher sensitivity of the detection antibody and lower production cost.

(2) The method of the invention has stronger specificity when monoclonal antibody is used as capture antibody and polyclonal antibody is used as detection antibody, and lower color value of color development background.

(3) The method can quantitatively detect the content of the IL-6 in the sample, and has the advantages of high sensitivity and wide detection range, the lowest detection value is 1.17ng/mL, and the detection range is 1.17 ng/mL-300 ng/mL.

(4) Compared with the currently common Radioimmunoassay (RIA), RT-PCR and other methods, the method has the advantages of short detection period, simple and convenient operation, low requirement on technical level, simple equipment and capability of realizing rapid detection of a large number of samples.

Drawings

FIG. 1PCR identification of recombinant bacteria liquid of the present invention;

FIG. 2 is a diagram showing the double restriction enzyme digestion identification of the recombinant plasmid pET-IL6 of the present invention;

FIG. 3 SDS-PAGE analysis of expression products of the invention;

FIG. 4 Western-bioting analysis of expression products of the present invention;

FIG. 5 solubility analysis of expression products of the present invention;

FIG. 6 shows the washing and solubilization identification of inclusion bodies in the present invention;

FIG. 7 SDS-PAGE analysis of pET-IL6 expression products at different induction times in the present invention;

FIG. 8 SDS-PAGE analysis of pET-IL6 expression products at different induction temperatures according to the present invention;

FIG. 9 SDS-PAGE analysis of pET-IL6 expression products induced by different IPTG concentrations in the present invention;

FIG. 10 is a graph showing the relationship between the natural logarithm of the concentration of pET-IL6 antigen and the OD value in the present invention;

FIG. 11 is a standard graph of the canine IL-6ELISA kit of the present invention;

FIG. 12 shows the IL-6 profile in the sera of the immunorabies vaccine and the non-immunorabies vaccine of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 preparation of pET-IL6 protein

1. Design of specific primers

A pair of specific primers is designed according to the determined conserved nucleotide sequence by referring to the whole gene sequence of the canine IL-6 in GenBank and utilizing Primer auxiliary design software Primer5 for amplification of the IL-6 gene. BamHI and HindIII sites (underlined in the primer sequences SEQ ID NO.3 and SEQ ID NO.4) were inserted into the upstream and downstream of the primers, respectively, and protected bases were added thereto, and the primers were synthesized by Producer corporation.

The specific primers are as follows:

upstream primer P1(SEQ ID NO. 3): 5' -CGGGATCCATGAACTCCCTCTCCACAAGC-3’；

Downstream primer P2(SEQ ID NO. 4): 5' -CCAAGCTTCATTATCCGAACAGC-3’。

2. Construction of expression vectors

The primers (the upstream primer P1 and the downstream primer P2) are used for PCR amplification of an IL-6 gene, rHEP-CaIL6 plasmid (see the Chinese patent application No. 201710087252.6 for details) stored in the experiment is used as a template, and an amplification system is shown in Table 1:

TABLE 1PCR reaction System Components content

The length of the target gene of the amplified IL-6 gene is 624bp, and the reaction program comprises the following steps: pre-denaturation at 98 ℃ for 1 min; and (3) circulation: 15s at 98 ℃, 20s at 64 ℃ and 3min at 72 ℃ for 35 cycles; extension at 72 ℃ for 10 min.

Wherein the nucleotide sequence of the IL-6 gene is shown as SEQ ID NO. 2:

ATGAACTCCCTCTCCACAAGCGCCTTCTCCCTGGGGCTGCTCCTGGTGATGGCTACTGCTTTCCCTACCCCGGGACCCCTGGCAGGAGATTCCAAGGATGATGCCACTTCAAATAGTCTACCACTCACCTCTGCAAACAAAGTGGAAGAACTGATTAAGTACATCCTCGGCAAAATCTCTGCACTGAGAAAGGAGATGTGTGACAAGTTTAACAAGTGTGAAGACAGCAAAGAGGCACTGGCAGAAAATAACCTACATCTTCCCAAACTGGAGGGAAAAGATGGATGCTTCCAATCTGGGTTCAATCAGGAGACCTGCTTGACAAGAATCACTACCGGTCTTGTGGAGTTTCAGCTACACCTGAATATCCTCCAGAACAACTATGAGGGTGATAAGGAAAATGTCAAGTCTGTGCACATGAGTACCAAGATCCTGGTCCAGATGCTAAAGAGCAAGGTAAAGAATCAGGATGAAGTGACCACTCCTGACCCAACCACAGACGCCAGCCTGCAGGCTATCTTGCAGTCGCAGGATGAGTGGCTGAAGCACACAACAATTCACCTCATCCTGCGGAGTCTGGAGGATTTCCTGCAGTTCAGTCTGAGGGCTGTTCGGATAATGTAG。

the IL-6 gene and a vector pET-32a (+) are subjected to enzyme digestion by BamH I and Hind III, and are connected by T4 ligase, wherein the connection system is shown in Table 2, and the components are blown, beaten, uniformly mixed and connected in a water bath at 16 ℃ for 8 hours to obtain the pET-IL6 recombinant plasmid.

TABLE 2 contents of the ingredients in the connection System

And (3) transforming the pET-IL6 recombinant plasmid into competent BL21 escherichia coli to obtain pET-IL6 recombinant bacterium liquid. And (3) carrying out PCR identification on the pET-IL6 recombinant bacterial liquid, namely taking PCR of the pET-IL6 recombinant bacterial liquid as a template. PCR amplification of bacterial suspension was performed using the upstream primer P1 and the downstream primer P2.

The PCR reaction system of the bacterial liquid is shown in Table 3, the components are blown and uniformly mixed, and the following conditions are operated on a PCR amplification instrument: pre-denaturation at 96 ℃ for 5 min; and (3) circulation: 25s at 96 ℃, 20s at 64 ℃ and 3min at 72 ℃ for 35 cycles; extension at 72 ℃ for 5 min.

The positive bacterial liquid (shown in figure 1) is obtained by detecting 4 bright bands by nucleic acid electrophoresis, and the size of the positive bacterial liquid is consistent with the expected size of 624 bp. The successful transformation of the recombinant plasmid into E.coli is demonstrated.

TABLE 3 contents of each component in PCR reaction system of bacterial liquid

After the pET-IL6 recombinant bacterial liquid is subjected to PCR detection, plasmid extraction is carried out on the bacterial liquid, and the plasmid extraction is carried out according to the specification of a plasmid extraction kit of magenta company. And carrying out double enzyme digestion detection on the extracted plasmid. The plasmid double digestion reaction system is shown in table 4:

TABLE 4 contents of the respective components in the enzyme digestion reaction system

After all the components are added, the mixture is blown evenly by a gun, enzyme digestion is carried out for 5 hours in water bath at 37 ℃, 3 mu L of enzyme digestion products are taken for nucleic acid electrophoresis detection, and the result is observed under a gel illumination instrument. The PCR of the bacterial liquid is used as a control, the upper band and the lower band are respectively at 5900bp and 624bp, the result is shown in figure 2 and is consistent with the expectation, and the result indicates that the pET-IL6 recombinant plasmid is successfully constructed.

And (3) sending the pET-IL6 recombinant plasmid which is verified to be correct by PCR and double enzyme digestion to a company for sequencing, and comparing and analyzing sequencing data by using SnapGene and DNAStar software. According to the sequencing result, the IL-6 gene is correctly connected to a pET-32a (+) vector, and the synonymous mutation of 3 bases (the mutation of G at 387bp of the target sequence to A, the mutation of G at 528bp to A, and the mutation of C at 545bp to G) is found, but the amino acid change is not caused, so that the sequencing verification is correct. The pET-IL6 plasmid is transformed into BL21 escherichia coli to obtain pET-IL6 recombinant bacteria.

3. Inducible expression of a protein of interest

The identified pET-IL6 recombinant strain is streaked and cultured for 12h, and a single colony is picked out the next day and added into the recombinant strain containing 6mL of Amp⁺Shaking in LB test tube at 37 deg.C for 12h with constant temperature shaking table (210r/min) to obtain first-stage seed; first seed 60. mu.L (in a ratio of 1: 100) was added to a solution containing 6mL Amp⁺Shaking in LB test tube at 37 deg.C for 3h with constant temperature shaking table (210 r/min); adding IPTG with the final concentration of 1mM, shaking at 37 ℃ for 4h in a constant temperature shaking table (210r/min), and collecting the bacteria. 20 μ L of 5 Xprotein loading buffer was pipetted into 80 μ L of induction product, heated in a metal bath for 15min, and subjected to SDS-PAGE protein electrophoresis, or stored at-20 ℃. And simultaneously setting a non-induction expression control, a no-load control and an expression bacteria self control.

The SDS-PAGE result shows that the induced band is obviously clearer than the non-induced band, the position band size is consistent with the expected 38.28KD, and the induced non-loaded pET-32a (+), BL21 escherichia coli has no specific band, which is shown in figure 3. The result shows that the pET-IL6 recombinant plasmid is successfully expressed in the escherichia coli, and the expression quantity of the recombinant bacteria after IPTG induction is higher than that of the recombinant bacteria without induction.

Western-blotting analysis is carried out on pET-IL6 recombinant bacteria liquid after induction expression, and the result also shows that the induced band is obviously clearer and more obvious than the non-induced band, the size of the position band is consistent with the expected 38.28KD, no specific band appears in the no-load pET-32a (+), BL21 escherichia coli after induction, and the result in a graph 4 is consistent with the expected result. The result shows that the pET-IL6 recombinant plasmid is successfully expressed in the escherichia coli, and the expression quantity of the recombinant bacteria after IPTG induction is higher than that of the recombinant bacteria without induction.

4. Solubility analysis of protein of interest

Adding 200 mu L (1: 100) of the pET-IL6 recombinant bacterium solution into 20mL of Amp⁺In LB, shake bacteria for 8h as first seed. Taking first-class seeds according to the proportion of 1: 100 to 2L Amp⁺In LB, induction was performed according to the method explored in step 2.13.4: centrifuging at 8000r/min for 15min at 4 deg.C, collecting bacterial sludge, discarding culture medium, and obtaining bacterial sludge of one tube per 300mL to obtain bacterial sludge of 6 tubes; adding 15mL of PBS to each tube of the bacterial sludge to suspend the bacteria, and adding 1% to the tubeThe PMSF (permanent magnet synchronous motor) is uniformly mixed by using a vortex instrument in a shaking way, and meanwhile, BL (DE3) bacterial liquid containing pET-32a (+) is set as a negative control; placing the re-suspended thallus on ice for ultrasonication (parameters are set as the power is 400w, every 4s of ultrasonication, the interval is 9s, and the ultrasonication time is 34 min); the supernatant and the precipitate were collected and analyzed by SDS-PAGE gel electrophoresis.

The results are shown in FIG. 5 (M: protein prestained; 1-4: supernatant after cell disruption; 5-8: precipitate after cell disruption), the bands of the supernatant after disruption are lighter, and the precipitates after disruption show obvious specific protein bands, which indicates that pET-IL6 protein is mainly expressed in the form of inclusion bodies.

Washing the inclusion body (pET-IL6 protein) with a washing solution (formula shown in table 5), repeating twice, washing the inclusion body once with Tris-HCl, finally dissolving the inclusion body with a dissolving solution (formula shown in table 6) at 4 ℃ for 10-12 h, collecting supernatant and precipitate samples after each washing and dissolving, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, and analyzing the change of the protein amount in each dissolving and washing process. The supernatant obtained in the first three times of washing has the presence of impure protein, which indicates that the supernatant obtained in the third time of washing has the impurity-removed protein, the dissolved supernatant has no impure protein, the amount of protein in the dissolved precipitate is also large, and the precipitate can be dissolved for the second time, as shown in FIG. 6 (M: protein pre-staining; 1: supernatant obtained by washing inclusion bodies for the first time; 2: precipitate obtained by washing inclusion bodies for the first time; 3: supernatant obtained by washing inclusion bodies for the second time; 4: precipitate obtained by washing inclusion bodies for the second time; 5: supernatant obtained by washing inclusion bodies for the third time; 6: precipitate obtained by washing inclusion bodies for the third time; 7: supernatant obtained by dissolving inclusion bodies; 8: precipitate obtained by dissolving inclusion bodies). The washing and dissolving effects are better, and the protein purity after washing and dissolving is higher.

The inclusion body wash buffer: the reagents were added according to Table 5, ddH₂O to 1000mL, mixed well by shaking, adjusted to pH 8.0 with HCl and stored in a refrigerator at 4 ℃ until needed.

TABLE 5 formulation of inclusion body wash

The inclusion body solubilization buffer: according to Table 6 addAdding various reagents, ddH₂O to 1000mL, mixed well by shaking, adjusted to pH 8.0 with HCl and stored in a refrigerator at 4 ℃ until needed.

TABLE 6 formulation of inclusion body solubilization solution

5. Purification of proteins of interest

The pET-IL6 protein is purified by a dialysis bag dialysis purification method, and the specific operation method is as follows:

(1) cut dialysis bags and cut the required length according to the amount of protein (10mL protein requires about 14 cm).

(2) Cleaning a 2L container, adding appropriate amount of ddH₂And O, putting the cut dialysis bag into a container, and boiling for 15 min.

(3) Cooling the dialysis bag with ddH₂And O, cleaning the dialysis bag for 2-3 times.

(4) One person clamps one end with a clamp and opens the other end, one person carefully adds IL-6 protein into the open end with a gun, removes air bubbles and clamps the open end, places the clamped dialysis bag in a container with 6M urea TGE, and dialyzes for 8h at 4 ℃ in a refrigerator.

(5) 6M Urea TGE was discarded from the beaker and 4M Urea TGE was added and dialyzed at 4 ℃ for 12h in a refrigerator.

(6) Discarding 4M urea TGE in the beaker, changing to 2M urea TGE, and dialyzing in refrigerator at 4 deg.C for 12 h.

(7) The 2M urea TGE in the beaker was changed to 0M urea TGE and dialyzed in a refrigerator at 4 ℃ for 12 h.

(8) 0M urea TGE in the beaker was discarded and replaced with PBS and dialyzed at 4 ℃ for 12h in a refrigerator.

(9) The purified pET-IL6 protein after dialysis was obtained.

Wherein 6M urea dialysate (TGE): the reagents were added as in Table 7, followed by 360.36g urea, ddH2O to 1000mL, mixed well with a magnetic stirrer, adjusted to pH 8.0 with HCl and left at 4 ℃.

Dialysate (TGE) for 4M urea: the reagents were added as in Table 7, followed by 240.24g of urea, ddH2O to 1000mL, mixed well with a magnetic stirrer, adjusted to pH 8.0 with HCl and left at 4 ℃.

Dialysate (TGE) for 2M urea: the reagents were added as in Table 7, followed by 120.12g of urea, ddH2O to 1000mL, mixed well with a magnetic stirrer, adjusted to pH 8.0 with HCl and left at 4 ℃.

Dialysate (TGE) with 0M urea: the reagents were added as in Table 7, followed by 0g of urea, ddH2O to 1000mL, mixed well with a magnetic stirrer, adjusted to pH 8.0 with HCl and left at 4 ℃.

TABLE 7 dialysate formulations

The purified pET-IL6 protein is sent to a manufacturer for protein mass spectrum identification by adopting LC-MS/MS mass spectrometry. 7 peptide fragments DDATSNSLPLTSANKVEELIK, EALAENNLHLPKLEGK, SVHMSTKILVQMLK, DGCFQSGFNQETCLTRITTGLVEFQLHLNILQNNYEGDKENVK, AVRIM, SKVK and HTTIHLILRSLEDFLQFSLR with higher confidence coefficient are matched by peptide fingerprint identification and partial strong signal peptide fragment sequencing, and the peptide fragment with the confidence coefficient of more than or equal to 95 percent accounts for 24.15 percent, so the mass spectrum result has higher confidence coefficient.

The successful preparation of the target pET-IL6 protein is shown, and the amino acid sequence of the pET-IL6 protein is shown in SEQ ID NO. 1:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAMNSLSTSAFSLGLLLVMATAFPTPGPLAGDSKDDATSNSLPLTSANKVEELIKYILGKISALRKEMCDKFNKCEDSKEALAENNLHLPKLEGKDGCFQSGFNQETCLTRITTGLVEFQLHLNILQNNYEGDKENVKSVHMSTKILVQMLKSKVKNQDEVTTPDPTTDASLQAILQSQDECVKHTTIHLILRSLEDFLQFSLRAVRIM

example 2 Effect of Induction time, temperature and IPTG concentration on expression of proteins of interest

1. Induction time

The pET-IL6 recombinant strain was expressed according to the procedure of "3. inducible expression of target protein" in example 1, and when the concentration of the bacterial solution reached OD600 to 0.6, IPTG was added to the final concentration of 1.0mmol/L, and the resultant was induced and cultured at 37 ℃ for 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours, respectively, and the induced products were taken and examined by Coomassie staining. SDS-PAGE analysis shows that the amount of the induced 4h protein is large, and the result is shown in figure 7 (in the figure, M: protein prestained; 1: 2h induction; 2: 3h induction; 3: 4h induction; 4: 5h induction; 5: 6h induction).

2. Induction temperature

The pET-IL6 recombinant strain was expressed according to the procedure of "3. inducible expression of target protein" in example 1, when the concentration of the bacterial solution reached OD600 to 0.6, IPTG was added to the final concentration of 1.0mmol/L, and the resultant was induced and cultured at 16 deg.C, 22 deg.C, 28 deg.C, 37 deg.C, and 42 deg.C for 4 hours, and the respective induced products were taken out and examined by Coomassie staining to compare the differences in protein expression levels at different induction temperatures. SDS-PAGE analysis showed that the amount of protein was higher at 37 ℃ induction, and the results are shown in FIG. 8 (M: protein prestained; 1: 16 ℃ induction; 2: 22 ℃ induction; 3: 28 ℃ induction; 4: 37 ℃ induction; 5: 42 ℃ induction).

IPTG concentration

The pET-IL6 recombinant strain was expressed according to the procedure of "3. inducible expression of target protein" in example 1, and when the concentration of the bacterial solution reached OD600 to 0.6, IPTG was added to the bacterial solution at final concentrations of 0.1mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L and 1.5mmol/L, and the mixture was induced at 37 ℃ for 4 hours, and the induced products were taken out and examined for differences in protein expression levels by Coomassie staining. SDS-PAGE analysis showed that the concentrations were not very different, and IPTG was used at a final concentration of 0.1mmol/L, as shown in FIG. 9 (M: protein prestained; 1: 0.1mmol/L induction; 2: 0.5mmol/L induction; 3: 0.8mmol/L induction; 4: 1.0mmol/L induction; 5: 1.2mmol/L induction; 6: 1.5mmol/L induction).

EXAMPLE 3 preparation of pET-IL6 antibody

1. Preparation of monoclonal antibodies

BALB/c mice were immunized by emulsifying purified pET-IL6 protein (prepared in example 1) in combination with adjuvants until a serum titer of 1: 10 was reached⁵Taking mouse spleen cells and myeloma cells with round cell shape, good light transmittance and uniform sizeCell fusion was performed.

The specific operation is as follows:

BALB/c mice were immunized with the purified pET-IL6 protein (prepared in example 1) mixed with an adjuvant and emulsified, the first immunization, and the injection was performed at multiple points subcutaneously on the back of the mouse neck; carrying out second and third immunizations after fully mixing and emulsifying the equivalent volume of IL-6 protein by Freund incomplete adjuvant (IFA) at 14d and 28d respectively; after the first immunization for 32 days, blood is collected from the orbit, the supernatant is centrifuged, and the serum antibody level is detected when the positive serum titer reaches 1: 10⁵Then preparing for cell fusion; the mice are injected with non-emulsified antigens in the abdominal cavity 3d before fusion for boosting immunity, and the splenocytes of the mice are taken aseptically for fusion of SP2/0 cells 3-4 d, wherein the specific immunization program is shown in Table 8.

TABLE 8BABL/c mouse immunization procedure

Mouse splenocytes and SP2/0 cells were prepared in advance, and mouse myeloma SP2/0 cells were mixed with splenocytes at a ratio of 1: (5-10) (cell number), fusing under PEG1450, washing, centrifuging, suspending with HAT culture medium, placing in 96-well culture plate, and culturing at 37 deg.C in the presence of 5% CO₂After 3 days of culture in an incubator, the HAT culture medium is used for changing the culture medium, the HT culture medium is used for changing the culture medium on the 9 th day, and the indirect ELISA method is used for screening positive holes of the cells, and the specific operation is as follows:

antigen coating: the IL-6 protein prepared in example 1 was diluted with coating solution at 8. mu.g/mL, 100. mu.L per well, added to the well, and refrigerated overnight at 4 ℃;

washing the plate: discarding the liquid in the plate, spin-drying, adding 200 μ L PBST per hole, placing in a micro oscillator, shaking for 60s, spin-drying, and washing the plate for 4 times;

and (3) sealing: add 100. mu.L/well of blocking solution to the well, put at 37 ℃ for 2h (or 4 ℃ overnight block);

the confining liquid is as follows: adding 25g of Bovine Serum Albumin (BSA) into 500mL of PBST, fully mixing, subpackaging into a centrifuge tube, and placing in a refrigerator at-20 ℃;

washing the plate: discarding liquid in the plate, spin-drying water, carrying out PBST with 200 mu L/hole, spin-drying water, and washing the plate for 4 times;

hatching primary antibody: adding the cell supernatant of the fusion well and 100 mu L of positive, negative and blank controls into an enzyme label plate, and acting for 70min at 37 ℃;

washing the plate: discarding cell supernatant in fusion holes in the plate, spin-drying water, PBST with 200 muL/hole, spin-drying liquid, and washing for 4 times;

hatching a secondary antibody: the reaction solution was washed with PBS 1: 10000-diluted HRP-goat anti-mouse IgG, 100 mu L of diluted secondary antibody is added into each hole, and the mixture acts for 40min at the temperature of 37 ℃;

washing the plate: discarding the secondary antibody in the plate, spin-drying water, carrying out PBST with 200 mu L/hole, spin-drying water, and washing the plate for 4 times;

color development: allowing 100 μ L of color developing solution to act at 37 deg.C for 10min (operation in dark place);

and (4) terminating: the reaction was stopped by adding 50. mu.L of diluted sulfuric acid to each well and reading was performed as quickly as possible using a microplate reader.

The screened positive cell wells are further identified and screened by an indirect ELISA method (using purified pET-32a (+) no-load protein, pET-IL6 protein and other fusion protein expressed by pET-32a (+) no-load as antigen to carry out coating and screening out false positive), the limited dilution method is adopted to clone to about less than 1 cell per well, and after 10 days, the cell strains obtained from the single cloning wells which are detected to be positive and have better competition are the cell strains secreting monoclonal antibodies. After indirect ELISA screening and three times of subcloning, hybridoma cell strains (shown in the table 9 as subcloned to 9 monoclonal holes, and the shaded part is the monoclonal hole) for stably secreting monoclonal antibodies are obtained and used for preparing IL-6 monoclonal antibodies.

TABLE 9 subcloning of Positive hybridoma cells

2. Preparation and characterization of monoclonal antibodies

The ascites is prepared by taking positive hybridoma cells with strong antibody secretion capacity and good growth state through an in vivo induction method. And (3) sensitizing the mice by Freund's incomplete adjuvant, and injecting 500 mu L of the mixture into the abdominal cavity of each female BALB/c mouse for 7-10 days for later use. Blowing the cells from the large dish, washing the cells with 1640 for 2-3 times, and adjusting the number to 1 × 10⁶Cells per mL were injected intraperitoneally into sensitized mice. After 1-2 weeks, when the abdomen of the mouse is obviously enlarged, ascites is extracted, centrifugation is carried out for 15min at 10000-12000 r/min, supernatant is taken out, subpackaging and storing at-80 ℃ to obtain the monoclonal antibody.

Western-blotting and IFA verification prove that the obtained monoclonal antibody is a conformational epitope, which indicates that the anti-IL-6 monoclonal antibody of the mouse is successfully prepared.

3. Preparation of polyclonal antibodies

The pET-IL6 protein prepared in example 1 is mixed and emulsified with adjuvant, and then immunized with New Zealand white rabbits, after three times of immunization (1 mg/one is mixed and emulsified with the same amount of adjuvant), blood is collected, centrifuged and serum is taken, thus obtaining pET-IL6 polyclonal antibody, and the titer is measured by an indirect ELISA method, which comprises the following steps:

washing the plate: discarding the liquid in the plate, spin-drying, adding 200 μ L PBST per hole, placing in a micro-oscillator, oscillating for 60s, spin-drying, and washing the plate for 4 times;

the confining liquid is as follows: taking 25g of Bovine Serum Albumin (BSA), adding 500mL of PBST, fully mixing, subpackaging into a centrifuge tube, and placing in a refrigerator at-20 ℃ for later use;

incubating primary antibody: adding the rabbit serum and 100 μ L of positive, negative and blank control into enzyme labeling plate, and allowing to act at 37 deg.C for 70 min;

washing the plate: discarding rabbit serum in the plate, spin-drying water, PBST with 200 muL/hole, spin-drying liquid, and washing for 4 times;

hatching a secondary antibody: the reaction solution was washed with PBS 1: diluting HRP-goat anti-mouse IgG by 10000, adding 100 mu L of diluted secondary antibody into each hole, and acting for 30-40 min at 37 ℃;

color development: acting 100 mu L of color development liquid in each hole for 5-10 min at 37 ℃ (operation in dark place);

The titer is shown in Table 10, which indicates that the antibody titer is high, and the pET-IL6 polyclonal antibody can be used as a detection antibody.

TABLE 10 Rabbit serum titer assay

EXAMPLE 4 establishment of ELISA kit for detecting Canine IL-6

Composition of ELISA kit

The ELISA kit comprises:

pET-IL6 monoclonal antibody (prepared in example 3);

pET-IL6 polyclonal antibody (prepared in example 3);

an ELISA plate;

sealing liquid: 5% of skimmed milk powder (mass ratio);

goat anti-rabbit enzyme-labeled secondary antibody;

color development liquid: tetramethylbenzidine (TMB);

stopping liquid: 2mol/L of H₂SO₄；

Coating liquid: 0.05M carbonate buffer (pH9.6, Na)₂CO₃ 1.59g，NaHCO₃ 2.93g)；

Washing liquid: pbs (pbst) buffer containing 0.05% tween 20.

Second, use of ELISA kit

Use of an ELISA kit comprising the steps of:

s1, coating: diluting pET-IL6 monoclonal antibody to 48 mu g/mL by using coating solution, adding 100 mu L/hole into the plate, and refrigerating at 4 ℃ overnight;

s2, washing a plate: discarding the liquid in the ELISA plate, and oscillating and rinsing for 4 times and once for 60s according to 200 mu L/hole of PBST (phosphate Tween buffer solution);

s3, sealing: adding 100 μ L of sealing solution into each hole, sealing in a thermostat at 37 deg.C for 60min, and washing plate in the same manner as S2;

s4, sample adding: incubating 100. mu.L of sample per well at 37 ℃ for 1.5h, and washing the plate as in S2;

s5, adding a detection antibody: blocking solution for pET-IL6 polyclonal antibody is prepared according to the following steps of 1: diluting with 100. mu.L of diluted IL-6 polyclonal antibody per well, performing action at 37 deg.C for 60min, and washing the plate with S2;

s6, adding a secondary antibody: add 1: 500, diluting the goat anti-rabbit enzyme-labeled secondary antibody with 100 mu L of blocking solution, incubating for 60min at 37 ℃, and washing the plate with S2;

s7, color development: adding color developing solution in a dark environment, performing reaction at 37 deg.C for 10min with a 100 μ L/hole;

s9, reading: OD450 values were read for each well using a microplate reader.

Example 5 selection of blocking solution in ELISA kit

First, experiment method

Blocking was performed with 2% BSA, 5% BSA, 2% skim milk powder, and 5% skim milk powder, respectively, instead of the blocking solution in the kit of example 4, and a 8. mu.g/mL pET-IL6 protein sample was detected using the kit.

Second, experimental results

Finally, the P/N value is compared to find that the P/N value is the largest when 5% skimmed milk powder is used for sealing, and the data result shows that the effect is the best when 5% skimmed milk powder is used for sealing under the same condition, and the table 11 shows that the P/N value is the largest when the skimmed milk powder is used for sealing.

TABLE 11 selection of blocking fluids

Example 6 determination of the optimal concentration of pET-IL6 antibody in an ELISA kit

First, experiment method

The concentrations of the capture antibody and the detection antibody were investigated by the checkerboard method, and the capture antibody pET-IL6 (prepared in example 3) was diluted with a coating solution to 48. mu.g/mL, 24. mu.g/mL, 12. mu.g/mL, 6. mu.g/mL, 3. mu.g/mL, and 1.5. mu.g/mL instead of pET-IL6 in the kit of example 4. A pET-IL6 protein sample (prepared in example 1) was diluted to 8. mu.g/mL with a blocking solution and used as a test sample. The detection antibody, pET-IL6 polyclonal antibody (prepared in example 3), was applied to a blocking solution in a 1: 100. 1: 500. 1: 1000. 1: 5000. 1: 10000. 1: 50000 dilutions, 100 μ L per well, were made in place of pET-IL6 polyclonal antibody in the kit of example 4. The above different concentrations of antibody combinations were tested using the kit of example 4. The P/N values were used to determine the optimal working concentrations of capture and detection antibodies.

Second, experimental results

According to the data in Table 12, pET-IL6 monoclonal antibody concentration is 48 μ g/mL, pET-IL6 polyclonal antibody is as 1: the P/N value is maximum when 100 is diluted, and the detection effect of the sandwich ELISA is optimal.

TABLE 12 determination of optimal concentrations of Capture and detection antibodies

Example 7 determination of blocking time in ELISA kits

First, experiment method

The kit of example 4 was used, and the blocking time in S3 was used as the kit for 1h, 1.5h, and 2h, respectively. Samples of pET-IL6 protein at 8. mu.g/mL were tested with different blocking times, respectively.

Second, experimental results

As a result, as shown in Table 13, the P/N ratio was maximized at a blocking time of 1 hour, and the optimum blocking time was selected to be 1 hour.

TABLE 13 determination of the closure time

Example 8 determination of incubation time of samples with pET-IL6 Capture antibody in ELISA kits

First, experiment method

The kit of example 4 was used, 1h, 1.5h, 2h, respectively, as the incubation time in step S4. Samples of pET-IL6 protein at 8. mu.g/mL were assayed with different incubation times, respectively.

Second, experimental results

As shown in Table 14, the duration of antigen action was 1.5 hours, since the P/N ratio was the highest at 1.5 hours.

TABLE 14 determination of antigen action time

Example 9 determination of the dilution of goat anti-rabbit enzyme-labeled Secondary antibody in ELISA kit

First, experiment method

Using the kit of example 4, goat anti-rabbit enzyme-labeled secondary antibody was added at a ratio of 1: 500. 1: 1000. 1: 5000. 1: 10000 dilution, respectively replacing goat anti-rabbit enzyme labeled secondary antibody in the kit, and detecting the pET-IL6 protein sample of 8 mug/mL.

Second, experimental results

The results are shown in Table 15, 1: the P/N of the 500 diluted secondary antibody is highest, so that the secondary antibody can be expressed as 1: and (5) diluting by 500 percent.

TABLE 15 selection of enzyme-labeled Secondary antibody dilution

EXAMPLE 10 creation of Standard Curve

First, experiment method

IL-6 standard protein was diluted to 600ng/mL, 300ng/mL, 150ng/mL, 75ng/mL, 37.5ng/mL, 18.75ng/mL, 9.38ng/mL, 4.69ng/mL, 2.34ng/mL, 1.17ng/mL, 0.59ng/mL, each concentration was duplicated, 100. mu.L/well was added to the plate, a protein-free control group was set, and each assay was performed using the kit of example 4, and a standard curve was drawn based on the results.

Second, experimental results

The standard curve is plotted as shown in FIGS. 10 and 11, and the results show that the linear relationship between the natural logarithm of the IL-6 protein concentration and the OD value is good when the IL-6 protein concentration is between 1.17ng/mL and 300ng/mL, so that the standard curve can be plotted, and the regression equation is as follows: 0.2001x +0.1258, R²0.9912, x ln (x). The detection range is 1.17 ng/mL-300 ng/mL, and the lower detection limit is 1.17 ng/mL.

Example 11 clinical application of ELISA kits

First, experiment method

Using the kit of example 4, the change of IL-6 in dogs immunized with rabies vaccine and dogs not immunized with rabies vaccine in Zengcheng dog farm was examined within 11 months, 3 samples of each month were taken from the immunized group and the non-immunized group, and the serum was sampled and analyzed for the results.

Second, experimental results

The average OD value of the samples per month was taken as the ordinate and the month as the abscissa, to prepare FIG. 12. As can be seen from FIG. 12, IL-6 is secreted in the dog body after immunization of rabies vaccine and increases in endocrine compared with the serum of non-immunized rabies vaccine, the IL-6 level is in a slow decline trend along with the increase of time, the IL-6 content is highest in 1 month, the IL-6 content reaches 3.17ng/mL, and the IL-6 content gradually declines backwards until reaching the normal level. The IL-6 content in the serum of the non-immunized rabies vaccine is always at the same level, and the fluctuation is small. The rabies vaccine still stimulates an organism to generate antibodies within one year after immunization, and the kit has good detection effect and high detection sensitivity.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

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Ala Met Asn Ser Leu Ser Thr Ser Ala Phe Ser Leu Gly Leu Leu Leu

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Claims

1. An ELISA kit for detecting canine IL-6, wherein the ELISA kit comprises: the kit comprises a pET-IL6 monoclonal antibody, a pET-IL6 polyclonal antibody, an enzyme label plate, a confining liquid, a goat anti-rabbit enzyme labeled secondary antibody, a developing liquid and a stop solution;

2. The ELISA kit of claim 1 wherein the blocking solution is one or more of 2% to 5% BSA, 2% to 5% skimmed milk powder; wherein the percentage is the mass ratio percentage.

3. The ELISA kit of claim 1, wherein the pET-IL6 protein is prepared by the following method:

transforming the plasmid into BL21 escherichia coli to obtain pET-IL6 recombinant bacteria; inducing the pET-IL6 recombinant bacteria by using 0.1-1.5 mmol/L IPTG, carrying out induced culture at 22-42 ℃ for 2-6 h, collecting bacterial liquid, centrifuging, removing supernatant, taking supernatant after precipitation, washing and dissolving the obtained protein, and purifying to obtain pET-IL6 protein;

wherein, the nucleotide sequence of the IL-6 gene is shown in SEQ ID NO. 2.

4. The ELISA kit of claim 1, wherein the concentration of the pET-IL6 monoclonal antibody is 24-48 μ g/mL, and the concentration of the pET-IL6 polyclonal antibody is 1: 500-1: 100 dilution.

5. The ELISA kit of claim 1, wherein the dilution of the goat anti-rabbit enzyme-labeled secondary antibody is 1: 1000-1: 500.

6. the ELISA kit of claim 1, wherein the color-developing solution is tetramethylbenzidine.

7. The ELISA kit of claim 1 wherein the stop solution is H₂SO₄。

8. The ELISA kit of any one of claims 1 to 7, characterized in that its method of use comprises the following steps:

s1, coating: adding the pET-IL6 monoclonal antibody into an ELISA plate at a concentration of 100 mu L/hole, and putting the ELISA plate into a refrigerator overnight;

s2, washing a plate: discarding the liquid in the ELISA plate in S1, and washing the plate by using PBST;

s3, sealing: adding 100 mu L of sealing liquid into each hole of the ELISA plate of S2, sealing in a constant temperature box at 37 ℃, and washing the plate after sealing in the same step as S2;

s4, sample adding: adding 100 mu L of sample into each well of the ELISA plate of S3, incubating at 37 ℃, and washing the plate as with S2;

s5, adding a detection antibody: diluting pET-IL6 polyclonal antibody with blocking solution, adding 100 μ L of diluted pET-IL6 polyclonal antibody into each hole of an ELISA plate of S4, acting for 60min at 37 ℃, and washing the plate with S2;

s6, adding a secondary antibody: adding 100 mu L of diluted goat anti-rabbit enzyme-labeled secondary antibody into each hole of the enzyme-labeled plate of S5, incubating for 60min at 37 ℃, and washing the plate with S2;

s7, color development: adding color developing solution into the ELISA plate of S6 in a dark environment, performing reaction at a temperature of 37 ℃ for 10min at a concentration of 100 mu L/hole;

s8, terminating: adding 50 mu L of stop solution into each hole of the ELISA plate of S7 to stop the reaction;

s9, reading: the OD450 values of each well of the microplate in S8 were read using a microplate reader.

9. The ELISA kit of claim 8, wherein in step S3, the blocking time is 1-2 h.

10. The ELISA kit of claim 8, wherein in step S4, the incubation time of the sample is 1-2 h.