阅读说明：本技术 一种猪源胞内劳森菌ipma抗原检测方法及其应用 (Swine lawsonia intracellularis IPMA antigen detection method and application thereof ) 是由 范红结 李敏雪 肖宁 李剑男 于 2020-07-17 设计创作，主要内容包括：本发明涉及一种猪源胞内劳森菌免疫过氧化物酶细胞单层试验(IPMA)抗原检测方法。利用纯化的重组SodC蛋白作为免疫原,制备了兔抗胞内劳森菌SodC蛋白多克隆抗体,并检测其效价和反应原性。以猪胞内劳森菌阳性菌为包被原,制备的多克隆抗体为一抗,建立了一种胞内劳森菌IPMA抗原检测方法。本发明方法具有良好的特异性、敏感性和重复性,不需要荧光倒置显微镜、酶标仪、PCR仪等昂贵仪器,操作简便,判断更为直观,染色后的微孔板可以长时间保存,适合大量样品的筛查,为胞内劳森菌在感染细胞中的定位及检测提供了新手段。(The invention relates to a swine lawsonia intracellularis immunoperoxidase cell monolayer assay (IPMA) antigen detection method. The rabbit anti-lawsonia intracellularis SodC protein polyclonal antibody is prepared by using the purified recombinant SodC protein as immunogen, and the titer and the reactogenicity of the rabbit anti-lawsonia intracellularis SodC protein polyclonal antibody are detected. The lawsonia intracellularis IPMA antigen detection method is established by taking the lawsonia intracellularis positive bacteria as the coating antigen and the prepared polyclonal antibody as the primary antibody. The method has good specificity, sensitivity and repeatability, does not need expensive instruments such as a fluorescence inverted microscope, an enzyme labeling instrument, a PCR instrument and the like, is simple and convenient to operate and more intuitive to judge, can preserve the dyed microporous plate for a long time, is suitable for screening of a large number of samples, and provides a new means for positioning and detecting Lawsonia intracellularis in infected cells.)

1. A lawsonia intracellularis IPMA antigen detection method is characterized by comprising the following steps:

(1) obtaining a rabbit anti-lawsonia intracellularis SodC protein polyclonal antibody as a primary antibody, and using HRP-labeled goat anti-rabbit IgG as a secondary antibody;

(2) preparing a lawsonia intracellularis antigen sample to be detected;

(3) preparation of an IPMA detection reaction plate: inoculating host cells of Lawsonia intracellularis into a cell culture plate, culturing by adopting DMEM cell culture solution containing 10% FBS by volume to form monolayer cells, removing the culture solution, and taking out the cell culture plate; adding the lawsonia intracellularis antigen sample to be detected prepared in the step (2) into a cell culture plate, setting a negative control containing no lawsonia intracellularis antigen sample, and adding a DMEM culture solution with the same amount as the lawsonia intracellularis antigen sample to be detected into a negative control hole; culturing the cell culture plate, and fixing and cleaning the cell culture plate after the culture is finished to obtain the IPMA detection reaction plate for later use;

preferably, the host cell of Lawsonia intracellularis is a McCoy cell, or an IPEC-J2 cell, or an IEC-18 cell;

(4) IPMA detection of lawsonia intracellularis: taking the IPMA detection reaction plate prepared in the step (3), adding Triton-100 solution with the volume percentage of 0.5%, permeabilizing at room temperature, and washing with PBS;

adding the primary antibody obtained in the step (1) into an IPMA detection reaction plate after dilution, incubating and washing by PBS;

diluting the secondary antibody obtained in the step (1), adding an IPMA detection reaction plate, incubating, and washing with PBS;

adding an AEC substrate into each hole, developing at room temperature, throwing off the AEC substrate, washing with PBS, adding hematoxylin staining solution, and washing with PBS;

observing the result by using an optical microscope after the IPMA detection reaction plate is completely dried, wherein when the Lawsonia intracellularis antigen sample to be detected is positive, the nucleus is blue, and the cytoplasm is brownish red; when the lawsonia intracellularis antigen sample to be detected is negative, the nucleus is blue, and the cytoplasm is not colored.

2. The method for detecting lawsonia intracellularis IPMA antigen of claim 1, wherein said primary antibody of step (1) is prepared by the following method:

(1-1) designing a specific primer pair for amplification by taking a swine lawsonia intracellularis strain attenuated vaccine culture as a template;

preferably, the sequences of the specific primer pairs are as follows:

SF：5’-GGATCCATGGAATAAAACAGAGTATAGG-3’(SEQ ID NO.1)；

SR：3’-CTCGAGCTAGTTTGGTATAACACCAC-5’(SEQ ID NO.2)；

(1-2) connecting the amplified product in the step (1) by taking pGex-6p-1 as a vector to construct a pGex-6p-1-sodc recombinant plasmid, and transforming BL21 to obtain a recombinant bacterium pGex-6p-1-sodc/BL 21;

(1-3) inoculating the recombinant bacterium pGex-6p-1-SodC/BL21 obtained in the step (1-2) into an LB liquid culture medium containing ampicillin and chloramphenicol, expressing Lawsonia intracellularis SodC protein, centrifuging to collect supernatant, and purifying the SodC protein by using a GST affinity chromatography column;

mixing the purified Lawsonia intracellularis SodC protein with an oil adjuvant according to the volume ratio of 1:1, emulsifying to prepare an immunogen, injecting the immunogen into a white rabbit, and measuring the serum titer after the immunization;

preferably, the method for determining the serum titer comprises: the coating concentration of SodC protein is 5-20ug/ml, and the titer serum to be detected is diluted to 1:100 x 2³⁰OD determination after TMB development_450nmAbsorbance of the sample to be assayed for OD of the titer serum well_450nmValue/negative serum well OD_450nmThe value is not less than 2.1, and the product is judged to be positive;

preferably, the Lawsonia intracellularis SodC protein expression conditions in steps (1-3) are: the recombinant strain pGex-6p-1-sodc/BL21 is inoculated into LB liquid culture medium containing ampicillin and chloramphenicol, and shake culture is carried out at 37 ℃ and 180rpm until OD is reached_600nmWhen the value is 0.4-0.6, adding 0.5mM IPTG to the final concentration, inducing at 16 ℃ and 90r/min for 18-24h, and centrifuging at 6000-10000rpm and 4 ℃ for 10-15min to collect thalli, washing the thalli for 3-5 times by PBS, finally suspending and precipitating by PBS, repeatedly freezing and thawing at-80 ℃ for more than or equal to 1h, carrying out ultrasonic crushing and cracking, and centrifuging at 4 ℃ and 10000-12000rpm for 20-30 min; the supernatant was collected.

3. The method for detecting lawsonia intracellularis IPMA antigen of claim 1, wherein said lawsonia intracellularis antigen sample to be detected in step (2) is a bacterial culture or a porcine ileum tissue homogenate.

4. The method for detecting Lawsonia intracellularis IPMA antigen as claimed in claim 1, wherein the density of the host cell inoculated in step (3) is 1X 10⁴1X 10 per mL⁵Per mL;

the culture conditions for culturing the host cells of the Lawsonia intracellularis to form monolayer cells are as follows: 37 ℃ and 5% CO₂Culturing for 24h in an incubator;

the cell culture plate culture operation is as follows: centrifuging the cell culture plate at the room temperature of 2000g for 10min, placing the cell culture plate in a three-gas culture box for culturing for 3h, then replacing the cell culture plate with a DMEM cell culture solution containing vancomycin, neomycin and amphotericin B and containing 10% FBS in volume ratio, placing the cell culture plate in the three-gas culture box for culturing for 5 days, taking out the cell culture plate, and washing with PBS; preferably, the concentration of vancomycin in the DMEM cell culture solution containing 10% FBS by volume is 100ug/mL, the concentration of neomycin is 50ug/mL, and the concentration of amphotericin B is 2 ug/mL;

fixing the reaction plate by using pre-cooled paraformaldehyde fixing liquid with a volume ratio of 4%, and fixing for 10-20 min, preferably for 15min, at room temperature;

the addition amount of the Lawsonia intracellularis host cell, the Lawsonia intracellularis antigen sample to be detected prepared in the step (2) and precooled paraformaldehyde fixing solution with the volume percentage of 4% are all 100 uL/hole.

5. The method for detecting Lawsonia intracellularis IPMA antigen as claimed in claim 1, wherein the addition amount of Triton-100, diluted primary antibody, diluted secondary antibody and AEC substrate in step (4) is 100 uL/well in volume ratio.

6. The method for detecting Lawsonia intracellularis IPMA antigen as claimed in claim 2 or 3 wherein the primary dilution method in step (4) is: the dilution is carried out by using PBST, the volume ratio of primary antibody to PBST is 1:100-1:3200, and preferably, the volume ratio of primary antibody to PBST is 1: 1600;

the incubation time of the diluted primary antibody is 30min-120min, preferably, the incubation time of the primary antibody is 45 min; the primary incubation temperature was 37 ℃;

the method for diluting the secondary antibody comprises the following steps: the dilution is carried out by using PBST, the volume ratio of the secondary antibody to the PBST is 1:1000-1:5000, and preferably, the volume ratio of the secondary antibody to the PBST is 1: 2000;

the incubation time of the diluted secondary antibody is 30min-120min, preferably, the incubation time of the secondary antibody is 1h, and the incubation temperature of the secondary antibody is 37 ℃.

7. The method for detecting lawsonia intracellularis IPMA antigen of claim 3, wherein said porcine ileum tissue homogenate is prepared by the following method: collecting porcine ileum sample, dissecting intestine, scraping intestinal mucus into a centrifuge tube, adding trypsin solution into the centrifuge tube, and digesting at 37 ℃; adding SPG solution into the intestinal mucus, homogenizing, filtering the homogenized intestinal mucus with filter paper and filter membrane with pore diameter of 1.2 μm and 0.65 μm, respectively, collecting filtrate, and adding DMSO with final concentration of 10% by volume into the filtrate to obtain porcine ileum tissue homogenate.

8. Use of the lawsonia intracellularis IPMA antigen detection method of any one of claims 1-7 in laboratory separation and identification of lawsonia intracellularis, wherein said lawsonia intracellularis antigen sample to be tested is a bacterial culture.

9. Use of the lawsonia intracellularis IPMA antigen detection method according to any one of claims 1-7 for preventing and treating lawsonia intracellularis-induced porcine proliferative enteritis, wherein said lawsonia intracellularis antigen sample to be detected is porcine ileum tissue homogenate.

Technical Field

The invention belongs to the technical field of bacteria detection, and particularly relates to a lawsonia intracellularis IPMA antigen detection method and application thereof.

Background

Porcine Proliferative Enteritis (PPE), also known as porcine ileitis, is an infectious intestinal disease caused by Lawsonia Intracellularis (LI). The strain generally infects a conservation swinery, the pathological change part mainly occurs at the tail end of an ileum, and the strain is clinically mainly characterized by hyperplasia and mucosa thickening, can cause the growth and development retardation of pigs and the specific gravity reduction of feed meat, and causes serious economic loss to a pig farm. The disease takes three forms of acute type, subacute type and chronic type, clinically, the subacute type is mainly used, and the death of infected pigs is generally not caused. The disease was first reported in 1931, is widely seen in europe, north america, australia and some countries in asia, and is prevalent in a world scale. In recent years, the incidence of the disease in large-scale pig farms in China is on an increasing trend. Lawsonia intracellularis is a strict intracellular parasitic bacterium, has high culture requirements, cannot be cultured in vitro, cannot grow on chick embryos, needs a special cell culture technology, has no report on the separation and culture of Lawsonia intracellularis at present in China, and is the key for the separation of the Lawsonia intracellularis successfully detected.

At present, the antigen detection methods such as PCR, qPCR, IFA and the like of the strain are established at home and abroad. Although the PCR can realize the rapid detection of clinical samples, the PCR is only limited to the detection of the fecal samples in the period of bacteria elimination of infected pigs. In addition, the feces contain a low content of lawsonia intracellularis and a PCR inhibitor, which may cause a certain false negative. The IFA result needs to be judged by using an expensive fluorescence microscope, the detection result can also have false negative, no detection can not represent no LI, and the IFA result is difficult to popularize in all laboratories. The IPMA detection has the advantages that expensive instruments such as a fluorescence inverted microscope, a microplate reader, a PCR instrument and the like are not needed, the result can be observed only by a simple optical microscope, the operation is simple and convenient, the judgment is more visual, the specificity is high, the dyed microporous plate can be stored for a long time, and the IPMA detection is suitable for screening of a large number of samples.

The invention aims to establish an IPMA detection method aiming at lawsonia intracellularis antigens, and provides an effective technical means for the subsequent laboratory separation and identification of lawsonia intracellularis, thereby providing a certain reference basis for further controlling and preventing porcine ileitis.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an IPMA (immunoperoxidase cell monolayer test) detection method aiming at lawsonia intracellularis antigens, which has the advantages of simple and convenient operation, intuitive judgment, strong specificity, high sensitivity, good repeatability and the like, can detect the disease materials which can not be detected by the conventional PCR, and provides a technical support for early prevention and control of the disease.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention aims to provide a lawsonia intracellularis IPMA antigen detection method, which comprises the following steps:

(1) obtaining a polyclonal antibody of rabbit anti-lawsonia intracellularis superoxide dismutase C protein (SodC protein) as a primary antibody, and using goat anti-rabbit IgG marked by HRP (horse radish peroxidase) as a secondary antibody;

(2) preparing a lawsonia intracellularis antigen sample to be detected;

(3) preparation of an IPMA detection reaction plate: inoculating host cells of Lawsonia intracellularis into a cell culture plate, culturing by adopting DMEM cell culture solution containing 10% FBS by volume to form monolayer cells, removing the culture solution, and taking out the cell culture plate; adding the lawsonia intracellularis antigen sample to be detected prepared in the step (2) into a cell culture plate, infecting host cells of lawsonia intracellularis, setting a negative control containing no lawsonia intracellularis antigen sample, and adding a DMEM culture solution with the same amount as the lawsonia intracellularis antigen sample to be detected into a negative control hole; culturing the cell culture plate, and fixing and cleaning the cell culture plate after the culture is finished to obtain the IPMA detection reaction plate for later use;

preferably, the host cell of Lawsonia intracellularis is a McCoy cell, or an IPEC-J2 cell, or an IEC-18 cell;

(4) IPMA detection of lawsonia intracellularis: taking the IPMA detection reaction plate prepared in the step (3), adding Triton-100 solution with the volume percentage of 0.5%, permeabilizing at room temperature, and washing with PBS;

adding the primary antibody obtained in the step (1) into an IPMA detection reaction plate after dilution, incubating and washing by PBS;

diluting the secondary antibody obtained in the step (1), adding an IPMA detection reaction plate, incubating, and washing with PBS;

adding 3-amino-9-ethylimidazole (AEC) substrate into each hole, developing at room temperature, throwing off the AEC substrate, washing with PBS, adding hematoxylin staining solution, and washing with PBS;

observing the result by using an optical microscope after the IPMA detection reaction plate is completely dried, wherein when the Lawsonia intracellularis antigen sample to be detected is positive, the nucleus is blue, and the cytoplasm is brownish red; when the lawsonia intracellularis antigen sample to be detected is negative, the nucleus is blue, and the cytoplasm is not colored.

Further, the primary antibody in the step (1) is prepared by the following method:

(1-1) designing a pair of specific primers to amplify by taking a swine lawsonia intracellularis strain attenuated vaccine culture as a template;

preferably, the sequences of the specific primer pairs are as follows:

SF：5’-GGATCCATGGAATAAAACAGAGTATAGG-3’(SEQ ID NO.1)；

SR：3’-CTCGAGCTAGTTTGGTATAACACCAC-5’(SEQ ID NO.2)；

the Lawsonia intracellularis SodC gene full length 543bp (NCBI genbank: AB218757.1), the invention regards Lawsonia intracellularis strain attenuated vaccine culture of pig as template, use the specificity primer to intercept 115-one 543 site amino acid expression protein, the software analysis shows, the SodC protein antigenicity and hydrophilicity expressed by the invention truncate are all stronger than SodC gene full length expression protein, therefore Lawsonia intracellularis SodC protein polyclonal antibody prepared is specific and strong, except reacting with Lawsonia intracellularis, it does not react with common enteric bacteria and virus such as salmonella, porcine epidemic diarrhea virus, etc..

(1-2) connecting the amplified product in the step (1) by taking pGex-6p-1 as a vector to construct a pGex-6p-1-sodc recombinant plasmid, and transforming BL21 to obtain a recombinant bacterium pGex-6p-1-sodc/BL 21;

(1-3) inoculating the recombinant bacterium pGex-6p-1-SodC/BL21 obtained in the step (1-2) into LB liquid medium containing ampicillin and chloramphenicol, expressing Lawsonia intracellularis SodC protein, centrifuging to collect the supernatant, and purifying the SodC protein by using a GST affinity chromatography column.

Preferably, the volume ratio of the recombinant bacterium pGex-6p-1-sodc/BL21 to the LB liquid medium containing ampicillin and chloramphenicol is 1: 100;

preferably, the lawsonia intracellularis SodC protein expression conditions are: the recombinant strain pGex-6p-1-sodc/BL21 is inoculated into LB liquid culture medium containing ampicillin and chloramphenicol, and shake culture is carried out at 37 ℃ and 180rpm until OD is reached_600nmWhen the value is 0.4-0.6, adding 0.5mM IPTG into the mixture, inducing the mixture at 16 ℃ and 90r/min for 18-24h, 6000 plus 10000rpm and 4 ℃ for centrifugation for 10-15min, collecting thalli, washing the thalli for 3-5 times by PBS, finally suspending and precipitating the thalli by PBS, repeatedly freezing and thawing at-80 ℃ for more than or equal to 1h, carrying out ultrasonic disruption and lysis, centrifuging the mixture at 4 ℃ and 10000 plus 12000 multiplied by rpm for 20-30min, and collecting supernatant.

Mixing the purified Lawsonia intracellularis SodC protein with an oil adjuvant according to the proportion of 1:1, emulsifying to prepare an immunogen, injecting the immunogen into a white rabbit, and measuring the serum titer after the immunization; preferably, the method for determining the serum titer comprises: SodC protein coating concentration is 520ug/ml, the titre serum to be assayed is separated from the serum in a volume ratio of 1:100 times diluted to 1:100 x 2³⁰OD determination after TMB development_450nmAbsorbance of the sample to be assayed for OD of the titer serum well_450nmValue/negative serum well OD_450nmThe value is not less than 2.1, and the product is judged to be positive;

preferably, the rabbit is immunized by subcutaneous multipoint injection at the back, the immunization dose is 1 mg/rabbit, the 14 th and 21 th booster immunization is carried out once after the first immunization, and the immunization dose and the immunization route are immunized firstly; and (3) performing auricular vein blood sampling on the rabbit at 7 days after the three-immunization, determining the serum titer by adopting a conventional ELISA method, wherein the SodC protein coating concentration is 5-20ug/ml, and performing pretreatment on the serum to be detected from 1:100 times diluted to 1:100 x 2³⁰After dilution, OD was measured after TMB color development_450nmAbsorbance of the sample to OD of the well to be assayed_450nmValue/negative serum well OD_450nmThe value ≧ 2.1 is judged as positive.

Further, the lawsonia intracellularis antigen sample to be detected in the step (2) is a bacterial culture or pig ileum tissue homogenate.

Further, the cell density of the Lawsonia intracellularis host inoculated in the step (3) is 1X 10⁴1X 10 per mL⁵Per mL;

the culture conditions for culturing the Lawsonia intracellularis host cells to form monolayer cells are as follows: 37 ℃ and 5% CO₂Culturing for 24h in an incubator;

the cell culture plate culture operation is as follows: centrifuging the cell culture plate at the room temperature of 2000g for 10min, placing the cell culture plate in a three-gas culture box for culturing for 3h, then replacing the cell culture plate with a DMEM cell culture solution containing vancomycin, neomycin and amphotericin B and containing 10% FBS in volume ratio, placing the cell culture plate in the three-gas culture box for culturing for 5 days, taking out the cell culture plate, and washing with PBS; preferably, the concentration of vancomycin in the DMEM cell culture solution containing 10% FBS by volume is 100ug/mL, the concentration of neomycin is 50ug/mL, and the concentration of amphotericin B is 2 ug/mL;

fixing the reaction plate by using pre-cooled paraformaldehyde fixing liquid with a volume ratio of 4%, and fixing for 10-20 min, preferably for 15min, at room temperature;

the addition amount of the Lawsonia intracellularis host cell added in the step (3), the Lawsonia intracellularis antigen sample to be detected prepared in the step (2) and precooled paraformaldehyde fixing solution with the volume ratio of 4% are all 100 uL/hole.

Further, in the step (4), the addition amount of the Triton-100, the diluted primary antibody, the diluted secondary antibody and the AEC substrate in the volume ratio of 0.5% is 100 uL/hole.

Further, the primary anti-dilution method in the step (4) is as follows: the dilution is carried out by using PBST, the volume dilution ratio of primary antibody to PBST is 1:100-1:3200, and preferably, the volume ratio of the primary antibody to the PBST is 1: 1600, the incubation time of the diluted primary antibody is 30min-120min, preferably, the incubation time is 45min, and the incubation temperature is 37 ℃;

the method for diluting the secondary antibody comprises the following steps: the dilution is carried out by adopting PBST, the volume dilution ratio of the secondary antibody to the PBST is 1:1000-1:5000, and preferably, the volume ratio of the secondary antibody to the PBST is 1: 2000, the incubation time of the diluted secondary antibody is 30min-120min, preferably, the incubation time is 1h, and the incubation temperature is 37 ℃.

Further, the porcine ileum tissue homogenate is prepared by the following method: cutting open the intestines of the collected porcine ileum sample by using sterile scissors, scraping intestinal mucus into a centrifugal tube, adding a trypsin solution into the centrifugal tube, and digesting at 37 ℃; adding SPG solution into the intestinal mucus, homogenizing in a tissue homogenizer, filtering the homogenized intestinal mucus with filter paper and filter membrane with pore diameter of 1.2 μm and 0.65 μm, respectively, collecting filtrate, and adding DMSO with final concentration volume ratio of 10% into the filtrate to obtain porcine ileum tissue homogenate.

The second purpose of the invention is to provide the application of the lawsonia intracellularis IPMA antigen detection method in the separation and identification of lawsonia intracellularis in a laboratory, and the lawsonia intracellularis antigen sample to be detected is a bacterial culture.

In one embodiment, the bacterial culture is a lawsonia intracellularis culture prepared by:

taking McCoy cells out of liquid nitrogen, rapidly thawing in 37 deg.C water, adding DMEM containing 10% fetal calf serum into the thawed cellsSugar medium, 5% CO at 37 ℃₂Culturing in an incubator, after the cells grow into a monolayer, removing the culture solution, rinsing with PBS for 3 times, digesting with pancreatin with the mass ratio of 0.25%, stopping digestion when a small amount of cells fall off, adding DMEM cell culture solution containing FBS with the volume ratio of 10%, repeatedly beating for several times until the cells are dispersed into a single cell, and sucking a proper amount of cell sap into another cell bottle for subculture.

Discarding the culture solution when the McCoy is cultured to about 30% of density, adding the attenuated vaccine positive bacteria of the pig lawsonia intracellularis diluted by 10 times of the inoculation culture solution of the lawsonia intracellularis, centrifuging the cell culture plate at room temperature of 2000g for 10min, placing the cell culture plate in a three-gas culture box for culturing for 3h, replacing the cell culture plate with a DMEM cell culture solution containing 10% of FBS in volume ratio of vancomycin (100ug/mL), neomycin (50ug/mL) and amphotericin B (2ug/mL), placing the cell culture plate at 37 ℃ and 8% of O₂、8.8％CO₂And 83.2% N₂The three-gas culture box is used for continuously culturing for 7 days, and the culture solution is replaced every 2-3 days.

The third purpose of the invention is to provide the application of the lawsonia intracellularis IPMA antigen detection method in preventing and treating porcine proliferative enteritis caused by lawsonia intracellularis, wherein the lawsonia intracellularis antigen sample to be detected is porcine ileum tissue homogenate.

Further, the porcine ileum tissue homogenate is prepared by the following method: cutting open the intestines of the collected porcine ileum sample by using sterile scissors, scraping intestinal mucus into a centrifugal tube, adding a trypsin solution into the centrifugal tube, and digesting at 37 ℃; adding SPG solution into the intestinal mucus, homogenizing in a tissue homogenizer, filtering the homogenized intestinal mucus with filter paper and filter membrane with pore diameter of 1.2 μm and 0.65 μm respectively, collecting filtrate, and adding DMSO with final concentration volume ratio of 10% into the filtrate to obtain porcine ileum tissue homogenate.

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

(1) the Lawsonia intracellularis SodC gene has the full length of 543bp, the invention intercepts 115-plus 543 site amino acid expression protein, and software analysis shows that the antigenicity and the hydrophilicity of the truncated and expressed SodC protein are both stronger than those of the SodC gene full-length expression protein, so that the prepared Lawsonia intracellularis SodC protein polyclonal antibody has strong specificity, and does not react with common enterobacteria and viruses such as salmonella, porcine epidemic diarrhea virus and the like except the Lawsonia intracellularis.

(2) The IPMA detection methods established at home and abroad are all used for detecting the lawsonia intracellularis antibody, the lawsonia intracellularis attenuated vaccine is used as a coating antigen, and serum to be detected is used as a primary antibody, so that the antibody level in the serum is detected; the invention relates to a first IPMA method for establishing lawsonia intracellularis antigen detection, a coating source is a sample to be detected, and a detection means is mainly provided for separation and identification of lawsonia intracellularis in a laboratory and determination of distribution and positioning of lawsonia intracellularis in cells.

(3) The sensitivity of the IPMA detection method established by the invention can reach as high as 10³The specificity and repeatability detection results show that the method has good specificity and higher repeatability, the result can be observed only by an optical microscope, and the established IPMA detection method is simple, visual and effective and can be directly used for separation and identification of lawsonia intracellularis in a laboratory and clinical detection.

Drawings

FIG. 1 is a SDS-PAGE analysis of the expression product of the recombinant expression plasmid pGex-6p-1-sodc/BL21 of example 1;

m: protein molecular weight standards; pGex-6p-1 empty vector plasmid; 2. an uninduced recombinant expression plasmid; 3. inducing the expressed whole bacteria liquid; 4. supernatant after induction expression; 5. inducing the expressed inclusion body; 6. purified SodC.

FIG. 2 is a Western-blot analysis chart of SodC protein;

m: prestained protein molecular mass standard; SodC purified protein; GST tag protein.

FIG. 3 is a view showing the judgment of the IPMA reaction result;

a: reacting rabbit anti-lawsonia intracellularis SodC protein polyclonal antibody with bacteria infected cells; b: the rabbit anti-lawsonia intracellularis SodC protein polyclonal antibody reacts with healthy cells.

FIG. 4 is a graph showing the results of the specificity test of the IPMA detection method;

A：LI；B：S.Cholerasuis；C：PRV；D：PEDV；E：TGEV；F：PCV。

FIG. 5 shows the sensitivity test of the IPMA detection method;

A：1×10⁷per mL; b: 1X 10⁶Per mL; c: 1X 10⁵Per mL; d: 1X 10⁴Per mL; e: 1X 10³Per mL; f: 1X 10²one/mL.

Detailed Description

Unless otherwise specified, the raw materials and chemical reagents used in the examples are all conventional commercial products, and the technical means used are conventional means known to those skilled in the art.

The material sources used in the invention are as follows:

DMEM high-glucose medium and fetal bovine serum were purchased from GIBCO.

TMB color developing solutions were purchased from bi yun tian;

mouse fibroblast-like cells (McCoy) were purchased from ATCC;

lawsonia intracellularis positive strain from bouling invager.

HRP-labeled goat anti-rabbit antibodies purchased from Strobilanthes Wuhan, Dreher bioengineering, Inc.;

the AEC color development kit is a product of Nanjing Biotechnology GmbH.

Diaminobenzidine (DAB) developing solution and hematoxylin staining solution were purchased from Kai-based biotechnology Ltd;

other reagents not listed are commercially available from regular sources;