CN114163522A - Nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application - Google Patents

Abstract

The invention provides a nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, wherein the nano antibody targets a preferred epitope fusion protein of gB protein and gC protein of PRV, and is named as PRV-BC21, and the amino acid sequence of the nano antibody is shown as SEQ ID No. 3. A contrast test proves that the nano antibody PRV-BC21 is more sensitive than a commercial monoclonal antibody in PRV detection, the nano antibody PRV-BC21 is combined with a colloidal gold labeling technology, the sensitivity is high, and the detection limit of PRV detection can reach 1 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

Nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application

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 gB and gC 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 and BUK strain, 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.

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 development of a rapid and simple PRV virus detection method 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 gB protein and gC protein of PRV, which is named PRV-BC21, and whose amino acid sequence is shown in SEQ ID No. 3.

Further, the invention provides a preparation method of the nano antibody PRV-BC21, which comprises the following steps:

(1) construction of recombinant eukaryotic expression vectors: connecting a DNA sequence of a coded nano antibody PRV-BC21 to an expression vector through PCR amplification and enzyme digestion to obtain a positive plasmid;

(2) and (3) transforming the positive plasmid in the step (1) into a host cell, and inducing and expressing the nano antibody PRV-BC 21.

Further, the expression vector may be a eukaryotic expression vector or a prokaryotic expression vector, and preferably, the expression vector is a eukaryotic expression vector, and the expression vector may be selected from: pCRII, pCR3, and pcDNA3.1(Invitrogen, San Diego, CA), pB SII (Stratagene, La Jolla, CA), pET 15(Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N1(Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO90/14363), and pFastBacDual (Gibco-BRL, Grand Island, NY), and the like.

The host cell is escherichia coli, yeast or eukaryotic cell, preferably the host cell is eukaryotic cell, and can be selected from, for example: CHO in Chinese hamster ovary cells, COS in monkey kidney cells, HEK-293 in human embryonic kidney cells, HELA in human cervical cancer cells, etc.

Furthermore, the invention provides a test strip for rapidly detecting porcine pseudorabies virus, which adopts a mode of combining the nano antibody PRV-BC21 and a colloidal gold test strip to rapidly detect the porcine pseudorabies virus, thereby realizing rapid detection, shortening the original time-consuming two-day immunodifferencing test into rapid detection finished in a few minutes, and realizing the primary application of the nano antibody PRV-BC21 in the aspect of rapid detection.

Further, the test paper strip for rapidly detecting the porcine pseudorabies virus provided by the invention comprises a bottom plate, water absorption packing paper, an NC membrane, a gold pad and a sample chromatography pad, wherein the water absorption packing paper, the NC 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-BC21 marked by colloidal gold is arranged on the gold pad;

the NC 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 a nano antibody PRV-BC21 marked by colloidal gold.

Further, the invention provides a preparation method of the test strip for rapidly detecting the porcine pseudorabies virus,

1) purifying the nanometer antibody PRV-BC21, and dialyzing for later use;

2) the colloidal gold is labeled with a nano antibody PRV-BC 21;

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 an NC film;

7) drying at room temperature and low humidity; sequentially adhering absorbent paper, an NC 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 nano antibody PRV-BC21 for specifically recognizing PRV and discloses an amino acid sequence of the nano antibody. Sequencing results of the specific nano antibody provided by the invention show that the sequence of the nano antibody PRV-BC21 is highly similar to human VH, and ELISA results show that the specific nano antibody has high binding force, presumably VH-HcAbs naturally existing in camels, and probably has great application prospect. The nano antibody can specifically recognize PRV, can be used as a material for immunological detection, and has a very wide application prospect.

A contrast test proves that the nano antibody PRV-BC21 is more sensitive than a commercial monoclonal antibody in PRV detection, the nano antibody PRV-BC21 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 3D epitope mimic diagram of gB and gC epitope fusion proteins of PRV

FIG. 2 shows the expression and purification of gB-gC epitope fusion protein of PRV, wherein M is marker, 1-4 are SDS-PAGE identification images (different loading wells) of PET-28a-BC recombinant positive plasmid, and a specific positive band with a molecular weight equivalent to the expected molecular weight appears at about 27kD position, thus proving the successful expression of gB-gC epitope fusion protein of PRV;

FIG. 3 shows the ELISA method for detecting bactrian camel serum titer, wherein the negative control is pre-immune camel serum;

FIG. 4 shows the fusion protein specific nanobody ELISA results after 3 rounds of panning;

FIG. 5 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 an NC membrane, and 5 is absorbent pad paper;

fig. 6 is a cross-reactivity test of different strains of the PRV-BC21 colloidal gold test strip, wherein 1 is a negative control, 2 is PRV JS2012 strain, 3 is PRV HNB strain, 4 is PRV JL15(2021) strain, and 5 is vaccine strain Bartha-K61.

FIG. 7 shows the sensitivity test of the PRV-BC21 colloidal gold test strip, in which 1-6 corresponds to the test results at dilutions of 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000, respectively, and 7 is a negative control.

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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within 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 gB and gC epitope screening of PRV and expression of fusion proteins

Firstly, carrying out homology analysis on gB genes of a porcine pseudorabies virus JS-2012 strain, a porcine pseudorabies HeN1 strain, an NVDC-PRV-BJ strain, an NVDC-PRV-HEB strain, an NVDC-PRV-SD strain, a PRV TJ strain, a porcine pseudorabies virus variant PRV-ZJ01, a porcine pseudorabies virus variant HN1201 strain, a porcine pseudorabies virus variant HN1202 strain, a porcine pseudorabies virus Fa strain, a porcine pseudorabies virus Bartha strain, a porcine pseudorabies virus Kaplan strain and a porcine pseudorabies virus Becker strain, screening candidate regions of the antigen fragment by using a biological analysis means, finally determining and selecting the sequence shown by the amino acids from the 76 th position to the 135 th position and the amino acids from the 576 th position to the 640 th position of the gB-containing protein, and according to bioinformatics analysis, partial amino acid residues in the gB epitope peptide are replaced or optimized (see figure 1), and finally the amino acid sequence of the gB epitope peptide is determined to be shown in SEQ ID NO. 1.

Optimized design of porcine pseudorabies virus gC protein epitope polypeptide fragment

As mentioned above, the identity analysis and computer simulation are carried out on the known porcine pseudorabies virus gC protein, the screened gC protein epitope polypeptide is in series connection of 52-113 bit and 401-458 bit, and corresponding optimization is carried out, and finally the amino acid sequence of the gC protein epitope polypeptide is determined to be shown as SEQ ID NO. 2.

Construction of tandem expression cassettes

Amplifying the polypeptide sequence by using an overlap PCR method, directly fusing and connecting gB and gC sequences in series to form fusion protein, constructing a PRVBC fusion protein expression cassette, connecting the PRVBC fusion protein expression cassette to PET-28a (purchased from Invitrogen company, a product number A11499) by using double enzyme digestion, selecting a single clone to identify the insertion direction, sending a plasmid with the correct insertion direction to the Invitrogen company for sequencing, and naming the plasmid with the correct sequencing as PET-28 a-BC. The PET-28a-BC recombinant positive plasmid is expressed, and after being verified by SDS-PAGE (shown in figure 2), the soluble PRV-BC fusion protein is obtained by purifying through a nickel column chromatography.

Example 2 screening and identification of PRV-BC fusion protein specific Nanobody

After the purified soluble PRV-BC fusion protein is fully emulsified with Freund adjuvant, the camel is immunized by subcutaneous injection at the neck, and 6 times of immunization are carried out in total at intervals of 2 weeks between every two immunizations. After blood serum is separated after the 6 th immunization, PRV-BC fusion protein is used as antigen to measure the antibody titer, and the immune effect is monitored. The negative control is camel serum before immunization, the serum after the immunization is centrifuged is taken, the antibody titer of the serum is detected by an ELISA method (figure 3), and the result shows that the antibody titer in the serum reaches 1: 256000. Lymphocyte RNA was extracted using a kit (purchased from Invitrogen corporation), and oligo (dT)20 primer reverse transcriptase was used using the extracted RNA as a template: (

III) cDNA was synthesized and further amplified VHH fragments by nested PCR. Cloning the amplified VHH gene fragment into the cleaved pCANTAB 5E phage display vector through a cleavage site. The ELISA result of screening the PRV-BC fusion protein specific nano antibody is increased from 0.15 to 3.25 by using phage display technology through 3 rounds of panning, which shows that the PRV-BC fusion protein specific nano antibody is obviously enriched (figure 4). Positive as determined by ELISA test result(more than 3 times of the negative value) of the monoclonal bacteria solution, and sequencing. The sequencing results were aligned and sorted by the software MegAlign. According to the sequence classification result, detecting the screened crude extract of the nano antibody by an ELISA method according to the sequence classification result of 1: 2. diluted 1:20, 1:200, 1:2000 fold and the titer was determined again. The specificity and the affinity of the nano antibody are analyzed by ELISA, and the finally screened amino acid sequence of the nano antibody of the PRV-BC fusion protein aiming at the PRV is shown as SEQ ID NO. 3. This nanobody was named PRV-BC 21.

Expression and purification of Nanobody PRV-BC21

1) With primer F1: 5'-CATCGCTAGTTACACGTTATAGC-3' (SEQ ID NO. 4); r1: 5'-AAGGCTAAGCTGACCCGATCACG-3' (SEQ ID NO.5), amplifying PRV-BC21, recovering enzyme, connecting to prokaryotic expression vector pET28a (+), screening single colony sequencing and screening positive plasmid;

2) the screened pET28a (+) -BC21 plasmid is transfected into escherichia coli BL21(DE3) competent cells, the cells are amplified and cultured in LB culture medium, the cells are cultured at 250rpm and 37 ℃ until OD600 is 0.5-0.8, and 100 mu L of IPTG solution with the concentration of 1.0M is added for induction overnight.

3) Centrifuging at 4 deg.C and 10000rpm for 15min, removing supernatant carefully in sterile operating platform, and extracting periplasmic protein from thallus precipitate with bacterial protein extraction kit (Clontech Technology) to obtain crude extract of protein. The crude protein extract was dialyzed overnight against equilibration buffer (50mM phosphate, 300mM sodium chloride, 20mM imidazole; pH 7.4).

4) Antibody purification using His60 nickel column (Clontech Technology): washing the nickel column with 10 column volumes of an equilibration buffer, introducing the dialyzed supernatant protein in the step (3) into a His60 nickel column (Clontech Technology) for antibody purification, washing the column with 10 column volumes of an elution buffer (50mM phosphate, 300mM sodium chloride, 40mM imidazole; pH 7.4), eluting the nanobody PRV-BC21 with 10 column volumes of an elution buffer (50mM phosphate, 300mM sodium chloride, 300mM imidazole; pH 7.4), collecting the eluate, filling into a dialysis bag, dialyzing with 0.01M phosphate buffer (pH 7.4) for 3-4 times, concentrating, packaging and storing at-20 ℃ for later use.

Example 3 detection of Properties of Nanobody PRV-BC21

The antibody specificity of the nanometer antibody PRV-BC21 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, drawing competitive ELISA curves of nano antibodies PRV-BC21 in sequence, calculating the standard product concentration when the inhibition rate is 50%, expressing the standard product concentration by IC50, and calculating the cross reaction rate according to the following calculation formula: the cross-reactivity rate (%) (nanobody PRV-BC21 IC 50/analog IC50) × 100%, giving nanobody PRV-BC21 a 50% inhibitory concentration IC50 to PRV of 1.03 ng/mL; the cross reaction rate with PRRSV, TGEV, PEDV, PPV, SFV, NDV, CDV and HEV is less than 0.1%. Therefore, the nano antibody PRV-BC21 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 4 Assembly of the PRV-BC21 colloidal gold test strip

(1) Taking the purified nanometer antibody PRV-BC21 protein, and dialyzing for 3-4 h for later use by using a phosphate buffer solution with the pH value of 7.4 and 0.01M;

(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 a nano antibody PRV-BC21 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 BSA of 0.1-0.5 wt%, sucrose of 2-4 wt% and pH7.2, repeating the step for 2-3 times, retaining precipitate, re-suspending the precipitate with 1/10 volume of the original colloidal gold, and standing at 4 deg.C;

(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) the PRV-BC fusion protein was diluted to 1mg/mL with 0.01mol/L, pH 7.2.2 phosphate buffer, and coated on a test line (T line) on a nitrocellulose membrane (NC membrane) with a Bio dot streaking apparatus in an amount of 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 16h for later use.

(8) According to the section structure of the test strip shown in the attached figure 5, the water absorption packing paper, the NC membrane, the gold pad and the sample chromatographic pad are sequentially adhered to the PVC bottom plate, wherein the gold pad is covered by the sample chromatographic pad from the starting end with an 1/3 area, the sample diffusion efficiency can be improved due to the increase of the interface, and the sample is favorably 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 NC membrane, the tail end of the NC membrane 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 NC 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 strip into small strips with proper width by a machine, placing the small strips in a specially-made card shell made of plastics, sealing the card shell with an aluminum foil bag, and storing the small strips in an environment at 4-30 ℃ until the validity period reaches 12 months.

Example 5 Property detection of PRV-BC21 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 are shown in figure 6, wherein the blood samples of the serum 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), a positive result can still be detected, and the sensitivity is very high (FIG. 7).

3) Stability test

4 ℃ storage stability test: and (2) sealing and packaging the prepared PRV-BC21 colloidal gold test strip and a drying agent together by using an aluminum foil bag, placing the packaged test strip and the drying agent in 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 a strip, the degree of placing a gold-labeled antibody 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 89 parts of the 108 samples are positive samples, and 19 parts of the 108 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 (SEQ ID NO.6) and a downstream primer RPRV GTCCACGCCCC GCTTGAAGCT (SEQ ID NO. 7). Reaction system (20 μ L): sample 1. mu.L, 1. mu.L of each of the forward and reverse primers, DNA-MIX 10. mu.L, ddH₂O7 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. As proved by PCR, 90 of 120 samples are positive samples, and 18 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-BC21 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-BC21 colloidal gold test strip is used, the pretreatment time of the sample is short, and the detection limit of the standard product 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> nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 125

<212> protein

<213> gB epitope polypeptide

<400> 1

ATPDGFSAEESLEEIDGAVSPGPSDAPDGEEGDLDARTAVRAVATERDRFYRLNPSEVATAALGQRVSARVLADVMAISRCVEVRGAVYVQNSMRVPGERGTCYSRPLVTFEHNGTAVIEGQLGD

<210> 2

<211> 120

<212> protein

<213> gC epitope polypeptide

<400> 2

STPEPVSGTTGAEASTPAAVSTPRVPAPSVSRRKPQRNGNRTRVHGDKATSHGRKPIVCRERAEHAGLLNVRSARPLSDLDGPVDYTCRLEGMPSVLPIFEDTQRYDASPTSESWPVVTS

<210> 3

<211> 136

<212> PROTEIN

<213> Nanobody PRV-BC21

<400> 3

EVQLVEAPRGVSPKGSLRLSCEWAGGKRPAMSWISPKPEKRLEYSDAGHPPARKILQETRYYKVGSSNATWYQQKPGGQSPLLAYRFVAPHSWYGVPDWGGQGEDSPAKTGVQQYGSYPLYWGAGQGTQVTVSSAA

<210> 4

<211> 23

<212> DNA

<213> primer F1

<400> 4

CATCGCTAGTTACACGTTATAGC

<210> 5

<211> 23

<212> DNA

<213> primer R1

<400> 5

AAGGCTAAGCTGACCCGATCACG

<210> 6

<211> 20

<212> DNA

<213> upstream primer FPRV

<400> 6

CAGGAGGACGAGCTGGGGCT

<210> 7

<211> 21

<212> DNA

<213> downstream primer RPRV

<400> 7

GTCCACGCCCC GCTTGAAGCT

Claims (7)

1. A nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity targets a preferred epitope fusion protein of gB protein and gC protein of PRV, is named as PRV-BC21, and has an amino acid sequence shown as SEQ ID No. 3.

2. The method for preparing the nanobody PRV-BC21 of claim 1, comprising the steps of:

(1) construction of recombinant eukaryotic expression vectors: connecting a DNA sequence of a coded nano antibody PRV-BC21 to an expression vector through PCR amplification and enzyme digestion to obtain a positive plasmid;

(2) and (3) transforming the positive plasmid in the step (1) into a host cell, and inducing and expressing the nano antibody PRV-BC 21.

3. The method according to claim 2, wherein the expression vector is eukaryotic or prokaryotic, and preferably the expression vector is eukaryotic and is selected from the group consisting of: pCRII, pCR3, and pcDNA3.1(Invitrogen, San Diego, CA), pB SII (Stratagene, La Jolla, CA), pET 15(Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N1(Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO90/14363), and pFastBacDual (Gibco-BRL, Grand Island, NY), and the like. The host cell is escherichia coli, yeast or eukaryotic cell, preferably the host cell is eukaryotic cell, and can be selected from, for example: CHO in Chinese hamster ovary cells, COS in monkey kidney cells, HEK-293 in human embryonic kidney cells, HELA in human cervical cancer cells, etc.

4. A test strip for rapidly detecting porcine pseudorabies virus is characterized by comprising a bottom plate, water-absorbing packing paper, an NC membrane, a gold pad and a sample chromatography pad, wherein the water-absorbing packing paper, the NC 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-BC21 marked by colloidal gold is arranged on the gold pad; the NC 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 a nano antibody PRV-BC21 marked by colloidal gold.

5. A method for preparing a test strip for rapidly detecting porcine pseudorabies virus,

1) purifying the nanometer antibody PRV-BC21, and dialyzing for later use;

2) the colloidal gold is labeled with a nano antibody PRV-BC 21;

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 an NC film;

7) drying at room temperature and low humidity; sequentially adhering absorbent paper, an NC 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.

6. The use of the test strip for rapid detection of porcine pseudorabies virus according to claim 4 in the rapid detection of non-disease diagnosis of porcine pseudorabies virus.

7. The use of the nanobody of claim 1 for the high-precision and sensitive identification of porcine pseudorabies virus for the rapid detection of non-disease diagnosis of porcine pseudorabies virus.

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