CN114989306B - Pig pseudorabies virus gE and gI nano antibody, preparation method and application - Google Patents
Pig pseudorabies virus gE and gI nano antibody, preparation method and application Download PDFInfo
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Abstract
The invention provides a pig pseudorabies virus gE and gI nano antibody, a preparation method and application, wherein the nano antibody targets a PRV gE protein and gI protein preferential epitope fusion protein. The contrast test proves that the nanometer antibody PRV-nanometer is more sensitive than the commercial monoclonal antibody in PRV detection, the nanometer antibody PRV-nanometer is combined with the colloidal gold labeling technology, the sensitivity is high, and the detection limit of detecting PRV can reach 10ng/mL. The specificity is strong, and the cross reaction is less. The first developed colloidal gold test strip realizes the rapid detection, shortens the original two-day immune identification test to the rapid detection completed in a few minutes, and realizes the primary application of the rapid detection of PRV.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a specificity detection method of porcine pseudorabies virus (Porcine Pseudoraibies Virus, PRV), an active unit of which is a nano antibody targeting gE and gI proteins of porcine pseudorabies virus (Porcine Pseudoraibies Virus, PRV), a preparation method and application.
Background
Porcine pseudorabies (Porcine Pseudoraibies, PR) is an acute infectious disease of pigs, also known as Aujeszky disease, caused by porcine pseudorabies virus (Porcine Pseudoraibies Virus, PRV). Pigs as the natural host of PRV are more susceptible to viral infection and are the only animal species that survive acute infections and have latent infectivity. After infection, newborn piglets are characterized by high mortality and nervous system disorder, pregnant sows have abortion and reproductive disorders, elderly pigs are mainly characterized by respiratory diseases, the mortality of piglets characterized by nervous symptoms is more than 20%, the probability of infection of pigs with wild viruses is as high as 50%, and the pig industry loss in China is huge.
PRV belongs to a member of the Herpesviridae family (Herpesviridae), the alpha-Herpesviridae subfamily (Alpha Herpesvirinae), and has a genome of linear double-stranded DNA of about 150kb in size. PRV is currently known to have only one serotype, and PRV encodes more than 70 proteins altogether, with the main protein being the envelope structural protein except the gG protein, and the remaining 10 (gB, gC, gD, gE, gH, gI, gK, gL, gM, gN). gB. The gH, gL and gM proteins are relatively conserved in the herpesviridae family, and gB, gH and gL are essential for replication of all herpesviruses in cell culture. The gB protein is capable of inducing the production of neutralizing antibodies, associated with immune protection. The gE protein was originally called the gI protein. gE gene deletion exists in PRV vaccine strains such as Bartha strain, BUK strain and the like screened and prepared in 1960. Constructing a gE gene deleted PRV mutant strain by a DNA recombination technology in the middle 80 th century; on this basis, deletion of the TK gene further attenuated the PRV virus. Many gE gene deleted vaccines are capable of preventing or alleviating clinical symptoms caused by a challenge infection, allowing swine herds to use gE gene deleted vaccines in many countries.
Pseudorabies can be controlled by vaccination, so it becomes very important to distinguish between immunized and infected animals. Detection of vaccine immune antibodies and naturally infected antibodies can be achieved by vaccination with a gene-deleted vaccine. The differential diagnosis method is a diagnosis method which is applied on the basis of using a gene deletion vaccine. DNA homologous recombination defective, gene deleted and naturally defective vaccines can be used to identify immune antibodies from naturally infected antibodies due to the lack of specific glycoproteins (gG, gE or gC). Because of the presence of multiple nonessential glycoprotein genes in PRV, virus mutants lacking these genes are unable to produce glycoproteins encoded by the deleted genes, but do not affect the proliferation and immunogenicity of the virus on cells. After injecting such a gene-deleted vaccine into an animal, the animal cannot produce antibodies against the deleted proteins. Therefore, the serological positive pigs infected with the wild toxin naturally can be identified from the injected pigs by a serological method.
The deleted glycoprotein genes of the deletion vaccine are mainly gE and gI genes, when the immune antibody level of a farm immunized by using the gE and gI gene double-deletion vaccine is detected, the wild virus identification kit is matched for screening, the source of positive antibodies is determined, immune antibodies and natural infection antibodies are identified and distinguished, recessive infection pigs are screened out, wild virus positive pigs are eliminated in time, a healthy negative pig group is established, but most of the common commercial identification kits on the market are gE single-gene deletion ELISA kits, and the clinical detection requirement cannot be met. Since the gE protein antibody is currently generally considered as the most important infection index among various specific glycoproteins, but there are individual differences in the time of production and response level of the gE antibody, and the gE antibody or the time delay of production of the gE antibody cannot be detected in an infected pig organism, the detection of porcine pseudorabies virus by a kit coated with gE protein alone may result in false negative and low accuracy. Also, detection of a single protein antibody is inaccurate because different protein antibodies will appear at different times after infection of the animal. Therefore, for diagnosing the porcine pseudorabies, at least more than one protein antibody should be detected to improve the detection accuracy. For those animals that have been immunized and infected, replication of the virus is slow due to neutralizing antibodies, resulting in very low concentrations of specific proteins in the body, but commercial ELISA kits are less sensitive, making these low concentrations of specific proteins difficult to detect, resulting in missed detection of partially infected animals.
The isolation and identification of virus is the gold standard for detecting PRV in terms of sensitivity and specificity, but the time spent for separation (at least 2-3 days) is low in sensitivity and requires a high level of cell culture, and the virus culture process is also prone to detailed misoperation to finally influence the diagnosis result, so that the virus is difficult to popularize in basic veterinary units. Virus neutralization assay (VNT) is one of the methods commonly used in PRV detection, which is efficient and sensitive. However, this method is less sensitive in the acute phase of infection and tends to make the result false negative. The indirect sandwich ELISA and other immunological diagnosis methods have high sensitivity and strong specificity, but require more pure protein, require fine operation and are easy to generate false positives, and particularly depend on detection antibodies with high sensitivity and specificity, so that the property of the detection antibodies directly determines the accuracy and precision of detection. Although these methods can detect PRV virus and achieve a certain effect in practice, they have the disadvantages of complicated experimental operation, long time consumption, need of detection reagents and related instruments matched with each other, and are limited to laboratory operation, and are difficult to popularize. Therefore, the rapid and simple PRV virus detection method is developed and has important significance for real-time monitoring of virus proliferation.
Disclosure of Invention
In view of the above-mentioned shortcomings in the prior art, the present invention aims to provide a rapid specific detection method for porcine pseudorabies virus (Porcine Pseudoraibies Virus, PRV) to solve the above-mentioned problems of the prior art.
In order to achieve the above object, the present invention provides a nanobody capable of recognizing porcine pseudorabies virus with high accuracy and sensitivity, wherein the nanobody targets a preferred epitope fusion protein of gE protein and gI protein of PRV, which is named PRV-NANOEI, and the amino acid sequence thereof is as follows: QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQYAVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSSAAAY (SEQ ID NO. 1).
Furthermore, the invention provides a rapid detection test strip for the porcine pseudorabies virus, which adopts a combination mode of a nano antibody PRV-NANOEI and a colloidal gold test strip to realize rapid detection for the porcine pseudorabies virus, so that the rapid detection is realized, the original two-day immune identification test is shortened to be completed in a few minutes, and the preliminary application of the nano antibody PRV-NANOEI in the rapid detection is realized.
Furthermore, the rapid detection test strip for the porcine pseudorabies virus provided by the invention adopts a nitrocellulose membrane (CN 95) as a chromatographic material, adopts a glass cellulose membrane as a sample pad, and is assembled with a PVC bottom plate, absorbent paper and the like to form the test strip.
Further, the rapid detection test strip comprises a PVC bottom plate, water absorption pad paper, a glass cellulose membrane, a gold pad and a sample chromatographic pad, wherein the water absorption pad paper, the glass cellulose membrane, the gold pad and the sample chromatographic pad are sequentially adhered to the PVC bottom plate from top to bottom, and a colloidal gold-labeled nano antibody PRV-nano I is arranged on the gold pad;
the glass cellulose membrane is provided with a detection area and a quality control area which are mutually separated, the detection area is sprayed with PRV coating antigen, and the quality control area is sprayed with an antibody which is specifically combined with a colloidal gold labeled nano antibody PRV-nano.
Furthermore, the invention provides a preparation method of the quick test paper strip for detecting porcine pseudorabies virus,
1) Purifying nanometer antibody PRV-NANOEI, and dialyzing for use;
2) Colloidal gold labeled nanobody PRV-nanoEI;
3) BSA blocking reaction;
4) After centrifugation, the precipitate was washed with 1% BSA in PBS;
5) Spraying a gold mark pad after re-dissolution;
6) Coating a glass cellulose film;
7) Drying at room temperature and low humidity; sequentially adhering water absorbing paper, a glass cellulose film, a gold pad and a sample chromatographic pad from top to bottom on a bottom plate, and cutting for later use;
8) And (3) performing application test on the colloidal gold test strip to obtain the detection limit of the colloidal gold test strip at the level of 10ng/mL, and meeting the requirement of rapidly detecting PRV.
Advantageous effects
The invention provides a nanometer antibody PRV-NANOEI for specifically recognizing PRV, and discloses an amino acid sequence of the nanometer antibody. The nano antibody can specifically identify PRV, can be used as a material for immunological detection, and has a very wide application prospect.
The rapid detection test strip for the porcine pseudorabies virus has the advantages that: the rapid detection test strip can be used for determining the source of antigen, distinguishing immune samples from natural infection samples, judging whether the detected animal is infected with virus or vaccinated, has good sensitivity, specificity and stability, and is particularly suitable for the detection of low-concentration samples and the early diagnosis of epidemic diseases.
The contrast test proves that the nanometer antibody PRV-nanometer is more sensitive than the commercial monoclonal antibody in PRV detection, the nanometer antibody PRV-nanometer is combined with the colloidal gold labeling technology, the sensitivity is high, and the detection limit of PRV detection can reach 1ng/mL. The test strip disclosed by the invention has strong specificity and less cross reaction, and is negative to cross reactions of PRRSV antibody positive serum, TGEV antibody positive serum, PEDV antibody positive serum, PPV antibody positive serum and SFV antibody positive serum of porcine epidemic diarrhea virus, and NDV (newcastle disease virus) positive serum, CDV (canine distemper virus) positive serum and HEV hepatitis virus positive serum of rabbit. The first developed colloidal gold test strip realizes the rapid detection, shortens the original two-day immune identification test to the rapid detection completed in a few minutes, and realizes the primary application of the rapid detection of PRV.
Drawings
FIG. 1 is a 3D epitope mimetic diagram of gI and gE epitope fusion proteins of PRV
FIG. 2 is a cross-sectional structure of a test strip, wherein 1 is a PVC bottom plate, 2 is a sample chromatographic pad, 3 is a gold pad, 4 is a glass cellulose membrane, and 5 is absorbent pad paper;
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
Example 1 gE and gI epitope screening and fusion of PRV
Firstly, carrying out homology analysis on gE and gI genes of a porcine pseudorabies virus JS-2012 strain, a porcine pseudorabies virus HeN1 strain, an NVDC-PRV-BJ strain, an NVDC-PRV-HEB strain and an NVDC-PRV-SD strain, a PRV TJ strain, a porcine pseudorabies virus variant strain PRV-ZJ01, a porcine pseudorabies virus variant strain HN1201, a porcine pseudorabies virus variant strain HN1202, a porcine pseudorabies virus Fa strain, a porcine pseudorabies virus Bartha strain, a porcine pseudorabies virus Kaplan strain, a porcine pseudorabies virus Becker strain, and screening antigen fragment candidate regions by utilizing a biological analysis means, meanwhile, the deletion of gE and gI gene information in the existing deletion vaccine strain (construction and application of virus strain and cell porcine pseudorabies gene deletion inactivated vaccine JS-2012-DeltagI/gE strain (Wu Tong, et al A live, attenuated pseudorabies virus strain JS-2012 reduced for gE/gI protects against both classical and emerging strain. Anti Research,2016, 130:110-117), gI/gE deletion virus vPRV-DgE/gI (Wang Tao, construction and application of artificial chromosome clone of pseudorabies virus variant JS-2012 infectious bacteria, 2017.5, academy of sciences of Chinese agricultural sciences) are referred to, and partial amino acid residues in the strain are replaced or optimized according to bioinformatics analysis (see figure 1), so that the amino acid sequence of the gE and gI fusion antigen epitope polypeptide is finally determined as shown in SEQ ID NO.2 (VSVTTVCFETACHPDLVLGRACVPEAPEMGIGDYLYVTVIKELTAPARAPGTPWGPGGGDDAIYVDGVTTPAPPARPWRLRREEGGMGIGDYASQSPNAKNTSPVMVELLRLDPKRACYTREYAAEYDLCPRVHHEAFRMVLNASVVSRVLLAAANATAGARGPGKIAMVLGPTIVVLLIFLGGIACVARRCARNRIYRPRPGRGSAVHAAPPR).
After the determined antigen fusion protein is synthesized and purified by Beijing Yisen Biotechnology Co., ltd, screening and preparing qualified nano antibody PRV-NANOEI, and determining the amino acid sequence as follows: QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQYAVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSSAAAY (SEQ ID NO. 1).
Example 2 detection of Properties of nanobody PRV-nanoEI
The antibody specificity of nanobodies PRV-nanoEI was determined by indirect competition ELISA, specifically described by cross-reactivity, as follows: eight different standard substance storage solutions such as PRRSV antibody positive serum, TGEV antibody positive serum, PEDV antibody positive serum, PPV antibody positive serum and SFV antibody positive serum are diluted to ten different working concentrations by 10% methanol/PBS gradient, and are measured by adopting an indirect competition ELISA method under the same condition, competition ELISA curves of nano-antibodies PRV-NANOEI are sequentially drawn, the standard substance concentration when the respective inhibition rate is 50% is calculated, the standard substance concentration is expressed by IC50, and the cross reaction rate is calculated according to the following calculation formula: cross reaction rate (%) = (nanobody PRV-NANOEI IC 50/analog IC 50) ×100%, giving nanobody PRV-NANOEI 1.03ng/mL for 50% inhibitory concentration IC50 against PRV; the cross-reactivity with PRRSV, TGEV, PEDV, PPV, SFV, NDV, CDV, HEV was less than 0.1%. Therefore, the nanobody PRV-nanoEI is a high-specificity nanobody aiming at PRV, and can be applied to research and development of detection reagents for specifically recognizing PRV.
EXAMPLE 3 detection of ELSIA antibody drug of nanobody PRV-nanoEI
A. Preparation of a coated plate in a pseudorabies virus ELISA detection kit:
according to the sequence shown in SEQ ID NO.1, a short peptide with purity of more than 95% is prepared by a chemical synthesis method conventional in the art, and the short peptide is a detection antibody. The antibody was dissolved and diluted to 2. Mu.g/mL with carbonate buffer, and then added to a 96-well ELISA plate at 100. Mu.L per well, and left overnight at 4℃to allow the antibody to adsorb in the ELISA plate. The next day the wells were discarded, and 150. Mu.L of phosphate buffer (0.5% BSA) was added to each well and the wells were discarded after 2 hours in a 37℃incubator. And (5) drying.
B. Other components of the pseudorabies virus ELISA antibody detection kit are prepared:
the other components of the kit also comprise an enzyme marker, a sample diluent, a positive control, a negative control, a chromogenic liquid A, a chromogenic liquid B, a washing liquid and a stop solution. The enzyme marker is goat anti-pig secondary antibody, the sample diluent is phosphate buffer solution, the positive control is pig positive control serum immunized by the pseudorabies virus vaccine, and the negative control is healthy pig negative serum without the pseudorabies virus vaccine. The washing solution is PBS buffer solution containing 0.05% Tween-20; the color development liquid A is a citrate buffer solution containing 50mg/mL urea hydrogen peroxide, and the color development liquid B is a citric acid/sodium citrate buffer solution containing 0.2mg/mL TMB and having pH of 5.0. The stop solution was a 0.25% hydrofluoric acid solution by volume.
C. The pseudorabies virus ELISA detection kit comprises the following operation steps:
1) And taking out the detection plate pre-coated with the nano antibody from the kit, adding 100 mu L of diluted serum to be detected (diluted 1:40) into the antigen coating plate, and simultaneously setting negative and positive control holes, wherein each hole is provided with 2 holes, and 100 mu L of each hole.
2) The samples in the wells were gently shaken and incubated at 37℃for 60 minutes. The solution in the plate holes is thrown off, 200 mu L/hole of washing solution is added, the plate is washed 5 times, and finally the plate is patted dry on absorbent paper.
3) Each well was incubated with 100. Mu.L of the enzyme label at 37℃for 30 minutes. Washing 5 times, and the method is the same as the step 2.
4) Adding 50 mu L of each of the developing solution A and the developing solution B into each hole, uniformly mixing, and developing at room temperature (18-26 ℃) for 10 minutes in a dark place. 50 mu L of stop solution is added to each well, and the OD450nm reading value of each well is measured by an enzyme label instrument within 10 minutes.
The judgment standard of the kit is as follows: the test is established under the condition that the difference between the average OD450nm value of the negative control wells and the average OD450nm value of the positive control wells is greater than or equal to 0.5.S = sample well OD450nm value, N = negative control well average OD450nm value. If the S/N ratio is greater than 2.1, the sample is judged to be positive for the pseudorabies virus antibody. If the S/N ratio is less than or equal to 2.1, the sample is judged to be negative to the pseudorabies virus antibody.
D. Application of pseudorabies virus ELSIA detection kit:
1. the ELISA detection kit for the pseudorabies virus for the specificity test is used for detecting standard positive serum and pseudorabies virus negative serum of classical swine fever virus, pseudorabies virus, swine foot-and-mouth disease (O type), porcine parvovirus, swine influenza, porcine reproductive and respiratory syndrome and the like, the S/N value of the standard positive serum of the pseudorabies virus is obviously more than 2.1, the S/N value of the rest serum is less than 2.1, and the kit accords with the judgment standard of the negative serum, thus the method has good specificity (see table 1, wherein the listed numbers are OD450nm values, and experiments 1 and 2 are two groups of double-blind parallel tests).
TABLE 2 serum specificity assay
2. The test kit for sensitivity detects the positive serum of pseudorabies virus at different dilutions, and even if the positive serum of pseudorabies virus is diluted by 1280 times, the positive serum of pseudorabies virus is still detected as positive, which indicates that the sensitivity of the kit is very high (two groups of double blind parallel tests are adopted in experiments 1 and 2).
TABLE 2 serum sensitivity assay
| 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | Positive control | Negative control | |
| Experiment 1 | 1.805 | 1.759 | 1.138 | 0.892 | 0.492 | 0.307 | 1.926 | 0.112 |
| |
1.857 | 1.697 | 1.140 | 0.903 | 0.502 | 0.311 | 1.879 | 0.109 |
3. Repeatability experiments
Detecting by using ELISA conditions established by optimized conditions, wherein the obtained inter-group variation coefficient is between 1.13% and 5.03%; the inter-group variation coefficient is between 1.21% and 5.98%, which shows that the established ELISA has good repeatability.
4. Accuracy test 130 serum from clinic was tested with nanobody PRV-NANOEI, and the results are shown in Table 3. It can be seen from the table that rapid and accurate detection can be achieved by using the nanobody PRV-NANOEI of the present application.
Table 3 comparison of homemade pseudorabies virus ELISA detection kit with commercial pseudorabies virus detection kit (yaji biological porcine pseudorabies gE antibody detection kit)
In conclusion, the ELISA kit established by the specific epitope of the pseudorabies virus prepared by the method has the advantages of strong specificity, good repeatability and high sensitivity, and can be used for detecting clinical pseudorabies virus samples. Example 4 assembly of PRV-NANOEI colloidal gold test strip
(1) Taking purified nano antibody PRV-NANOEI protein, and dialyzing with 0.01M phosphate buffer solution with pH value of 7.4 for 3-4 hours for later use;
(2) Preparing colloidal gold, diluting 1% chloroauric acid into 0.01% (mass fraction) by double-distilled deionized water, placing 100mL into a conical flask, heating to boil by a constant-temperature electromagnetic stirrer, adding 1.5mL of 1% trisodium citrate under continuous high temperature and continuous stirring, continuing to stir and heat at a constant speed until the solution is transparent red, stopping cooling to room temperature, recovering to the original volume by deionized water, and preserving at 4 ℃. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight. Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution for standby;
(3) Washing 1.5mL of centrifugal tube with ultrapure water twice, sucking 1mL of colloidal gold, slowly adding into the tube, dropwise adding nano antibody PRV-NANOEI with the total amount of 20 mug through magnetic stirring, and uniformly mixing and reacting for 40min;
(4) Adding 90 mu L of 8% BSA, and performing a blocking reaction for 40min;
(5) Centrifuging at 14000rpm/min for 35min at low temperature, removing supernatant, washing with phosphate buffer solution containing BSA 0.1-0.5 wt%, sucrose 2-4 wt% and pH7.2 of 0.02mol/L, repeating the steps for 2-3 times, retaining precipitate, re-suspending the precipitate with buffer solution with volume 1/10 of initial colloidal gold volume, and standing at 4deg.C for use;
(6) The gold pad was soaked in 0.02mol/L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, uniformly soaked for 2h, and dried at 37℃for further use. Uniformly spraying the prepared colloidal gold marker on a gold pad by using a Bio dot film-drawing instrument, spraying 0.01mL of the colloidal gold marker on each 1cm of gold pad, placing the gold pad in a 37 ℃ environment (humidity is less than 20%) for 2 hours, taking out the gold pad, and placing the gold pad in a dry environment (humidity is less than 20%) for storage for later use;
(7) The fusion protein was diluted to 1mg/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a detection line (T line) on a nitrocellulose membrane (glass cellulose membrane) in an amount of 1.0. Mu.L/cm using a Bio dot-based film cutter; the goat anti-mouse antibody was diluted to 200. Mu.g/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 1.0. Mu.L/cm using a Bio dot-based membrane cutter. And (5) drying the coated reaction film for 16 hours at 37 ℃ for standby.
(8) According to the cross-section structure of the test strip shown in figure 2, sequentially adhering water-absorbing pad paper, a glass cellulose film, a gold pad and a sample chromatographic pad on a PVC base plate, wherein a 1/3 area of the gold pad is covered by the sample chromatographic pad from the initial end, the interface is increased to improve the sample diffusion efficiency, and the sample is beneficial to being diffused from the sample chromatographic pad to the gold pad under the action of gravity; the tail end of the gold pad is connected with the initial end of the glass cellulose film, the tail end of the NC is connected with the initial end of the water absorption pad paper, the initial end of the sample chromatographic pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad paper is aligned with the tail end of the PVC bottom plate; the glass cellulose film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped belts which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the sample chromatographic cushion; the control line is located on the side remote from the end of the conjugate sample chromatographic pad. Wherein the sample chromatographic pad is a glass fiber membrane treated by phosphate buffer solution, and the phosphate buffer solution is phosphate buffer solution containing 1-5% BSA and surfactant.
(9) Cutting the test paper strip into small strips with proper width by a machine, arranging the small strips in a specially-made plastic card shell, arranging a sample adding hole and an observation hole on the card shell, sealing the card shell by an aluminum foil bag, and storing the card shell in an environment with the temperature of 4-30 ℃ for 12 months.
Example 5 property detection of PRV-NANOEI colloidal gold test strip
1) Cross-talk validation of different strains
In order to verify whether the test strip can be used for corresponding detection in practical application occasions such as pig farms and the like, and simultaneously verify whether cross reaction can be generated on PRV strains from different sources, blood samples of PRV JS2012 strain, PRV HNB strain, PRV JL15 (2021) strain and vaccine strain Bartha-K61 infected pigs are respectively detected by the test strip, and test results show that serum samples of PRV JS2012 strain, PRV HNB strain, PRV JL15 (2021) strain and vaccine strain Bartha-K61 infected pigs are positive. The results show that the test strip can be applied to antibody detection of various PRV virus strains in clinic.
2) Sensitivity test
To verify the sensitivity of the nanobody screened in the application, PRV-BC fusion protein was fixed to a concentration of 1mg/mL, diluted in a ratio of 1:10,1:100,1:1000,1:10000,1:100000,1:1000000, and tested, and repeated three times. The detection result shows that when the serum dilution ratio is 1:100000 (namely, the minimum detection limit is 10 ng/mL), the positive result can be detected, and the sensitivity is high.
3) Stability test
Storage stability test at 4 ℃): sealing and packaging the prepared PRV-NANOEI colloidal gold test strip and a drying agent together by using an aluminum foil bag, taking out 2 PRV-BC fusion protein standard series solutions with a visible detection limit concentration every two months in a refrigerator at the temperature of 4 ℃, and observing stability test results (comprising the presence or absence of a detection line and a quality control line, the definition of the strip, the gold-labeled antibody placing degree of a gold-labeled pad, the sensitivity of the test strip and the like). The result proves that the test strip can still keep good detection effect after being stored for more than 8 months at the temperature of 4 ℃.
4) Clinical actual detection
108 inactivated clinical samples (including serum samples, organ tissue samples, wherein the organ tissue samples were measured by centrifugation after homogenization and grinding with PBS buffer using laboratory conventional means) were measured from pig farm samples near Chongqing. The test strip is used for detection, and the detection result shows that 101 samples in 157 samples are positive samples and 56 samples are negative serum. The primer (synthesized by Chengdu Biotechnology Co., ltd.) is designed with reference to GB/T18641 pseudorabies diagnostic technique, and has the sequence of an upstream primer FPRV CAGGAGGACGAGCTGGGGCT and a downstream primer RPRV GTCCACGCCCC GCTTGAAGCT. Reaction system (20 μl): 1. Mu.L of sample, 1. Mu.L of each of the upstream and downstream primers, 10. Mu.L of DNA-MIX, ddH 2 O7. Mu.L. PCR reaction conditions: denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 40 cycles; extending at 72℃for 10min. The PCR shows that 112 out of 157 samples are positive samples and 45 samples are negative serum. Therefore, the test strip established by the experiment can be reliably applied to clinical detection, and can not achieve 100% consistency with the PCR detection result, but can be directly, widely and effectively applied to clinical sample detection due to the characteristics of high speed, high efficiency, sensitivity and reliability.
The PRV-NANOEI colloidal gold test strip has the characteristics of high specificity, high sensitivity, high accuracy and the like, and has the advantages of wide detection range, low false positive rate and reliable detection result. When the PRV-NANOEI colloidal gold test strip is used, the pretreatment time of the sample is short, and the detection limit of the standard substance is 10ng/mL. The detection method is suitable for clinical sample detection or epidemic prevention detection in pig farms; a large number of samples can be detected in a short time, excluding a large number of negative samples. The sample treatment is simple and easy to implement, and expensive instruments and equipment are not needed for detection, so that the method is suitable for popularization and use in basic-level inspection and quarantine units.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
SEQUENCE LISTING
<110> Chongqing city animal epidemic disease prevention control center
<120> a pig pseudorabies virus gE and gI nano antibody, preparation method and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 122
<212> protein
<213> PRV-NANOEI nanobody
<400> 1
QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQYAVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSSAAAY
<210> 2
<211> 214
<212> protein
<213> gE and gI epitope fusions
<400> 2
VSVTTVCFETACHPDLVLGRACVPEAPEMGIGDYLYVTVIKELTAPARAPGTPWGPGGGDDAIYVDGVTTPAPPARPWRLRREEGGMGIGDYASQSPNAKNTSPVMVELLRLDPKRACYTREYAAEYDLCPRVHHEAFRMVLNASVVSRVLLAAANATAGARGPGKIAMVLGPTIVVLLIFLGGIACVARRCARNRIYRPRPGRGSAVHAAPPR
Claims (4)
1. The porcine pseudorabies virus gE and gI epitope fusion protein is characterized in that the amino acid sequence of the epitope fusion protein is shown as SEQ ID NO. 2.
2. An antibody specifically aiming at the porcine pseudorabies virus gE and gI epitope fusion protein according to claim 1, which is characterized in that the antibody is a nano antibody PRV-NANOEI, and the amino acid sequence of the antibody is shown as SEQ ID NO. 1.
3. A rapid detection test strip for porcine pseudorabies virus, which is characterized by comprising a bottom plate, water-absorbing pad paper, a glass cellulose membrane, a gold pad and a sample chromatographic pad, wherein the water-absorbing pad paper, the glass cellulose membrane, the gold pad and the sample chromatographic pad are sequentially adhered from top to bottom on the bottom plate, and the gold pad is provided with a colloidal gold-labeled nano antibody PRV-NANOEI as claimed in claim 2; the glass cellulose membrane is provided with a detection area and a quality control area which are mutually separated, the detection area is sprayed with PRV coating antigen, and the quality control area is sprayed with an antibody which is specifically combined with the nanometer antibody PRV-nanometer of claim 2 marked by colloidal gold.
4. The application of the antigen epitope fusion protein of claim 1, the antibody of claim 2 and the rapid detection test strip of the porcine pseudorabies virus in the rapid detection of the non-disease diagnosis of the porcine pseudorabies virus.
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| CN102608315A (en) * | 2012-02-21 | 2012-07-25 | 武汉科前动物生物制品有限责任公司 | Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof |
| CN103792373A (en) * | 2014-03-12 | 2014-05-14 | 武汉中博生物股份有限公司 | Colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and preparation method |
| CN103983781A (en) * | 2014-05-27 | 2014-08-13 | 武汉中博生物股份有限公司 | Porcine pseudorabies virus gE IgM antibody colloidal gold immunochromatograohic assay test strip as well as preparation method and application thereof |
| CN104597255B (en) * | 2015-02-04 | 2017-07-21 | 华中农业大学 | The test card and preparation method and application of pseudorabies virus gE protein antibodies in a kind of detection Swine serum |
| CN105087506B (en) * | 2015-03-20 | 2020-06-30 | 普莱柯生物工程股份有限公司 | Porcine pseudorabies virus weakening method, porcine pseudorabies virus weakening virus strain, porcine pseudorabies virus vaccine composition and application of porcine pseudorabies virus weakening virus strain |
| CN110156878B (en) * | 2019-05-24 | 2021-06-29 | 北京标驰泽惠生物科技有限公司 | Porcine pseudorabies virus gE-gI protein, expression plasmid thereof, preparation method and application |
| CN113201507B (en) * | 2020-07-10 | 2022-02-22 | 浙江海隆生物科技有限公司 | Recombinant pseudorabies virus and vaccine composition thereof |
| CN113985030A (en) * | 2021-10-28 | 2022-01-28 | 重庆市动物疫病预防控制中心 | Immune colloidal gold test paper for rapidly detecting porcine pseudorabies antibody and preparation method thereof |
| CN114163522A (en) * | 2021-12-07 | 2022-03-11 | 重庆市动物疫病预防控制中心 | Nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application |
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