CN113063942A - Indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting Morganella morganii antibody and application thereof - Google Patents
Indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting Morganella morganii antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an indirect ELISA detection kit for detecting Morganella morganii antibodies and application thereof. The indirect ELISA detection kit takes Morganella morganii recombinant LPP protein as a coating antigen; the preparation method of the Morganella morganii recombinant LPP protein comprises the following steps: the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system. The invention establishes an indirect ELISA detection method for detecting the Morganella morganii antibody for the first time, the detection method has the characteristics of simplicity, convenience, rapidness, stability, strong specificity, high sensitivity and the like, can be used for detecting the Morganella morganii antibody, and provides a foundation for epidemiological investigation and early prevention and treatment of the Morganella morganii.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to an indirect ELISA detection kit for detecting Morganella morganii antibodies and application thereof.
Background
Morganella morganii (Morganella morganii) belongs to the genus Morganella, which has only one species of Morganella morganii. Morganella morganii is often parasitic in the intestinal tracts of humans and animals, and is a clinically unusual but important opportunistic pathogen. Morganella morganii can cause infection of respiratory tract, wound, urinary tract, gallbladder, blood and eye. Meanwhile, the Morganella morganii has natural drug resistance to penicillin, oxacillin, cephalosporin, cefpodoxime, lincosamide antibiotics, glycopeptides, fosfomycin and the like, so that the selection of drugs is limited when the Morganella morganii is infected and treated. Therefore, the early detection of the Morganella morganii is of great significance to the clinical prevention and treatment of the Morganella morganii.
Because the separation rate of Morganella morganii in clinic is lower, the research on the biological characteristics, pathogenic mechanism and the like of Morganella morganii is still in the initial stage, and the technical maturity is lower. At present, the detection of the Morganella morganii is mainly carried out by adopting a PCR detection method, but the PCR detection has higher requirements on equipment and is not suitable for basic-level farms.
The enzyme-linked immunosorbent assay (ELISA) is a detection technology that known antigen or antibody is coated on the surface of a solid phase carrier, enzyme-labeled antigen-antibody reaction is carried out on the surface of the solid phase, free components in a liquid phase are washed away by a washing method, a corresponding enzyme substrate is added to carry out color reaction, whether corresponding immunoreaction exists or not is judged by the color reaction of the substrate, and the content of the corresponding antibody or antigen in a sample is detected by the shade of the color reaction. The ELISA detection has the characteristics of simple and convenient operation, high sensitivity, good specificity, economy, safety and the like, is widely applied in clinic, and is particularly suitable for basic detection. However, as Morganella morganii is difficult to separate, the clinical separation rate is only 0.5 percent of that of gram-negative bacteria at home, and if the whole mycoprotein of Morganella morganii is used as the coating antigen for ELISA detection, the large-scale application is difficult to realize; in addition, there are few studies and analyses on functions of genes and proteins related to Morganella morganii, it is difficult to select which protein of Morganella morganii is used as a coating antigen, and it is difficult to prepare the coating antigen, and there are problems that the protein is not expressed or the expression level is low, and the protein purification is difficult. Therefore, there has been no report of detecting Morganella morganii antibodies by indirect ELISA.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide an indirect ELISA detection kit for detecting Morganella morganii antibodies and application thereof. The antibody of the Morganella morganii can be quickly and effectively detected to monitor the epidemic situation of the Morganella morganii in a farm, so that the clinical prevention and treatment of the Morganella morganii are facilitated.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides the application of Morganella morganii recombinant LPP protein as a coating antigen in the preparation of an ELISA detection kit for detecting Morganella morganii antibody;
the Morganella morganii recombinant LPP protein is prepared by the following method:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
In a second aspect of the invention, an indirect ELISA detection kit for detecting Morganella morganii antibodies is provided, wherein the indirect ELISA detection kit takes Morganella morganii recombinant LPP protein as a coating antigen;
the preparation method of the Morganella morganii recombinant LPP protein comprises the following steps:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
Preferably, the recombinant expression vector is constructed by the following method: the pcDNA3.1(+) vector and the gene which is shown in SEQ ID NO.1 and codes LPP protein are subjected to double enzyme digestion treatment by using restriction endonucleases Nhe I and EcoR I, and then products after enzyme digestion are connected by using T4DNA ligase.
Preferably, the coating antigen is carbonate solution as a coating solution, the coating concentration of the Morganella morganii recombinant LPP protein is 2 mug/mL, and the coating condition is 4 ℃ and 12 h.
Further, the indirect ELISA detection kit further comprises: enzyme-labeled secondary antibody, negative control serum, positive control serum, substrate color development liquid and stop solution.
Preferably, the enzyme-labeled secondary antibody is an HRP goat anti-mouse enzyme-labeled secondary antibody. Different enzyme-labeled secondary antibodies can be determined according to the source of a test sample during actual detection.
The application of the indirect ELISA detection kit in the epidemiological investigation of Morganella morganii is also the protection scope of the invention.
In a third aspect of the present invention, an indirect ELISA method for detecting an antibody against morganella morganii is provided, comprising the following steps:
(1) coating: diluting an antigen by taking Morganella morganii recombinant LPP protein expressed by eukaryotic cells as a coating antigen, adding the diluted antigen into a hole of an enzyme-labeled plate, and placing the plate for 12 hours at 4 ℃;
(2) and (3) sealing: adding 200 μ L of 5% skimmed milk powder into each well, standing at 4 deg.C for 12 hr, washing for 5 times, and discarding liquid in the plate after 3 min;
(3) adding a primary antibody: adding diluted serum to be detected, adding 100 μ L of the diluted serum to each well, and reacting at 37 deg.C for 1 h;
(4) adding an enzyme-labeled secondary antibody: and adding 100 mu L of diluted HRP enzyme-labeled secondary antibody into each hole, wherein the dilution concentration of the enzyme-labeled secondary antibody is 1:40000, and reacting at 37 deg.C for 1 hr;
(5) substrate color development: adding 100 μ L of TMB color developing solution into each well, and developing for 45min in dark;
(6) adding a stop solution: add 100. mu.L of 2M H per well2SO4The mixture was shaken and mixed, and the OD450nm value was measured within 5 min.
In the step (1), the Morganella morganii recombinant LPP protein is prepared by the following method:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
The invention has the beneficial effects that:
the invention establishes an indirect ELISA detection method for detecting the Morganella morganii antibody for the first time, the detection method has the characteristics of simplicity, convenience, rapidness, stability, strong specificity, high sensitivity and the like, can be used for detecting the Morganella morganii antibody, and provides a foundation for epidemiological investigation and early prevention and treatment of the Morganella morganii.
Drawings
FIG. 1: the result of the PCR amplification of the LPP protein gene; lane M, DNA Marker; lane 1: a fragment of interest.
FIG. 2: electrophoresis picture of recombinant plasmid double restriction enzyme products; lane M, DNA Marker; lane 1: target fragments and vector fragments.
FIG. 3: expression of recombinant LPP protein; lane M: a protein Marker; lane 1: transfecting a cellular protein; lane 2: cell proteins were not transfected.
FIG. 4: purifying the recombinant LPP protein; lane M: a protein Marker; lanes 1-5 are: transfection cell protein, flow-through liquid, washing liquid and purified protein.
FIG. 5: western Blot analysis of purified proteins; lane M: protein Marker lane 1: and (5) purifying the protein.
FIG. 6: and (5) detecting the titer of the polyclonal antibody.
FIG. 7: and (4) detecting the specificity of the polyclonal antibody.
FIG. 8: western Blot detects the reactogenicity of the polyclonal antibody and the recombinant protein.
FIG. 9: and (4) screening the optimal coating solution.
FIG. 10: screening for optimal coating conditions.
FIG. 11: and (4) screening the optimal confining liquid.
FIG. 12: and (4) screening the optimal sealing conditions.
FIG. 13: screening for optimal incubation time of primary antibody.
FIG. 14: and (5) screening results of enzyme-labeled secondary antibody dilution.
FIG. 15: and (5) screening results of incubation time of the enzyme-labeled secondary antibody.
FIG. 16: screening for optimal incubation time of substrate.
FIG. 17: specific assay results (P/N values).
FIG. 18: specific detection results (Cut off values).
FIG. 19: and (3) detecting the stability of an indirect ELISA detection method.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Description of terms:
the "Morganella morganii" and "Morganella morganii" referred to in the present invention mean the same bacteria, Morganella morganii (Morganella morganii), which are Morganella.
As described in the background section, Morganella morganii causes more and more infections, but Morganella morganii is difficult to separate, the clinical separation rate is only 0.5 percent of that of gram-negative bacteria in China, and the Morganella morganii is rarely researched at present. Morganella genus is a conditioned pathogen, has potential threat to cow breeding industry, and once outbreak occurs, the death rate is high, but at present, in large-scale outbreak, the cases which generate higher death rate are relatively few, and the attention of people is not enough.
Therefore, the research on the detection of Morganella morganii needs to be strengthened. Although ELISA detection is a conventional detection method, indirect ELISA detection of Morganella morganii antibody has a plurality of technical difficulties. Specifically, the method comprises the following steps:
due to the difficulty in separation of Morganella morganii, it is difficult to directly adopt the whole Morganella morganii protein as a coating antigen for large-scale ELISA detection. Although the recombinant protein is adopted as the coating antigen, large-scale detection application can be realized, the research and analysis on related genes and protein functions of Morganella morganii are less, the support of related theoretical basis is lacked, and the selection of proper protein as the coating antigen for the detection of the Morganella morganii antibody is difficult; in addition, the preparation of the coating antigen is difficult, the problems of protein non-expression or low expression quantity, optimization of protein expression conditions, purification of expressed protein and the like exist, and when the protein expressed by pronucleus is used as the coating antigen, the specificity is poor, and the coating antigen can generate cross reaction with other epidemic disease positive serum.
Therefore, there has been no report of detecting Morganella morganii antibodies by indirect ELISA.
Based on the above, the invention selects the Morganella morganii recombinant LPP protein expressed by eukaryotic cells as the coating antigen for indirect ELISA detection of the Morganella morganii antibody, and the recombinant protein has excellent reactogenicity with the Morganella morganii polyclonal antibody and can be used for detecting the Morganella morganii antibody.
On the basis of Morganella morganii recombinant LPP protein, the invention further optimizes a detection reaction system for detecting Morganella morganii antibody by indirect ELISA, realizes simple, quick and specific detection of the Morganella morganii antibody, and is particularly suitable for basic-level farms.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. Part of reagents and components used in the invention are as follows:
preparation of phosphate buffer solution
8.00g NaCl, 1.44g Na were weighed2HPO4·12H2O，0.25g KCl，0.25g KH2PO4Adding the mixture into a 1000mL constant volume bottle, fixing the volume, adjusting the pH value to about 7.2, and subpackaging for later use.
Preparation of carbonate buffer
0.75g of Na was weighed2CO3，1.46g NaHCO3And putting the mixture into 500mL of deionized water, adjusting the pH value to about 9.6, and subpackaging for later use.
TMB color development liquid:
buffer A: 66.5063g of sodium citrate are weighed out and dissolved in 800mL of distilled water and the pH is adjusted to 4.0 with concentrated HCl, H is added2O is constant volume to 1L and stored at 4 ℃;
buffer B: 0.2956g of Tetramethylbenzidine (TMB) and 0.0633g of tetrabutylammonium borohydride (TBABH) were weighed out and dissolved in 30ml of Dimethylacetamide (DMA), and stored at 4 ℃ in the dark;
when in use, the buffer A and the buffer B are mixed according to the volume ratio of 39:1, and the mixture is prepared as it is.
2M H2SO4Stopping liquid: adding concentrated H2SO4Adding distilled water according to the volume ratio of 1:5, uniformly mixing and cooling to room temperature to obtain 2M H2SO4And (4) stopping the solution.
In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.
Example 1: preparation of coating antigen
1. The preparation method comprises the following steps:
1.1 optimization design of coding gene sequence of Morganella morganii LPP protein:
in order to enable the Morganella morganii LPP protein to be expressed better in a eukaryotic expression system, the invention carries out optimization design on the coding gene sequence of the Morganii morganii LPP protein on the basis of M.morganii prolipoprotein gene and 5'flank (GenBank: K00084.1), and the Kozak sequence is added at the 5' end of the sequence, so that the expression can be increased theoretically; the His tag is added at the 3' end, so that the purification is convenient; NheI-EcoRI is selected as enzyme cutting sites at both ends, the size of an expected amplification sequence is about 276bp, and the optimized sequence is shown as SEQ ID NO. 1; the method comprises the following specific steps:
GCTAGCGCCGCCACCATGGGCAGGTCCAAGATCGTGCTGGGCGCCGTGGTGCTGGCCAGCGCTCTGCTGGCTGGCTGCTCCTCCAACGCCAAGTTCGATCAGCTGGACAACGACGTGAAGACACTGAATGCCAAGGTGGACCAGCTGAGCAATGACGTGAATGCCATCAGGGCCGATGTGCAGCAGGCCAAGGATGAGGCCGCCAGAGCCAACCAGAGACTGGATAACCAGGTGAGGTCCTACAAGAAGCACCACCACCACCATCACTGAGAATTC。
1.2 gel recovery of amplified fragment of LPP protein gene:
designing corresponding primers for the optimized Morganella morganii LPP protein coding gene sequence to carry out conventional PCR amplification. And (3) carrying out gel electrophoresis experiments on the amplified LPP gene fragments, cutting off the gel containing the target gene fragments after gel running is finished, and putting the gel into a centrifugal tube. And (4) recovering the purified PCR product according to the instructions of the gel recovery kit, carrying out gel electrophoresis detection after recovery, storing the rest at-20 ℃, and carrying out subsequent experiments.
The primers are as follows
F1：5’-GCTAGCGCCGCCACCATG-3’；(SEQ ID NO.2)
R1：5’-GAATTCTCAGTGATGGTGGTGGTG-3’。(SEQ ID NO.3)
Amplification conditions:
pre-denaturation: 94 ℃ for 5 min; denaturation: 94 ℃ for 30 s; annealing: 50 ℃ for 30 s; extension: 72 deg.C, 1min 30 s;
and (3) circulation: circulating for 32 times; extension: 72 deg.C, 10 min.
The agarose gel electrophoresis detection method specifically comprises the following steps:
(1) the rubber bed and the comb are brushed clean under running water by a glass brush, then the clean comb and the rubber bed are arranged in a rubber making device, and then the whole device is placed on a horizontal table board.
(2) The 10 XTAE was diluted to 1 XTAE with deionized water and poured into an electrophoresis tank.
(3) 1g of agarose was weighed and poured into a conical flask to prepare 2% agarose.
(4) Shaking, heating with microwave oven for about 30s to dissolve agarose completely, heating once if not until solid agarose is completely dissolved, cooling to slightly scald hands at room temperature, adding 10 μ l nucleic acid dye at about 50 deg.C, and shaking the flask to mix well.
(5) And quickly pouring the agarose solution into the rubber groove in which the comb is inserted, removing bubbles, and ensuring that the gel is smooth and flat and has no bubbles.
(6) Naturally solidifying for more than 20min at room temperature, after the gel solution is completely coagulated into solid, pulling out the comb, then putting the gel into the liquid in the electrophoresis tank, ensuring that the electrophoresis liquid is over 2mm above the gel, ensuring that one end of the gel with holes horizontally faces to the negative electrode (black) of a power supply, and if bubbles exist in the sample holes, slightly sucking the bubbles by using a gun head.
(7) Uniformly mixing 10 mu l of sample, and slowly adding the mixture into the gel sample adding hole; while an equal amount of Marker was added to another well of the gel.
(8) And switching on a power supply, carrying out electrophoresis at a direct current voltage of 100V, switching off the power supply after the Marker runs to a corresponding position (generally 45min), and stopping electrophoresis.
(9) Taking the rubber gloves to take out the gel from the electrophoresis solution, putting the gel in a preservative film, transferring the gel into a dark room of a gel imager, opening the device, observing the electrophoresis strip and the position of the DNA sample in an exposure page, carefully comparing the electrophoresis strip with a nucleic acid molecular weight standard Marker, and calculating the size of a target strip.
1.3 double enzyme digestion of target fragment and eukaryotic expression vector:
the pcDNA3.1(+) vector and LPP gene were subjected to double digestion with restriction enzymes Nhe I and EcoRI at 37 ℃ in a water bath for 3.5 hours, and the results were examined by 2% agarose gel electrophoresis. The double enzyme digestion reaction system of the target fragment (LPP gene amplification product) is shown in Table 1; the double digestion reaction system of the eukaryotic expression vector (pcDNA3.1(+) vector) is shown in Table 2.
Table 1: target fragment double-enzyme digestion reaction system
Table 2: eukaryotic expression vector double enzyme digestion system
Two sterile PCR tubes were placed on ice, and the enzyme digestion systems were placed in the tubes, respectively. The samples were mixed and centrifuged briefly to allow the sample to settle to the bottom of the tube. The centrifuge tube was incubated at 37 ℃ for 3 hours and 10. mu.L of stop solution was added to terminate the digestion reaction. And taking the enzyme digestion product for agarose gel electrophoresis analysis.
1.4 construction of Morganella morganii LPP gene eukaryotic expression vector:
and (2) using T4DNA ligase to carry out overnight treatment on the fragments recovered by the glue in the step 1.3 at 37 ℃, then transforming the overnight ligation products into a competent cell DH5 alpha, constructing a pCDNA3.1-His-LPP vector, screening white colonies through blue-white spots, inoculating the white colonies into a liquid culture medium prepared in advance and containing Amp, shaking the bed at 37 ℃ for 12h, extracting the plasmid again, carrying out Nhe I and EcoR I double enzyme digestion identification and PCR identification, and handing the plasmid to Shanghai biological engineering Limited company for sequencing.
The ligation reaction system of T4DNA ligase is shown in Table 3.
Table 3: connection system
1.5 transfer of the recombinant plasmid into competent cells:
(1) and (5) turning on an ultraviolet lamp of the sterile workbench in advance for at least 30min for disinfection. Meanwhile, the electric heating constant-temperature water bath tank is opened in advance, and the temperature of the water bath tank rises slowly and is set to 42 ℃ for later use.
(2) Quickly taking a 50 mu LE DH5 alpha competent cell cryopreservation tube from an ice bank at the temperature of-80 ℃, putting the tube on ice, sucking 10 mu L of uniformly mixed ligation reaction solution by using a pipette gun, slowly adding the uniformly mixed ligation reaction solution into the competent cell, then flicking the tube wall to uniformly mix, and putting the tube into the ice for ice bath for 30 min.
(3) The cells were quickly transferred to a 42 ℃ water bath for 90s by heat shock and then quickly transferred to an ice bath for 4 min.
(4) And adding 500 mu L of sterile LB culture medium (without double antibody) into the competent cells, fully and uniformly mixing, and placing the mixture in a constant-temperature shaking table with the mode of 37 ℃ and 220rpm for shake culture for 1h to fully recover the bacteria.
(5) And taking a proper amount of bacterial liquid, coating the bacterial liquid on an LB solid culture medium containing 100 mu g/mL of ampicillin, and incubating overnight in a constant-temperature incubator. And selecting colonies for identifying positive bacteria.
1.6 recombinant plasmid amplification:
3-5 positive colonies were picked, inoculated into LB liquid medium containing 100. mu.g/mL ampicillin, labeled, and placed in a shaker at 37 ℃ overnight at 220 rpm.
1.7 recombinant plasmid extraction:
plasmids were extracted using an endotoxin-free plasmid extraction kit.
1.8 recombinant plasmid double restriction enzyme identification
The LPP recombinant protein plasmid pCDNA3.1-His-LPP stored in a laboratory is subjected to double enzyme digestion by using Nhe I and EcoR I restriction endonucleases, and then agarose electrophoresis analysis is carried out, wherein the enzyme digestion system is shown in a table 4:
table 4: double enzyme digestion identification system
1.9 eukaryotic expression and expression protein purification of recombinant plasmid pCDNA3.1-His-LPP:
(1) inoculating 293T cells with good state and 70% monolayer cells into a six-well plate according to a cell passage method, wherein 1-3 multiplied by 10 cells are inoculated into each well plate5Adding 2mL of culture medium into each cell, placing at 37 ℃ and 5% CO2The culture was carried out overnight in an incubator.
(2) When the monolayer cells in the six-hole plate are observed to grow to 60% -70% under a microscope, the operation of transfection plasmids can be carried out.
(3) Mu.g of plasmid DNA was diluted with 200. mu.L of Opti-MEM to a final plasmid concentration of greater than 0.01. mu.g/. mu.L and gently mixed to prepare DNA dilutions.
(4) mu.L of transfection reagent was added directly to the DNA-containing dilution and gently mixed.
(5) And incubating the compound of the transfection reagent and the DNA for 5min at room temperature, and preparing the transfection compound.
(6) The cell culture dish was removed and the transfection complex was added dropwise to the cells.
(7) After transfection, 293T cells were cultured for 4 hours, the medium was changed, and the total protein was extracted after further culturing in the incubator for 24 hours to detect the expression of the fusion protein.
1.10 purification of recombinant LPP protein:
purification of tagged recombinant LPP protein was extracted using HIS tag protein purification kit.
2. The experimental results are as follows:
2.1 PCR amplification results of LPP protein Gene:
the result of agarose gel electrophoresis analysis shows that 276bp LPP protein gene (FIG. 1) is successfully amplified by PCR, which is consistent with the expected size of the band.
2.2 recombinant plasmid pCDNA3.1-His-LPP double enzyme digestion identification results:
the agarose gel electrophoresis analysis showed that 276bp and 5158bp fragments were obtained after double digestion (FIG. 2), which are consistent with the expected band size.
2.3 expression of recombinant LPP protein:
after the induction expression of the recombinant plasmid, SDS-PAGE gel electrophoresis shows that an obvious target band is present at 12KD (shown in figure 3), which indicates that the induction expression of the recombinant plasmid is successful.
2.4 purification of recombinant LPP protein:
after the induction expression of the recombinant plasmid is successful, the target protein is purified by using the kit, and SDS-PAGE gel electrophoresis shows that an obvious target band and no impurity band exist at the position of 12KD, which indicates that the protein is successfully extracted and has higher purity (figure 4). Western Blot analysis using antibodies against the HIS tag protein showed a clear band of interest at 12kD (FIG. 5).
Example 2: preparation and titer detection of Morganella morganii polyclonal antibody
1. Preparation of polyclonal antibody:
firstly, immunizing a mouse by using antigens (the antigens are whole mycoprotein of Morganella morganii and two antigens of inactivated bacteria, and are immunized by different groups), wherein the process comprises the following steps: firstly, selecting 20 healthy and nonimmunized SPF mice, emulsifying an antigen and complete Freund's adjuvant, and injecting 50 mu g/mouse in a mode of injecting 0.5mL into an abdominal cavity of the mice; after two weeks of first immunization, the antigen is emulsified with incomplete Freund's adjuvant and then 0.5mL of the antigen is injected into the abdominal cavity with 50 mug/mouse; after 10d, the third immunization is carried out in the same way; after the three immunizations are finished, the antigen is used for immunization at 10d, and the dosage is 100 mu g/mouse. And (4) after 15 days of boosting, tail breaking and blood collecting are carried out on the experimental mice, and serum is collected.
2. And (3) detecting the titer of the polyclonal antibody:
diluting antigen with antigen coating solution, coating 100 μ L thallus whole protein diluent and 0.4% formaldehyde inactivated Morganella 3.1 × 10 per well of enzyme label plate7CFU/mL, 100. mu.L per well, PBS was used as a negative control. Left to act at 4 ℃ overnight. And washing and patting dry. Adding 200 μ L of 5% skimmed milk powder sealing solution into each well, standing at 37 deg.C for 1 hr, washing, and drying. Sera were obtained from 1: diluting at 100 times, adding 100 μ L of serum to be detected into each well, and reacting at 37 deg.C for 1 h. After washing, 100. mu.L of diluted goat anti-mouse enzyme-labeled secondary antibody was added to each well and allowed to act at 37 ℃ for 1 hour. After washing, 100. mu.L of TMB developing solution was added to each well, and the mixture was developed in the dark for 30 min. Adding a stop solution: add 100. mu.L of 2M H per well2SO4Shaking and mixing, and determining OD within 5min450nm value and calculating P/N value.
The result shows that the serum titer of the mouse immunized by the Morganella morganii holoprotein and the inactivated Morganella morganii can reach 1: 12800 or more, and up to 1: above 204800 (FIG. 6), the antibody levels were high and could be used in subsequent experiments.
3. Detecting the specificity of the polyclonal antibody:
(1) the specific detection method for detecting the specificity of the polyclonal antibody comprises the following steps: the method for testing agglutination of glass slide is characterized by that it uses the prepared polyclonal antibody as diagnosis serum, and adds a drop of said polyclonal antibody and tested particle antigen bacterial suspension on the glass slide respectively, and uniformly mixes them, after several minutes, the agglutination result can be observed by naked eye, and the agglutination of the particles is positive reaction.
(2) The results of antibody specificity detection of a polyclonal antibody prepared by using the whole protein of the bacterium as an antigen, a polyclonal antibody prepared by using inactivated Morganella as an antigen, and a mixed antibody of antibodies prepared by using the two antigens are consistent with the above results.
(3) The prepared polyclonal antibody was used as a diagnostic serum to perform a slide agglutination experiment with different bacteria.
The results showed that only Morganella showed aggregated particles and the reaction results were positive, indicating that the prepared antibody did not cross-react with other bacteria and was capable of specifically binding to Morganella, and that the antibody was specific (FIG. 7).
4. And (3) determining the reactogenicity of the polyclonal antibody and the recombinant LPP protein:
western Blot for detecting reactogenicity of polyclonal antibody and recombinant LPP protein
SDS-PAGE electrophoresis
(1) Rubber plate: a1.5 mm slab rubber was selected, washed with detergent and wiped dry. The glue plates were aligned first and then fixed on a plate rack for clamping, deionized water was added before pouring the glue and left for half an hour, if there was no leakage, the water was poured out and sucked dry with absorbent paper and then used again.
(2) Glue preparation and glue filling: the 12% split glue concentration was selected and formulated as in table 2.5. Immediately after the addition of the reagents, the gel was shaken, blown and mixed to prevent coagulation, the separation gel was added to the slab and absolute ethanol was added to flatten the gel line.
(3) After standing for half an hour and the separation gel solidified, the absolute ethanol was discarded and blotted dry with absorbent paper. Preparing 5% concentrated gel, adding related reagents in sequence, shaking continuously to prevent premature coagulation, adding into gel plate, and inserting comb with acceptable thickness to prevent air bubble from entering. It was left at room temperature for about half an hour to gel solid.
(4) Protein electrophoresis: and taking out the comb, taking down the rubber plate fixed on the frame, putting the rubber plate into an electrophoresis tank, adding a proper amount of electrophoresis liquid, and adding a sample to be detected and the protein marker. The protein band required for this experiment was obtained by running at 80V for about 3 h.
Rotary film
(1) Cutting the glue: after electrophoresis, the gel plates were removed and carefully separated. Cutting off the target protein gel block according to the size of Maker, marking, and placing into a membrane transferring solution to prevent drying.
(2) Preparation of filter paper and PVDF membrane: the same size filter paper and PVDF film were cut according to the size of the block cut. The filter paper is immersed in the membrane transfer solution for 3min to completely soak the filter paper and activate the PVDF membrane for later use.
(3) Film transfer: the filter paper, the rubber block and the PVDF membrane were correctly placed in the order from negative to positive to remove air bubbles. Placing the film transferring clamp into a film transferring groove; and (5) putting the film rotating tank into ice water, and rotating the film for 50min at the voltage of 70V.
Antibody incubation
(1) Washing: the PVDF membrane was removed, placed in TBST, washed for 10min and repeated 3 times.
(2) And (3) sealing: putting the PVDF membrane into a sealing solution, sealing on a horizontal shaking table for 90min, and cleaning for 3 times according to the method after sealing.
(3) Incubation of primary antibody: PVDF membrane was placed in a primary antibody diluted in proportion and incubated overnight at 4 ℃. After incubation was complete, washing was performed 3 times as described above.
(4) And (3) incubation of the secondary antibody: the PVDF membrane is put into a secondary antibody which is diluted according to the proportion, and incubated for 1 hour at room temperature. After incubation was complete, washing was performed 3 times as described above. Antibody incubation
(5) Protein development: PVDF membrane was developed using ECL (super luminescent liquid).
The result shows that the polyclonal antibody has better combination effect with the recombinant LPP protein, but has no reaction with the empty carrier protein. Indicating that the polyclonal antibody has excellent reactogenicity with the recombinant LPP protein (fig. 8).
Example 3: establishment of indirect ELISA detection method and optimization of reaction conditions
The indirect ELISA detection method mainly comprises the following steps: coating, sealing, adding primary antibody, adding enzyme-labeled secondary antibody, developing substrate, adding stop solution and the like. This example optimizes the reaction conditions for each step. The method comprises the following specific steps:
1. the optimization method comprises the following steps:
1.1 selection of optimal antigen coating concentration and optimal polyclonal antibody dilution concentration:
the best conditions were selected using square matrix titration. Wherein the transverse row (1-12) of the ELISA plate is set as serum dilutionAnd the vertical rows (A-H) are set as antigen coating concentration, wherein the horizontal rows are sequentially diluted by multiple ratios to be 100-1: 204800. the vertical rows are as follows: 100-1: 12800 dilution, optimal serum dilution and optimal antigen coating concentration of the corresponding hole with maximum P/N (P is positive serum OD)450nm reading, N is negative serum OD450nm reading).
1.2 selection of optimal coating solution for antigen:
the antigen is coated, the selection of coating liquid is 3, which are respectively deionized water, carbonate solution (carbonate buffer solution) and phosphate solution (phosphate buffer solution), under the same other conditions, the optimal coating liquid of the antigen is selected according to the maximum value of P/N.
1.3 selection of optimal coating time for antigen:
the optimal coating time of the antigen is 3, namely 1h at 37 ℃, 2h at 37 ℃ and overnight at 4 ℃, and under the same other conditions, the optimal coating time of the antigen is selected according to the maximum value of P/N.
1.4 selection of optimal blocking solution:
blocking agents (all in mass percent) containing 1% BSA, 3% BSA, 5% BSA, 1% skim milk, 3% skim milk and 5% skim milk were prepared, and then the blocking agents were used to block the prepared ELISA plates. In determining the optimal blocking agent, it should be ensured that the other conditions are the same, and it is selected according to the maximum value of P/N.
1.5 selection of optimal blocking time:
under the same conditions, the mixture is sealed by sealing liquid, the action time is 1h and 2h, the temperature is 37 ℃, and the temperature is 4 ℃ for 12 h. The closing time should be determined according to the maximum value of P/N.
1.6 selection of optimal action time of polyclonal antibody:
after ensuring the other conditions to be the same, respectively diluting the positive serum and the negative serum, and selecting the optimal proportion of the dilution proportion. The action time of the diluted serum is respectively 30min, 45min, 60min and 75min, and the action temperature is 37 ℃. The optimal action time should be selected according to the maximum value of P/N.
1.7 selection of optimal dilution of enzyme-labeled antibody:
and diluting the enzyme-labeled secondary antibody at a dilution ratio of 1:5000, 1:10000, 1:20000, 1:40000 and 1:80000 after ensuring the same other conditions. The optimal dilution factor should be chosen according to the maximum value of P/N.
1.8 selection of optimal action time of enzyme-labeled antibody:
after ensuring the same other conditions, adding the diluted enzyme-labeled secondary antibody into each hole, respectively selecting the action time from 30min, 45min, 60min and 75min, and placing the mixture at 37 ℃ for action. The optimum reaction time is selected based on the maximum P/N.
1.9 selection of optimal action time of the substrate:
according to the selected conditions, after other conditions are ensured to be the same, adding a substrate color development liquid TMB, and respectively selecting the action time for 15min, 30min and 45min, wherein the action temperature is room temperature. The optimum reaction time should be chosen according to the maximum value of P/N.
1.10 determination of the decision criterion:
OD was determined by examining 36 samples which were confirmed to be negative by microscopic examination and PCR detection450The value is obtained. Determining the critical value of the positive sample as the average value of the negative sample +3 SD; the cut-off value of the negative samples was determined as the mean value of negative samples +2 SD.
2. And (4) optimizing the result:
2.1 screening results of recombinant protein, optimal dilution of polyclonal antibody:
screening the recombinant protein and the optimal dilution of the polyclonal antibody by using a matrix titration method, and when the recombinant protein coating concentration is 1: 1600, concentration of polyclonal antibody 1: 12800, the P/N value is highest, so the optimal coating concentration of the recombinant protein is 1: 1600, calculating the concentration of the recombinant protein to be 2 ug/mL; the optimal working concentration of the polyclonal antibody is 1: 12800.
table 5: screening for optimal dilution of protein and polyclonal antibody
2.2 screening results of optimal coating solution:
the experimental result shows that when the coating liquid is carbonate, the P/N value is the highest, so the best coating liquid is carbonate.
Table 6: screening of optimal coating solution
2.3 screening results for optimal coating conditions:
the experimental results show that the P/N value is highest when the coating conditions are 4 ℃ for 12h, so the optimal coating conditions are 4 ℃ for 12 h.
Table 7: screening for optimal coating conditions
2.4 screening results of optimal blocking solution:
the experimental results show that when the confining liquid is 5% skimmed milk powder, the P/N value is the highest, so the best confining liquid is 5% skimmed milk powder.
Table 8: screening of optimal blocking solutions
2.5 screening results for optimal blocking conditions:
the experimental results show that the P/N value is highest when the blocking condition is 4 ℃ for 12h, so the optimal blocking condition is 4 ℃ for 12 h.
Table 9: screening for optimal blocking conditions
2.6 screening results for optimal incubation time of multiple antibodies:
the experimental results show that when the primary antibody is incubated for 60min, the P/N value is the highest, so that the optimal primary antibody is incubated for 60 min.
Table 10: screening for optimal incubation time
2.7 screening results for optimal dilution of enzyme-labeled secondary antibody:
the experimental result shows that when the dilution concentration of the enzyme-labeled secondary antibody is 1:40000, the P/N value is highest, so the optimal dilution concentration of the enzyme-labeled secondary antibody is 1: 40000.
table 11: results of screening for enzyme-labeled Secondary antibody dilution
2.8 screening results of optimal incubation time of enzyme-labeled secondary antibody:
the experimental result shows that when the incubation time of the secondary antibody is 60min, the P/N value is the highest, so that the optimal incubation time of the secondary antibody is 60 min.
Table 12: screening results of enzyme-labeled Secondary antibody incubation time
2.9 screening results for optimal incubation time of substrate:
according to the selected conditions, after other conditions are ensured to be the same, adding a substrate color development liquid TMB, and respectively selecting the action time for 15min, 30min and 45min, wherein the action temperature is room temperature. The experimental results show that the P/N value is highest when the substrate incubation time is 45min, so that the optimal substrate incubation time is 45 min.
Table 13: screening for optimal incubation time of substrate
2.10 determination of the criteria (Cut off value)
And coating an enzyme label plate according to the determined optimal condition, detecting 36 negative serums, and judging a critical value standard. The mean value was 0.075 and the Standard Deviation (SD) was 0.0147, and the OD of serum was judged according to the statistical principle450Value of nm>0.119 judged as positive, OD450Value of nm<When the value is 0.104, the result is judged to be negative, and the result is suspicious between the two values.
Table 14: negative serum OD450Value of nm
The optimized detection conditions of the indirect ELISA are as follows:
(1) coating: according to the following steps of 1: 1600 ratio the recombinant protein was diluted with coating solution, each well of a microtiter plate was coated with 100. mu.L of recombinant protein (concentration of recombinant protein was 2. mu.g/mL), and left at 4 ℃ for 12 h. When the water-absorbing plate is used, the water-absorbing plate is washed for 5 times, once for 3min, liquid in the plate is thrown off, the water-absorbing plate is flapped on clean paper with strong water absorption, and no residual liquid is left in each hole.
(2) And (3) sealing: adding 200 μ L of 5% skimmed milk powder into each well, standing at 4 deg.C for 12 hr, washing for 5 times, 3min once, removing liquid from the plate, beating on clean paper with strong water absorption to ensure no residual liquid in each well.
(3) Adding a primary antibody: adding diluted serum to be detected, adding 100 μ L per well, and reacting at 37 deg.C for 1 h. Washing for 5 times, once for 3min, throwing off the liquid in the plate, beating on clean paper with strong water absorption, and ensuring that no residual liquid exists in each hole.
(4) Adding an enzyme-labeled secondary antibody: and adding 100 mu L of diluted HRP enzyme-labeled secondary antibody into each hole, wherein the dilution concentration of the enzyme-labeled secondary antibody is 1:40000, and standing at 37 deg.C for 1 hr. Washing for 5 times, once for 3min, throwing off liquid in the plate, beating on clean paper with strong water absorption, and ensuring that no residual liquid exists in each hole.
(5) Substrate color development: 100 mu L of TMB color developing solution is added into each hole, and color development is carried out for 45min in a dark place.
(6) Adding a stop solution: add 100. mu.L of 2M H per well2SO4The mixture was shaken and mixed, and the OD450nm value was measured within 5 min.
Example 4: methodology investigation
The indirect ELISA detection method established in example 3 was examined methodically and specifically as follows:
1. and (3) specificity test:
the established method is used for detecting pathogenic serum of salmonella, bacillus subtilis, staphylococcus aureus, escherichia coli, pasteurella and the like, and whether the established indirect ELISA detection method has cross reaction with the pathogens is judged according to the Cut off value.
The results showed that the established indirect ELISA detection method did not cross-react with the above pathogens (FIGS. 17 and 18).
2. And (3) repeatability test:
using the same batch of coated ELISA plates, detecting 12 Morganella infection positive serum samples and 12 Morganella infection negative serum samples according to the optimized indirect ELISA method, repeating each sample for 3 times, and performing statistical analysis on the test result to evaluate the batch repeatability of the indirect ELISA test method. And 3 different batches of enzyme-labeled coated plates were used for indirect ELISA detection. To assess the reproducibility of the indirect ELISA test method between batches.
The results show that: the intra-batch CV values were between 0.6% and 13.2%, and the inter-batch CV values were between 0.6% and 10.3%, all less than 15%, indicating good reproducibility stability of the process (fig. 19).
Example 5: clinical application
476 serum samples and indirect ELISA tests were carried out in 12 dairy farms in Shandong province, Jintai, Weifang, Zizhuang, Tai' an, Linyi, Texas, Dongying, chat, Zibo, Neze, and Jinan, and the results showed that 28 positive sera accounted for 5.9% of the total.
Table 15: indirect ELISA detection method for detecting serum sample
The experiment uses the enzyme label plate coated by the Morganella thallus to carry out contrast detection, and the results of the two are consistent, which shows that the method can be used for clinical detection of actual samples and the accuracy of the detection result is good.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> indirect ELISA detection kit for detecting Morganella morganii antibody and application thereof
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Claims (9)
1. The application of the Morganella morganii recombinant LPP protein as a coating antigen in preparing an ELISA detection kit for detecting Morganella morganii antibodies;
the Morganella morganii recombinant LPP protein is prepared by the following method:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
2. An indirect ELISA detection kit for detecting Morganella morganii antibody is characterized in that the indirect ELISA detection kit takes Morganella morganii recombinant LPP protein as a coating antigen;
the preparation method of the Morganella morganii recombinant LPP protein comprises the following steps:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
3. The indirect ELISA detection kit of claim 2, wherein the recombinant expression vector is constructed by the method comprising: the pcDNA3.1(+) vector and the gene which is shown in SEQ ID NO.1 and codes LPP protein are subjected to double enzyme digestion treatment by using restriction endonucleases Nhe I and EcoR I, and then products after enzyme digestion are connected by using T4DNA ligase.
4. The indirect ELISA detection kit of claim 2 wherein the coating antigen is a carbonate solution as a coating solution, the coating concentration of Morganella morganii recombinant LPP protein is 2 μ g/mL, and the coating conditions are 4 ℃ and 12 h.
5. The indirect ELISA detection kit of any one of claims 2-4, wherein said indirect ELISA detection kit further comprises: enzyme-labeled secondary antibody, negative control serum, positive control serum, substrate color development liquid and stop solution.
6. The indirect ELISA detection kit of claim 5 wherein the enzyme-labeled secondary antibody is an HRP goat anti-mouse enzyme-labeled secondary antibody.
7. Use of the indirect ELISA detection kit of any one of claims 2-6 in epidemiological investigations of M.
8. An indirect ELISA detection method of Morganella morganii antibody is characterized by comprising the following steps:
(1) coating: diluting an antigen by taking Morganella morganii recombinant LPP protein expressed by eukaryotic cells as a coating antigen, adding the diluted antigen into a hole of an enzyme-labeled plate, and placing the plate for 12 hours at 4 ℃;
(2) and (3) sealing: adding 200 μ L of 5% skimmed milk powder into each well, standing at 4 deg.C for 12 hr, washing for 5 times, and discarding liquid in the plate after 3 min;
(3) adding a primary antibody: adding diluted serum to be detected, adding 100 μ L of the diluted serum to each well, and reacting at 37 deg.C for 1 h;
(4) adding an enzyme-labeled secondary antibody: and adding 100 mu L of diluted HRP enzyme-labeled secondary antibody into each hole, wherein the dilution concentration of the enzyme-labeled secondary antibody is 1:40000, and reacting at 37 deg.C for 1 hr;
(5) substrate color development: adding 100 μ L of TMB color developing solution into each well, and developing for 45min in dark;
(6) adding a stop solution: add 100. mu.L of 2M H per well2SO4The mixture was shaken and mixed, and the OD450nm value was measured within 5 min.
9. The indirect ELISA detection method of claim 8 wherein in step (1), the Morganella morganii recombinant LPP protein is prepared by the following method:
the nucleotide sequence shown in SEQ ID NO.1 is used as a gene for coding LPP protein, a recombinant expression vector is constructed, and Morganella morganii recombinant LPP protein is expressed by a eukaryotic expression system.
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