Novel chicken circovirus type 3 strain and detection system based on same
阅读说明：本技术 一株鸡新型圆圈病毒3型毒株及基于该病毒的检测体系 (Novel chicken circovirus type 3 strain and detection system based on same ) 是由 成子强 张世成 严天行 周德方 张利 郝小静 于 2021-08-16 设计创作，主要内容包括：本发明涉及分子生物学、免疫学和病毒学领域,具体提供了一株鸡新型圆圈病毒3型(GyV3)毒株及基于该病毒的检测体系,该病毒毒株生物保藏编号为CCTCC NO:V202061,其VP1基因的核苷酸序列如SEQ ID NO.1所示,其氨基酸序列如SEQ ID NO.2所示,同时发明人还提供了该病毒的ELISA抗体检测方法和鸡新型GyV3的实时荧光定量PCR检测方法组成检测体系,二者结合可实现对该GyV3病毒安全、特异、快速、灵敏、简单的快速检测,从而为鸡新型环状病毒GyV3的流行病学、早期诊断提供参考依据,具有重要的意义,填补了国内外相关领域空白。(The invention relates to the fields of molecular biology, immunology and virology, and particularly provides a novel chicken circovirus type 3 (GyV3) strain and a detection system based on the virus, wherein the biological preservation number of the virus strain is CCTCC NO: V202061, the nucleotide sequence of a VP1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2, and meanwhile, the invention also provides a detection system consisting of an ELISA antibody detection method of the virus and a novel chicken GyV3 real-time fluorescent quantitative PCR detection method, and the combination of the two can realize safe, specific, rapid, sensitive and simple rapid detection of the GyV3 virus, thereby providing a reference basis for epidemiology and early diagnosis of the novel chicken circovirus GyV3, having important significance and filling up the blank of related fields at home and abroad.)
1. A novel chicken circovirus type 3 (GyV3) strain is characterized in that: the biological preservation number of the virus strain is CCTCC NO. V202061, the nucleotide sequence of the VP1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
2. The method for detecting the ELISA antibody of the novel circovirus type 3 (GyV3) of chicken of claim 1, wherein the ELISA antibody comprises: the protein is capsid protein VP1 fusion protein, and is prepared by connecting a novel circovirus type 3 VP1 gene with a prokaryotic expression vector pET32a through enzyme digestion and transforming escherichia coli competent cells BL 21.
3. The fluorescent quantitative PCR detection method of the novel chicken circovirus type 3 (GyV3) of claim 1, which is characterized in that: the primer sequences used were as follows:
an upstream primer GyV3 Forward: 5'-ACCGGGACTTGGACACC-3', the nucleotide sequence of which is shown in SEQ ID NO. 3;
downstream primer GyV3 Reverse: 5'-AGCCAGGAAGCGATACG-3', the nucleotide sequence is shown in SEQ ID NO. 4.
4. The real-time fluorescent quantitative PCR detection method of chicken novel circovirus type 3 (GyV3) according to claim 3, which is characterized in that: the method comprises the following steps:
(1) real-time fluorescent quantitative PCR reaction: extracting the genome DNA of the object to be detected, performing PCR amplification by using specific primers, wherein the specific primers comprise an upstream primer and a downstream primer,
the reaction system of the PCR amplification is as follows: 20. mu.L of reaction solution per tube, containingGreen Premix Pro Taq HS qPCR Mix 10. mu.L, GyV3 forward 10. mu. mol/. mu.L, GyV3 reverse 0.4. mu.L each, RNase Free ddH2O8.2 mu L,1 mu L of sample DNA template to be detected;
the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles;
after the amplification is finished, making a dissolution curve to obtain an amplification kinetic curve; deducing a standard curve by taking the common logarithm of the initial copy number of the standard substance as an abscissa and taking a cycle number threshold as an ordinate, and obtaining sensitivity test data of the standard curve; T-GyV3 is used as a positive standard plasmid; setting blank as negative control;
(2) and (4) judging a result:
and (3) describing and judging results: no Ct value and no amplification curve, and the sample is judged to be negative; ct value is less than or equal to 36.86, and a typical amplification curve appears, and the sample is judged to be positive.
Technical Field
The invention relates to the fields of molecular biology, immunology and virology, and relates to a novel chicken circovirus type 3 (GyV3) strain and a detection system based on the same.
Background
Circovirus type 3 (Gyrovirus 3, GyV3), the third circovirus member following the Chicken infectious anemia virus (Chicken infectious anaemia virus, CAV or CIAV), is classified in the family dactyloviridae (angioviridae) of the single-stranded Circular DNA virus phylum (Circular replication-associated protein-encoding single-stranded DNA viruses) that encodes replication-related proteins. GyV3 is found by macroviromics identification in acute gastroenteritis diarrhea feces with unknown etiology of chile children at the earliest in 2012, and then is found in diarrhea feces of different ages of human, ferret diarrhea feces and market chicken, and is a pathogen causing infectious proventriculitis in recent years. The infectious proventriculitis brings great harm to the poultry industry, can be generated in laying hens and broilers of different varieties and is wide in popularity, the morbidity is generally 7% -27%, and the mortality is generally 30% -50%. Clinically, emaciation, swollen glandular stomach and excessive stool are the main symptoms. At present, the virus is the second reported single-stranded circular DNA virus, and the first reported single-stranded circular DNA virus is the chicken infectious anemia virus. GyV3 the virus can cause the symptoms of avian immunosuppression, anemia, and adenogastric enlargement, and the pathogenicity experiment later makes it clear that it can infect mice, realizes the horizontal transmission between two groups, and the cross-species transmission phenomenon indicates the potential public health significance, and the harm of GyV3 to human needs to be researched urgently. In view of the harm caused by the virus, a quick and sensitive detection system is urgently needed to be established for early diagnosis, accurately judging the infection state of organisms, timely eliminating sick chickens, effectively protecting uninfected animals and providing guarantee for the healthy development of poultry industry and public health service.
The Real-time fluorescent quantitative PCR method (Real time PCR) is a method for detecting products of Polymerase Chain Reaction (PCR) in each cycle by using fluorescent chemical substances as indicators in DNA amplification reaction. The real-time fluorescence quantitative PCR is to detect the PCR process in real time through a fluorescence signal in the PCR amplification process. However, in the exponential phase of PCR amplification, the Ct value of the template in the sample to be detected and the initial copy number of the template have a linear relationship, so that the method can rapidly and sensitively detect the initial copy number of the virus in the sample to be detected, thereby rapidly detecting the nucleic acid of the sample to be detected.
The indirect Enzyme-linked immunosorbent assay (ELISA) is a detection method for detecting an unknown antibody by using a known antigen in an antigen-antibody binding reaction. The detection method is characterized in that the qualitative and semi-quantitative detection is carried out on the antibody to be detected through the optimized dilution times of the coated antigen. And in the process of reacting the antibody to be detected with the antigen, combining the enzyme-labeled anti-antibody with the antibody to be detected, and judging the level of the antibody of the organism through reading of an enzyme-labeling instrument. GyV3, the antibody level measured by the method is the real antibody level of the organism because no effective vaccine exists in clinic.
In conclusion, a more effective detection method is provided for newly discovered GyV3 virus, so that the infection state of chicken flocks is comprehensively and systematically evaluated, diseased chickens are eliminated in time, uninfected chicken flocks are protected from causing larger loss, and the healthy development of poultry industry is ensured, which becomes an urgent problem to be solved in the field
Disclosure of Invention
The invention provides a novel chicken circovirus type 3 (GyV3) and a detection system based on the same, aiming at the blank of the prior art, the biological preservation number of the virus strain is CCTCC NO: V202061, the nucleotide sequence of the VP1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2, and simultaneously, the invention also provides a detection system consisting of an ELISA antibody detection method of the virus and a novel chicken GyV3 fluorescence quantitative PCR detection method, and the combination of the detection system and the ELISA antibody detection method can realize safe, specific, rapid, sensitive, simple and convenient rapid detection of the GyV3 virus, thereby providing a reference basis for epidemiological investigation and early diagnosis of the novel chicken GyV3 and having important clinical diagnosis value and public health significance.
The specific technical scheme of the invention is as follows:
the inventor firstly provides a novel GyV3 virus strain named as SDAU-2 strain (Gyrovirus 3, SDAU-2) of circovirus type 3, and the inventor performs biological preservation on the strain, wherein the biological preservation number is CCTCC NO: V202061;
the inventor firstly provides a novel GyV3 VP1 protein which is prepared by connecting a novel GyV3 VP1 gene with a prokaryotic expression vector pET32a through enzyme digestion and transforming escherichia coli competent cells BL 21; wherein the nucleotide sequence of the novel chicken circovirus type 3 VP1 gene is shown as SEQ ID NO.1, the amino acid sequence thereof is shown as SEQ ID NO.2, and the VP1 fusion protein is VP190-463A protein.
GyV3 virus belongs to single strand circular DNA virus, which is composed of three open reading frames, namely VP1(1392bp), VP2(720bp) and VP3(378bp), based on that VP1 protein plays a very key role in virus infection and epitope recognition, the invention selects capsid protein VP1 of GyV3 as target protein, in order to realize large-scale expression in colibacillus, analyzes the amino acid sequence, because 65-82 th amino acid is transmembrane region, has no signal peptide sequence, has good hydrophilicity, and has large influence on whole protein expression, therefore, 374 th amino acid in total from 90 th to 463 th amino acid is selected as antigen region to express, so as to prepare VP1 fusion protein.
Further, the inventors provide a detection method for the above virus, specifically as follows:
carrying out induction expression, thallus fragmentation, protein purification, protein renaturation and the like on the obtained novel GyV3 VP1 protein strain to obtain novel GyV3 VP1 protein with epitope; then mixing the novel GyV3 VP1 protein with antigen coating solution to obtain antigen for ELISA coating;
the ELISA antibody detection method of the virus, wherein the protein is the VP1 protein, and the detailed steps and the used reagents are as follows:
1) antigen coating: diluting the VP1 protein to a proper concentration by using an antigen coating solution, adding 100 mu L of the antigen coating solution into a 96-well enzyme label plate, incubating overnight in a refrigerator at 4 ℃, and removing liquid in the wells after the overnight incubation is finished;
2) and (3) sealing: washing with PBST buffer solution for 3-5 times, adding 200 μ L skim milk dissolved in PBST buffer solution 5% into each well, incubating at 37 deg.C for 1h, and removing liquid from the wells;
3) primary antibody incubation: washing with PBST buffer solution for 3-5 times, adding diluted serum to be detected 100 μ L into each well, incubating at 37 deg.C for 1h, and discarding the liquid in the well;
4) and (3) secondary antibody incubation: washing with PBST buffer solution for 3-5 times, adding diluted 100 μ L of goat-anti-chicken labeled with horseradish peroxidase into each well, incubating at 37 deg.C for 1h, and discarding the liquid in the well;
5) color development: washing with PBST buffer solution for 3-5 times in dark place, adding 100 μ L of TMB color development solution into each well, and incubating at 37 deg.C in dark place for 10 min;
6) adding a stop solution: adding 50 mu L of 2M sulfuric acid solution into each hole, and stopping color development;
7) reading: OD450nm values were detected in a microplate reader within 5 min.
The ELISA antibody detection method is the biggest difference from the prior art that the adopted protein is novel chicken GyV3 capsid protein VP1, which fills the blank of the field, and the adopted primary antibody is verified GyV3 positive serum; in addition, the reagents adopted by the invention are cheap and easily available products in the market, and the conditions of all detection parameters are optimal results after comparison, so that the detection time is further shortened, and the detection result is accurate and reliable.
In combination with the above ELISA antibody detection method, the inventors also provide a corresponding fluorescent quantitative PCR detection method, comprising the steps of:
(1) real-time fluorescent quantitative PCR reaction: extracting the genome DNA of the object to be detected, performing PCR amplification by using specific primers, wherein the specific primers comprise an upstream primer and a downstream primer,
the corresponding primer sequences are as follows:
an upstream primer GyV3 Forward: 5'-ACCGGGACTTGGACACC-3', the nucleotide sequence of which is shown in SEQ ID NO. 3;
downstream primer GyV3 Reverse: 5'-AGCCAGGAAGCGATACG-3', the nucleotide sequence of which is shown in SEQ ID NO. 4;
the reaction system of the PCR amplification is as follows: 20. mu.L of reaction solution per tube, containingGreen Premix Pro Taq HS qPCR Mix 10. mu.L, GyV3 forward 10. mu. mol/. mu.L, GyV3 reverse 0.4. mu.L each, RNase Free ddH2O8.2 mu L,1 mu L of sample DNA template to be detected;
the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles;
after the amplification is finished, making a dissolution curve to obtain an amplification kinetic curve; and (3) deriving a standard linear regression equation (standard curve) Y-2.8546X +36.82 (Y: Ct value, X: template initial copy number) and a regression coefficient R by taking the common logarithm (lgC) of the initial copy number of the standard as an abscissa and a cycle threshold (Ct value) as an ordinate2Obtaining sensitivity test data of the strain as 0.99; by T-GyV3Is a positive standard plasmid; setting blank as negative control;
(2) and (4) judging a result:
quality control standard: negative controls had no Ct values and no amplification curves; the Ct value of the positive control is less than or equal to 28.86, and a specific amplification curve appears; if the negative control and the positive condition do not satisfy the above conditions, the experiment is regarded as invalid;
and (3) describing and judging results: no Ct value and no amplification curve, and the sample is judged to be negative; ct value is less than or equal to 36.86, and a typical amplification curve appears, and the sample is judged to be positive.
The fluorescence quantitative method provided by the method is a faster, accurate and sensitive method after conditions are optimized, and adoptsGreen Premix Pro Taq HS qPCR Mix can quickly and accurately judge 96 samples.
In conclusion, the invention further provides an ELISA detection method and a fluorescent quantitative PCR detection method for antibody detection on the basis of obtaining a novel chicken GyV3 virus strain, and the specific application steps are as follows:
collecting chicken cloaca cotton swabs and serum, and using fluorescent quantitative PCR detection to accurately judge chicken infection GyV3 and virus detoxification conditions; the situation of the antibody in the chicken can be accurately judged GyV3 by collecting the chicken serum;
the results of the two are combined to judge the dynamic situation of the antigen and the antibody of the chicken flocks, thereby accurately judging the infection state of the chicken flocks. The inventor of the invention combines the two methods for the first time to form a detection system, which can quickly and accurately judge the state, the detoxification rule and the epidemiology of the circovirus type 3 infected chicken flock, can provide reference basis for the early diagnosis, prevention and control of the novel GyV3 chicken, has important clinical diagnosis significance, fills the blank at home and abroad, reduces the loss of poultry industry to the maximum extent and ensures the healthy development of the poultry industry.
Preservation information
Preservation time: 10 and 1 month in 2020
The name of the depository: china center for type culture Collection
The preservation number is: CCTCC NO: V202061
The address of the depository: wuhan university of Wuhan, China
Classification nomenclature of circovirus type 3 SDAU-2 strain (Gyrovir 3 SDAU-2)
Drawings
FIG. 1 is a transmission electron micrograph of the new circovirus type 3 of example 1,
FIG. 2 is the electrophoresis chart of the VP1 gene of the new circovirus type 3 in example 2:
in the figure, M is DL2000 Marker, and lanes 1-3 are electrophoresis pictures of the novel circovirus type 3 VP1 gene;
FIG. 3 shows the restriction identification electrophoretogram of PET32a-VP1 in example 2:
in the figure, M is DL2000 Marker, lane 1 is a novel circovirus type 3 VP1 gene and empty vector electrophoretogram, and two bands are obvious at 1125bp (target fragment) and 2700bp (empty load);
FIG. 4 is a SDS-PAGE result of the VP1 protein of the new circovirus type 3 in example 2:
in the figure, M is 170kDa Marker, and a lane 1 is unpurified novel circovirus type 3 VP1 protein;
FIG. 5 is a SDS-PAGE confirmation of the VP1 protein purified in example 2:
in the figure, M is 170kDa Marker, and FIG. 5A is anti-His tag antibody for verifying VP1 protein, wherein lane 1 is unpurified prokaryotic expression VP1, lane 2 is purified protein column effluent, lane 3 is purified protein washing solution, and lane 4 is purified VP1 protein;
FIG. 5B shows the effect of anti-VP 1 polyclonal antibody on VP1 renaturation, wherein lane 1 shows the non-purified prokaryotic expression of VP1, and lane 2 shows the purified VP1 protein;
FIG. 6 is a Western blot identification chart of the VP1 protein in example 2:
in the figure, M is 170kDa Marker, lane 6A:1 is unpurified novel circovirus type 3 VP1 protein, lane 2 is purified protein column effluent, lane 3 is purified protein washing solution, lane 4 is purified VP1 protein;
FIG. 6B, lane 1, is unpurified novel circovirus type 3 VP 1; lane 2 is the novel circovirus type 3 VP1 protein after renaturation;
FIG. 7 shows the determination of ELISA cut-off values in example 3;
FIG. 8 shows ELISA sensitivity profiles in example 3;
FIG. 9 is a specific search of ELISA in example 3;
FIG. 10 is a chart of the epidemiological survey in example 3;
FIG. 11 is a graph showing a combination of a melting curve, an amplification curve and a melting curve in the quantitative fluorescence PCR in example 4.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are intended to illustrate the invention and not to limit it. In this embodiment, except for the special description, the others are all completed by using the prior art.
Example 1 VP190-463Construction of prokaryotic expression vector
1. Identification of circovirus type 3
The glandular stomach tissue of the circovirus type 3 regressed animal chicken is subjected to tissue grinding treatment, the glandular stomach tissue grinding fluid is subjected to differential centrifugation after ultrasonic crushing, and the virus is purified by adopting a sucrose density gradient centrifugation method after the supernatant is collected. And the size and the shape of the virus particles are observed by an electron transmission electron microscope in combination with a tungsten phosphate negative staining method. It was observed that the virions had a circular or icosahedral symmetric structure, no envelope, and a particle size of about 26-28nm in diameter (as shown in FIG. 1).
2. Cloning of the fragment of interest
The inventors designed primers as follows:
F-CGAGCTCGACACAACTAAAGGCAA, the nucleotide sequence of which is shown in SEQ ID NO. 5;
R-TTGCGGCCGCCTAGTCTGCGGGGACGC, the nucleotide sequence of which is shown in SEQ ID NO. 6;
weighing 7mg glandular stomach tissue of determined novel GyV3 infected chicken, extracting tissue DNA, amplifying target band according to the above primer,
the reaction system is as follows: 10.5 μ L of water, 12.5 μ L of dNTP mix, and 1 μ L of template of 0.5 μ L, DNA for each of the upstream and downstream primers;
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 40s, and 35 cycles; further extension for 10min at 72 ℃; the obtained PCR product was subjected to gel electrophoresis to obtain a single desired band of 1125bp in size (see FIG. 2).
3. The construction of the novel GyV3-VP1 protein prokaryotic expression vector comprises the following steps:
3.1 obtaining GyV3-VP1 gene according to the whole genome of the novel circovirus type 3 strain, selecting amino acids from 90 th to 463 th positions for expression through transmembrane region analysis, signal peptide analysis, hydrophobicity analysis and antigenicity analysis, and selecting Sac I and Not I as upstream and downstream enzyme cutting sites respectively for whole gene synthesis.
3.2 Synthesis of GyV3 VP11125The gene is double digested by Sac I and Not I and then connected with the corresponding restriction enzyme site of a pMD 18-T Vector, transformed into a competent cell DH5 alpha, plasmids are extracted, the extracted plasmids and a prokaryotic expression Vector pET32a (+) are double digested by Sac I and Not I enzymes, and the double digested fragment VP1 obtained in the last step1125Connecting with pET32a (+), transforming into competent cell BL21(DE3), extracting plasmid, sending to Huahua big gene for sequencing, and obtaining the sequencing result and GyV3 VP11125The gene comparison result shows complete matching, and the recombinant plasmid is successfully constructed.
The prokaryotic expression vector constructed above is transformed into a competent cell BL21, and the transformation steps are as follows:
1) thawing competent cells BL 21100. mu.L in ice bath, adding 10. mu.L of the connected recombinant plasmid, and gently mixing;
reacting for 30min in ice bath; after 90s of water bath at 42 ℃ (time is strictly controlled), the mixture is quickly transferred to an ice bath for 2min, and shaking is strictly prohibited during the period;
2) adding 400 μ L LB medium (without ampicillin) and culturing at 37 deg.C and 200rpm/min in constant temperature shaking table for 1 h; spreading 200 μ L of the suspension on LB solid culture plate containing AMP, and culturing overnight at 37 deg.C;
3) picking single colony to be placed in a test tube containing 10mL LB liquid culture medium with 100ng/L AMP to be cultured for 6h at 37 ℃ and 200 rpm/min;
4) 200 mu L of bacterial liquid is taken and sent to Huada gene for sequencing, and the sequencing result shows that the recombinant plasmid is successfully constructed.
4. In vitro expression and identification of recombinant plasmids
Activating the correctly sequenced bacterial liquid, shaking the bacterial liquid, adding 1.0mmol/L IPTG to induce expression when the bacterial liquid is in a logarithmic growth phase, collecting 1mL of bacterial liquid 4h after induction, carrying out ultrasonic crushing, carrying out SDS-PAGE gel electrophoresis detection on the inclusion body, and displaying that the recombinant protein is expressed in a large amount in the inclusion body, thereby proving that the constructed recombinant plasmid can correctly express the circovirus type 3 VP1 protein.
The in vitro expression comprises the following specific steps:
adding 1000 μ L LB culture medium containing ampicillin into the tube, shaking at 37 deg.C for 2.5h, then 6000rpm, centrifuging for 10min, discarding 800 μ L, and placing the rest 200 μ L coated plate in 37 deg.C constant temperature incubator overnight for culture;
picking single colony, shaking the bacteria for 8h, sending to Huada sequencing, and measuring VP190-463The nucleotide sequence is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2; and (4) carrying out double enzyme digestion identification on the plasmid with correct sequencing. The digested lane 1 shows pET-32a (+) no-load band and 1125bp target band VP190-463As a result, the recombinant plasmid was successfully constructed (shown in FIG. 3), and the recombinant plasmid was transformed into BL21 competent cells and expressed as described above.
Example 2 expression, purification and renaturation of VP1
1. Inducible expression
Selecting and transforming into a novel plasmid pET-32a-VP190-463BL21 competent cells were cultured overnight in a 37 ℃ incubator. And (3) on the second day, mixing the bacterial liquid according to the proportion of 1: inoculating 50% of the strain into a 2L conical flask, culturing in a shaker at 37 deg.C to OD 600nm of about 0.6, taking out 10mL of non-induced bacterial liquid as control, adding IPTG (final concentration of 1mmol/L) for induction expression, culturing for 5h in the shaker, then 6000rpm, and centrifuging for 10min to collect the thallus. Adding 40ml PBS to resuspend the thallus, resuspending for 3 times to remove residual culture medium and impurities, adding 1% PMSF, ultrasonic crushing in ice bath, crushing for 5S, cooling for 5S, circulating at 99 × 5, centrifuging at 12000rpm for 10min, collecting inclusionThe supernatant was discarded from the pellet.
2. VP1 protein purification and renaturation
1) The precipitate was resuspended in 9-fold volume of Wash I (500mmol/L Tris-Cl (pH 8.0), 100mmol/L NaCl, 100mmol/L EDTA), and after standing for 15min, 12000rpm was applied, and the supernatant was discarded after 15 min.
2) Resuspend the precipitate with 9 times the volume of Wash II (2M urea was added to Wash I), let stand for 15min, 12000rpm, 15min, and discard the supernatant.
3) Resuspend the precipitate with 9 times the volume of Wash III (4M urea was added to Wash I), let stand for 5min, 12000rpm, 15min, and discard the supernatant.
4) 20mL LE Buffer (8M urea was added to washing solution I) was added to the pellet, stirred and dissolved (about 7.5mL LE Buffer/mg inclusion body), centrifuged at 13400 Xg 4 ℃ for 30min, the pellet was removed, and the supernatant was collected as the dissolved inclusion body.
5) Performing protein purification by using a Ni-NTA affinity chromatography medium, adding an unpurified protein sample into Ni-NTA resin which is well balanced by using a balance buffer solution, slowly flowing out the sample, controlling the flow rate to be 0.5-1mL/min, and repeatedly or circularly loading for 3-4 times; washing the filler by using LE Buffer, and repeating for 3-5 times; washing the heteroprotein on the column with 2 column volumes of washing solution; eluting with as little eluent as possible, wherein the collected eluent is the purified protein. As a result, as shown in FIGS. 4 and 5, it was found that the number of bands before purification was large and only one band appeared after purification, indicating that the purification effect was good.
6) The supernatant is put into a dialysis bag and is sequentially put into a gradient solution containing 6mol,4mol, 2mol,1mol and 0mol of urea for overnight dialysis at 4 ℃ to renature the protein.
7) And detecting the purity of the purified target protein by an SDS-PAGE method. After expression and purification, a single target protein with the size of 57kD can be obtained, namely the target protein VP190-463A protein.
The antibody of anti-His label is used as a primary antibody and the polyclonal antibody is further identified by using Western blot, a mouse anti-His label monoclonal antibody (pET32a vector with His label) and rabbit anti-VP 1 protein are used as primary antibodies, and a positive signal appears at a position of 57kDa through detection, and the result shows that the GyV3-VP1 protein can be specifically combined with the rabbit anti-VP 1 protein polyclonal antibody (as shown in figure 6), which indicates that the renaturation effect is good, and the antigenic determinant is obviously exposed. The concentration of VP1 protein was 0.576mg/mL as determined spectrophotometrically.
Example 3 ELISA antibody detection method of novel Chicken circovirus type 3 (GyV3)
The ELISA antibody detection method, wherein the protein used is VP1 protein after renaturation (namely the ELISA antigen used), comprises the following specific steps:
1) antigen coating: diluting the VP1 protein to a proper concentration by using an antigen coating solution, adding 100 mu L of the antigen coating solution into a 96-well enzyme label plate, incubating overnight in a refrigerator at 4 ℃, and removing liquid in the wells after the overnight incubation is finished;
2) and (3) sealing: washing with PBST buffer solution for 3-5 times, adding 200 μ L skim milk dissolved in PBST buffer solution 5% into each well, incubating at 37 deg.C for 1h, and removing liquid from the wells;
3) primary antibody incubation: washing with PBST buffer solution for 3-5 times, adding diluted chicken GyV3 positive serum 100 μ L into each well, incubating at 37 deg.C for 1h, and discarding the liquid in the well;
4) and (3) secondary antibody incubation: washing with PBST buffer solution for 3-5 times, adding diluted 100 μ L of goat-anti-chicken labeled with horseradish peroxidase into each well, incubating at 37 deg.C for 1h, and discarding the liquid in the well;
5) color development: washing with PBST buffer solution for 3-5 times in dark place, adding 100 μ L of TMB color development solution into each well, and incubating at 37 deg.C in dark place for 10 min;
6) adding a stop solution: adding 50 mu L of 2M sulfuric acid solution into each hole, and stopping color development;
7) reading: OD450nm values were detected in a microplate reader within 5 min.
In the method, the specific parameter searching and determining process is as follows:
1. antigen-antibody dilution multiple groping
Diluting the antigen (VP1 protein) and antibody prepared after renaturation respectively in a multiple ratio, diluting the antigen with carbonate buffer (determining the dilution range according to the protein concentration), coating an ELISA plate, 100 mu L of each well, incubating overnight in a refrigerator at 4 ℃, discarding the coating solution, washing with 300 mu L of PBST buffer solution for 3 times, incubating at 37 ℃ for 2h, discarding, washing with 300 mu L of PBST buffer solution for 3 times, diluting the antibody with PBST buffer solution (range 1:100-1:12800), adding 100 mu L of antibody into each well, performing ELISA detection, incubating at 37 ℃ for 1.5h, washing with 300 mu L of PBST after incubation, washing with 3 times, adding 100 mu L of PBST diluted goat anti-chicken with horseradish peroxidase, incubating at 37 ℃ for 1h, washing with 300 mu L of PBST after incubation, washing for 3 times, adding 100 mu L of TMB substrate into each well, and (3) performing color development in an incubator at 37 ℃ in a dark place for 30min, adding 50 mu L of 2mol/L stop solution into each well to terminate the reaction, reading an OD450nm value on an enzyme linked detector within 5min, calculating a P/N value, and determining the optimal dilution times of the antigen and the antibody according to the P/N value. The results show that: the optimal dilution factor for the antibody was 1:640, and the optimal coating concentration for the antigen was 0.89. mu.g/mL (as shown in Table 1).
TABLE 1 search for optimal dilution factor of antigen antibody
2. Selection of optimal coating solution
Using the following six solutions as coating solutions respectively, and diluting the antigen to the optimal concentration for detecting CBS, wherein the concentration of the antigen is 0.1mol/L and the pH value is 9.2 carbonate buffer solution; TBST 20mmol/L Tris-HCl with pH 8.4; PBS 0.01mol/L pH7.2-7.4 phosphate buffer solution; PBST, pH7.4 containing 0.05% Tween 20 phosphate buffer; w is distilled water; s, physiological saline, each coating liquid is 3 technically repeated. And respectively calculating the P-mean value, the N-mean value and the P/N value, and determining the optimal coating liquid according to the maximum P/N value. The results show that: PBS was the best protein coating (as shown in table 2).
TABLE 2 best coating solution grope
3. Optimum confining liquid concentration groping
Diluting the antigen to an optimal concentration by using an optimal coating solution, preparing 8 skimmed milk with different gradient concentrations such as 0.5% -7% from the most common and most easily available skimmed milk powder in a laboratory by using a PBST buffer solution, repeating 3 technical steps for each gradient, and determining the concentration of the optimal blocking solution to the maximum according to a P/N value. As a result: the most preferred confining liquid is skim milk at 5% (as shown in table 3).
TABLE 3 optimum blocking concentration groping
4. Best closing time groping
And (3) sealing the ELISA plate by using the optimal sealing liquid concentration obtained by the experiment, respectively detecting the sealing time of 5 gradients such as 0.5h, 1h, 1.5h, 2h and 2.5h, repeating the technical process of 3 gradients, and determining the optimal sealing time to the maximum according to the P/N value. As a result: the optimal blocking time was 1h at 37 deg.C (as shown in Table 4).
TABLE 4 best seal time groping
5. The optimal action time of antiserum is searched
After the positive serum antibody and the negative control serum are diluted by the optimal multiple, 100 mu L of the positive serum antibody and the negative control serum antibody are added into each hole and divided into 4 groups, each group is respectively incubated for different time of 0.5h, 1h, 1.5h and 2h, the P/N value is respectively calculated, and the optimal action time of the antiserum is determined according to the maximum P/N value. As a result: the optimal action time of the chicken anti-novel GyV3 serum is 1h at 37 ℃ (shown in Table 5).
TABLE 5 time of best primary antibody
6. Second antibody optimal dilution groping
The secondary antibody is a goat anti-chicken secondary antibody (HRP marked) of Beijing Boaosen, the antibody is respectively diluted by 5 gradients of 1:5000, 1:10000, 1:20000, 1:40000, 1:80000 and the like, detection is respectively carried out, each gradient is repeated by 3 technical means, the P/N value is respectively calculated, and the optimal dilution multiple of the secondary antibody is determined by the maximum P/N value. As a result: the optimal dilution factor for the HRP-labeled goat anti-chicken antibody was 1:5000 (as shown in table 6).
TABLE 6 optimal Secondary antibody concentration
7. Second antibody action time exploration
After the goat anti-chicken secondary antibody (marked by HRP) is diluted by the optimal multiple, 4 gradients of 0.5h, 1h, 1.5h, 2h and the like are set for the secondary antibody incubation time, the secondary antibody is respectively acted on an ELISA plate, each gradient is repeated by 3 technical means, the P/N value is calculated, and the optimal action time of the secondary antibody is determined at the maximum P/N value. As a result: the optimal action time of the secondary antibody is 1h at 37 ℃ (as shown in Table 7).
TABLE 7 optimal secondary antibody action time
8. Best color development time groping
Setting 4 groups of TMB single-component substrate color developing solution for 10min, 20min, 30min, 40min and the like for detection respectively, repeating 3 technical methods for each gradient, calculating the P/N value of each gradient, and determining the optimal acting time of the substrate color developing solution to the maximum by the P/N value. As a result: the optimal development time of the TMB substrate was 10min at 37 deg.C (as shown in Table 8).
TABLE 8 best color development time groping
9. Determination of a threshold value
42 SPF chicken sera were randomly drawn and tested by the optimized ELISA method, and the mean value (x) and standard deviation SD of the mean OD450nm of the 42 sera were calculated, with the positive and negative cut-off value being x +3 SD. When the detected sample OD450nm value is more than or equal to x +3SD, the sample can be judged to be positive at the level of 99.9%. The results show that: the optimized ELISA negative-positive cutoff value was 0.184 (as shown in figure 7).
10. Sensitivity detection by indirect ELISA method
The GyV3 positive serum and GyV3 negative serum which are verified before are respectively diluted by 1:100-25600 times, the sensitivity of the serum is determined by an optimized ELISA method, and the ELISA sensitivity is determined by the dilution which is more than a critical value. As a result: the ELISA sensitivity was 1:3200 (as shown in FIG. 8).
11. Specificity detection by Indirect ELISA method
ALV-J, REV, MDV, NDV, CAV and GyV3 positive sera were assayed according to the optimized ELISA protocol, 3 technical replicates per serum, and specificity assays were performed. As a result: GyV3-VP1 ELISA did not bind to other viruses, but only specifically bound to GyV3, indicating that the ELISA specificity was good (as shown in FIG. 9).
12. In-plate reproducibility detection of indirect ELISA method
Coating 1 quick ELISA plate with VP1 protein prepared from the same batch, detecting 3 parts of GyV3 positive serum sample and 3 parts of non-infected GyV3 SPF chicken serum sample in the same ELISA plate, repeating 6 wells for each serum sample, calculating the average value, standard deviation and Coefficient of Variation (CV)), and obtaining the CV (standard deviation/average value). The results show that: the reproducibility maximum was 9.41% < 15%, indicating that the ELISA has good in-plate reproducibility (as shown in table 9).
TABLE 9 in-plate repeatability test
13. Indirect ELISA method for plate-to-plate repeatability detection
Coating 6 enzyme label plates with the prepared VP1 protein of the same batch, detecting 3 parts of GyV3 positive serum samples and 3 parts of non-infected GyV3 SPF chicken serum samples in the 6 enzyme label plates respectively under the same condition, repeating 6 holes for each serum sample, and calculating the average value, standard deviation and Coefficient of Variation (CV) of each serum sample, wherein CV is the standard deviation/average value. As a result: the maximum reproducibility was 10.19% < 15%, indicating that the ELISA had good reproducibility between plates (as in table 10).
TABLE 10 repeatability test between plates
14. Epidemiological investigation
Serum samples from 7 cities collected from Shandong were tested by optimized ELISA and subjected to GyV3 epidemiological investigation. As a result: antibody levels reached a maximum at GyV3 day 5 of infection of the chicken, followed by a gradual decline in titer and finally a plateau (as shown in figure 10).
After the ELISA conditions are optimized, reagents used in experiments are cheaper and easily obtained, the required time is further shortened, the infection state of chicken flocks can be efficiently and accurately determined, accurate judgment can be made under clinical simple conditions after the ELISA kit is popularized to breeding companies, and the ELISA kit is beneficial to prevention and control of novel chicken GyV3 and limitation of propagation of the novel chicken GyV 3.
EXAMPLE 4 establishment of fluorescent quantitative PCR detection method of 4 GyV3
The corresponding primer sequences are as follows:
an upstream primer GyV3 Forward: 5'-ACCGGGACTTGGACACC-3', the nucleotide sequence of which is shown in SEQ ID NO. 3;
downstream primer GyV3 Reverse: 5'-AGCCAGGAAGCGATACG-3', the nucleotide sequence of which is shown in SEQ ID NO. 4;
the reaction system of the PCR amplification is as follows: 20. mu.L of reaction solution per tube, containingGreen Premix Pro Taq HS qPCR Mix 10. mu.L, 10. mu. mol/. mu.L GyV3 Forward, GyV3 Reverse each 0.4. mu.L, RNase Free ddH2O8.2 mu L,1 mu L of sample DNA template to be detected;
the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles;
selecting DNA sample identified as positive by GyV3 identified primer as template, carrying out PCR amplification,
the amplification product was identified by agarose gel (1.0%) electrophoresis, and purified and recovered using agarose gel DNA recovery kit (Tiangen). The purified DNA product was ligated into pMD 18-T Vector to construct a novel plasmid, which was ligated for 50min at 16 ℃. Constructing a novel plasmid, diluting the plasmid connected with the GyV 3-mesh segment by 10 times, performing fluorescent quantitative PCR amplification to generate a fluorescent quantitative amplification curve and a dissolution curve, and constructing a fluorescent quantitative standard curve; and (3) deriving a standard linear regression equation (standard curve) Y-2.8546X +36.82 (Y: Ct value, X: template initial copy number) and a regression coefficient R by taking the common logarithm (lgC) of the initial copy number of the standard as an abscissa and a cycle threshold (Ct value) as an ordinate2Obtaining sensitivity test data of the strain as 0.99; T-GyV3 is used as a positive standard plasmid; blank as negative control (as shown in fig. 11);
(2) and (4) judging a result:
quality control standard: negative controls had no Ct values and no amplification curves; the Ct value of the positive control is less than or equal to 28.86, and a specific amplification curve appears; if the negative control and the positive condition do not satisfy the above conditions, the experiment is regarded as invalid;
and (3) describing and judging results: no Ct value and no amplification curve, and the sample is judged to be negative; ct value is less than or equal to 36.86, and a typical amplification curve appears, and the sample is judged to be positive.
(3) Specific detection examples are as follows:
236 parts of national serum and cloaca cotton swabs are collected in 2020, and the corresponding detection is carried out by using fluorescent quantitative PCR and indirect ELISA. The results show that: the fluorescence quantitative positive rates of the serum and cloaca cotton swab are 5.5% and 5.08%, the ELISA antibody positive rate is 5.93%, and the comparison of the results of the two is almost the same, which indicates that the detection system has good detection effect (shown in Table 11).
TABLE 11 test results of the test system
Sequence listing
<110> Shandong university of agriculture
<120> novel chicken circovirus type 3 strain and detection system based on virus
<150> 2021104703754
<151> 2021-04-29
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ggctgctggc catacagtac acaaatgtca tcatctagac agccaggggc atggccttca 240
gaatggtggc gatgggcact tcttcttatg catcctagat ccaatgtacg attcttcgga 300
tccccgaaac tgatgaccct accacaaata ggacagttcc tggggggctg gcaactattc 360
acccacagat tcacaaaatt ccgtgtgctt gcaactaaga gcagagaatc gttctccccg 420
gtcgcgagcc tgcttgtaca agacaattac tttgcaagaa gagagggtgc agggccacca 480
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gaaagcaatg ctccagctaa tgaaaccaca ataccatcca tgccaccaga cccaccccaa 600
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tccttcaaca aacatgtcat tagaggcatg ggtgacccac aaggcaaaaa atggctcacc 840
ctggtaccga aagaacaaga atggatcaat tcggactcaa tgacaaagtc agaactggac 900
acggacatag ctacattgta cctagctcaa ggaacaagca gagcaaacag ctacaaattc 960
aacacattcc acgaggtaat ggtacaagac cccatgaatg tagccccctg ggcagtcgtc 1020
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