CN112964871A

CN112964871A - Pig encephalomyocarditis indirect ELISA diagnostic kit and preparation method thereof

Info

Publication number: CN112964871A
Application number: CN202110169040.9A
Authority: CN
Inventors: 崔尚金; 梁瑞英; 梁琳; 韩若婵; 秦彤
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-06-15

Abstract

本发明涉及一种猪脑心肌炎间接ELISA诊断试剂盒及其制备方法。本发明所述试剂盒以猪脑心肌炎病毒2A纯化蛋白作为ELISA抗体检测板上的包被抗原。本发明公开了制备及纯化2A蛋白的方法，即利用猪脑心肌炎病毒HB10株基因组DNA为模板，用特异性引物进行PCR扩增，扩增产物经酶切后构建原核表达载体pET28a‑2A，再进行诱导表达2A蛋白。本发明的ELISA诊断方法特异性强，结果稳定可靠，可特异性地检测出猪脑心肌炎病毒抗体，完全适用于猪脑心肌炎病毒感染后的诊断。The invention relates to an indirect ELISA diagnostic kit for porcine encephalomyocarditis and a preparation method thereof. The kit of the present invention uses the purified protein of porcine encephalomyocarditis virus 2A as the coating antigen on the ELISA antibody detection plate. The invention discloses a method for preparing and purifying 2A protein, that is, using the genomic DNA of porcine encephalomyocarditis virus HB10 strain as a template, performing PCR amplification with specific primers, and the amplified product is digested with enzymes to construct a prokaryotic expression vector pET28a-2A, and then Induction was performed to express the 2A protein. The ELISA diagnostic method of the invention has strong specificity, stable and reliable results, can specifically detect the porcine encephalomyocarditis virus antibody, and is completely suitable for the diagnosis of porcine encephalomyocarditis virus infection.

Description

Pig encephalomyocarditis indirect ELISA diagnostic kit and preparation method thereof

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 screening_450nmLess 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:

(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 reader_450nmValue).

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

Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6	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

Sample 1	Sample 2	Sample 3	Sample 4	Sample 5
					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:

(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

Sample 1	Sample 2	Sample 3	Sample 4	Sample 5
					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

1. a porcine encephalomyocarditis ELISA diagnostic kit, is characterized in that, with porcine encephalomyocarditis virus 2A protein as the coating antigen on the ELISA antibody detection plate, the nucleotide sequence of described porcine encephalomyocarditis virus 2A protein is as SEQ ID NO .3 shown.

2 . The porcine encephalomyocarditis ELISA diagnostic kit according to claim 1 , wherein the primers for amplifying the nucleotide sequence are shown in SEQ ID NO. 1-2. 3 .

3. The porcine encephalomyocarditis ELISA diagnostic kit according to any one of claims 1-2, wherein the coating amount of the porcine encephalomyocarditis virus 2A protein is 0.5-1 μg/well.

4. porcine encephalomyocarditis ELISA diagnostic kit according to claim 3, is characterized in that, in described porcine encephalomyocarditis ELISA diagnostic kit, also comprises goat anti-mouse enzyme-labeled secondary antibody, washing solution, color developing solution, termination liquid.

5. porcine encephalomyocarditis ELISA diagnostic kit according to claim 4, is characterized in that, detection operation step is:

(1) Dilute the 10× concentrated washing solution 10 times to be the washing solution;

(2) The serum to be tested, the positive control serum and the negative control serum are all diluted with antibody diluent, added to the ELISA detection plate, and dried after incubation;

(3) add washing liquid to each hole, spin dry after washing;

(4) Add enzyme-labeled secondary antibody working solution to each well, and spin dry after incubation;

(5) add washing liquid to every hole, spin dry after washing;

(6) Add chromogenic solution to each well and incubate in the dark;

(7) Add stop solution to each well, and read the light absorption value with a microplate reader at a wavelength of 450 nm.

6. porcine encephalomyocarditis ELISA diagnostic kit according to claim 5, is characterized in that, the criterion of detection result is:

(1) If the OD ₄₅₀ of the sample to be tested is greater than or equal to 0.25, the sample is judged to be positive;

(2) If the OD ₄₅₀ of the sample to be tested is ≤0.20, the sample is judged to be negative;

(3) If the sample to be tested is 0.25 > OD ₄₅₀ > 0.20, the sample is determined to be a suspicious sample; if the suspicious sample is re-examined, if the suspicious sample OD ₄₅₀ ≥ 0.23, the suspicious sample is determined to be positive, and if the suspicious sample OD ₄₅₀ ≤ 0.23, the suspicious sample is determined to be suspicious. is negative.

7. the preparation method of the porcine encephalomyocarditis ELISA diagnostic kit described in any one of claim 1-6, is characterized in that, the preparation of porcine encephalomyocarditis virus 2A protein comprises:

Use the sequence shown in SEQ ID NO.3 to construct a prokaryotic expression vector pET28a-2A;

The prokaryotic expression vector pET28a-2A was transformed into BL21, and the positive recombinant bacteria were picked and inoculated in LB medium containing kanamycin for culture.

8. preparation method according to claim 7, is characterized in that, the induction expression method of described porcine encephalomyocarditis virus 2A protein comprises:

When the OD ₆₀₀ of the BL21 bacterial solution reached 0.6, IPTG with a final concentration of 0.1 mM was added to the LB medium, and expression was induced at 37° C. for 8 h.