CN115093467A - Recombinant porcine enterovirus G-type VP2 and VP3 protein polyclonal antibody and indirect ELISA detection method - Google Patents
Recombinant porcine enterovirus G-type VP2 and VP3 protein polyclonal antibody and indirect ELISA detection method Download PDFInfo
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Abstract
The invention discloses recombinant porcine enterovirus G-type VP2 and VP3 proteins which are respectively encoded by gene base sequences of sequence tables SEQ ID No.1 and SEQ ID No.2 or have amino acid sequences of SEQ ID No.3 and SEQ ID No. 4. Accordingly, polyclonal antibodies are also prepared, and two indirect ELISA detection methods are established based on VP2 and VP3 proteins. The obtained recombinant protein has the characteristics of good antigenicity and strong immunogenicity, and the ELISA detection method has the advantages of simple operation, strong specificity, good repeatability and high sensitivity, can be used for detecting and accurately judging the infection condition of the porcine enterovirus G type, and has good application prospect. In conclusion, the invention provides a technical means for epidemiological investigation of porcine enterovirus G type and provides a novel rapid serological detection method for porcine enterovirus G type infection.
Description
Technical Field
The invention belongs to the technical field of animal antibodies, and particularly relates to a recombinant porcine enterovirus G-type VP2 and VP3 protein polyclonal antibody and an indirect ELISA detection method.
Background
Porcine Enterovirus type G (Enterovirus G, EV-G) is ubiquitous in the herd and infecting piglets can affect their weight gain and cause neurological symptoms. Porcine enteroviruses are present in europe, america and asia and present a certain prevalence in the east of our country. EV-G infection is usually asymptomatic, the disease is often complicated with other diarrhea diseases, clinical symptoms such as diarrhea, muscle relaxation paralysis, reproductive disorders and the like are caused, and meanwhile, EV-G is found to be capable of infecting brain tissues and has wide tissue tropism, so that certain threats are caused to pig breeding and piglet health in China. At present, no porcine enterovirus G-type serum antibody detection kit exists in the market.
The diagnosis of porcine enterovirus type G mainly comprises pathogen separation, molecular diagnosis and serological detection, and the methods are generally antigen-specific detection, often consume a large amount of time and are inconvenient for the detection of porcine group EV-G antibodies and a large amount of clinical samples. The indirect ELISA method has the characteristics of simple operation, short time consumption and high sensitivity, can quickly and simultaneously detect a large quantity of clinical serum samples, and has few reports on the indirect ELISA method for detecting the EV-G antibody at home and abroad.
The EV-G genome is single-strand positive-strand RNA, has the total length of about 7.4kb, and only encodes one open reading frame, and comprises seven nonstructural proteins and four structural proteins VP 1-VP 4. Wherein VP 1-VP 3 form the complete capsid of the virus, which is located on the surface of the capsid of the virus and is subject to immunological pressure, and VP4 is located inside the capsid. The VP2 and VP3 proteins are located on the surface of the virion, are important components of the viral capsid, are located on the outermost layer of the virion, are highly exposed, are immunodominant proteins, and both VP2 and VP3 contain epitopes and participate in recognition of the receptor, binding and immune response of the host.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a recombinant porcine enterovirus G-type VP2 and VP3 protein polyclonal antibody and an indirect ELISA detection method.
In order to solve the technical problems, the invention adopts the following technical scheme:
recombinant porcine enterovirus G type VP2 and VP3 proteins are respectively encoded by gene base sequences of sequence tables SEQ.ID.No.1 and SEQ.ID.No.2 or have amino acid sequences of SEQ.ID.No.3 and SEQ.ID.No.4.
The preparation method of the recombinant porcine enterovirus G-type VP2 and VP3 protein comprises the following steps:
a. extracting the virus total RNA of a Guangxi isolate CH/17GXQZ/2017 of porcine enterovirus G, and performing reverse transcription to obtain cDNA;
b. respectively amplifying porcine enterovirus G-type VP2 and VP3 genes by taking cDNA as a template;
c. recovering two amplified products which are respectively 279bp, 738bp and 714 bp;
d. carrying out double enzyme digestion on the recovered fragment and a pET-32a vector at the same time, and recovering an enzyme digestion product;
e. connecting the target fragment with a vector under the action of T4 ligase at 16 ℃ overnight, transforming the target fragment into DH5 alpha competent cells, and selecting a single colony for culturing;
f. carrying out bacteria liquid PCR identification on the cultured monoclonal colonies to obtain positive bacteria liquid, selecting the positive bacteria, carrying out amplification culture, and extracting plasmids pET-32a-VP2 and pET-32a-VP 3;
g. respectively transforming the recombinant plasmids pET-32a-VP2 and pET-32a-VP3 to BL21(DE3) competent cells, selecting a single colony for culture, carrying out amplification culture on bacterial liquid identified as positive by PCR, and carrying out amplification culture on the bacterial liquid according to the ratio of 1: adding 4mL of positive bacterial liquid into 400mL of LB culture medium containing ampicillin according to the proportion of 100, carrying out shake culture until OD600 is 0.5, adding IPTG (isopropyl-beta-thiogalactopyranoside) with the final concentration of 0.2mmol/L, and continuing to culture overnight at 16 ℃; centrifuging to collect thallus, resuspending the thallus with PBS, ultrasonically cracking in an ultrasonic crusher, centrifuging at 4500rpm at 4 deg.C for 20min after ultrasonic treatment, collecting precipitate, and dissolving the precipitate with 8M urea at 4 deg.C overnight.
h. And purifying the recombinant protein by using Ni-NTA agarose resin to obtain the recombinant protein.
The rabbit polyclonal antibody of the recombinant porcine enterovirus G-type VP2 and VP3 proteins.
The preparation method of the rabbit polyclonal antibody uses the recombinant porcine enterovirus G-type VP2 and VP3 protein and Freund's adjuvant to emulsify and immunize rabbits to obtain polyclonal antibody serum.
The preparation method of the rabbit polyclonal antibody comprises the following steps: fully emulsifying the recombinant porcine enterovirus G-type VP2 and VP3 proteins with equal volume of Freund complete adjuvant at a dose of 1 mg/respectively, immunizing New Zealand white rabbits for the first time, boosting the immunity after 14 days, fully emulsifying with equal volume of Freund incomplete adjuvant at a dose of 0.5 mg/respectively, boosting the immunity for 4 times totally, collecting blood from the heart on the 7 th day after the immunity is finished, and collecting serum at 4 ℃ to obtain the polyclonal antibody.
The indirect ELISA antibody detection kit for the porcine enterovirus G-type VP2 and VP3 proteins comprises an enzyme-coated plate, wherein the enzyme-coated plate takes the recombinant porcine enterovirus G-type VP2 and VP3 proteins of claim 1 as coating antigens.
The indirect ELISA antibody detection kit further comprises porcine enterovirus G-type positive serum, porcine enterovirus G-type negative serum, HRP-goat anti-porcine IgG, coating liquid, washing liquid, confining liquid, developing liquid and stopping liquid; the coating solution is carbonate buffer solution, the washing solution is TBST washing solution, the confining solution is 5% skimmed milk powder, the developing solution is TMB developing solution, and the stop solution is 2% H 2 SO 4 A solution; the preparation of the washing liquid is as follows: 4.4g NaCl, 10mL Tris-HCl (pH 8.0), 500. mu.L Tween-20.
The indirect ELISA detection method for G-type VP2 and VP3 proteins of porcine enteroviruses of non-diagnostic purposes comprises the following steps:
antigen coating: diluting porcine enterovirus G-type VP2 and VP3 recombinant proteins with a coating solution, and coating for 12h at 4 ℃; then TBST is used for washing away unbound antigen and impurities;
and (3) sealing: adding BSA as a blocking solution, blocking at 37 ℃, then discarding the blocking solution, and washing away residual blocking solution by TBST;
adding serum to be detected: diluting the serum to be detected in a multiple ratio, incubating at 37 ℃, and washing with TBST after reaction;
adding enzyme-labeled secondary antibody: diluting HRP-goat anti-pig IgG, incubating at 37 ℃, reacting, and washing with TBST;
color development and measurement: adding TMB color development liquid, keeping out of the sun, developing, adding stop solution, and reading at 450nm wavelength of an enzyme-labeling instrument.
Coating the VP2 protein at 50 ng/hole, and diluting the serum to be detected at a ratio of 1: 200; coating VP3 protein at a concentration of 100 ng/well, and diluting the serum to be detected at a ratio of 1: 200;
the action time of the serum to be detected is 60min, and the action time of the enzyme-labeled secondary antibody is 45 min;
antigen coating conditions are 4 ℃ overnight, and the dilution of the secondary antibody is 1:2000 or 1: 4000;
the blocking solution is 1% BSA, and the blocking time is 2 h;
the color development time was 10 min.
Aiming at the problems existing in the detection of the porcine enterovirus G type, the inventor researches and designs recombinant porcine enterovirus G type VP2 and VP3 proteins which are respectively encoded by gene base sequences of sequence tables SEQ ID No.1 and SEQ ID No.2 or have amino acid sequences of SEQ ID No.3 and SEQ ID No. 4. Two structural proteins VP2 and VP3 of porcine enterovirus G type are used as capsid proteins and contain partial antigen epitopes, so the two segments of genes are selected for amplification and cloned to pET-32a vector for expression to obtain two recombinant proteins. According to Guangxi separating strain, a specific primer is designed to amplify a target fragment, prokaryotic expression plasmids of VP2 and VP3 are constructed, recombinant protein is obtained, a polyclonal antibody is prepared, and an indirect ELISA detection method is established based on VP2 and VP3 proteins. The obtained recombinant protein has the characteristics of good antigenicity and strong immunogenicity, and the ELISA detection method has the advantages of simple operation, strong specificity, good repeatability and high sensitivity, can be used for detecting and accurately judging the infection condition of the porcine enterovirus G type, and has good application prospect. In conclusion, the invention provides a technical means for epidemiological investigation of porcine enterovirus G type and provides a novel rapid serological detection method for porcine enterovirus G type infection.
Drawings
FIG. 1 is a graph showing the amplification results of VP2 and VP3, in which: a is the result of VP2 amplification; b is VP3 amplification result; m: DL2000 DNA Marker; 1: a VP2 fragment; 2: fragment VP 3.
FIG. 2 is a diagram showing the results of double digestion of recombinant plasmids, in which: a is the double digestion result of the pET-32a-VP2 recombinant plasmid; b is the double digestion result of the pET-32a-VP3 recombinant plasmid; m: DL5000 DNA Marker; 1: pET-32a-VP2 double enzyme digestion product; 2: pET-32a-VP3 double enzyme digestion product.
FIG. 3 is a graph showing the results of recombinant protein expression and purification of pET-32a-VP2, wherein: m: protein Marker; 1: induced pET-32 a; 2: uninduced pET-32a-VP 2; 3: induced pET-32a-VP 2; 4: pET-32a-VP2 supernatant; 5: pET-32a-VP2 precipitation; 6: purified pET-32a-VP 2.
FIG. 4 is a graph showing the results of recombinant protein expression and purification of pET-32a-VP3, wherein: m: a protein Marker; 1: induced pET-32 a; 2: uninduced pET-32a-VP 3; 3: induced pET-32a-VP 3; 4: pET-32a-VP3 supernatant; 5: precipitation of pET-32a-VP 3; 6: purified pET-32a-VP 3.
FIG. 5 is a Western-blotting analysis of recombinant proteins, in which the primary antibody is a murine anti-His monoclonal antibody and the secondary antibody is HRP-goat anti-mouse IgG, in which: a is the verification of pET-32a-VP2 recombinant protein; b is the verification of pET-32a-VP3 recombinant protein; m: a protein Marker; 1: VP2 recombinant protein; 2: VP3 recombinant protein.
FIG. 6 shows Western-blotting analysis of polyclonal antibodies, in which the primary antibody is a prepared rabbit anti-polyclonal antibody, and the secondary antibody is HRP-goat anti-rabbit IgG, in which: a is pET-32a-VP2 polyclonal antibody verification; b is the polyclonal antibody verification of pET-32a-VP 3; m: a protein Marker; 1: the VP2 recombinant protein reacts with VP2 polyclonal antibody; 2: the VP3 recombinant protein reacted with VP3 polyclonal antibody.
FIG. 7 is a graph showing the result of IFA validation of the VP2 polyclonal antibody, wherein: A. b, C: EV-G infection of MARC-145 cells verified the polyclonal antibody to VP2 (X200); D-F: EV-G infected MARC-145 cell negative serum control (x 200); G-I: uninfected MARC-145 cells demonstrated polyclonal antibody (X200) to VP 2.
FIG. 8 is a validation chart of the VP3 polyclonal antibody IFA, in which: A. b, C: EV-G infection of MARC-145 cells verified the polyclonal antibody to VP3 (X200); D-F: EV-G infected MARC-145 cell negative serum control (x 200); G-I: uninfected MARC-145 cells demonstrated polyclonal antibody (X200) to VP 3.
FIG. 9 is a diagram showing the results of the specificity test of the indirect ELISA detection method of the present invention, in which: a is a specific test result chart of the VP2 indirect ELISA detection method; b is a specific test result chart of the VP3 indirect ELISA detection method.
FIG. 10 is a graph showing the results of the sensitivity test in the indirect ELISA detection method of the present invention, in which: a is a sensitivity test result graph of the VP2 indirect ELISA detection method; b is a graph of the sensitivity test result of the VP3 indirect ELISA detection method.
Detailed Description
Materials and methods
Strains, cells and plasmids: the porcine enterovirus G-type isolated strain (consistent with the strain in the Chinese patent application "primers and kit for real-time fluorescent quantitative PCR detection of porcine enterovirus G-type" (application No. 2019110463799, published as 2019.12.24); "epidemiological survey of porcine enterovirus G-type and analysis of VP1 gene sequence" (2020. Chinese veterinary)), DH5 alpha, BL21 competent cells and pET-32a plasmid were all stored in the applicant's laboratory.
The main reagents are as follows: restriction enzymes BamH I, HindIII and SalI and protein molecular mass standards were purchased from Novozan Biotech Ltd; viral RNA extraction kit purchased from AxyPrep TM Body Fluid Viral RNA/DNA Miniprep Mix was purchased from corning life sciences (wujiang); the DNA fragment purification and recovery kit and the plasmid small-amount extraction kit are purchased from OMEGA company; ni sepharose was purchased from seikang as a century company; the ELISA plate was purchased from Laine Biotechnology Ltd; HRP-goat anti-pig IgG (H + L) was purchased from Solebao.
1. Amplification of porcine enterovirus G-type structural protein gene and construction of recombinant plasmid
The porcine enterovirus G-type isolate obtained by laboratory separation is taken as a template to amplify VP2 and VP3 genes, and specifically comprises the following steps:
the following primers were used as specific primers, and restriction sites (underlined) were introduced into the upstream and downstream primers, respectively, as shown in Table 1.
TABLE 1 amplification primer information
Firstly, RT-PCR amplification is carried out, and the 12.5 mu L RT-PCR amplification system is as follows:
TaKaRa: 5 × Buffer 2.5 μ L, TaKaRa: dNTP mix (2.5 mmol/. mu.L) 1. mu.L, TaKaRa: M-MLV Reverse Transcriptase (200U/. mu.L) 0.25. mu.L, Vazyme: RNase enzyme inhibitor (40U/. mu.L) 0.25. mu.L, downstream primer (for single strand positive strand RNA) or Oligo dT 0.5. mu.L, RNA 8. mu.L, at 42 ℃ for 1h to give cDNA;
then, PCR amplification is carried out, and the 25-microliter PCR amplification reaction system is as follows:
ddH 2 o8.5. mu.L, forward Primer 0.5. mu.L, reverse Primer 0.5. mu.L, Primer Star enzyme 12.5. mu.L, cDNA 3. mu.L; the amplification conditions were: 5min at 94 ℃; entering a circulation: 32 cycles of 94 ℃ for 40s, 58 ℃ for 40s, 72 ℃ for 50 s; the target fragments VP2 and VP3 were amplified at 72 ℃ for 10min to obtain target fragments 738bp and 714bp, respectively, which were consistent with the expected sizes, and the results are shown in FIG. 1.
And (3) performing gel recovery on the PCR product, and performing double enzyme digestion on the PCR product and a prokaryotic expression vector pET-32a, wherein the double enzyme digestion system is as follows: plasmid 33. mu.L, endonuclease 7. mu.L each, 10 XBuffer 10. mu.L, ddH 2 O43 mu L, performing double enzyme digestion in a water bath at 37 ℃ overnight, and recovering and purifying the enzyme digestion product.
The ligation of the digested product was performed by T4 ligase at 16 ℃ and 10. mu.L of ligase reaction system was: 1 mu L of T4 DNA ligase, 7 mu L of target fragment recovered by double digestion of recombinant plasmid, 1 mu L of target fragment recovered by double digestion of pET-32a vector, and 1 mu L of Buffer; mixing the components, centrifuging, and connecting at 16 deg.C for at least 8 hr. Obtaining recombinant expression vectors pET-32a-VP2 and pET-32a-VP3, sequencing the vectors and carrying out double-enzyme digestion identification, wherein the result is shown in figure 2, and sequencing proves that the recombinant plasmid is successfully constructed.
2. Induced expression and purification of recombinant protein
And (3) converting the recombinant plasmid with correct double enzyme digestion and sequencing results into BL21 competent cells, plating and culturing, then placing the competent cells on a horizontal shaking table at 37 ℃ for amplification, and continuing to perform shaking table induction at 37 ℃ for 6 hours according to the addition of IPTG (final concentration of 0.2mmol/L) when the OD value of the amplified bacterial liquid is 0.6. And centrifuging the induced bacterial liquid, carrying out ultrasonic crushing and the like, and carrying out SDS-PAGE electrophoresis to detect the expression condition and the reactionogenicity of the recombinant protein.
Reagents used for purification included:
binding Buffer: Tris-HCl (pH 8.0)20mM, imidazole 5mM, sodium chloride 0.5M, urea 8M;
elution Buffer: Tris-HCl (pH 8.0)20mM, imidazole 500mM, sodium chloride 0.5M, urea 8M.
Dissolving the collected precipitate in urea at 4 ℃ overnight after the completion of the ultrasonic treatment, centrifuging at 13000 rpm for 20min, and collecting the supernatant; filtering the sample by using a 0.22 mu m filter, adding the collected protein sample into a chromatographic column, controlling the flow rate to ensure that the protein sample is fully combined with Ni-NTA agarose resin, and collecting a flow-through peak; then adding 15mL Binding Buffer to wash out foreign protein, adding 10mL Elution Buffer to carry out Elution, and collecting Elution peak; after the elution is finished, adding 10mL Binding Buffer and 10mL deionized water to wash the column; finally, 6mL of 20% ethanol is added to seal the column and the column is stored at 4 ℃.
SDS-PAGE electrophoresis is carried out on the purified protein to detect whether the band is single as shown in figures 3-5.
3. Establishment of indirect ELISA method for detecting porcine enterovirus G-type antibody
3.1 determination of optimal coating concentration of antigen and serum dilution
Antigen coating: diluting porcine enterovirus G-type VP2 and VP3 recombinant proteins with coating solutions, wherein the concentrations are respectively 6.25 ng/well, 12.5 ng/well, 25 ng/well, 50 ng/well, 100 ng/well and 200 ng/well, and coating is carried out at 4 ℃ for 12 h; then TBST is used for washing away unbound antigen and impurities;
and (3) sealing: adding 5% skimmed milk powder as sealing solution, sealing at 37 deg.C for 2 hr, discarding the sealing solution, and washing off residual sealing solution with TBST;
adding serum to be detected: and (3) mixing the serum to be detected according to the ratio of 1: 100. 1: 200. 1: 400. 1: diluting by 800 times, incubating for 1h at 37 ℃, and washing by TBST after reaction;
adding enzyme-labeled secondary antibody: HRP-goat anti-porcine IgG was purified according to 1: diluting with 4000, incubating for 2h at 37 ℃, and washing with TBST after reaction;
color development and measurement: adding TMB color development liquid, keeping out of the sun, developing, adding stop solution, and reading at 450nm wavelength of an enzyme-labeling instrument. 3.2 determination of incubation time of the serum to be assayed and the enzyme-labeled Secondary antibody
And setting the incubation time of the serum to be detected and the enzyme-labeled secondary antibody to be 30min, 45min and 60min, and performing indirect ELISA detection on EV-G positive serum and negative blood.
3.3 antigen coating conditions and determination of dilution of Secondary antibodies
The coating time is set as 37 ℃ for 1h, 37 ℃ for 2h, 37 ℃ for 4h and 4 ℃ overnight, and the HRP-goat anti-pig IgG is set to have a dilution gradient of 1: 1000, 1:2000, 1:4000 and 1: 8000 for indirect ELISA detection.
3.4 determination of optimal blocking fluid and blocking time
And (3) respectively selecting 1% skimmed milk powder, 5% skimmed milk powder, 1% BSA and 5% BSA during blocking, setting the blocking time to be 37 ℃ for blocking for 1h and 2h, and carrying out ELISA detection.
3.5 determination of optimal color development time
Adding color developing solution, and reading D respectively at color development time of 5min, 10min, 15min, and 25min 450 And (5) carrying out indirect ELISA detection on the nm value.
3.6 determination of the threshold value
Performing indirect ELISA detection on 24 parts of EV-G negative serum under optimized conditions, repeating the detection three times, and determining D 450 nm value, calculating sample D according to the measured result 450 Mean (X) and Standard Deviation (SD) of the nm values. When D of the sample is detected 450 Positive at nm of X +3SD or more, D 450 The result is judged to be negative when the nm is less than or equal to X +2SD and the X +2SD is less than D 450 And the result that the nm is less than X +3SD is judged to be suspicious.
3.7 specificity test
Performing indirect ELISA detection on positive serum samples of FMDV-O, PRRSV, CSFV, AFSV, PCV-2, PRV, PEDV and PoRV under optimized conditions, repeating 3 times with positive and negative serum of EV-G as control, and reading D 450 The nm value analyzes the specificity of the detection method.
3.8 repeatability test
Applying optimized conditions to an indirect ELISA method established according to VP2 and VP3 recombinant proteinsDetection of positive and negative serum for EV-G, three replicates were set up, based on D determined 450nm Values, mean (X), Standard Deviation (SD) and Coefficient of Variation (CV) were calculated, and the indirect ELISA method was analyzed for plate-to-plate and plate-to-plate reproducibility.
3.9 sensitivity test
Selecting 3 parts of EV-G positive serum to dilute according to the ratio of 1:50, 1:100, 1:200, 1:400, 1:800, 1: 1600 and 1: 3200, adopting the established indirect ELISA method to detect, repeating each sample for 3 times, reading D 450nm The value is obtained.
3.10 compliance rate test
20 sera were compared to the results of the Western blot assay using indirect ELISA detection methods of VP2 and VP 3.
4. Results of the experiment
4.1 determination of optimal coating concentration of antigen and serum dilution
The optimal coating concentration of the VP2 recombinant protein and the dilution of the serum to be detected are determined by a matrix method, and the result is shown in Table 2, when the VP2 protein is coated in 50 ng/hole and the serum to be detected is diluted 1:200, the maximum P/N value is 12.106.
TABLE 2 determination of optimal coating concentration and serum dilution of the VP2 recombinant protein
The optimal coating concentration of the VP3 recombinant protein and the dilution of the serum to be detected are determined by a matrix method, and the result is shown in Table 3, when the VP3 protein is coated in 100 ng/hole and the serum to be detected is diluted 1:200, the maximum P/N value is 12.720.
TABLE 3 determination of optimal coating concentration and serum dilution of the VP3 recombinant protein
4.2 determination of incubation time of the serum to be assayed and the enzyme-labeled Secondary antibody
The optimal incubation time of the serum to be detected and the enzyme-labeled secondary antibody based on the indirect ELISA method of the VP2 protein is determined by a matrix method, the result is shown in Table 4, and when the action time of the serum to be detected is 60min and the action time of the enzyme-labeled secondary antibody is 45min, the maximum P/N value is 10.339.
TABLE 4 VP2 determination of incubation time of serum to be tested and enzyme-labeled secondary antibody in Indirect ELISA
The optimal incubation time of the serum to be detected and the enzyme-labeled secondary antibody based on the indirect ELISA method for the VP3 protein is determined by a matrix method, the result is shown in Table 5, and when the action time of the serum to be detected is 60min and the action time of the enzyme-labeled secondary antibody is 45min, the maximum P/N value is 11.143.
TABLE 5 VP 3-determination of incubation time of the test serum and the enzyme-labeled secondary antibody in an indirect ELISA
4.3 antigen coating conditions and determination of dilution of Secondary antibody
The optimal antigen coating conditions and the dilution of the secondary antibody based on the indirect ELISA method of VP2 protein were determined by a matrix method, and the results are shown in Table 6, wherein the maximum P/N value was 9.311 when the antigen coating conditions were 4 ℃ overnight and the dilution of the secondary antibody was 1: 2000.
TABLE 6 VP2 recombinant protein antigen coating conditions and determination of the dilution of the secondary antibody
The optimal coating conditions of the antigen based on the indirect ELISA method of VP3 protein and the dilution of the secondary antibody were determined by the matrix method, and the results are shown in Table 7, wherein when the antigen coating conditions were 4 ℃ overnight and the dilution of the secondary antibody was 1:4000, the maximum P/N value was 10.086.
TABLE 7 VP3 recombinant protein antigen coating conditions and determination of the dilution of the secondary antibody
4.4 determination of optimal sealing fluid and sealing time
The optimal blocking conditions based on the indirect ELISA method using VP2 protein were determined, and the results are shown in Table 8, wherein the maximum P/N was 10.813 when the blocking solution was 1% BSA and the blocking was performed for 2 h.
TABLE 8 determination of optimal blocking solution and blocking time for the VP2 recombinant protein
The optimal blocking conditions based on the indirect ELISA method using VP3 protein were determined, and the result is shown in Table 9, wherein the maximum P/N was 12.317 when the blocking solution was 1% BSA and the blocking was performed for 2 h.
TABLE 9 determination of optimal blocking solution and blocking time for the VP3 recombinant protein
4.5 determination of optimal color development time
Setting the color development time to 5, 10, 15, 25min, and reading D 450 nm value and calculating P/N value. As shown in Table 10, the two indirect ELISA methods established with the VP2 and VP3 recombinant proteins showed the maximum P/N at 10min of color development, with the P/N values of 8.006 and 8.464, respectively.
TABLE 10 determination of optimal color development time
4.6 determination of the threshold value
Uses VP2 recombinant protein as envelope antigen, when being coatedTest sample D 450 Positive at nm of 0.292 or more, and D 450 When nm is less than or equal to 0.244, the negative is judged, and when the nm is less than or equal to 0.244, D is more than 0.244 450 And when the nm is less than 0.292, the result is judged to be suspicious, and the result is shown in a table 11.
TABLE 11 determination of cut-off values for the recombinant protein VP2
Using VP3 recombinant protein as coating antigen, when the detected sample D is 450 Positive when nm is not less than 0.258, and D is 450 When nm is less than or equal to 0.217, the result is judged to be negative, and when nm is less than or equal to 0.217, D is more than 0.217 450 And when the nm is less than 0.258, the result is judged to be suspicious, and the result is shown in a table 12.
TABLE 12 determination of cut-off values for the recombinant protein VP3
4.7 specificity assay
Positive sera of FMDV-O, PRRSV, CSFV, AFSV, PCV-2, PRV, PEDV, PoRV and EV-G are respectively detected by using indirect ELISA methods based on VP2 and VP3 proteins, the results are respectively shown in figure 9, only detection results of EV-G positive sera are positive in the two indirect ELISA methods, and detection results of other virus positive sera and EV-G negative sera are negative, which indicates that the established indirect ELISA methods have good specificity.
4.8 repeatability test
The in-plate and inter-plate repeat results based on the indirect ELISA method using VP2 protein are shown in Table 13, the in-plate repeat variation coefficient is 1.9% -6.1%, the inter-plate repeat variation coefficient is 2.2% -8.6%, and both are less than 10%, which indicates that the method has better repeatability.
TABLE 13 VP 2-Indirect ELISA repeatability test results
The in-plate and inter-plate repeat results based on the indirect ELISA method using VP3 protein are shown in Table 14, the in-plate repeat variation coefficient is 2.8% -8.4%, the inter-plate repeat variation coefficient is 2.3% -8.1%, and both are less than 10%, which indicates that the method has good repeatability.
TABLE 14 VP 3-Indirect ELISA repeatability test results
4.9 sensitivity test
3 parts of EV-G positive serum are diluted according to a 1:50, 1:100, 1:200, 1:400, 1:800, 1: 1600 and 1: 3200 fold ratio, and two indirect ELISA methods are tested and repeated for three times. As shown in FIG. 10, the indirect ELISA method based on VP2 protein still showed positive results when serum samples were diluted 1: 1600 (FIG. 10A); when the serum samples were diluted 1:800, the detection result of the indirect ELISA method based on the VP3 protein was still positive (FIG. 10B), indicating that the indirect ELISA method based on the VP2 and VP3 proteins established in the present study has good sensitivity.
4.10 preliminary application of Indirect ELISA method based on VP2 and VP3 proteins
46 parts of pig serum is detected by applying established indirect ELISA methods of VP2 and VP3, wherein 26 parts of pig enterovirus G-type antibody positive serum and 20 parts of pig enterovirus G-type antibody negative serum are detected by adopting the VP2 indirect ELISA method; 24 positive serums and 22 negative serums of the porcine enterovirus G-type antibody are detected by a VP3 indirect ELISA method.
In conclusion, the invention provides porcine enterovirus G-type recombinant VP2 and VP3 proteins as antigens, and establishes two indirect ELISA methods, which have the advantages of strong specificity, high sensitivity, good repeatability and certain application prospect.
Sequence listing
<110> Guangxi university
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Claims (9)
1. Recombinant porcine enterovirus G-type VP2 and VP3 proteins are characterized in that the recombinant porcine enterovirus G-type VP2 and VP3 proteins are respectively encoded by gene base sequences of sequence tables SEQ ID No.1 and SEQ ID No.2 or have amino acid sequences of SEQ ID No.3 and SEQ ID No. 4.
2. The method for preparing recombinant porcine enterovirus G-type VP2, VP3 protein of claim 1, which is characterized by comprising the following steps:
a. extracting the virus total RNA of a Guangxi isolate CH/17GXQZ/2017 of porcine enterovirus G type, and carrying out reverse transcription to obtain cDNA;
b. respectively amplifying porcine enterovirus G-type VP2 and VP3 genes by taking cDNA as a template;
c. recovering two amplified products which are respectively 279bp, 738bp and 714 bp;
d. carrying out double enzyme digestion on the recovered fragment and a pET-32a vector at the same time, and recovering an enzyme digestion product;
e. connecting the target fragment with a vector under the action of T4 ligase at 16 ℃ overnight, transforming the target fragment into DH5 alpha competent cells, and selecting a single colony for culturing;
f. carrying out bacteria liquid PCR identification on the cultured monoclonal colonies to obtain positive bacteria liquid, selecting the positive bacteria, carrying out amplification culture, and extracting plasmids pET-32a-VP2 and pET-32a-VP 3;
g. the recombinant plasmids pET-32a-VP2 and pET-32a-VP3 are respectively transformed into BL21(DE3) competent cells, a single colony is selected for culture, and a bacterial solution which is identified as positive by PCR is subjected to amplification culture, wherein the bacterial solution is prepared according to the following steps of 1: adding 4mL of positive bacterial liquid into 400mL of LB culture medium containing ampicillin according to the proportion of 100, carrying out shake culture until OD600 is 0.5, adding IPTG (isopropyl-beta-thiogalactopyranoside) with the final concentration of 0.2mmol/L, and continuing to culture overnight at 16 ℃; centrifuging to collect thallus, resuspending the thallus with PBS, ultrasonically cracking in an ultrasonic crusher, centrifuging at 4500rpm at 4 ℃ for 20min after ultrasonic treatment, collecting precipitate, and dissolving the precipitate with 8M urea at 4 ℃ overnight.
h. And (3) purifying the recombinant protein by using Ni-NTA agarose resin to obtain the recombinant protein.
3. Rabbit polyclonal antibody of recombinant porcine enterovirus G-type VP2, VP3 protein of claim 1.
4. A method of producing a rabbit polyclonal antibody according to claim 3, wherein: polyclonal antibody serum is obtained by immunizing rabbits with the recombinant porcine enterovirus G-type VP2 and VP3 proteins of claim 1 after emulsification with Freund's adjuvant.
5. The method of claim 4, comprising the steps of: fully emulsifying the recombinant porcine enterovirus G-type VP2 and VP3 proteins with equal volume of Freund complete adjuvant at a dose of 1 mg/respectively, immunizing New Zealand white rabbits for the first time, boosting the immunity after 14 days, fully emulsifying with equal volume of Freund incomplete adjuvant at a dose of 0.5 mg/respectively, boosting the immunity for 4 times totally, collecting blood from the heart on the 7 th day after the immunity is finished, and collecting serum at 4 ℃ to obtain the polyclonal antibody.
6. The indirect ELISA antibody detection kit for the porcine enterovirus G-type VP2 and VP3 proteins comprises an enzyme-coated plate, and is characterized in that: the coated enzyme label plate takes the recombinant porcine enterovirus G-type VP2 and VP3 protein of claim 1 as a coated antigen.
7. The indirect ELISA antibody detection kit of claim 6, characterized by further comprising porcine enterovirus G-type positive serum, negative serum, HRP-goat anti-porcine IgG, coating solution, washing solution, blocking solution, developing solution and stop solution; the coating solution is carbonate buffer solution, the washing solution is TBST washing solution, the confining solution is 5% skimmed milk powder, the developing solution is TMB developing solution, and the stop solution is 2% H 2 SO 4 A solution; the preparation of the washing liquid is as follows: 4.4g NaCl, 10mL Tris-HCl (pH 8.0), 500. mu.L Tween-20.
8. The indirect ELISA detection method for G-type VP2 and VP3 proteins of porcine enteroviruses for non-diagnosis purposes is characterized by comprising the following steps:
antigen coating: diluting porcine enterovirus G-type VP2 and VP3 recombinant proteins with a coating solution, and coating for 12h at 4 ℃; then TBST is used for washing away unbound antigen and impurities;
and (3) sealing: adding BSA as a blocking solution, blocking at 37 ℃, then discarding the blocking solution, and washing away residual blocking solution by TBST;
adding serum to be detected: diluting the serum to be detected in a multiple ratio, incubating at 37 ℃, and washing with TBST after reaction;
adding enzyme-labeled secondary antibody: diluting HRP-goat anti-pig IgG, incubating at 37 ℃, reacting, and washing with TBST;
color development and measurement: adding TMB color development liquid, keeping out of the sun, developing, adding stop solution, and reading at 450nm wavelength of an enzyme-labeling instrument.
9. The indirect ELISA detection method of claim 8 wherein:
coating the VP2 protein by 50 ng/hole, and diluting the serum to be detected according to the ratio of 1: 200; coating VP3 protein at a concentration of 100 ng/well, and diluting the serum to be detected at a ratio of 1: 200;
the action time of the serum to be detected is 60min, and the action time of the enzyme-labeled secondary antibody is 45 min;
antigen coating conditions are 4 ℃ overnight, and the dilution of the secondary antibody is 1:2000 or 1: 4000;
the blocking solution is 1% BSA, and the blocking time is 2 h;
the color development time was 10 min.
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