CN107576791A - One boar lawsonia intracellularis ELISA detection kit - Google Patents

Abstract

The invention discloses an ELISA detection kit for Lawsonia intracellulare, the ELISA detection kit for Lawsonia intracellulare comprises a solid phase carrier and an antigen coated on the solid phase carrier, and the antigen is LsaA recombinant protein , the amino acid sequence of the LsaA recombinant protein is shown in SEQ ID NO.1. The kit has the characteristics of high sensitivity, strong specificity, and good stability. Moreover, it is convenient to use, simple to operate, short in detection time, and low in cost. It can be used for serological investigation of Lawsonia intracellulare suis infection in pigs , Antibody detection and evaluation of vaccine immune effects, etc., suitable for large-scale promotion and use.

Description

A kind of Lawsonia intracellulare ELISA detection kit

technical field

The invention belongs to the technical field of biological detection, in particular to an ELISA detection kit for Lawsonia intracellulare.

Background technique:

Lawsonia intracellularis (LI) is an obligate intracellular parasitic bacterium that mainly parasitizes in the epithelial cells of the intestinal mucosa of pigs and causes proliferative enteritis in pigs. Clinically, it leads to loss of appetite, diarrhea and growth retardation in affected pigs, causing huge economic losses to the pig industry. Since porcine proliferative enteritis was reported in 1931, it has existed and been prevalent in major pig-raising countries in the world. There is not much attention paid to Lawsonia intracellulare infection in my country, and there is no comprehensive and clear understanding of the prevalence of this bacteria in pig herds in China. The main reason for this situation is not only the lack of attention, but also the lack of detection methods. Serological diagnosis plays an important role in understanding the epidemic situation of the disease, detecting antibody levels, evaluating the immune effect of vaccines, and evaluating the effect of drug treatment. Serological diagnosis is a key step in controlling the epidemic.

For the serological diagnosis of Lawsonia intracellulare, some scholars have used immunoperoxidase monolayer assay (IPMA) and indirect immunofluorescence test (IFAT) to detect Lawsonia antibodies, but both methods require fluorescence. Microscopes have high requirements for personnel and equipment, and the judgment of test results is highly subjective. Boesen et al. used the sodium deoxycholate extract of cultured Lawsonia intracellulare as the coating antigen to establish an ELISA for detecting the specific antibody of Lawsonia intracellulare. The established ELISA method can be well used for pig proliferation Diagnosis of proliferative enteritis and evaluation of the prevalence of proliferative enteritis in swine herds. However, Lawsonia intracellulare is a strict intracellular parasitic bacterium, and its culture conditions are harsh. At present, only a few laboratories in the world have achieved the isolation and culture of Lawsonia intracellulare, and it is very difficult and costly to obtain coated antigens by culturing bacteria. Therefore, it is of great significance to prepare a new ELISA coating antigen that replaces the whole Lawsonia intracellularis, and then develop an ELISA detection kit for Lawsonia intracellularis with my country's independent intellectual sovereignty, and has broad prospects.

LsaA protein is a surface protein of Lawsonia intracellulare, which is synthesized during the infection of Lawsonia intracellulare and participates in the adhesion and invasion of Lawsonia intracellulare. The natural LsaA gene encoding the synthetic LsaA protein is 0.7kb in length, contains only one ORF, and the molecular weight of the encoded protein is about 27.4KDa. Because each organism has a large difference in the utilization of codons when translating genes, this difference directly affects the level of gene expression. Lawsonia intracellulare is an obligate intracellular parasitic bacterium that mainly parasitizes in the epithelial cells of the intestinal mucosa of pigs. Codon utilization analysis found that the codon utilization of LsaA protein was low in the prokaryotic expression system (E. low expression. In order to solve this problem, the present invention optimizes the gene sequence encoding the synthetic LsaA protein according to the codon preference of Escherichia coli, so that its codons have a high utilization rate in Escherichia coli, thereby realizing that the LsaA protein can be expressed at a high level in Escherichia coli , and then use the purified LsaA recombinant protein instead of the whole Lawsonia intracellularis extract as the coating antigen to prepare an ELISA detection kit for Lawsonia intracellularis.

Contents of the invention

Aiming at the problems existing in the prior art, the object of the present invention is to provide an ELISA detection kit for Lawsonia intracellularis. The Lawsonia intracellularis suis ELISA detection kit has strong specificity and good stability, and can be used to detect the antibody level of Lawsonia intracellularis suis in pigs to judge the infection of Lawsonia intracellularis suis in pigs and testing immune effects.

For realizing the purpose of the invention, the technical scheme adopted in the present invention is as follows:

An ELISA detection kit for Lawsonia intracellulare, the ELISA detection kit for Lawsonia intracellulare includes a solid phase carrier and an antigen coated on the solid phase carrier, the antigen is LsaA recombinant protein, and the LsaA The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

According to the aforementioned Lawsonia intracellularis suis ELISA detection kit, preferably, the LsaA recombinant protein is encoded by a gene having the nucleotide sequence shown in SEQ.ID.NO.2.

According to the above Lawsonia intracellularis ELISA detection kit, the preparation method of the LsaA recombinant protein is: synthesize the opt-LsaA gene, and clone the opt-LsaA gene into the pET-32a vector to obtain the recombinant expression vector pET-32a-opt -LsaA; transform the recombinant expression vector pET-32a-opt-LsaA into the host bacteria, select positive clones containing the recombinant expression vector pET-32a-opt-LsaA, and perform IPTG-induced expression on the positive clones; the expression product After being purified by Ni-NTA agarose affinity chromatography column, the purified LsaA recombinant protein is obtained; wherein, the nucleotide sequence of the opt-LsaA gene is shown in SEQ ID NO.2.

According to the above-mentioned Lawsonia intracellularis ELISA detection kit, preferably, the specific process of constructing the recombinant expression vector pET-32a-opt-LsaA is: synthesizing the opt-LsaA gene, and the opt-LsaA gene sequence at 5 NdeI restriction endonuclease recognition site is added to the 'end, and Xho I restriction endonuclease recognition site is added to the 3' end; opt-LsaA gene and pET-32a vector are respectively used NdeI and Xho I restriction endonuclease performs double digestion, and the opt-LsaA gene after digestion is connected to the pET-32a vector to obtain the recombinant expression vector pET-32a-opt-LsaA. More preferably, the host bacteria is Escherichia coli.

According to the aforementioned Lawsonia intracellularis suis ELISA detection kit, preferably, the solid phase carrier is an ELISA plate, and each well of the ELISA plate is coated with 0.2 μg of LsaA recombinant protein.

According to the above Lawsonia intracellularis ELISA detection kit, preferably, the Lawsonia intracellularis suis ELISA detection kit also includes enzyme-labeled antibody, negative control serum, positive control serum, washing solution, sample and antibody dilution solution, developing solution and stop solution. More preferably, the enzyme-labeled antibody is horseradish peroxidase-labeled rabbit anti-pig IgG; the positive control serum is collected from pig serum containing anti-LsaA recombinant protein antibody after immunizing pigs with LsaA recombinant protein, which is Positive control serum; the negative control serum is the serum of healthy pigs not infected with Lawsonia intracellulare.

The preparation of Lawsonia intracellularis ELISA detection kit of the present invention:

1. Preparation of reagents:

(1) Coating solution: 0.05M carbonate buffer solution with pH 9.6, which is prepared as follows: Dissolve 0.159g Na ₂ CO ₃ and 0.294g NaHCO ₃ with a small amount of sterile water, and then add sterile water to make up the volume to 100mL;

(2) Concentrated washing solution: 10 times washing solution (diluted 10 times with double distilled water as working solution), diluted 10 times with double distilled water, then it is 0.1M PBS buffer containing 0.5% (v/v) Tween-20 Liquid (pH7.2-7.4);

(3) Sample diluent: 0.1M PBS buffer (pH7.2-7.4) containing 5% (w/v) skimmed milk and 0.5% (v/v) Tween-20;

(4) Enzyme-labeled antibody: horseradish peroxidase-labeled rabbit anti-pig IgG (manufactured by Abcam), obtained by diluting with sample diluent at a volume ratio of 1:10000.

(5) Negative control serum: the serum of healthy pigs not infected with Lawsonia intracellularis was collected, which was the negative control serum.

(6) Positive control serum: After immunizing pigs with LsaA recombinant protein, collect pig serum containing anti-LsaA recombinant protein antibody, which is the positive control serum.

(7) Color-developing solution: The color-developing solution is composed of color-developing solution A and color-developing solution B; the preparation method of the color-developing solution A is: 200mg of tetramethylbenzidine is dissolved in the dehydrated alcohol of 100mL, and then Set the volume to 1000mL with double-distilled water to obtain the color-developing solution A; the preparation method of the color-developing solution B is: weigh 21g of citric acid and 28.2g of anhydrous sodium phosphate, fully dissolve them in 400ml of double-distilled water, and then add 0.75% hydrogen peroxide urea 6.4mL, then distill the volume to 1000mL with double distilled water, adjust the pH value to 4.5-5.0; chromogenic solution A and chromogenic solution B are both 60mL;

(8) Stop solution: 2M H ₂ SO ₄ solution.

2. Preparation of ELISA plate coated with LsaA recombinant protein:

The specific steps of the preparation method of the enzyme label plate coated with LsaA recombinant protein are as follows:

(1) Antigen dilution and coating: Dilute the purified LsaA recombinant protein with coating solution (0.05M carbonate buffer at pH 9.6) to a concentration of 2 μg/mL, and take the diluted LsaA recombinant protein Add 100 μL/well to a 96-well ELISA plate, incubate at 37°C for 1 hour, then overnight at 4°C;

(2) Washing: Discard the liquid in the wells of the 96-well ELISA plate, then add washing solution (10 times concentrated washing solution diluted 10 times with double distilled water) to the wells of the ELISA plate, 300 μL/well, repeat washing for 5 times, 5 minutes each time (discard the liquid in the microplate wells for each wash, and drain the remaining liquid on absorbent paper after discarding the washing liquid for the last wash);

(3) Sealing: Add 5% skimmed milk (300 μL/well) to the 96-well ELISA plate, block at 37° C. for 2 h, then wash according to step (2), and seal the ELISA plate with a self-adhesive sealing film. Packaged in a sealed bag and stored at 4°C.

3. The composition of the ELISA detection kit for Lawsonia intracellulare of the present invention:

(1) 5 microtiter plates coated with LsaA recombinant protein;

(2) Negative control serum and positive control serum 2 mL each;

(3) Enzyme-labeled antibody, the specification is 60mL;

(4) Concentrated washing solution, the specification is 200mL;

(5) Sample diluent, the specification is 150mL;

(6) Chromogenic solution A, the specification is 60mL;

(7) Chromogenic solution B, the specification is 60mL;

(8) Stop solution, the specification is 30mL.

The positive beneficial effect that the present invention obtains:

(1) According to the codon preference of Escherichia coli, the present invention conducts DNA analysis and RNA structure prediction on the Lawsonia intracellularis suis LsaA gene sequence (accession number AF498259) registered in GenBank, without changing the amino acid sequence of the LsaA protein The LsaA gene was artificially optimized to obtain the opt-LsaA gene, which made the codon of the LsaA protein have a high utilization rate in E. coli, thereby realizing the high-level expression of the LsaA protein of Lawsonia intracellulare in E. coli, effectively solving the problem of encoding The original LsaA gene for synthesizing LsaA protein is directly cloned into the problem that the expression level of LsaA protein in Escherichia coli is low.

(2) The present invention is based on the principle of indirect ELISA, and then uses the purified LsaA recombinant protein synthesized by the prokaryotic expression system to replace the whole bacterial extract of Lawsonia intracellulare as the coating antigen, and prepares a pig that is coated with the LsaA recombinant protein Lawsonia intracellulare ELISA detection kit, the kit has the characteristics of high sensitivity, strong specificity, good stability, etc., and it is easy to use, simple to operate, short detection time, low cost of use, and can be used for pig cells It is suitable for large-scale promotion and use in serological investigation of Lawsonia innerella infection, antibody detection and evaluation of vaccine immune effect.

Description of drawings

Figure 1 is the enzyme digestion and identification diagram of the recombinant expression vector pET-32a-opt-LsaA obtained by inserting the artificially optimized and synthesized opt-LsaA gene into the prokaryotic expression vector pET-32a; in which: lane 1 is DNA Marker DL15000; lane 2 is the recombinant The double restriction fragment of the expression vector pET-32a-opt-LsaA; Lane 3 is the recombinant expression vector pET-32a-opt-LsaA without digestion.

Figure 2 is the SDS-PAGE electrophoresis of the expression product of the positive clone bacteria containing the recombinant expression vector pET-32a-opt-LsaA induced by IPTG; wherein: Lane 1 is the low molecular weight protein Marker; Lane 2 is the uninduced Positive clones containing the recombinant expression vector pET-32a-opt-LsaA; lane 3 is the positive clones induced by IPTG; lane 4 is the supernatant of the positive clones induced by IPTG; lane 5 is the positive clones induced by IPTG The precipitate after bacterial destruction; Lane 6 is the purified LsaA recombinant protein.

Figure 3 is the antigenic identification diagram of the purified LsaA recombinant protein; among them: Swimming lane 1 is the pre-stained molecular weight protein Marker; Swimming lane 2 is the reaction between the purified LsaA recombinant protein and Lawsonia intracellularis positive serum; Swimming lane 3 is the purified LsaA recombinant protein The LsaA recombinant protein reacted with Lawsonia intracellulare-negative sera.

detailed description

The content of the present invention will be further described below in conjunction with specific examples, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention. Unless otherwise specified, the raw materials and chemical reagents used in the examples are conventional commercially available products, and the technical means used are conventional means known to those skilled in the art. Unless otherwise specified, the reagents and kits used in the present invention are commercially available.

Example 1: Synthesis of opt-LsaA gene and construction of recombinant expression vector pET-32a-opt-LsaA

According to the codon preference of Escherichia coli, the present invention conducts DNA analysis and RNA structure prediction on the LsaA gene sequence of Lawsonia intracellularis (accession number AF498259) registered in GenBank, and synthesizes the code without changing the amino acid sequence of the LsaA protein. The LsaA gene of the LsaA protein is artificially optimized to obtain the opt-LsaA gene, and the nucleotide sequence of the artificially optimized and synthesized opt-LsaA gene is shown in SEQ ID NO.2.

Add the NdeI restriction endonuclease recognition site at the 5' end of the opt-LsaA gene sequence artificially optimized and synthesized above, and add the XhoI restriction endonuclease recognition site at its 3'end; opt-LsaA The gene and the pET-32a vector were double-digested with NdeI and XhoI restriction endonucleases, and the digested opt-LsaA gene was ligated with the pET-32a vector with T4DNA ligase, and the ligated product was transformed into E. coli DH5α. 100μg·mL ^-1 Amp containing LB solid culture plate ^, cultured in a constant temperature incubator at 37°C overnight; pick the monoclonal colony on the LB culture plate and inoculate it on the LB solid culture plate containing 100μg·mL ^-1 Amp In the liquid medium, shake and culture overnight at 37°C. The kit extracts the bacterial fluid plasmids for PCR identification. The positive plasmids are then identified by double enzyme digestion. The positive plasmids identified by double enzyme digestion are sent to the sequencing company for sequencing, and finally sequenced. The correct recombinant expression vector pET-32a-opt-LsaA (Figure 1).

Example 2: Induced expression and purification of LsaA recombinant protein

The correct recombinant expression vector pET-32a-opt-LsaA sequenced in Example 1 was transformed into E.coli BL21 (DE3) competent cells, and then the transformed E.coli BL21 (DE3) was coated to contain 100 μg. LB solid culture plate of mL ^-1 Amp, cultivate overnight at 37°C. Pick a single colony on the next day in LB liquid medium containing 100μg·mL ^-1 Amp, culture at 37°C 250r·min ^-1 until the OD _600nm is about 0.8, add IPTG to a final concentration of 0.1mmol·L ^-1 , 37 ℃, 250r·min ^-1 shake induction culture for 6h. Centrifuge the induced bacterial solution at 4000rpm for 10min, discard the supernatant, and collect the bacterial pellet; resuspend the pellet with 0.1M PBS, sonicate for 5min (sonication for 9s, stop for 6s) to break the bacterial cell, and then centrifuge at 10000rpm at 4°C for 10min , collect the supernatant and the precipitate respectively, resuspend the precipitate with 0.1M PBS, and conduct SDS-PAGE electrophoresis to identify whether the protein is soluble or not. The results of SDS-PAGE electrophoresis identification are shown in Figure 2, from which it can be seen that the expression form of the LsaA recombinant protein is inclusion body. Because the LsaA recombinant protein was fused with a polyhistidine (6×His) tag, the Ni-NTA protein purification kit (Sangon Bioengineering (Shanghai) Co., Ltd.) was used to purify the LsaA recombinant protein in the expressed product. After purification, The LsaA recombinant protein with a size of 27 kDa and a single band was obtained ( FIG. 2 ).

Embodiment 3: Identification of LsaA recombinant protein

The antigenicity of the purified LsaA recombinant protein was analyzed by Western blot. The specific operation was as follows: the LsaA recombinant protein separated by SDS-PAGE electrophoresis was transferred to a nitrocellulose membrane, and the nitrocellulose membrane was placed in a 5 % (w/v) skimmed milk powder blocking solution overnight at 4°C. Porcine anti-Lawsonia intracellulare positive serum was diluted with PBS buffer containing 5% (w/v) skimmed milk and 0.5% (v/v) Tween-20 at a volume ratio of 1:200 as the primary antibody , incubate at 37°C for 2h, wash the membrane and dilute horseradish peroxidation with PBS buffer containing 5% (w/v) skimmed milk and 0.5% (v/v) Tween-20 according to the volume ratio of 1:5000 Phytosome-labeled rabbit anti-pig IgG was used as a secondary antibody, incubated at 37°C for 2 hours, and finally developed with a DAB color development kit (purchased from Wuhan Boster Bioengineering Co., Ltd.). The results showed that the purified LsaA recombinant protein had good antigenicity ( FIG. 3 ).

Embodiment 4: Preparation of Lawsonia intracellularis ELISA detection kit of the present invention

1. Preparation of reagents:

(1) Coating solution: 0.05M carbonate buffer solution with pH 9.6, which is prepared as follows: Dissolve 0.159g Na ₂ CO ₃ and 0.294g NaHCO ₃ with a small amount of sterile water, and then add sterile water to make up the volume to 100mL;

(2) Concentrated washing solution: 10 times washing solution (diluted 10 times with double distilled water as working solution), diluted 10 times with double distilled water, then it is 0.1M PBS buffer containing 0.5% (v/v) Tween-20 Liquid (pH7.2-7.4);

(3) Sample diluent: 0.1M PBS buffer (pH7.2-7.4) containing 5% (w/v) skimmed milk and 0.5% (v/v) Tween-20;

(4) Enzyme-labeled antibody: horseradish peroxidase-labeled rabbit anti-pig IgG (manufactured by Abcam), obtained by diluting with sample diluent at a volume ratio of 1:10000.

(5) Negative control serum: the serum of healthy pigs not infected with Lawsonia intracellularis was collected, which was the negative control serum.

(6) Positive control serum: After immunizing pigs with LsaA recombinant protein, collect pig serum containing anti-LsaA recombinant protein antibody, which is the positive control serum.

(7) Color-developing solution: The color-developing solution is composed of color-developing solution A and color-developing solution B; the preparation method of the color-developing solution A is: 200mg of tetramethylbenzidine is dissolved in the dehydrated alcohol of 100mL, and then Set the volume to 1000mL with double-distilled water to obtain the color-developing solution A; the preparation method of the color-developing solution B is: weigh 21g of citric acid and 28.2g of anhydrous sodium phosphate, fully dissolve them in 400ml of double-distilled water, and then add 0.75% hydrogen peroxide urea 6.4mL, then distill the volume to 1000mL with double distilled water, adjust the pH value to 4.5-5.0; chromogenic solution A and chromogenic solution B are both 60mL;

(8) Stop solution: 2M H ₂ SO ₄ solution.

2. Preparation of ELISA plate coated with LsaA recombinant protein:

The specific steps of the preparation method of the enzyme label plate coated with LsaA recombinant protein are as follows:

(1) Antigen dilution and coating: The purified LsaA recombinant protein obtained in Example 2 was diluted with coating solution to a concentration of 2 μg/mL, and the diluted LsaA recombinant protein was added to 96 Incubate at 37°C for 1 hour, then overnight at 4°C;

(2) Washing: Discard the liquid in the wells of the 96-well ELISA plate, then add washing solution (10 times concentrated washing solution diluted 10 times with double distilled water) to the wells of the ELISA plate, 300 μL/well, repeat washing for 5 times, 5 minutes each time (discard the liquid in the microplate wells for each wash, and drain the remaining liquid on absorbent paper after discarding the washing liquid for the last wash);

(3) Sealing: Add 5% skimmed milk (300 μL/well) to the 96-well ELISA plate, block at 37° C. for 2 h, then wash according to step (2), and seal the ELISA plate with a self-adhesive sealing film. Packaged in a sealed bag and stored at 4°C

3. The composition of the ELISA detection kit for Lawsonia intracellulare of the present invention:

(1) 5 microtiter plates coated with LsaA recombinant protein;

(2) Negative control serum and positive control serum 2 mL each;

(3) Enzyme-labeled antibody, the specification is 60mL;

(4) Concentrated washing solution, the specification is 200mL;

(5) Sample diluent, the specification is 150mL;

(6) Chromogenic solution A, the specification is 60mL;

(7) Chromogenic solution B, the specification is 60mL;

(8) Stop solution, the specification is 30mL.

Example 5: Reaction conditions and positive and negative thresholds of the ELISA detection kit for Lawsonia intracellulare of the present invention

value definition

1. Determination of the ELISA reaction conditions of the kit:

(1) Determination of the optimal coating amount of the antigen and the optimal dilution concentration of the serum to be tested:

The optimal coating amount of the antigen and the optimal dilution concentration of the serum to be tested were determined by square array titration. The specific operation is: using square array titration, dilute LsaA recombinant protein with coating solution to 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml, add 100 μl to each well, and make 1:1 for positive serum and negative serum respectively: 25, 1:50, 1:100, 1:200, 1:400 times dilution. Operate according to the conventional ELISA method, measure the absorbance at OD _450mm , calculate the P/N value (P/N value = positive serum OD _450mm / negative serum OD _450mm ), select the positive serum OD _450mm value close to 1, and the P/N value Antigen coating amount and serum dilution corresponding to the largest well. The final selected antigen amount was 2 μg/ml, and the serum dilution was 1:200. The results are shown in Table 1.

Table 1 The optimal coating amount of antigen and the optimal dilution of serum

(2) Determination of the best antigen coating conditions:

Coat the ELISA plate with the optimal antigen coating amount, and incubate under three conditions: overnight at 4°C, overnight at 4°C after incubation at 37°C for 1 hour, and overnight at 4°C after incubation at 37°C for 2 hours. Carry out indirect ELISA determination, calculate the P/N value (P/N value = positive serum OD _450mm / negative serum OD _450mm ), select the condition corresponding to the well with the positive serum OD _450mm value close to 1 and the largest P/N value as the best Optimum coating conditions, determined coating conditions: 1h at 37°C and overnight at 4°C, the results are shown in Table 2.

Table 2 Optimal coating time for antigen

Action time overnight at 4°C 1h at 37°C, overnight at 4°C 2h at 37°C, overnight at 4°C positive serum 0.985 0.968 1.252 negative serum 0.170 0.127 0.197 P/N 5.794 7.622 6.355

(3) Determination of the best closing time:

Use 5% skimmed milk as the blocking solution, block at 37°C for 1h, _1.5h , and _2h respectively, and perform indirect ELISA determination. The positive serum OD _450mm value is close to 1, and the blocking time corresponding to the well with the largest P/N value is the optimal blocking time. The optimal blocking time is determined to be 37°C, 2h.

Table 3 Optimal sealing conditions

Action time 37℃1h 37℃1.5h 37℃2h positive serum 1.076 0.940 1.101 negative serum 0.133 0.129 0.132 P/N 8.090 7.286 8.341

(4) Determination of the optimal action time of the primary antibody:

React the primary antibody at 37°C for 1h, 1.5h, and 2h, respectively, and perform indirect ELISA determination to calculate the P/N value (P/N value = positive serum OD _450mm /negative serum OD _450mm ), and select positive serum with an OD _450mm value close to 1 , and the time corresponding to the hole with the largest P/N value is the optimum action time of the primary antibody. The optimum action time of the primary antibody was determined to be 37°C, 2h. The results are shown in Table 4.

Table 4 Optimum action time of primary antibody

Action time 37℃1h 37℃1.5h 37℃2h positive serum 0.860 1.101 1.159 negative serum 0.138 0.132 0.124 P/N 6.232 8.341 9.347

(5) Determination of the optimal working concentration of the enzyme-labeled secondary antibody:

Dilute the HRP enzyme-labeled rabbit anti-pig IgG secondary antibody to 1:5000, 1:10000, 1:20000, 1:40000 respectively, and perform indirect ELISA determination. According to the P/N value (P/N value = positive serum OD _450mm / Negative serum OD _450mm ), select the well with the positive serum OD _450mm value close to 1, and the dilution corresponding to the well with the largest P/N value is the optimal working concentration of the secondary antibody, and determine the optimal working concentration of the enzyme-labeled secondary antibody as 1 :10000 dilution, the results are shown in Table 5.

Table 5 Optimum working concentration of enzyme-labeled secondary antibody

Secondary Antibody Dilution Concentration 1:5000 1:10000 1:20000 1:40000 positive serum 1.872 1.159 0.624 0.384 negative serum 0.192 0.124 0.103 0.090 P/N 0.750 9.247 6.058 4.267

(6) Determination of the optimal action time of the enzyme-labeled secondary antibody:

The enzyme-labeled secondary antibody was reacted at 37°C for 1h, 1.5h, and 2h, respectively, for indirect ELISA determination, and the P/N value was calculated (P/N value = positive serum OD _450mm /negative serum OD _450mm ), and the positive serum OD _450mm value was selected The time corresponding to the well with the largest P/N value close to 1 is the optimum action time of the secondary antibody. The optimum action time of the enzyme-labeled secondary antibody was determined to be 1.5h at 37°C. The results are shown in Table 6.

Table 6 Optimum action time of enzyme-labeled secondary antibody

Action time 37℃1h 37℃1.5h 37℃2h positive serum 0.678 1.102 1.781 negative serum 0.128 0.107 0.133 P/N 5.296 10.299 13.398

(7) Determination of the optimum action time of the chromogenic solution (TMB):

Carry out indirect ELISA assay with the determined optimal reaction conditions, after adding chromogenic solution (TMB), react at 37°C for 10min, 15min, 20min, 25min respectively, add stop solution (2M H ₂ SO ₄ ), within 10min the internal enzyme label Measure the absorbance at OD _450nm on the instrument, and calculate the P/N value (P/N value = positive serum OD _450mm /negative serum OD _450mm ). Select the well corresponding to the well whose positive serum OD _450mm value is close to 1 and has the largest P/N value. The time is the optimum color development time, and the optimum action time of the color development solution is determined to be 10 minutes, and the results are shown in Table 7.

Table 7 Optimal action time of chromogenic solution (TMB)

Color development time 10min 15min 20min 25min positive serum 1.017 1.635 1.791 2.337 negative serum 0.107 0.121 0.145 0.147 P/N 9.505 13.512 12.352 15.897

2. Determination of negative and positive critical values

Utilize the best reaction condition that above-mentioned optimization obtains, adopt Lawsonia intracellularis swine ELISA detection kit of the present invention to detect 40 parts of sera that have been identified as Lawsonia intracellularis antibody negative, read OD _450nm value (result see Table 8), the average value of the OD _450nm values of 40 Lawsonia intracellulare antibody-negative sera measured by calculation is 0.159, and its standard deviation SD value is 0.048, According to statistical analysis, when the OD _450nm value of the sample is ≥0.303, it is judged as positive; when the OD _450nm value is ≤0.255, it is judged as negative, and the test results of negative samples show a normal distribution; when the OD _450nm value is between the two , judged to be suspicious and should be repeated.

Table 8 OD _450nm value of 40 Lawsonia intracellulare antibody-negative sera

Embodiment 6: Operation steps of Lawsonia intracellularis suis ELISA detection kit of the present invention

(1) Dilute the serum sample to be tested 200 times with the sample diluent, take 100 μL of the diluted serum sample to be tested and add it to the microtiter plate, and set two wells of positive control serum and negative control serum at the same time, 100 μL per hole ( Among them, the positive control serum and the negative control serum were diluted 200 times with the sample diluent before use); shake lightly to fully mix the serum in the well, and incubate at 37°C for 2h;

(2) Discard the liquid in the wells of the microplate, add washing solution (300 μL/L) to the wells of the microplate, and repeat the washing 3 times for 5 minutes each time (discard the liquid in the wells of the microplate for each wash). Liquid, after discarding the washing liquid for the last wash, drain the remaining liquid on absorbent paper);

(3) Add 100 μL enzyme-labeled antibody to each well and incubate at 37°C for 1.5h;

(4) Discard the liquid in the wells of the microplate, add washing solution (300 μL/L) to the wells of the microplate, and repeat the washing 5 times for 5 minutes each time (discard the liquid in the wells of the microplate for each wash). Liquid, after discarding the washing liquid for the last wash, drain the remaining liquid on absorbent paper);

(5) Add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well in turn, shake slightly to mix, and develop color at room temperature for 10 minutes in the dark;

(6) Add 50 μL of stop solution to each well to stop the color development and terminate the reaction;

(7) Measure the OD _450nm value with a microplate reader, and judge the result according to the OD value.

Result judgment: when the sample OD _450nm value ≥ 0.303, it is judged as positive; when the sample OD _450nm value ≤ 0.255, it is judged as negative; when the sample OD _450nm value is between the two, it is judged as suspicious, and repeated measurements should be carried out .

Example 7: Specificity of the ELISA detection kit for Lawsonia intracellulare of the present invention

Adopt the ELISA detection kit described in the embodiment of the present invention 4 to detect Escherichia coli (E.coli), Salmonella (Salmonella), Streptococcus (Streptococcus), porcine epidemic diarrhea virus (PEDV) and Porcine transmissible gastroenteritis virus (TGEV) antiserum, set up anti-Lawsonia intracellulare negative, positive serum control simultaneously, evaluate the specificity of the ELISA detection kit of Lawsonia intracellularis of the present invention, the results are shown in Table 9, by As can be seen from Table 9, except for the anti-Lawsonia intracellulare positive serum, the OD _450nm values of the remaining serums are all lower than 0.255, which is judged as negative serum, thus showing the specificity of the Lawsonia intracellularis porcine ELISA detection kit established by the present invention good.

Table 9 The specificity test of the Lawsonia intracellularis suis ELISA detection kit of the present invention

serum OD _450nm value result TGEV 0.095 - PEDV 0.099 - E. coli 0.093 - Salmonella 0.147 - Streptococcus 0.227 - L. intracellularis (+) 0.933 + L. intracellularis (-) 0.117 -

Note: "+" means positive, "-" means negative

Embodiment 8: the sensitivity of the ELISA detection kit of Lawsonia intracellulare of the present invention

After diluting the antiserum of Lawsonia intracellulare 1:200, 1:400, 1:800, 1:1600, 1:3200, use the ELISA detection kit described in Example 4 of the present invention according to the embodiment The operation step of 6 detects the specific antibody in different dilution concentration antiserum, and each dilution concentration is done 3 repetitions, evaluates the sensitivity of the Lawsonia intracellularis suis ELISA detection kit of the present invention, 3 parts of sera at 1:800 The OD450nm values were all greater than the critical value, which was judged as positive, indicating that the established indirect ELISA method had better sensitivity. The results are shown in Table 10.

Table 10 Sensitivity test of Lawsonia intracellulare ELISA detection kit of the present invention

Embodiment 9: Repeat stability of Lawsonia intracellularis suis ELISA detection kit of the present invention

(1) Intra-batch repetition

The same batch of coated ELISA plates was used to perform repeated experiments on 6 serum samples, and each serum sample was used for 3 parallel experiments. The results are shown in Table 11. It can be seen from Table 11 that the intra-assay coefficient of variation is 0.352%-2.752%, all less than 5%, indicating that the Lawsonia intracellularis suis ELISA detection kit and detection method of the present invention have good intra-assay repeatability.

Table 11 Repeat test results in batches

(2) Inter-batch repetition

The inter-batch repeated experiments were performed on 6 serum samples with 3 different batches of coated ELISA plates, and 3 parallels were performed for each serum sample. The results are shown in Table 12. It can be seen from Table 12 that the coefficient of variation between batches is 0.877%-3.000%, all less than 5%, indicating that the Lawsonia intracellularis suis ELISA detection kit and detection method of the present invention have good inter-batch repeatability.

Repeated test results between batches of table 12

Example 10: Clinical application of the ELISA detection kit for Lawsonia intracellulare of the present invention

Using the Lawsonia intracellulare ELISA detection kit of the present invention, 232 serum samples from pig farms with clinical diarrhea symptoms submitted by pig farms in different regions of Henan Province were tested for the Lawsonia intracellulare antibody. The infection and prevalence of Lawsonia intracellulare in pig herds in the region, the test results are shown in Table 13. It can be known from Table 13 that 71 of the 232 swine serum samples were positive for Lawsonia intracellulare antibodies, with a positive rate ranging from 13.3% to 57.1% and an average positive rate of 30.6%.

Table 13 Test results of clinical serum samples

serum source Number of samples positive feminine positive rate Xinyang twenty four 4 20 16.7% Zhumadian 35 9 26 25.7% Nanyang 56 32 twenty four 57.1% Zhoukou 15 2 13 13.3% Shangqiu 20 3 17 15.0% Xinxiang 18 5 13 27.7% Luoyang twenty two 6 16 27.3% Pingdingshan 12 3 9 25.0% Zhengzhou 30 7 twenty three 23.3% total 232 71 161 30.6%

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

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Claims (7)

1. A 1. boar lawsonia intracellularis ELISA detection kit, it is characterised in that the pig lawsonia intracellularis ELISA detections examination Agent box includes solid phase carrier and the antigen being coated on solid phase carrier, and the antigen is LsaA recombinant proteins, the LsaA restructuring The amino acid sequence of albumen is as shown in SEQ ID NO.1.
2. 2. pig lawsonia intracellularis ELISA detection kit according to claim 1, it is characterised in that the LsaA restructuring Albumen is as the gene code of the nucleotide sequence shown in SEQ ID No.2.
3. 3. pig lawsonia intracellularis ELISA detection kit according to claim 1, it is characterised in that the LsaA restructuring The preparation method of albumen is：Opt-LsaA genes are synthesized, opt-LsaA gene clonings to pET-32a carriers are recombinantly expressed Carrier pET-32a-opt-LsaA；Recombinant expression carrier pET-32a-opt-LsaA is transformed into Host Strains, pick out containing Recombinant expression carrier pET-32a-opt-LsaA positive colony bacterium, IPTG induced expressions are carried out to positive colony bacterium；Will expression Product after purification, obtains the LsaA recombinant proteins of purifying by Ni-NTA agarose affinity chromatographies post；Wherein, the opt-LsaA The nucleotide sequence of gene is as shown in SEQ ID NO.2.
4. 4. pig lawsonia intracellularis ELISA detection kit according to claim 3, it is characterised in that the recombination expression Carrier pET-32a-opt-LsaA structure detailed process be：Opt-LsaA genes are synthesized, and in opt-LsaA gene orders 5 ' end add Nde I restriction endonuclease recognition sites, its 3 ' end plus Xho I restriction endonuclease identification Site；Opt-LsaA genes and pET-32a carriers are subjected to double digestion with Nde I and Xho I restriction endonuclease respectively, Opt-LsaA genes after digestion are connected with pET-32a carriers, that is, obtain recombinant expression carrier pET-32a-opt-LsaA.
5. 5. pig lawsonia intracellularis ELISA detection kit according to claim 1, it is characterised in that the solid phase carrier For ELISA Plate, ELISA Plate μ g of coating LsaA recombinant proteins 0.2 per hole.
6. 6. pig lawsonia intracellularis ELISA detection kit according to claim 1, it is characterised in that the pig intracellular labor Gloomy bacterium ELISA detection kit also includes enzyme labelled antibody, negative control sera, positive control serum, cleaning solution, sample dilution Liquid, nitrite ion and terminate liquid.
7. 7. pig lawsonia intracellularis ELISA detection kit according to claim 6, it is characterised in that the enzyme labelled antibody For the rabbit-anti pig IgG of horseradish peroxidase-labeled.