CN114989306A - Porcine pseudorabies virus gE and gI nano antibody, preparation method and application - Google Patents
Porcine pseudorabies virus gE and gI nano antibody, preparation method and application Download PDFInfo
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Abstract
The invention provides a porcine pseudorabies virus gE and gI nano antibody, a preparation method and application thereof, wherein the nano antibody targets a preferred epitope fusion protein of a gE protein and a gI protein of a PRV. A contrast test proves that the nano antibody PRV-NANOEI is more sensitive than a commercial monoclonal antibody in PRV detection, the nano antibody PRV-NANOEI is combined with a colloidal gold labeling technology, the sensitivity is high, and the detection limit of PRV detection can reach 10 ng/mL. Strong specificity and less cross reaction. The colloidal gold test strip developed for the first time realizes rapid detection, shortens the original two-day-consuming immune differential test into rapid detection finished in a few minutes, and realizes the primary application of PRV rapid detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a specificity detection method of Porcine pseudorabies Virus (PRV), wherein an active unit of the specificity detection method is a nano antibody of gE and gI proteins of targeted Porcine pseudorabies Virus (PRV), and a preparation method and application thereof.
Background
Porcine Pseudorabies (PR) is an acute infectious disease of pigs, also known as Aujeszky's disease, caused by Porcine pseudorabies Virus (PRV). Pigs, as the natural host for PRV, are more susceptible to viral infection and are the only animal species that can survive acute infection and have latent infectivity. The infected newborn piglet is characterized by high mortality and nervous system disorder, the pregnant sow has abortion and reproduction disorder, the old pigs mainly have respiratory diseases, the piglet mortality characterized by nervous symptoms is more than 20 percent, the probability of the pig infecting wild viruses is as high as 50 percent, and the loss of the pig industry in China is huge.
PRV belongs to the family of Herpesviridae (Herpesviridae), members of the sub-family of Alpha-Herpesviridae (Alpha Herpesviridae), and has a linear double-stranded DNA genome of about 150kb in size. PRV is currently known as only one serotype, and PRV encodes more than 70 proteins, and the main proteins (gB, gC, gD, gE, gH, gI, gK, gL, gM, gN) are envelope proteins except for gG protein. 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, which are associated with immune protection. The gE protein was first referred to as the gI protein. The PRV vaccine strains screened and prepared in 1960, such as Bartha strain, BUK strain and the like, have gE gene deletion. In the middle of the 80's of the 20 th century, a PRV mutant strain with deletion of the gE gene was constructed by a DNA recombination technique; on the basis, PRV is further weakened by deleting TK gene. Many gE gene-deleted vaccines are capable of preventing or alleviating clinical symptoms caused by challenge infection, and are permitted to be used in swine herds in many countries.
Pseudorabies can be controlled by vaccination and therefore it becomes very important to distinguish between immunized and infected animals. Detection of vaccine immunity antibodies and natural infection antibodies can be achieved by vaccination with gene deletion vaccines. Differential diagnosis methods are a type of diagnosis methods applied on the basis of using gene-deletion vaccines. DNA homologous recombination-defective, gene-deleted and naturally defective vaccines can be used to discriminate immune antibodies from naturally infected antibodies due to the lack of specific glycoproteins (gG, gE or gC). Due to the presence of multiple nonessential glycoprotein genes in PRV, viral mutants lacking these genes are unable to produce the glycoprotein encoded by the deleted genes, but do not affect viral proliferation and immunogenicity on cells. After injecting the gene-deleted vaccine into animals, the animals cannot produce antibodies against the deleted protein. Therefore, serological positive pigs and nursery pigs which are naturally infected with wild viruses can be identified by a serological method.
The glycoprotein genes deleted by the deletion vaccine are mainly gE and gI genes, for a farm immunized by the gE and gI gene double-deletion vaccine, a wild virus identification kit is matched for screening when the immune antibody level is detected, the source of a positive antibody is determined, the immune antibody and a natural infection antibody are identified and distinguished, a recessive infection pig is screened out, the wild virus positive pig is eliminated in time, a healthy negative pig group is established, but common commercialized identification kits in the market are mostly gE single-gene deletion ELISA kits, and the requirement of clinical detection can not be met frequently. Because the gE protein antibody is currently generally regarded as the most important infection index in various specific glycoproteins, but the generation time and the response level of the gE antibody are different among individuals, and the gE antibody or the time delay of the gE antibody generation cannot be detected in an infected pig organism, the gE protein alone coated kit is used for detecting the porcine pseudorabies virus, and the result shows false negative and is not high in accuracy. Also, detection of a single protein antibody is inaccurate because different protein antibodies appear at different times after infection of an animal. Therefore, for diagnosing porcine pseudorabies, at least more than one protein antibody should be detected to improve the detection accuracy. For those animals which are infected after immunization, the virus replication is slow due to the neutralizing antibody, so that the specific protein with low concentration is in vivo, but the sensitivity of the commercial ELISA kit is low, so that the specific protein with low concentration is difficult to detect, and the partial infected animals are missed to detect.
In terms of sensitivity and specificity, the isolation and identification of the virus are the gold standard for detecting PRV, but the time consumption is long (at least 2-3 days), the sensitivity is low, a high cell culture level is required, and the virus culture process is easy to have detailed misoperation so as to finally influence the diagnosis result, so that the virus culture method is difficult to popularize in basic veterinary units. Virus neutralization assay (VNT) is one of the commonly used methods in PRV detection, which is efficient and sensitive. However, this method is less sensitive during the acute phase of infection and is prone to false negatives. Although immunological diagnostic methods such as indirect sandwich ELISA have high sensitivity and strong specificity, the immunological diagnostic methods need more pure proteins, need fine operation and are easy to generate false positive, and particularly depend on detection antibodies with high sensitivity and specificity, so that the detection accuracy and precision are directly determined by the properties of the detection antibodies. Although the methods can detect PRV virus and achieve certain effect in practice, the methods have the disadvantages of complex experimental operation, long time consumption, need of mutually matched detection reagents and related instruments, are only carried out in a laboratory, and are difficult to popularize. Therefore, the rapid and simple PRV virus detection method is researched and developed, and has important significance for real-time monitoring of virus proliferation.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a rapid specific detection method for Porcine pseudorabies Virus (PRV), so as to solve the problems of the prior art.
In order to achieve the above objects, the present invention provides a nanobody capable of recognizing porcine pseudorabies virus with high accuracy and sensitivity, which targets a preferred epitope fusion protein of gE protein and gI protein of PRV, which is named as PRV-NANOEI, and has an amino acid sequence of: QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQY AVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSS AAAY (SEQ ID NO. 1).
Furthermore, the invention provides a test strip for rapidly detecting the porcine pseudorabies virus, which adopts a mode of combining the nano antibody PRV-NANOEI and the colloidal gold test strip to rapidly detect the porcine pseudorabies virus, thereby realizing rapid detection, shortening the original time-consuming two-day immune identification test into rapid detection finished in a few minutes, and realizing the primary application of the nano antibody PRV-NANOEI in the aspect of rapid detection.
Furthermore, the test paper strip for rapidly detecting the porcine pseudorabies virus provided by the invention adopts a nitrocellulose membrane (CN95) as a chromatography material, adopts a glass cellulose membrane as a sample pad, and is assembled together with a PVC (polyvinyl chloride) bottom plate, absorbent paper and the like to form the test paper strip.
Further, the rapid detection test strip comprises a PVC base plate, water absorption packing paper, a glass cellulose membrane, a gold pad and a sample chromatography pad, wherein the water absorption packing paper, the glass cellulose membrane, the gold pad and the sample chromatography pad are sequentially pasted on the PVC base plate from top to bottom, and a nano antibody PRV-NANOEI marked by colloidal gold is arranged on the gold pad;
the glass cellulose membrane is provided with a detection area and a quality control area which are separated from each other, 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 colloidal gold labeled nano antibody PRV-NANOEI.
Further, the invention provides a preparation method of the test strip for rapidly detecting the porcine pseudorabies virus,
1) purifying the nanometer antibody PRV-NANOEI, and dialyzing for later use;
2) labeling the nano antibody PRV-NANOEI by colloidal gold;
3) BSA blocking reaction;
4) after centrifugation, washing the precipitate by PBS containing 1% BSA;
5) spraying a gold mark pad after redissolution;
6) coating a glass cellulose membrane;
7) drying at room temperature and low humidity; sequentially adhering absorbent paper, a glass cellulose membrane, a gold pad and a sample chromatography pad from top to bottom on a bottom plate, and cutting for later use;
8) the colloidal gold test strip is subjected to application test, the detection limit of the colloidal gold test strip is 10ng/mL grade, and the requirement of rapidly detecting PRV can be met.
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 recognize PRV, can be used as a material for immunological detection, and has a very wide application prospect.
The test strip for rapidly detecting the porcine pseudorabies virus has the advantages that: the rapid detection test strip can determine the source of the antigen, distinguish the immune sample from the naturally infected sample, judge whether the detected animal is infected with the virus or inoculated with the vaccine, has good sensitivity, specificity and stability, and is particularly suitable for the detection of low-concentration samples and the early diagnosis of epidemic diseases.
A contrast test proves that the nano antibody PRV-NANOEI is more sensitive than a commercial monoclonal antibody in PRV detection, the nano antibody PRV-NANOEI is combined with a colloidal gold labeling technology, the sensitivity is high, and the detection limit of PRV detection can reach 1 ng/mL. The test strip has strong specificity and less cross reaction, and the test strip provided by the invention has negative cross reaction on PRRSV antibody positive serum, TGEV antibody positive serum, porcine epidemic diarrhea virus PEDV antibody positive serum, porcine parvovirus PPV antibody positive serum, swine fever virus SFV antibody positive serum, newcastle disease virus NDV disease positive serum, canine distemper virus CDV disease positive serum and rabbit E-HEV hepatitis virus positive serum. The colloidal gold test strip developed for the first time realizes rapid detection, shortens the original two-day-consuming immune differential test into rapid detection finished in a few minutes, and realizes the primary application of PRV rapid detection.
Drawings
FIG. 1 is a schematic diagram of the epitope mimic of gI and gE epitope fusion protein 3D of PRV
FIG. 2 is a cross-sectional structure of a test strip, wherein 1 is a PVC base 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 technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Example 1 gE and gI epitope screening and fusion of PRV
Firstly, carrying out homology analysis on gE and gI genes of porcine pseudorabies virus JS-2012 strain, porcine pseudorabies HeN1 strain, NVDC-PRV-BJ strain, NVDC-PRV-HEB strain and NVDC-PRV-SD strain, PRV TJ strain, porcine pseudorabies virus variant strain PRV-ZJ01, porcine pseudorabies virus variant strain HN1201 strain, porcine pseudorabies virus variant strain HN1202 strain, porcine pseudorabies virus Fa strain, porcine pseudorabies virus Bartha strain, porcine pseudorabies virus Kaplan strain and porcine pseudorabies virus Becker strain, screening an antigen fragment candidate region by using a biological analysis means, simultaneously referring to gE and gI gene information (viral strain and porcine pseudorabies virus gene deletion JS-delta-gI/gE strain, hanging, strain, porcine pseudorabies virus strain and virus strain) deleted in the existing deletion vaccine strain 2012 (strain, porcine pseudorabies virus vaccine strain, Tojessajou strain and strain, recovery. 2016,130: 110-.
The determined antigen fusion protein is synthesized and purified by Beijing Yishengbao biotechnology limited company, and then the qualified nanometer antibody PRV-NANOEI is screened and prepared, and the amino acid sequence of the protein is determined to be: QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQY AVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSS AAAY (SEQ ID NO. 1).
Example 2 Property testing of Nanobody PRV-NANOEI
The antibody specificity of the nanometer antibody PRV-NANOEI is determined by adopting an indirect competition ELISA method, which is described by cross reaction rate, and the test method is as follows: storing liquid of eight different standard products, such as PRRSV antibody positive serum of porcine reproductive and respiratory syndrome virus, TGEV antibody positive serum of porcine transmissible gastroenteritis virus, PEDV antibody positive serum of porcine epidemic diarrhea virus, PPV antibody positive serum of porcine parvovirus, SFV antibody positive serum of classical swine fever virus, positive serum of chicken newcastle disease virus NDV disease, CDV disease positive serum of canine distemper virus and positive serum of rabbit E-type HEV hepatitis virus, diluting the liquid to ten different working concentrations by using 10% methanol/PBS gradient, measuring by adopting an indirect competitive ELISA method under the same condition, sequentially drawing competitive ELISA curves of nano antibodies PRV-NANOEI, calculating the standard product concentration when the inhibition rate is 50%, expressing the standard product concentration by using IC50, and calculating the cross reaction rate according to the following calculation formula: the cross-reactivity (%) was (nanobody PRV-NANOEI IC 50/analog IC50) × 100%, giving nanobody PRV-NANOEI a 50% inhibitory concentration IC50 of 1.03ng/mL for PRV; the cross reaction rate with PRRSV, TGEV, PEDV, PPV, SFV, NDV, CDV and HEV is less than 0.1%. Therefore, the nano antibody PRV-NANOEI is a high-specificity nano antibody aiming at PRV, and can be applied to the development of a detection reagent for specifically recognizing PRV.
Example 3 ELSIA antibody drug detection of Nanobody PRV-NANOEI
A. Preparing a coating plate in the ELISA kit for detecting the pseudorabies virus:
according to the sequence shown in SEQ ID NO.1, the short peptide with the purity of more than 95 percent is prepared by utilizing the conventional chemical synthesis method in the field, and the short peptide is the detection antibody. The antibody was dissolved and diluted to 2. mu.g/mL with carbonate buffer, and then added to a 96-well microplate in an amount of 100. mu.L per well, and left overnight at 4 ℃ to adsorb the antibody to the microplate. The next day, the wells were discarded, 150. mu.L of phosphate buffer (containing 0.5% BSA) was added to each well, and the wells were incubated at 37 ℃ for 2 hours and discarded. Patting to dry.
B. Preparing other components of the pseudorabies virus ELISA antibody detection kit:
other components of the kit also comprise an enzyme marker, a sample diluent, a positive control, a negative control, a developing solution A, a developing solution B, a washing solution and a stop solution. The enzyme marker is goat anti-swine secondary antibody, the sample diluent is phosphate buffer solution, the positive control is swine positive control serum immunized by the pseudorabies virus vaccine, and the negative control is healthy swine negative serum of the non-immune pseudorabies virus vaccine. The washing solution is PBS buffer solution containing 0.05 percent Tween-20; the color developing solution A is a citrate buffer solution containing 50mg/mL of carbamide peroxide, and the color developing solution B is a citric acid/sodium citrate buffer solution containing 0.2mg/mL of TMB and having a pH value of 5.0. The stop solution is a hydrofluoric acid solution containing 0.25% by volume.
C. The method comprises the following steps:
1) taking out the detection plate pre-coated with the nano antibody from the kit, adding 100 mu L of diluted serum to be detected (1:40 dilution) into the antigen coated plate, and simultaneously arranging 2 holes of negative and positive control holes, wherein each hole is 100 mu L.
2) The well was gently shaken and incubated at 37 ℃ for 60 minutes. The solution in the plate hole was spun off, 200. mu.L/hole of washing solution was added, the plate was washed 5 times, and finally patted dry on absorbent paper.
3) Add 100. mu.L of enzyme label to each well and incubate at 37 ℃ for 30 minutes. Washing 5 times, the method is the same as step 2.
4) Adding 50 mu L of color development liquid A and 50 mu L of color development liquid B into each hole, uniformly mixing, and developing for 10 minutes in a dark place at room temperature (18-26 ℃). Stop solution was added at 50. mu.L per well, and OD450nm reading per well was measured by microplate reader within 10 minutes.
The kit provided by the invention has the following judgment standards: the test was established if the difference between the mean OD450nm value of the negative control wells and the mean OD450nm value of the positive control wells was greater than or equal to 0.5. S-sample well OD450nm value and N-negative control well mean OD450nm value. If the S/N ratio is more than 2.1, the sample is judged to be positive by the pseudorabies virus antibody. If the S/N ratio is less than or equal to 2.1, the sample is judged to be negative for the pseudorabies virus antibody.
D. The application of the pseudorabies virus ELSIA detection kit comprises:
1. the specificity test uses the ELISA detection kit of pseudorabies virus to detect the 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 flu, porcine reproductive and respiratory syndrome and the like, except that the S/N value of the pseudorabies virus standard positive serum is obviously more than 2.1, the S/N values of the rest sera are all less than 2.1, accord with the judgment standard of the negative serum, show that the specificity of the method is good (see table 1, the number listed therein is OD450nm value, and experiments 1 and 2 are two groups of double-blind parallel tests).
TABLE 2 serum specificity assays
2. The sensitivity test kit is used for testing the pseudorabies virus positive serum with different dilutions, and the pseudorabies virus positive serum still can be tested to be positive even if the pseudorabies virus positive serum is diluted by 1280 times in table 2, which indicates that the sensitivity of the kit is very high (experiments 1 and 2 are two groups of double-blind parallel tests).
TABLE 2 serum sensitivity assays
| 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 test
Detecting by using ELISA conditions established by the optimized conditions, wherein the obtained inter-group variation coefficient is between 1.13 and 5.03 percent; the coefficient of variation among groups is 1.21-5.98%, which indicates that the built ELISA has good repeatability.
4. Accuracy test 130 clinical sera were tested with the nanobody PRV-NANOEI and the results are shown in table 3. As can be seen from the table, rapid and accurate detection can be achieved by using the nanobody PRV-NANOEI of the present application.
TABLE 3 comparison of the self-made Pseudorabies virus ELISA kit with the commercial Pseudorabies virus detection kit (Yaji biological porcine pseudorabies gE antibody detection kit)
In conclusion, the ELISA kit established by using the pseudorabies virus specific epitope prepared by the invention 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 the purified nano antibody PRV-NANOEI protein, and dialyzing for 3-4 h by using a phosphate buffer solution with the pH value of 7.4 and 0.01M for later use;
(2) preparing colloidal gold, diluting 1% chloroauric acid to 0.01% (mass fraction) with double-distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant-temperature electromagnetic stirrer, adding 1.5mL of 1% trisodium citrate under continuous stirring at a high temperature, continuing stirring and heating at a constant speed until the solution is bright red, cooling to room temperature, recovering the volume with deionized water, and storing at 4 ℃. The prepared colloidal gold has pure appearance, is transparent and bright, has no sediment or floating objects, and is wine red when observed in sunlight. Under magnetic stirring, 0.2mol/L potassium carbonate solution is used for adjusting the pH value of the colloidal gold to 7.2 for later use;
(3) cleaning a 1.5mL centrifugal tube twice with ultrapure water, sucking 1mL colloidal gold, slowly adding into the tube, dropwise adding the nanometer antibody PRV-NANOEI with the total amount of 20 mug by magnetic stirring, uniformly mixing, and reacting for 40 min;
(4) adding 90 μ L of 8% BSA, and performing blocking reaction for 40 min;
(5) centrifuging at low temperature of 14000rpm/min for 35min, removing supernatant, washing with 0.02mol/L phosphate buffer solution containing 0.1-0.5% of BSA, 2-4% of sucrose and pH7.2, repeating the step for 2-3 times, retaining precipitate, re-suspending the precipitate with 1/10 (original volume of colloidal gold) of the buffer solution, and standing at 4 ℃ for use;
(6) soaking the gold pad in 0.02mol/L phosphate buffer solution containing 0.5% BSA, 5% sucrose and pH 7.4, soaking for 2h, and oven drying at 37 deg.C for use. Uniformly spraying the prepared colloidal gold marker on a gold pad by using a Bio dot film-scribing instrument, spraying 0.01mL of the colloidal gold marker on every 1cm of the gold pad, placing the gold pad in an environment (the humidity is less than 20%) at 37 ℃ for 2h, taking out the gold pad, and placing the gold pad in a dry environment (the humidity is less than 20%) for storage for later use;
(7) diluting the fusion protein to 1mg/mL by using 0.01mol/L, pH 7.2.2 phosphate buffer, and coating the fusion protein on a detection line (T line) on a nitrocellulose membrane (glass cellulose membrane) by using a Bio dot streaking machine, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01mol/L, pH 7.2.2 phosphate buffer and coated on a control line on nitrocellulose membrane (line C) with a Bio dot striping machine in an amount of 1.0. mu.L/cm. And (3) drying the coated reaction membrane at 37 ℃ for 16 hours for later use.
(8) According to the section structure of the test strip shown in the attached figure 2, the water absorption pad paper, the glass cellulose membrane, the gold pad and the sample chromatographic pad are sequentially adhered to the PVC base plate, wherein the gold pad is covered by the sample chromatographic pad from the beginning end in a 1/3 area, the interface is enlarged to improve the sample diffusion efficiency, and the sample is favorable for being diffused to the gold pad from the sample chromatographic pad under the action of gravity; the tail end of the gold pad is connected with the initial end of the glass cellulose membrane, the tail end of the NC is connected with the initial end of the water absorption packing paper, the initial end of the sample chromatography pad is aligned with the initial end of the PVC base plate, and the tail end of the water absorption packing paper is aligned with the tail end of the PVC base plate; the glass cellulose membrane 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 strips which are vertical to the long phase of the test strip; the detection line is positioned on one side near the end of the sample chromatography pad; the control line is located on the side away from the end to which the sample chromatography pad is attached. Wherein the sample chromatographic pad is a glass fiber membrane treated by a phosphate buffer solution, and the phosphate buffer solution is a phosphate buffer solution containing 1-5% BSA and a surfactant.
(9) Cutting the test paper strip into small strips with proper width by a machine, placing the small strips in a special plastic card shell, wherein the card shell is provided with a sample adding hole and an observation hole, sealing the card shell by an aluminum foil bag, and storing the card shell in an environment at 4-30 ℃, wherein the validity period is up to 12 months.
Example 5 Property detection of PRV-NANOEI colloidal gold test strip
1) Cross-validation of different strains
In order to verify whether the test strip can carry out corresponding detection in practical application occasions such as a pig farm and the like and simultaneously verify whether cross reaction can be generated on PRV strains from other different sources, blood samples of pigs infected by PRV JS2012 strain, PRV HNB strain, PRV JL15(2021) strain and vaccine strain Bartha-K61 are respectively detected by the test strip, and test results show that serum samples infected by the PRV JS2012 strain, PRV HNB strain, PRV JL15(2021) strain and vaccine strain Bartha-K61 are all positive. The results show that the test strip can be applied to antibody detection of various PRV virus strains clinically.
2) Sensitivity test
In order to verify the sensitivity of the nano antibody screened by the application, the PRV-BC fusion protein is diluted to a concentration of 1mg/mL at a ratio of 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000 respectively, and then the detection is carried out, and the steps are repeated for three times. The detection result shows that when the serum dilution ratio is 1:100000 (namely the lowest detection limit is 10 ng/mL), the positive result can be still detected, and the sensitivity is very high.
3) Stability test
4 ℃ storage stability test: and (2) sealing and packaging the prepared PRV-NANOEI colloidal gold test strip and the drying agent together by using an aluminum foil bag, putting the packaged test strip and the drying agent into a refrigerator at 4 ℃, taking out 2 test strips every two months, detecting PRV-BC fusion protein standard series solution with visible detection limit concentration, and observing stability test results (including the existence of a detection line and a quality control line, the definition of strips, the degree of gold-labeled antibodies placed in 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 4 ℃.
4) Clinical practice test
108 inactivated clinical samples (including serum samples, organ tissue samples, wherein the organ tissue samples were assayed by homogenization with PBS buffer and centrifugation after grinding using laboratory routine methods) were assayed from census house samples near Chongqing. When the test strip is used for detection, the detection result shows that 101 parts of 157 parts of samples are positive samples, and 56 parts of samples are negative serum. Primers (synthesized by Kyodo Condy biotechnology, Inc.) are designed according to GB/T18641 Pseudorabies diagnosis technology, and the sequences are an upstream primer FPRV CAGGAGGACGAGCTGGGGCT and a downstream primer RPRV GTCCACGCCCC GCTTGAAGCT. Reaction system(20. mu.L): sample 1. mu.L, 1. mu.L of each of the forward and reverse primers, DNA-MIX 10. mu.L, ddH 2 O7 μ L. And (3) PCR reaction conditions: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 40 cycles; extension at 72 ℃ for 10 min. According to the PCR verification, 112 of 157 samples are positive samples, and 45 of 157 samples are negative sera. Therefore, the test strip established by the experiment can be reliably applied to clinical detection, and although the test strip cannot achieve 100% consistency with the PCR detection result, the test strip can be directly, widely and effectively applied to clinical sample detection due to the characteristics of high speed, high efficiency, sensitivity and reliability.
The test proves that 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 10 ng/mL. The detection method is suitable for clinical sample detection or epidemic prevention detection in a pig farm; a large number of samples can be detected in a short time, and a large number of negative samples can be excluded. Because the sample treatment is simple and easy, and the detection does not need expensive instruments and equipment, the method is suitable for popularization and use in primary inspection and quarantine units.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
SEQUENCE LISTING
<110> animal epidemic disease prevention control center in Chongqing city
<120> porcine 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> fusion of gE and gI epitopes
<400> 2
VSVTTVCFETACHPDLVLGRACVPEAPEMGIGDYLYVTVIKELTAPARAPGTPWGPGGGDDAIYVDGVTTPAPPARPWRLRREEGGMGIGDYASQSPNAKNTSPVMVELLRLDPKRACYTREYAAEYDLCPRVHHEAFRMVLNASVVSRVLLAAANATAGARGPGKIAMVLGPTIVVLLIFLGGIACVARRCARNRIYRPRPGRGSAVHAAPPR
Claims (5)
1. An antigen epitope fusion protein of porcine pseudorabies virus gE and gI, which is characterized in that the amino acid sequence of the antigen epitope fusion protein is VSVTTVCFETACHPDLVLGRACVPEAPEMGIGDYLYVTVIKELTAPARAPGTPWGPGGGDDAIYVDGVTTPAPPARPWRLRREEGGMGIGDYASQSPNAKNTSPVMVELLRLDPKRACYTREYAAEYDLCPRVHHEAFRMVLNASVVSRVLLAAANATAGARGPGKIAMVLGPTIVVLLIFLGGIACVARRCARNRIYRPRPGRGSAVHAAPPR (SEQ ID NO. 2).
2. An antibody specific for the fusion protein of porcine pseudorabies virus gE and gI epitopes of claim 1.
3. The antibody of claim 2, characterized in that it is a nanobody targeting a preferred epitope fusion protein of the gB and gC proteins of PRV, designated PRV-NANOEI, with the amino acid sequence QVQLQESGGSPNSGSGVACVSQSPNSGGKRGKEREKVAHSSTSTYYDKSATSTYGGSVQYAVKSPTDSPAKNANKTSPVMQMNNSGSSLKPEVDTAMNFGDIWWYWKTGVYGYNQSSSAAAY (SEQ ID No. 1).
4. A test strip for rapidly detecting porcine pseudorabies virus is characterized by comprising a bottom plate, water-absorbing pad paper, a glass cellulose membrane, a gold pad and a sample chromatography pad, wherein the water-absorbing pad paper, the glass cellulose membrane, the gold pad and the sample chromatography pad are sequentially stuck on the bottom plate from top to bottom, and a nano antibody PRV-NANOEI marked by colloidal gold is arranged on the gold pad; the glass cellulose membrane is provided with a detection area and a quality control area which are separated from each other, the detection area is sprayed with PRV coating antigen, and the quality control area is sprayed with antibody which is specifically combined with the nano antibody PRV-NANOEI marked by colloidal gold.
5. The use of the epitope fusion protein according to claim 1, the antibody according to claim 2 or 3, or the test strip for rapid detection of porcine pseudorabies virus according to claim 4 for rapid detection of non-disease diagnosis of porcine pseudorabies virus.
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