CN112964871A - Pig encephalomyocarditis indirect ELISA diagnostic kit and preparation method thereof - Google Patents
Pig encephalomyocarditis indirect ELISA diagnostic kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a pig encephalomyocarditis indirect ELISA diagnostic kit and a preparation method thereof. The kit provided by the invention takes the porcine encephalomyocarditis virus 2A purified protein as the coating antigen on the ELISA antibody detection plate. The invention discloses a method for preparing and purifying 2A protein, which comprises the steps of using genome DNA of porcine encephalomyocarditis virus HB10 strain as a template, carrying out PCR amplification by using a specific primer, carrying out enzyme digestion on an amplification product to construct a prokaryotic expression vector pET28a-2A, and carrying out induction expression on the 2A protein. The ELISA diagnosis method of the invention has strong specificity and stable and reliable result, can specifically detect the porcine encephalomyocarditis virus antibody, and is completely suitable for diagnosis after the porcine encephalomyocarditis virus infection.
Description
Technical Field
The invention relates to the field of ELISA diagnostic kits, in particular to a pig encephalomyocarditis indirect ELISA diagnostic kit and a preparation method thereof.
Background
Encephalomyocarditis virus (EMCV) is a single-stranded small RNA virus without a membrane. Although the virus has only one serotype, the host range of the virus is wide, and the virus can cause acute infectious diseases which are mainly characterized by encephalitis and myocarditis of some mammals, rodents and even human beings. Wherein, the pig is the most susceptible animal, and can cause the reproductive disturbance diseases such as piglet acute lethal encephalitis, myocarditis or myocarditis around the heart, abortion of pregnant sows, stillbirth, weak fetus, mummy fetus and the like, and bring serious economic loss to the pig industry; isolation has also been reported in animals such as dogs. Because EMCV is an important zoonosis pathogen, pigs are an important source for human tissue organ transplantation, dogs are companion animals of human beings and are in close contact with the human beings, the research on pathogenic mechanisms and scientific prevention and treatment of the zoonosis pathogen is particularly important.
Typical encephalomyocarditis viruses can be diagnosed based on clinical symptoms combined with pathological changes and epidemiological analysis, however, there is a lack of effective diagnostic methods for invisibly infected animals or atypical cases, whose diagnosis must be performed in the laboratory. The existing diagnostic methods for encephalomyocarditis virus are few, and mainly include methods such as electron microscope observation, PCR detection, genomics detection and the like of virus particles. However, these methods have the disadvantages of complicated operation, high price, low sensitivity and specificity, etc. Enzyme-linked immunosorbent assay (ELISA) has been widely used for detecting many pathogenic antigens or antibodies due to its advantages of convenient operation, rapidity, sensitivity, strong specificity, etc. The 2A protein is an important virulence factor of the encephalomyocarditis virus and is a hotspot for researching pathogenic mechanisms of the encephalomyocarditis virus. The 2A protein is used as the detection focus to establish an ELISA diagnostic method, which has important significance. At present, a stable and reliable serological diagnosis method for the encephalomyocarditis virus does not exist in the world, which is the most urgent problem to be solved in the current prevention and control of the encephalomyocarditis virus, and a rapid and effective diagnosis method for the encephalomyocarditis virus is established, so that the method has very important significance for preventing and controlling and eradicating the encephalomyocarditis virus.
Disclosure of Invention
The invention aims to provide a pig encephalomyocarditis ELISA diagnostic kit, which takes pig encephalomyocarditis virus 2A protein as a coating antigen on an ELISA antibody detection plate, and the nucleotide sequence of the pig encephalomyocarditis virus 2A protein is shown as SEQ ID NO. 3.
The primers for amplifying the porcine encephalomyocarditis virus 2A protein of the porcine encephalomyocarditis ELISA diagnostic kit provided by the invention are shown in SEQ ID NO. 1-2.
According to the ELISA diagnostic kit for porcine encephalomyocarditis, the coating amount of the porcine encephalomyocarditis virus 2A protein is 0.5-1 mug/hole;
preferably, the coating amount of the porcine encephalomyocarditis virus 2A protein is 0.5 mug/hole.
The ELISA diagnostic kit for pig brain myocarditis provided by the invention further comprises a goat anti-mouse enzyme-labeled secondary antibody, a washing solution, a developing solution and a stop solution.
The detection operation steps of the ELISA diagnostic kit for porcine brain myocarditis provided by the invention are as follows:
(1) diluting 10 times of the concentrated washing solution to obtain a washing solution;
(2) diluting the serum to be detected, the positive control serum and the negative control serum by using an antibody diluent, adding the diluted antibody into an ELISA detection plate, incubating and then drying;
(3) adding a washing liquid into each hole, and drying by spin-drying after washing;
(4) adding an enzyme-labeled secondary antibody working solution into each hole, incubating and then drying;
(5) adding a washing liquid into each hole, and drying by spin-drying after washing;
(6) adding a color development liquid into each hole, and incubating in a dark place;
(7) stop solution was added to each well and the absorbance was read with a microplate reader at a wavelength of 450 nm.
The detection result judgment standard of the ELISA diagnostic kit for porcine brain myocarditis provided by the invention is as follows:
(1) if the sample to be tested is OD450Judging the sample to be positive if the sample is more than or equal to 0.25;
(2) if the sample to be tested is OD450Less than or equal to 0.20Determining the sample as negative;
(3) if the sample to be detected is 0.25 & gtOD450Judging the sample as a suspicious sample when the sample is more than 0.20; rechecking the suspicious sample if the suspicious sample OD450Judging the suspicious sample to be positive if the suspicious sample OD is more than or equal to 0.23450And judging the suspicious sample to be negative if the sample is less than or equal to 0.23.
The second aspect of the invention provides a preparation method of the ELISA diagnostic kit for porcine brain myocarditis, wherein the preparation of the coating antigen comprises the following steps:
constructing a prokaryotic expression vector pET28a-2A by using a sequence shown in SEQ ID NO. 3;
the prokaryotic expression vector pET28a-2A is transformed into BL21(Transetta), and the positive recombinant bacteria are selected and inoculated into LB culture medium containing kanamycin for culture.
In the preparation method, the induced expression method of the porcine encephalomyocarditis virus 2A protein is to use BL21(Transetta) bacterial liquid OD600When the concentration reaches 0.6, IPTG with the final concentration of 0.1mM is added into the LB culture medium, and the porcine encephalomyocarditis virus 2A protein is obtained after the induced expression is carried out for 8 hours under the condition of 37 ℃.
By adopting the preparation method provided by the invention, about 80% of the porcine encephalomyocarditis virus 2A protein presents soluble expression.
The invention also claims the application of the pig brain myocarditis ELISA diagnostic kit or the preparation method thereof in pig epidemic prevention detection and pork pig breeding.
The invention has the beneficial effects that:
(1) the ELISA diagnosis method has strong specificity and stable and reliable result, can specifically detect the porcine encephalomyocarditis virus antibody, and is completely suitable for diagnosis after the porcine encephalomyocarditis virus is infected;
(2) the 2A recombinant protein adopted by the invention is expressed in the supernatant, has effective biological activity, the 2A gene can be stably and efficiently expressed in a pET prokaryotic expression system, and the recombinant protein carrying histidine marks is easy to purify;
(3) the kit provided by the invention has the advantages that the result judgment is convenient, sensitive, accurate and reliable, the TMB substrate is adopted for color development, the OD value is measured by an enzyme-labeling instrument, and the detection result of the sample to be detected is judged; the difference of the negative and positive results is obvious, and the color development is more sensitive, reliable and stable than that of OPD;
(4) the kit is simple, convenient and quick to operate, can complete sample detection within 1.5h, is short in time consumption and low in cost, and is very suitable for detection of a large number of animal serum samples.
Drawings
FIG. 1 is an electrophoretogram of the 2A gene amplified by the primer sequences provided in example 1 of the present invention; wherein lane 1 is a nucleic acid of the 2A gene; lane 2 is Marker.
FIG. 2 is an electrophoretogram of 2A recombinant protein obtained by amplification of the primer sequences provided in example 1 of the present invention; wherein lane 1 is Marker; lane 2 is vector; lane 3 is the protein expression supernatant transformed with BL21(DE 3); lane 4 is protein expression pellet transformed with BL21(DE 3); lane 5 is the protein expression supernatant of transformed BL21 (Transetta); lane 6 is the protein expression pellet transformed with BL21 (Transetta).
FIG. 3 shows 2A protein obtained by constructing prokaryotic expression vector with the primers provided in comparative example 1; wherein lane 1 is Marker; lane 2 is purified 2A recombinant protein; lane 3 is the 2A recombinant protein product before purification; lane 4 is an empty vector for IPTG inducible expression of pET28 a.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.
Example 1 Indirect ELISA diagnostic kit for porcine brain myocarditis
The embodiment provides a preparation method of a pig encephalomyocarditis indirect ELISA diagnostic kit and a specific experimental result of the ELISA diagnostic kit.
1. Cloning of encephalomyocarditis virus 2A gene and construction of prokaryotic expression vector
The specific primer of the 2A gene segment is designed by referring to JQ864080 in GenBank,
an upstream primer: CGGGATCCAGTCCAAATGCCCTAGACAT (SEQ ID NO. 1);
a downstream primer: CGCTCGAGttaTTGGGTCTGGAAAACCTGTT (SEQ ID NO. 2);
obtaining a 471bp 2A specific target gene (shown as SEQ ID NO. 3) through PCR amplification,
AGTCCAAATGCCCTAGACATTTCAAGAACATACCCCACGTTACATGTTCTCATTCAATTCAACCATAGAGGTTTGGAGGTTAGATTGTTTAGACATGGACAATTTTGGGCTGAAACACGTGCGGACGTGATTCTGAGATCGAAGACCAAACAGGTCTCTTTCCTGAGCAACGGGAACTACCCGTCAATGGACTCTAGAGCTCCCTGGAATCCTTGGAAGAATACCTACCAGGCGGTTCTAAGAGCAGAACCATGTAGAGTGACCATGGATATATACTATAAGAGAGTCAGGCCTTTTAGACTGCCCCTGGTTCAGAAGGAATGGCGCGTGCGAGAGGAGAACGTTTTCGGTTTGTACCGGATCTTCAATGCCCACTACGCTGGTTACTTTGCGGACCTACTGATTCATGACATTGAGACAAATCCAGGGCCCTTCATGTTTAGACCAAGGAAACAGGTTTTCCAGACCCAA are provided. The 2A specific target gene nucleic acid electrophoresis diagram is shown in FIG. 1.
2. High-efficiency expression and purification of 2A recombinant protein
Transforming a prokaryotic expression vector pET28a-2A into BL21(Transetta) competent cells, selecting positive recombinant bacteria, inoculating the positive recombinant bacteria into an LB culture medium containing kanamycin, performing shake culture at 37 ℃, and performing OD (microbial inoculum density) culture on the bacterial liquid600When the concentration reaches 0.6, adding IPTG with the final concentration of 0.1mM, carrying out induction expression for 8h at 37 ℃, centrifuging, collecting a large amount of expressed host bacteria, carrying out ultrasonic lysis, expressing most of protein in a soluble form, collecting supernatant, carrying out magnetic bead purification, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification to show that the size of the 2A recombinant protein is about 25kD, wherein an electrophoretogram of the 2A recombinant protein is shown in figure 2.
FIG. 2 shows that the soluble expression of 2A recombinant protein obtained by inducing expression for 8h at 37 ℃ using BL21(Transetta) competent cells and adding IPTG at a final concentration of 0.1mM is more than 80%.
The antibody content in the serum is low at the initial stage of pig infection with EMCV or after a period of time after the pig is infected with EMCV vaccine; in this case, a coating antigen having higher sensitivity and better specificity is required for detecting EMCV antibodies in serum. The preparation method provided by the invention can obtain the soluble 2A recombinant protein with higher expression quantity for preparing the ELISA diagnostic kit, and is beneficial to accurately reflecting the EMCV antibodies in the serum of sick pigs by using the ELISA diagnostic kit at the initial infection stage or after the EMCV vaccine is inoculated for a period of time.
3. Optimal antigen coating amount and coating method for ELISA detection method
The recombinant proteins were subjected to a matrix test at coating concentrations of 0.01. mu.g, 0.03. mu.g, 0.0625. mu.g, 0.125. mu.g, 0.25. mu.g, 0.5. mu.g, 1. mu.g and 2. mu.g per well, and the results are shown in Table 1.
TABLE 1 optimal antigen coating amount
The matrix test in Table 1 shows that the optimal coating concentration of the antigen is 0.5. mu.g/well, the purified 2A recombinant protein is diluted to 5. mu.g/mL using carbonate buffer solution with pH 9.6 as the coating solution, added to the ELISA plate at 100. mu.L/well, and coated overnight at 4 ℃. Washing the plate with PBST four times, adding 200 μ L of 1 × blocking b μ ffer into each well, sealing at 37 deg.C for 1 hr, washing the plate with PBST four times, air drying at room temperature, filling into desiccant, vacuum packaging, and storing at 4 deg.C.
4. Preparation of 2A Standard Positive and negative sera
Preparation of standard positive sera: selecting healthy SPF mice of 1.5 months old, injecting 200 mg of purified 2A protein subcutaneously on the back, performing boosting immunization once every 2 weeks after first immunization by adopting the same method, performing eyeground venous blood collection on the 5 th day after fourth immunization, adding one ten thousandth of thimerosal for antisepsis, and performing sterile filtration; the negative control serum is negative mouse serum (OD) obtained by screening450nmLess than or equal to 0.20), adding thimerosal one in ten thousand for antisepsis, and sterile filtering.
5. Reagent kit detection operation program
The detection procedure is as follows:
(1) diluting 10 times of the concentrated washing solution to obtain a washing solution;
(2) diluting the serum to be detected, the positive control serum and the negative control serum by 1: 100 times with an antibody diluent (1 Xblocking b [ mu ] ffer), adding 100 mu L of the diluted antibody into an ELISA detection plate per hole, incubating at 37 ℃ for 40min, and drying;
(3) adding 200 μ L of washing solution into each hole, washing for 4 times, each time for 1min, and spin-drying;
(4) adding 100 mu L of enzyme-labeled secondary antibody working solution into each hole, incubating at 37 ℃ for 40min, and drying;
(5) adding 200 μ L of washing solution into each hole, washing for 4 times, each time for 1min, and spin-drying;
(6) adding 100 μ L of TMB color development solution into each well, and incubating at 37 deg.C in dark for 5 min;
(7) add 50. mu.L of stop solution into each well, and read the light absorption value (OD) at 450nm with microplate reader450nmValue).
6. Determination criteria of detection results
30 negative samples with 4-7 months of age and without history of encephalomyocarditis infection were subjected to antibody detection, and the detection results are shown in Table 2. The c μ t-off value of the ELISA assay was obtained by calculating the negative mean and standard deviation (X. + -. 3 SD). The judgment criteria for the sample to be tested are defined by the calculated c μ t-off values as: when the sample to be tested is OD450Judging the sample to be positive if the sample is more than or equal to 0.25; OD450The sample is judged to be negative when the sample is less than or equal to 0.20; when 0.25 > OD450If the sample is suspicious above 0.20, the sample needs to be rechecked once, if the OD is not higher than450More than or equal to 0.23 is judged as positive, OD450If the value is less than or equal to 0.23, the result is judged to be negative.
TABLE 2 negative sample antibody test results
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|
|
|
|
|
Sample 7 | Sample 8 | Sample 9 | Sample 10 |
| 0.112 | 0.132 | 0.020 | 0.109 | 0.023 | 0.089 | 0.102 | 0.113 | 0.085 | 0.045 |
| Sample 11 | Sample 12 | Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 |
| 0.154 | 0.100 | 0.112 | 0.057 | 0.106 | 0.127 | 0.108 | 0.050 | 0.105 | 0.089 |
| Sample 21 | Sample 22 | Sample 23 | Sample 24 | Sample 25 | Sample 26 | Sample 27 | Sample 28 | Sample 29 | Sample 30 |
| 0.085 | 0.115 | 0.134 | 0.076 | 0.134 | 0.067 | 0.100 | 0.109 | 0.080 | 0.118 |
Table 2 results show the OD of 30 negative samples450Are all less than 0.20, i.e., the specificity provided by this exampleThe 2A protein prepared by the primer can stably identify the porcine brain myocarditis antibody and is not interfered by other antibodies in serum.
7. Specificity test
(1) The specific test of the diagnosis method is carried out by using 10 African swine fever, 10 pig foot-and-mouth disease and 3 pig aphtha positive serum, and the result detects the OD of the sample450Are all less than 0.25.
(2) Detection of clinical samples: the invention has carried on antibody detection to nearly 200 kinds of boars in three different pig farms, these pig farms raise the norm, have formulated and carried out and had comparatively strict immunization program, the management of disinfecting and epidemic prevention is all done excellently. The detection results show that all three pig farms are negative.
(3) Serum detection of clinically challenging pigs: the clinical symptoms and the anatomical identification of the offending pigs are 5 sick pig serum samples of the porcine encephalomyocarditis virus, and the detection data are shown in a table 3.
TABLE 3 serum sample test results of sick pigs
| |
|
|
|
|
| 2.787 | 1.985 | 2.980 | 2.658 | 2.700 |
The results in table 3 show that the 2A recombinant protein obtained by the provided preparation method has better solubility, and the detection result is more accurate when detecting porcine encephalomyocarditis virus.
Comparative example 1 ELISA diagnostic kit for porcine encephalomyocarditis with different 2A proteins
The comparative example provides preparation of a pig encephalomyocarditis indirect ELISA diagnostic kit and a specific experimental result thereof.
The comparative example only differs from example 1 in that a prokaryotic expression vector pET28a-2A is transformed into BL21(DE3), a positive recombinant bacterium is selected and inoculated into LB culture medium containing kanamycin for shaking culture at 37 ℃, and when the bacterium liquid OD is600When the concentration reaches 0.6, adding IPTG with the final concentration of 0.1mM, carrying out induced expression for 16h at 16 ℃, centrifugally collecting a large amount of expressed host bacteria, carrying out ultrasonic lysis, collecting supernatant, carrying out magnetic bead purification, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification to show that the size of the 2A recombinant protein is about 25kD, wherein an electrophoretogram of the 2A recombinant protein is shown in figure 3.
30 negative samples with 4-7 months of age and without history of encephalomyocarditis infection were subjected to antibody detection, and the detection results are shown in Table 4.
TABLE 4 negative sample antibody test results
Specificity test obtained in this comparative example:
(1) the specific test of the diagnosis method is carried out by using 10 African swine fever, 10 pig foot-and-mouth disease and 3 pig aphtha positive serum, and the result detects the OD of the sample450Are all less than 0.25.
(2) Detection of clinical samples: the invention carries out antibody detection on nearly 200 pigs in three different pig farms, and the feeding specifications of the pig farms are established and executed with stricter immune programs, so that the disinfection and epidemic prevention management is well done. The detection results show that all three pig farms are negative.
(3) Serum detection of clinically challenging pigs: the clinical symptoms and the anatomical identification of the offending pigs are 5 sick pig serum samples of the porcine encephalomyocarditis virus, and the detection data are shown in a table 5.
TABLE 5 serum sample test results of sick pigs
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| 1.943 | 1.020 | 2.581 | 2.232 | 2.173 |
The results in Table 5 show that the ELISA diagnostic kit used in this example can detect EMCV antibodies in porcine serum, but the detection result is not as stable as the ELISA diagnostic kit for porcine brain myocarditis provided in example 1.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (8)
1. The pig encephalomyocarditis ELISA diagnostic kit is characterized in that pig encephalomyocarditis virus 2A protein is used as a coating antigen on an ELISA antibody detection plate, and the nucleotide sequence of the pig encephalomyocarditis virus 2A protein is shown as SEQ ID No. 3.
2. The ELISA diagnostic kit for pig brain myocarditis of claim 1, wherein primers for amplifying the nucleotide sequence are shown in SEQ ID No. 1-2.
3. The ELISA diagnostic kit for pig brain myocarditis according to any one of claims 1 to 2, wherein the amount of the porcine brain myocarditis virus 2A protein coated is 0.5 to 1 μ g/well.
4. The ELISA diagnostic kit for pig brain myocarditis of claim 3, characterized in that the ELISA diagnostic kit for pig brain myocarditis further comprises goat anti-mouse enzyme-labeled secondary antibody, a washing solution, a developing solution and a stop solution.
5. The ELISA diagnostic kit for pig brain myocarditis according to claim 4, characterized in that the detection operation steps are as follows:
(1) diluting 10 times of the concentrated washing solution to obtain a washing solution;
(2) diluting the serum to be detected, the positive control serum and the negative control serum by using an antibody diluent, adding the diluted antibody into an ELISA detection plate, incubating and then drying;
(3) adding a washing liquid into each hole, and drying by spin-drying after washing;
(4) adding an enzyme-labeled secondary antibody working solution into each hole, incubating and then drying;
(5) adding a washing liquid into each hole, and drying by spin-drying after washing;
(6) adding a color development liquid into each hole, and incubating in a dark place;
(7) stop solution was added to each well and the absorbance was read with a microplate reader at a wavelength of 450 nm.
6. The ELISA diagnostic kit for pig brain myocarditis according to claim 5, wherein the determination criteria for the detection result are:
(1) if the sample to be tested is OD450Judging the sample to be positive if the sample is more than or equal to 0.25;
(2) if the sample to be tested is OD450Judging the sample to be negative if the sample is less than or equal to 0.20;
(3) if the sample to be detected is 0.25 & gtOD450Judging the sample as a suspicious sample when the sample is more than 0.20; rechecking the suspicious sample if the suspicious sample OD450Judging the suspicious sample to be positive if the suspicious sample OD is more than or equal to 0.23450And judging the suspicious sample to be negative if the sample is less than or equal to 0.23.
7. The method for preparing ELISA diagnostic kit for pig brain myocarditis according to any one of claims 1 to 6, wherein the preparation of pig brain myocarditis virus 2A protein comprises:
a prokaryotic expression vector pET28a-2A is constructed by using a sequence shown in SEQ ID NO. 3;
and (3) converting the prokaryotic expression vector pET28a-2A into BL21, and selecting a positive recombinant bacterium to inoculate the positive recombinant bacterium in an LB culture medium containing kanamycin for culture.
8. The method for preparing the porcine encephalomyocarditis virus 2A protein according to claim 7, wherein the method for inducing expression of the porcine encephalomyocarditis virus 2A protein comprises the following steps:
bacterial liquid OD in BL21600When the concentration reached 0.6, IPTG was added to the LB medium at a final concentration of 0.1mM, and expression was induced at 37 ℃ for 8 hours.
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