CN112649606A - Antibody ELISA detection method based on Salmonella pullorum X protein coating - Google Patents
Antibody ELISA detection method based on Salmonella pullorum X protein coating Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/255—Salmonella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses an antibody ELISA detection method based on Salmonella pullorum X protein coating, and relates to the field of poultry bacterial disease detection; the invention prepares and purifies the SPF chicken-derived high-affinity polyclonal antibody of the salmonella pullorum, screens and identifies the electron carrier in an electron transfer chain and the dominant antigen X protein with the activity of the oxidoreductase by adopting a co-immunoprecipitation technology; constructing, expressing and purifying the exocrine dominant antigen X protein by a specific mild method; an indirect ELISA method for detecting the salmonella pullorum antibody is established. The invention can effectively detect the positive antibody of the salmonella pullorum, and improves the specificity and the sensitivity of the prior detection technology; provides a powerful tool for the purification of salmonella pullorum in enterprises.
Description
Technical Field
The invention discloses an antibody ELISA detection method based on Salmonella pullorum X protein coating, and relates to the field of poultry bacterial disease detection.
Background
Pullorum Disease (PD) is an infectious disease of poultry caused by Salmonella Pullorum (SP). SP mainly affects chicks of 2-3 weeks old, is mainly characterized by white diarrhea, septicemia, myocarditis and encephalitis, and can cause a large number of deaths. After the adult chicken is infected, the adult chicken mostly presents chronic process or recessive toxic, and the vertical transmission causes the offspring chicken to be infected and die. In addition, SP host cells are immune cells-macrophages, and severe immunosuppression is caused after infection, so that direct death and panning loss and indirect economic loss caused by PD are huge. At present, the purification of salmonella pullorum is basically realized in developed countries in Europe and America, but the infection of salmonella pullorum is wide and serious in China, so that the purification work of salmonella pullorum is the key point for disease prevention and control of various large-scale chicken raising companies.
At present, a pullorum disease purification strategy is mainly to detect antibody positive chickens which eliminate salmonella pullorum disease, and for purification work, the greatest risk point is that 'positive chickens which show false negative in serum reaction' are missed and exist in chicken flocks for a long time. Therefore, the selection of scientific and reliable detection technology is a key point. The detection method used by the current large poultry breeding company for purification is mainly a whole blood/serum glass plate agglutination method. The existing research shows that the agglutination antibody detected by agglutination reaction is generated quickly in vivo, but the maintaining time is relatively short, and false negative chickens 'negative of agglutination antibody and positive of ELISA antibody' are inevitably existed in the later period. For these false negative chickens, an ELISA antibody detection technique may be selected.
The detection antigen coated by the conventional ELISA kit at present is a bacterial lipopolysaccharide component, the antigen inducing the long-term immune response in the chicken also comprises more bacterial structural proteins and functional non-structural proteins generated by the interaction between bacteria and host cells in the bacterial infection process, and potential immunogens which can be used for detection exist in the protein library. Therefore, an ELISA detection method for salmonella pullorum antibody based on the protein, which has good detection specificity and high sensitivity, is required to be established so as to accelerate the eradication of pullorum infection in chicken farms and finally achieve the purification purpose.
Disclosure of Invention
Aiming at the problems, the invention provides an ELISA detection method for an antibody based on Salmonella pullorum X protein coating.
The technical scheme of the invention is as follows: an ELISA detection method of an antibody based on Salmonella pullorum X protein coating comprises the following specific operation steps:
the method comprises the following steps of (1) screening dominant antigen X protein by a co-immunoprecipitation method through preparing and purifying an SPF chicken-derived high-affinity polyclonal antibody of salmonella pullorum;
comparing dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry;
step (3), construction, expression and purification of dominant antigen X protein;
and (4) establishing an indirect ELISA method.
Further, in the step (1),
the specific operation steps for screening the dominant antigen X protein by adopting a co-immunoprecipitation method are as follows:
(1.1) preparation of negative chicken serum: collecting SPF chicken serum as negative chicken serum for later use;
(1.2) preparing positive chicken serum: resuscitating and expanding LHSP001 Salmonella pullorum, orally infecting SPF chicken by 10-8CFU/ML, collecting blood, and collecting chicken serum positive to thin plate agglutination antibody; and glass plate agglutinates positive serum;
(1.3) antibody purification: purifying the positive and negative chicken serum by saturated ammonium sulfate method, dialyzing and desalting, and storing at-70 deg.C;
(1.4) preparation of antigen complex: resuspending and washing expanded LHSP001 Salmonella pullorum with 1xPBS, centrifuging, adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, and adding RNase A and DNase I after acting for 30min at 4 ℃; acting at 4 deg.C for 30min, adding PMSF, centrifuging, collecting supernatant, and storing at-70 deg.C;
(1.5), co-immunoprecipitation:
(1.5.1), adding 50ul of ProteinA/G into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 500ul of 40ug/ml rabbit anti-chicken antibody, incubating upside down for 30min, adding purified chicken IgG, and incubating upside down at 4 ℃ for 8 h;
(1.5.2) synchronously taking 50ul of protein A/G, adding into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 1ml of prepared antigen compound, and performing reverse incubation at 4 ℃ for 4 hours to obtain the antigen compound without non-specific binding;
(1.5.3) carrying out magnetic separation on the incubated sample, adding 500ul of 1XPBS for washing, repeating for 2 times, adding the antigen complex after removing the non-specific binding, and carrying out reverse incubation for 8h at 4 ℃;
(1.5.4), after magnetic separation, discarding the supernatant, adding 500ul1XPBS for washing, magnetic separation, repeating for 2 times, adding 200ul 0.1Mglycine, PH2.5, incubating for 5-7min, and collecting the supernatant by magnetic separation;
(1.5.5), extracting 30ul of running protein electrophoresis gel for qualitative analysis and recording; performing mass spectrum identification on the residual protein running electrophoresis gel;
(1.5.6), performing mass spectrum result combing analysis of mass spectrum identification, performing bioinformatics analysis on the protein obtained by co-immunoprecipitation, and finally screening out dominant antigen X protein.
Further, in the step (2),
the specific operation steps of comparing dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry are as follows:
(2.1) inoculating 10 mu L of LHSP001 Salmonella pullorum glycerobacteria into 5ml LB liquid culture medium, and shaking overnight at 37 ℃;
(2.2) extracting a genome according to the bacterial genome DNA extraction kit and then performing sequencing;
(2.3) comparing the dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry with a sequencing sequence, wherein the results are consistent.
Further, in the step (3),
the specific operation steps for constructing, expressing and purifying the dominant antigen X protein are as follows:
(3.1) synthesizing a target gene, subcloning, constructing and identifying expression;
(3.2) amplifying protein expression and purification:
(3.2.1), resuscitation: inoculating 50ul of successfully constructed strain into 5ml of TB liquid culture medium containing kanamycin, and carrying out inoculation at 37 ℃ for 200r for 4 h;
(3.2.2) mass propagation: taking 5ml of the bacterial liquid cultured for 4 hours to be placed in 250ml of TB liquid culture medium containing kanamycin, and measuring the OD of the bacterial liquid to be 0.6-0.8 at 37 ℃ at 200r for about 2 hours;
(3.2.3), inducing: adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5M into the bacterial liquid, and carrying out induced expression for 4h at 37 ℃;
(3.2.4), crushing: centrifuging at 4 ℃ for 15min at 5000r, resuspending and washing the 1XPBS once, adding 5ml of bacterial lysate into each gram of wet bacteria, and crushing the bacteria mildly; adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, respectively adding RNase A and DNase I after 30min at 4 ℃, and finally adding PMSF after 30min at 4 ℃; centrifuging and taking a supernatant;
(3.2.5), purification: purifying with His tag resin; filling resin into the upper column, allowing the balance liquid to pass through the column by 4-8 times of the column volume, allowing the sample to pass through the column for 2 times, allowing the washing liquid to pass through the column by 8 times of the column volume, measuring A280 to maintain balance, allowing the eluent to pass through the column by 4-8 times of the column volume, and measuring A280 to maintain balance; controlling the flow rate to be 1min/ml in the whole purification process; after the protein content was determined, the cells were stored at-70 ℃.
Further, in the step (4),
the specific operation steps for establishing the indirect ELISA method are as follows:
(4.1), antigen coating content concentration exploration: diluting protein with coating solution, respectively, at concentrations of 2, 1, 0.50, 0.25, 0.125ug/ml, overnight at 4 deg.C, washing with 1XPBST for 2 times, sealing the plate with 5% skim milk, incubating at 37 deg.C for 2h, washing with 1XPBST for 2 times, and drying;
(4.2), sample dilution concentration exploration: 1.1 and 1.2 of 1XPBST are used for preparing the negative and positive antibodies of the salmonella pullorum, and the dilution is 1:100, 1:200,1:400,1:800,1: 1600,1:3200,1:6400, incubating for 1h at room temperature, washing for 3 times, and drying;
(4.3), concentration of enzyme-labeled secondary antibody and searching: diluting the secondary antibody with 1XPBST at the concentration of 1:5000 and 1:10000 respectively, incubating for 30min, washing for 4 times, and patting dry;
(4.4), developing with TMB for 15min, and stopping with 1% SDS;
(4.5) and the reading OD630nm, and determining the result.
The invention has the beneficial effects that: according to the invention, dominant antigen X protein is screened by a co-immunoprecipitation method, an ELISA detection method is established by utilizing the X protein, and the result is judged by reading, so that the artificial judgment error is reduced, the detection sensitivity and specificity are improved, and a good salmonella pullorum purification tool is provided for breeding enterprises.
Drawings
FIG. 1 is a framework flow diagram of the present invention;
FIG. 2 is a schematic diagram of protein electrophoresis of the present invention, wherein NC is an unexpressed bacterial solution, and numbers 1 and 2 are induced expression protein X proteins;
FIG. 3 is a schematic diagram of Western blot of the present invention, wherein NC is unexpressed bacterial liquid, and the numbers 1 and 2 are induced expression protein X proteins;
FIG. 4 is a graph showing the results of a sensitivity test of the detection method of the present invention;
FIG. 5 is an analysis chart of the results of the specificity test of the detection method of the present invention.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings:
as shown in FIG. 1, an ELISA detection method for an antibody based on Salmonella pullorum X protein coating is characterized by comprising the following specific operation steps:
the method comprises the following steps of (1) screening dominant antigen X protein by a co-immunoprecipitation method through preparing and purifying an SPF chicken-derived high-affinity polyclonal antibody of salmonella pullorum;
comparing dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry;
step (3), construction, expression and purification of dominant antigen X protein;
and (4) establishing an indirect ELISA method.
Further, in the step (1), the pullorum disease dominant antigen X protein is an electron carrier in an electron transfer chain and is a dominant antigen with oxidoreductase activity. The protein is obtained by preparing and purifying an SPF chicken-derived high-affinity polyclonal antibody of salmonella pullorum;
the specific operation steps for screening the dominant antigen X protein by adopting a co-immunoprecipitation method are as follows:
(1.1) preparation of negative chicken serum: collecting SPF chicken serum as negative chicken serum for later use;
(1.2) preparing positive chicken serum: resuscitating and expanding LHSP001 Salmonella pullorum, orally infecting SPF chicken with 10^8CFU/ML, and collecting serum of chicken positive to thin plate agglutination antibody after 3 weeks; and glass plate agglutinates positive serum;
(1.3) antibody purification: purifying the positive and negative chicken serum by saturated ammonium sulfate method, dialyzing and desalting, and storing at-70 deg.C;
(1.4) preparation of antigen complex: resuspending and washing expanded LHSP001 Salmonella pullorum with 1xPBS, centrifuging, adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, and adding RNase A and DNase I (final concentration of 10ug/ml) after acting at 4 ℃ for 30 min; acting at 4 deg.C for 30min, adding PMSF (final concentration of 1mM), centrifuging, collecting supernatant, and storing at-70 deg.C;
(1.5), co-immunoprecipitation:
(1.5.1), adding 50ul of ProteinA/G into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 500ul of 40ug/ml rabbit anti-chicken antibody, incubating upside down for 30min, adding purified chicken IgG, and incubating upside down at 4 ℃ for 8 h;
(1.5.2) synchronously taking 50ul of protein A/G, adding into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 1ml of prepared antigen compound, and performing reverse incubation at 4 ℃ for 4 hours to obtain the antigen compound without non-specific binding;
(1.5.3) carrying out magnetic separation on the incubated sample, adding 500ul of 1XPBS for washing, repeating for 2 times, adding the antigen complex after removing the non-specific binding, and carrying out reverse incubation for 8h at 4 ℃;
(1.5.4), after magnetic separation, discarding the supernatant, adding 500ul1XPBS for washing, magnetic separation, repeating for 2 times, adding 200ul 0.1Mglycine, PH2.5, incubating for 5min, and collecting the supernatant by magnetic separation;
(1.5.5), extracting 30ul of running protein electrophoresis gel for qualitative analysis and recording; performing mass spectrum identification on the residual protein running electrophoresis gel;
(1.5.6), combing and analyzing the mass spectrum result of the mass spectrum identification, and analyzing biological information of the protein obtained by the co-immunoprecipitation, and finally screening out dominant antigen X protein;
further, in the step (2),
the specific operation steps of comparing dominant antigen X protein obtained by co-immunoprecipitation mass spectrometry are as follows:
(2.1) inoculating 10 mu L of LHSP001 Salmonella pullorum glycerobacteria into 5ml LB liquid culture medium, and shaking overnight at 37 ℃;
(2.2) extracting a genome according to the bacterial genome DNA extraction kit and then performing sequencing;
(2.3) comparing the dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry with a sequencing sequence, wherein the results are consistent.
Further, in the step (3),
the specific operation steps for constructing, expressing and purifying the dominant antigen X protein are as follows:
(3.1) synthesizing a target gene, subcloning, constructing and identifying expression; protein electrophoresis gel picture 2 and Western blot picture 3;
(3.2) amplifying protein expression and purification:
(3.2.1), resuscitation: inoculating 50ul of successfully constructed strain into 5ml of TB liquid medium containing kanamycin (50ug/ml), and culturing at 37 deg.C for 4 hr at 200 r;
(3.2.2) mass propagation: taking 5ml of the bacterial liquid after 4h of culture in 250ml of TB liquid culture medium containing 50ug/ml of kanamycin, measuring the OD of the bacterial liquid to be 0.7 at 37 ℃ at 200r for about 2 h;
(3.2.3), inducing: adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5M into the bacterial liquid, and carrying out induced expression for 4h at 37 ℃;
(3.2.4), crushing: centrifuging at 4 ℃ for 15min at 5000r, resuspending and washing the 1XPBS once, adding 5ml of bacterial lysate into each gram of wet bacteria, and crushing the bacteria mildly; adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, respectively adding RNase A and DNase I (final concentration is 10ug/ml) after acting for 30min at 4 ℃, and finally adding PMSF (final concentration is 1mM) after acting for 30min at 4 ℃; centrifuging and taking a supernatant;
(3.2.5), purification: purifying with His tag resin; filling resin into the upper column, allowing the balance liquid to pass through the column by 4-8 times of the column volume, allowing the sample to pass through the column for 2 times, allowing the washing liquid to pass through the column by 8 times of the column volume, measuring A280 to maintain balance, allowing the eluent to pass through the column by 5 times of the column volume, and measuring A280 to maintain balance; controlling the flow rate to be 1min/ml in the whole purification process; after the protein content was determined, the cells were stored at-70 ℃.
Further, in the step (4),
the specific operation steps for establishing the indirect ELISA method are as follows:
(4.1), antigen coating content concentration exploration: diluting protein with coating solution, respectively, at concentrations of 2, 1, 0.50, 0.25, 0.125ug/ml, overnight at 4 deg.C, washing with 1XPBST for 2 times, sealing the plate with 5% skim milk, incubating at 37 deg.C for 2h, washing with 1XPBST for 2 times, and drying;
(4.2), sample dilution concentration exploration: 1.1 and 1.2 of 1XPBST are used for preparing the negative and positive antibodies of the salmonella pullorum, and the dilution is 1:100, 1:200,1:400,1:800,1: 1600,1:3200,1:6400, incubating for 1h at room temperature, washing for 3 times, and drying;
(4.3), concentration of enzyme-labeled secondary antibody and searching: diluting the secondary antibody with 1XPBST at the concentration of 1:5000 and 1:10000 respectively, incubating for 30min, washing for 4 times, and patting dry;
(4.4), developing with TMB for 15min, and stopping with 1% SDS;
(4.5) reading OD630nm, and judging the result;
further, the method comprises the following steps of; the indirect ELISA method of the invention has the following optimal reaction parameters: the preferable specific operation steps are as follows:
firstly, coating: coating the dominant antigen X with the content of 1ug/ml, standing overnight at 4 ℃, washing for 2 times by 1XPBST, sealing the plate with 5% skim milk, incubating for 2 hours at 37 ℃, washing for 2 times by 1XPBST, and patting to dry;
II, serum to be detected: according to the following steps: diluting with 100 deg.C, incubating at 37 deg.C for 1h, washing for 3 times, and drying;
third, second antibody: diluting the goat anti-chicken marked by HRP (horse radish peroxidase) by 1:10000, incubating for 30min at 37 ℃, washing for 4 times, and patting dry;
fourthly, color development: color development with TMB for 15min, 1% SDS stop;
fifthly, reading (result judgment): ELISA detection is carried out by an ELISA reader OD630nm according to the detection method, 30 parts of negative serum is detected, statistical analysis is carried out on the obtained result, the calculated average value (x) is 0.161, the Standard Deviation (SD) is 0.047, OD is less than or equal to x +3SD, the result is judged to be negative, OD is less than or equal to 0.30 and the OD value is more than 0.3.
Furthermore, the invention adopts an enzyme label plate for coating the pullorum disease dominant antigen protein X, a goat anti-chicken secondary antibody marked by HRP, a washing solution, a TMB color development solution, a stop solution, a negative serum control and a positive serum control.
Further, the application of the pullorum disease dominant antigen protein X in the detection of the pullorum disease salmonella antibody.
Further, the sensitivity experiment and the specificity experiment of the invention have the following specific operation steps:
firstly, under the optimal reaction condition, diluting standard positive serum into 1: 100. 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1:6400 the detection is carried out respectively to determine the sensitivity of the method. Synchronously detecting and comparing the serum with each dilution degree by using a Dutch BioChek salmonella D group antibody detection kit;
secondly, detecting the serum of a plurality of common bacteria such as Escherichia coli serotypes O7, O66, O78 and O157, Salmonella enteritidis CVCCW3377, Citrobacter, Shigella flexneri 51571, Enterobacter cloacae 1.241, Proteus 49027 and the like under the optimal reaction condition, and evaluating the specificity of the bacteria;
thirdly, sensitivity experiment results:
as shown in fig. 4, the prepared salmonella pullorum standard positive serum was expressed in a ratio of 1:100, 1:200,1:400,1:800,1: 1600,1:3200,1:6400, wherein the detection result of the ELISA method established by the invention shows that the lowest detection serum dilution is 1:800, and the lowest detection serum dilution of the Dutch BioChek Salmonella group D antibody kit is 1:200 (the kit judgment standard S/P is more than or equal to 0.5), and the sensitivity of the ELISA kit established by the invention for detecting the pullorum disease Salmonella antibody is 4 times higher than that of the Dutch BioChek Salmonella group D antibody kit; specifically, as shown in table 1:
TABLE 1
| 1:100 | 1:200 | 1:400 | 1:800 | 1:1600 | 1:3200 | 1:6400 | 1*PBST | |
| LH-X (negative serum) | - | - | - | - | - | - | - | - |
| LH-X (positive serum) | + | + | + | + | - | - | - | - |
| BioChek (Positive serum) | + | + | - | - | - | - | - | - |
Remarking: 1. LH-X is negative when the determination standard OD630nm value is less than or equal to 0.30 and positive when the OD630nm value is more than 0.3 in the detection method established by the salmonella pullorum dominant protein X;
2. BioChek is a salmonella D group antibody, and the judgment standard S/P of the kit is more than or equal to 0.5;
fourthly, specific experimental results:
as shown in fig. 5, positive sera of a salmonella pullorum standard strain CVCC526 and a salmonella pullorum variant CVCC531, and sera of a plurality of common bacteria such as salmonella enteritidis CVCCW3377, escherichia coli serotype O7, O66, O78, O157, citrobacter, shigella flexneri 51571, enterobacter cloacae 1.241, proteus 49027 are respectively detected, and the detection result of the ELISA method established by the invention shows that the positive sera of the salmonella pullorum standard strain and the variant can be specifically detected; the detection method and the Holland BioChek Salmonella D group antibody kit have difference on the detection result of the Salmonella enteritidis (the detection method does not react with the Salmonella enteritidis antibody), and the sera generated by other common strains are negative; specifically, as described in table 2;
TABLE 2
Remarking: 1. LH-X is negative when the determination standard OD630nm value is less than or equal to 0.30 and positive when the OD630nm value is more than 0.3 in the detection method established by the salmonella pullorum dominant protein X;
2. BioChek is a salmonella D group antibody, and the judgment standard S/P of the kit is more than or equal to 0.5.
Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of embodiments of the present invention; other variations are possible within the scope of the invention; thus, by way of example, and not limitation, alternative configurations of embodiments of the invention may be considered consistent with the teachings of the present invention; accordingly, the embodiments of the invention are not limited to the embodiments explicitly described and depicted.
Claims (5)
1. An ELISA detection method for an antibody based on Salmonella pullorum X protein coating is characterized by comprising the following specific operation steps:
the method comprises the following steps of (1) screening dominant antigen X protein by a co-immunoprecipitation method through preparing and purifying an SPF chicken-derived high-affinity polyclonal antibody of salmonella pullorum;
comparing dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry;
step (3), construction, expression and purification of dominant antigen X protein;
and (4) establishing an indirect ELISA method.
2. The ELISA detection method based on the Salmonella pullorum X protein coat antibody according to claim 1, wherein in the step (1),
the specific operation steps for screening the dominant antigen X protein by adopting a co-immunoprecipitation method are as follows:
(1.1) preparation of negative chicken serum: collecting SPF chicken serum as negative chicken serum for later use;
(1.2) preparing positive chicken serum: resuscitating and expanding LHSP001 Salmonella pullorum, orally infecting SPF chicken, collecting blood, and collecting serum of chicken positive with thin plate agglutination antibody; and glass plate agglutinates positive serum;
(1.3) antibody purification: purifying the positive and negative chicken serum by saturated ammonium sulfate method, dialyzing and desalting, and storing at-70 deg.C;
(1.4) preparation of antigen complex: resuspending and washing expanded LHSP001 Salmonella pullorum with 1xPBS, centrifuging, adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, and adding RNase A and DNase I after acting for 30min at 4 ℃; acting at 4 deg.C for 30min, adding PMSF, centrifuging, collecting supernatant, and storing at-70 deg.C;
(1.5), co-immunoprecipitation:
(1.5.1), adding 50ul of ProteinA/G into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 500ul of 40ug/ml rabbit anti-chicken antibody, incubating upside down for 30min, adding purified chicken IgG, and incubating upside down at 4 ℃ for 8 h;
(1.5.2) synchronously taking 50ul of protein A/G, adding into an EP tube, adding 500ul of 1XPBS for washing, magnetically separating, repeating for 2 times, adding 1ml of prepared antigen compound, and performing reverse incubation at 4 ℃ for 4 hours to obtain the antigen compound without non-specific binding;
(1.5.3) carrying out magnetic separation on the incubated sample, adding 500ul of 1XPBS for washing, repeating for 2 times, adding the antigen complex after removing the non-specific binding, and carrying out reverse incubation for 8h at 4 ℃;
(1.5.4), after magnetic separation, discarding the supernatant, adding 500ul1XPBS for washing, magnetic separation, repeating for 2 times, adding 200ul 0.1Mglycine, PH2.5, incubating for 5-7min, and collecting the supernatant by magnetic separation;
(1.5.5), extracting 30ul of running protein electrophoresis gel for qualitative analysis and recording; performing mass spectrum identification on the residual protein running electrophoresis gel;
(1.5.6), performing mass spectrum result combing analysis of mass spectrum identification, performing bioinformatics analysis on the protein obtained by co-immunoprecipitation, and finally screening out dominant antigen X protein.
3. The ELISA detection method based on the Salmonella pullorum X protein coat antibody according to claim 1, wherein in the step (2),
the specific operation steps of comparing dominant antigen X protein obtained by co-immunoprecipitation mass spectrometry are as follows:
(2.1) inoculating 10 mu L of LHSP001 Salmonella pullorum glycerobacteria into 5ml LB liquid culture medium, and shaking overnight at 37 ℃;
(2.2) extracting a genome according to the bacterial genome DNA extraction kit and then performing sequencing;
(2.3) comparing the dominant antigen X protein obtained by the co-immunoprecipitation mass spectrometry with a sequencing sequence, wherein the results are consistent.
4. The ELISA detection method based on the Salmonella pullorum X protein coat antibody according to claim 1, wherein in the step (3),
the specific operation steps for constructing, expressing and purifying the dominant antigen X protein are as follows:
(3.1) synthesizing a target gene, subcloning, constructing and identifying expression;
(3.2) amplifying protein expression and purification:
(3.2.1), resuscitation: inoculating 50ul of successfully constructed strain into 5ml of TB liquid culture medium containing kanamycin, and carrying out inoculation at 37 ℃ for 200r for 4 h;
(3.2.2) mass propagation: taking 5ml of the bacterial liquid cultured for 4 hours to be placed in 250ml of TB liquid culture medium containing kanamycin, and measuring the OD of the bacterial liquid to be 0.6-0.8 at 37 ℃ at 200r for about 2 hours;
(3.2.3), inducing: adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5M into the bacterial liquid, and carrying out induced expression for 4h at 37 ℃;
(3.2.4), crushing: centrifuging at 4 ℃ for 15min at 5000r, resuspending and washing the 1XPBS once, adding 5ml of bacterial lysate into each gram of wet bacteria, and crushing the bacteria mildly; adding 4ml of bacterial lysate into 1g of wet bacteria for mild lysis, respectively adding RNase A and DNase I after 30min at 4 ℃, and finally adding PMSF after 30min at 4 ℃; centrifuging and taking a supernatant;
(3.2.5), purification: purifying with His tag resin; filling resin into the upper column, allowing the balance liquid to pass through the column by 4-8 times of the column volume, allowing the sample to pass through the column for 2 times, allowing the washing liquid to pass through the column by 8 times of the column volume, measuring A280 to maintain balance, allowing the eluent to pass through the column by 4-8 times of the column volume, and measuring A280 to maintain balance; controlling the flow rate to be 1min/ml in the whole purification process; after the protein content was determined, the cells were stored at-70 ℃.
5. The ELISA detection method based on the Salmonella pullorum X protein coat antibody according to claim 1, wherein in the step (4),
the specific operation steps for establishing the indirect ELISA method are as follows:
(4.1), antigen coating content concentration exploration: diluting protein with coating solution, respectively, at concentrations of 2, 1, 0.50, 0.25, 0.125ug/ml, overnight at 4 deg.C, washing with 1XPBST for 2 times, sealing the plate with 5% skim milk, incubating at 37 deg.C for 2h, washing with 1XPBST for 2 times, and drying;
(4.2), sample dilution concentration exploration: 1.1 and 1.2 of 1XPBST are used for preparing the negative and positive antibodies of the salmonella pullorum, and the dilution is 1:100, 1:200,1:400,1:800,1: 1600,1:3200,1:6400, incubating for 1h at room temperature, washing for 3 times, and drying;
(4.3), concentration of enzyme-labeled secondary antibody and searching: diluting the secondary antibody with 1XPBST at the concentration of 1:5000 and 1:10000 respectively, incubating for 30min, washing for 4 times, and patting dry;
(4.4), developing with TMB for 15min, and stopping with 1% SDS;
(4.5) and the reading OD630nm, and determining the result.
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