A nano-antibody with neutralizing activity against porcine delta coronavirus and its application

Abstract

The present invention provides a nano antibody with neutralizing activity against porcine delta coronavirus and its application. The present invention constructs a nano library of PDCoV S protein using phage display technology, and screens and obtains specific nano antibodies Nb1, Nb3 or Nb8 that have high affinity with PDCoV and S protein. Further, based on Nb3, a competitive ELISA method for rapid and efficient clinical detection of PDCoV antibodies was established. In addition, the results of in vivo and in vitro neutralization activity evaluation showed that the neutralization titer of Nb3 against PDCoV can reach 1:128. Oral administration of 5 mg / ml Nb3 to piglets with milk before and after the attack can significantly reduce the incidence of diarrhea in piglets and protect 3 / 5 of the piglets from the attack of strong strains of PDCoV. Therefore, the anti-PDCoV specific nano antibody Nb3 screened by the present invention will provide a technical and material support for the clinical prevention and control of PDCoV.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a nano antibody with neutralizing activity against porcine delta coronavirus. The present invention also relates to the use of the nano antibody in establishing a porcine delta coronavirus diagnostic method and preparing an oral anti-porcine delta coronavirus drug. Background Art

[0002] Porcine Deltacoronavirus (PDCoV) is one of the important pathogens that cause viral diarrhea in piglets at home and abroad. PDCoV is a single-stranded positive-strand RNA virus belonging to the Coronaviridae family and the Deltacoronavirus genus. The virus particles are spherical, with a diameter of about 100nm, an envelope and many protruding fibers at the end. The genome size is about 25.4kb, making it the smallest member of the coronavirus discovered so far. PDCoV contains 5'UTR, 3'UTR and 8 open reading frames (ORFs), which encode two polyproteins pp1a and pp1b, four structural proteins S (spike glycoprotein), E (envelopeprotein), M (membrane protein), N (nucleoprotein protein) and two non-structural proteins NS6 and NS7. The coronavirus S protein is a type I membrane glycoprotein that exists in the form of spikes on the surface of virus particles. It can be hydrolyzed by host proteases into two subunits, S1 and S2. The S1 subunit can recognize and bind to host cell receptors, and the S2 subunit amino terminus contains a membrane fusion peptide that mediates the membrane fusion of virus particles and host cells. Therefore, the S protein is the key protein for PDCoV to invade host cells. It plays a key role in viral virulence, species, tissue tropism, and inducing the host body to produce neutralizing antibodies. It is the preferred target antigen for vaccine development.

[0003] Since it was first discovered and reported in 2012, PDCoV has been reported worldwide to seriously endanger the pig farming industry in many countries. my country first reported the presence of PDCoV in domestic pig farms in 2015, and traceability tests found that the virus had existed as early as 2004. Subsequently, relevant research reports have shown that PDCoV exists in pig herds in most provinces in my country, including Taiwan, and seriously threatens the safety of pig farming in my country. PDCoV can infect pigs of different ages and show clinical symptoms such as diarrhea, vomiting, and dehydration similar to PEDV. Many relevant research reports and the applicant's research results show that the current positive rate of PDCoV in diarrhea piglets is second only to porcine epidemic diarrhea virus (PEDV). At present, the detection and diagnosis of the disease still mainly rely on conventional laboratory diagnostic methods, including virus isolation and identification, cytopathic observation, real-time fluorescence quantitative PCR, indirect fluorescent antibodies, immunohistochemistry, in situ hybridization and other technologies. However, these traditional technical means are not only time-consuming and labor-intensive, but also have low specificity and sensitivity, and require professional technicians and special experimental instruments. Serological detection methods are also commonly used for the diagnosis of viral infections. The combination of serological methods and pathogen-based detection technologies can complement each other and improve the accuracy of detection. Especially for latently infectious animals, ELISA is considered to be the most sensitive and specific method for serum antibody detection. However, there is currently no commercially available ELISA detection kit for specific antibodies against porcine delta coronavirus S protein. Therefore, the establishment of a fast, sensitive and specific porcine delta coronavirus S protein antibody detection method is of great significance for the clinical diagnosis and immune prevention and control of PDCoV.

[0004] Camelids have a type of heavy-chain-only antibodies (HCAbs) that naturally lack the constant regions of the light chain and heavy chain CH1. It is composed of the constant regions of the heavy chain CH2 and CH3, the hinge region, and the variable region of the heavy chain (VHH). Cloning VHH can produce a single-domain antibody (sdAb) consisting only of the variable region of the heavy chain. Its crystal diameter is 2.5nm, its length is 4nm, and its relative molecular mass is only about 15kDa. Therefore, it is also called a nanobody (Nb). It is the smallest fragment that can bind to an antigen and retains complete antigen binding ability. The CDR1 and CDR3 of Nb are slightly longer than those of traditional monoclonal antibody VH, and can penetrate deep into the interior of the antigen. Compared with the flat or concave antigen binding structure region of traditional monoclonal antibodies, Nb can better bind to the antigen. Nb has a simple structure and is easy to express in large quantities in microbial expression systems such as Escherichia coli and yeast, and can be industrially mass-produced. In addition, Nb is easy to modify and has the characteristics of low immunogenicity, high stability, high specificity, and high affinity, so it has broad application prospects. It has received extensive attention in the fields of human disease diagnosis, treatment, and research, and has been clinically applied in the treatment of new coronaviruses. However, up to now, no specific nanoantibodies against the S protein of porcine delta coronavirus have been reported. Summary of the invention

[0005] Based on the above, the purpose of the present invention is to provide an anti-swine delta coronavirus neutralizing active nanoantibody with low immunogenicity, high stability, high specificity, high affinity, etc., which can be industrially produced for the clinical diagnosis and immune prevention and control of PDCoV.

[0006] Another object of the present invention is to provide the use of the nanobody in establishing a competitive ELISA method for detecting PDCoV antibodies and in preparing anti-swine delta coronavirus drugs.

[0007] To achieve its purpose, the present invention adopts the following technical solution:

[0008] The present invention provides a nano antibody with neutralizing activity against porcine delta coronavirus, which is labeled as Nb1, Nb3 or Nb8, and its amino acid sequence is shown as SEQ ID No.1-SEQ ID No.3 respectively; preferably Nb3.

[0009] The present invention provides a competitive ELISA method for detecting PDCoV antibodies, comprising the following steps:

[0010] (1) labeling the nanobody Nb3 with HRP and diluting it with serum diluent at a volume ratio of 1:20000 to obtain an enzyme-labeled antibody working solution;

[0011] (2) Dilute the PDCoV S protein to 2 μg / mL with carbonate buffer, then add 100 μL / well to the ELISA plate and place it in a 37°C constant temperature incubator for coating for 2 h; after coating, wash with PBST buffer and pat dry; add 100 μL / well of ELISA plate stabilizer to each well, block at 37°C for 2 h, discard the blocking solution, gently pat dry, and dry at 37°C for 2 h;

[0012] (3) Add 25 μL of the serum sample to be tested to each well of the serum dilution plate, and set up two wells of negative serum and positive serum as controls, 25 μL / well; then add 50 μL of the enzyme-labeled antibody working solution in step (1) to each well, shake and mix, then transfer to each well of the enzyme-labeled reaction plate with a dispenser, and incubate at 37°C for 2 h;

[0013] (4) Shake dry the plate, wash five times with 1x PBST, pat dry, add 50 μL of TMB colorimetric reagent A solution to each well, then add 50 μL of TMB colorimetric reagent B solution, and incubate at 37°C in the dark for 10 min. Finally, add 100 μL of stop solution to each well to terminate the reaction, and read the OD value using a microplate reader. 450nm The blocking rate PI was calculated according to the formula PI = (1-positive serum OD450nm / negative serum OD450nm) × 100%. When the sample PI value was ≥50.02%, it was PDCoV positive; when the sample PI value was ≤40.29%, it was PDCoV negative; and when the sample PI value was between 40.29% and 50.02%, it was suspicious.

[0014] Furthermore, in step (2), the ELISA plate stabilizer is a protein-free rapid blocking solution.

[0015] Furthermore, in step (4), the stop solution is 2M H2SO4.

[0016] The anti-swine delta coronavirus neutralizing activity nanoantibodies provided by the present invention can be used to prepare a porcine delta coronavirus detection reagent or a detection kit.

[0017] Similarly, the anti-swine delta coronavirus neutralizing activity nanobodies provided by the present invention can be used to prepare drugs for preventing or treating porcine delta coronavirus infection; preferably oral drugs.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses phage display technology to construct a specific nano antibody library for PDCoV S protein, and further screens and obtains a specific nano antibody Nb3 with high affinity to PDCoV and S protein. Further, based on the nano antibodies obtained by the above screening, a competitive ELISA method for rapid and efficient clinical detection of PDCoV antibodies was established. This method has the characteristics of low cost, high sensitivity and strong specificity. In addition, the results of in vitro and in vivo neutralization activity evaluation showed that the neutralization titer of nano antibody Nb3 against PDCoV can reach 1:128. Oral administration of 5 mg / ml Nb3 to piglets with milk before and after the attack can significantly reduce the incidence of diarrhea in piglets and protect 3 / 5 of the piglets from the attack of strong strains of PDCoV. In general, the anti-PDCoV specific nano antibody Nb3 screened by the present invention will provide a new technical and material support for the clinical prevention and control of PDCoV in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression, purification and western blot results of the PDCoV S recombinant protein in Example 1 are as follows: A is the expression and purification result; B is the western blot result;

[0021] Figure 2 The agarose gel electrophoresis detection results of the PCR products in Example 2: A is the agarose gel electrophoresis detection result of the VHH fragment amplification product; B is the agarose gel electrophoresis detection result of the VHH gene library fragment amplification product;

[0022] Figure 3 The results of the ampicillin-resistant plate counts in Example 2 are as follows;

[0023] Figure 4 The sequencing results of 30 monoclonal clones in Example 2;

[0024] Figure 5 The amino acid sequence comparison analysis results of the VHH gene library fragments in Example 2;

[0025] Figure 6 is the neighbor-joining evolutionary tree of the VHH gene library fragments in Example 2;

[0026] Figure 7 The results of ELISA screening of phages in Example 3;

[0027] Figure 8 The results of the ELISA retest of the positive clones in Example 3 are as follows;

[0028] Fig. 9 The results of soluble ELISA screening in Example 3;

[0029] Fig.10 The SDS-PAGE test results after the induction expression of the nanobody in Example 4;

[0030] Fig.11 The SDS-PAGE test results of Nanobody Nb3 after Ni column affinity chromatography purification in Example 4;

[0031] Fig.12 The specific detection result of Nanobody Nb3 in Example 4;

[0032] Fig.13 The immunofluorescence test results of the pig serum samples in Example 5;

[0033] Fig.14 The specificity and sensitivity test results of the competitive ELISA method of the present invention in Example 5: A is the specificity test result; B is the sensitivity test result;

[0034] Fig.15 The results of the neutralization titer determination and challenge test of Nanobody Nb3 in Example 6: A is the neutralization titer determination result; B is the challenge test result. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments with reference to the accompanying drawings, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and these modifications and replacements all fall within the protection scope of the present invention.

[0036] The materials and sources used in the embodiments of the present invention are as follows:

[0037] ExpiCHO-S cells, expression medium and transfection reagents were purchased from Thermo Fisher Scientific; pcDNA3.1(+) expression vector, pComb3XTT, E. coli TG1, Helper phage M13KO7 and other experimental materials were preserved by the National Key Laboratory of Animal Disease Prevention and Control, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HRP labeling kit (BF06095S) was purchased from Biolong Biotechnology Co., Ltd.; BCA protein concentration determination kit (T9300A) was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., and gel recovery kit was purchased from Omega Bio-Tek.

[0038] The applicant's laboratory stored 300 pig serum samples, including 120 PDCoV-infected piglet sera, 80 inactivated PDCoV immune sera and 100 PDCoV-uninfected and non-immune sera.

[0039] Positive sera for porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine respiratory and reproductive syndrome virus (PRRSV) and porcine circovirus (PCV2) are stored by the applicant's laboratory.

[0040] The alpacas used in the present invention were from a farm in Kangle County, Gansu Province, China. Alpaca IgG (H+L) HRP was purchased from Jackson ImmunoResearch.

[0041] Example 1: Expression, purification and western blot detection of PDCoV S recombinant protein

[0042] Expression of PDCoV S recombinant protein and purification by nickel column affinity chromatography:

[0043] Referring to the literature (Yu R, Zhang L, Zhou P, Zhang Z, Liu X, Wang Y, Guo H, Pan L, Liu X. Evaluation of the immunoprotective effects of porcine deltacoronavirus subunit vaccines. Virology. 2024 Feb; 590: 109955. doi: 10.1016 / j.virol.2023.109955. Epub 2023 Dec 2. PMID: 38070302.), the pCDNA3.1 (+) -PDCoV-S recombinant plasmid was transfected into ExpiCHO-S cells and cultured in an incubator at 32 ° C with 5% CO2 in air for 10 days. The culture solution was then centrifuged at 4000 r / min for 30 min using a refrigerated centrifuge, and the supernatant was filtered with a 0.22 μm filter. The filtered supernatant was bound to a Ni-NTA affinity chromatography column (Qiagen, Germany) at 4°C for 2 h, and then non-specifically bound impurities were eluted with 1×PBS, and the target protein was eluted with 1×PBS containing 500 mM imidazole. The high-purity protein samples were collected and the protein concentration was determined using a BCA protein concentration assay kit. The samples were diluted to 1 mg / ml, filtered with a 0.22 μm filter, and aseptically packaged and stored at -80°C for later use.

[0044] After the purified protein sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie Brilliant Blue staining revealed a clear target protein band between 150 kDa and 250 kDa. The protein size was consistent with expectations and the purity was high, with almost no impurities ( Figure 1A).

[0045] Western blot detection of PDCoV S recombinant protein:

[0046] To further determine the specificity of the recombinant protein, it was transferred to a nitrocellulose membrane (Pall, USA) for immunoblotting (western blot) analysis, using Mouse Anti-PDCoV-S mAb (1:5000 dilution, Zhongshan Jinqiao, Beijing, China) as the primary antibody, HRP-labeled goat anti-mouse IgG (1:10000 dilution, Zhongshan Jinqiao, Beijing, China) as the secondary antibody, and developed with a chemiluminescent substrate (ECL, Thermo Scientific, USA). The results showed that the expressed and purified PDCoV-S recombinant protein can be specifically recognized by Mouse Anti-PDCoV-S mAb ( Figure 1 B).

[0047] Example 2: Construction of phage display VHH library

[0048] Alpacas immunized with PDCoV S protein:

[0049] The present invention adopts the general standard immunization scheme of protein antigens to immunize two 2-year-old alpacas (Alpaca). Specifically, blood was collected before immunization to prepare 2mL of alpaca negative serum. For the first immunization, Freund's complete adjuvant was mixed with PDCoV-S antigen (1mg) 1:1, and multiple subcutaneous injections were performed after emulsification. Three booster immunizations were performed on the 21st, 42nd and 63rd days after the first immunization. During the booster immunization, PDCoV-S antigen (0.5mg) was mixed with Freund's incomplete adjuvant in equal amounts and injected. After the booster immunization was completed, the indirect ELISA method detected the IgG (H+L) titer against PDCoV-S in the alpaca serum to be 1:9841500, indicating that the alpaca produced a strong immune response and met the conditions for the next step of library construction.

[0050] VHH library construction:

[0051] On the 70th day after the first immunization, 50 mL of alpaca peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were separated. The total RNA of PBMCs was extracted using TRIzol, and further reverse transcribed to obtain cDNA for the construction of the nanoantibody library. Reference literature (Ma Z, Wang T, Li Z, Guo X, Tian Y, Li Y, XiaoS. A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus. JNanobiotechnology. 2019Sep 16;17(1):96.doi:10.1186 / s12951-019-0531-x.PMID:31526383;PMCID:PMC6745792;Zhao Y,Yang J,Niu Q,Wang J,Jing M,Guan G,Liu M,Luo J,Yin H,LiuZ.Identification and Characterization of Nanobodies from aPhage DisplayLibrary and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus.J Clin Microbiol.2023Jun20;61(6):e0119722.doi:10.1128 / jcm.01197-22.Epub 2023May 8.PMID:37154731;PMCID:PMC10281114.) The cDNA was used as a template and the alpaca VHH primers were used for polymerase chain reaction (PCR) to amplify the VHH fragment. The PCR product was subjected to 1% agarose gel electrophoresis. The results were as follows: Figure 2 As shown in A.

[0052] Figure 2 A results show that the PCR product has two bands. The molecular weight of the target band is about 500-750bp (indicated by the arrow in the figure), and there is also a non-target band (traditional antibody fragment) at 900bp.

[0053] The target fragment (500-750 bp size band) was purified and recovered by gel excision using a gel recovery kit. The recovered product was used as a template for PCR and the VHH gene library fragment was amplified using the aforementioned alpaca VHH primers. The results are as follows: Figure 2 As shown in B.

[0054] Figure 2 B The result showed that the PCR amplification obtained a target band with a size consistent with the expected size (about 400 bp).

[0055] The Spe I restriction site was introduced at the 3′ end of the VHH gene library fragment, and the Sac I restriction site was introduced at the 5′ end. The phage display vector pComb3XTT after restriction digestion was ligated with the VHH gene library fragment. After purification, the ligation product was electrotransformed into Escherichia coli TG1 to obtain 10 mL of transformation product. 10 μL was taken for 10-fold gradient dilution, and 10 -4 , 10 -5 and 10 -6 A total of three gradients were used to count the ampicillin-resistant plates to estimate the library capacity (library capacity = number of clones × dilution factor × total volume of transformation product (mL)). Figure 3 As shown, the average storage capacity is: (300×10×10 4+2 +29×10×10 5+2 )=2.95×10 9 .

[0056] The next day, 30 single clones were randomly picked from the gradient dilution plate of the library and sequenced using the forward sequencing primer pComb3XTT-F. A total of 29 valid sequencing clones were obtained (no band in electrophoresis No. 1) ( Figure 4 ). The amino acid sequences of the VHH gene library fragments were compared and analyzed, and the results showed that 28 of the 29 clones contained antibody fragments with the correct reading frame ( Figure 5 The total accuracy of the library is about 28÷30×100%=93.3%. The neighbor-joining tree of the VHH gene library fragments was constructed using MEGA software. The results are as follows: Figure 6 As shown, it can be seen that the VHH gene library fragments have good diversity and the quality of the library meets the relevant requirements.

[0057] Example 3: Screening and identification of PDCoV-S protein-specific nanoantibody library

[0058] Screening of recombinant phage specific for PDCoV-S protein

[0059] Purified PDCoV-S protein was used as a coating antigen to screen specific recombinant phages. During the screening process, the enrichment effect of specific VHH recombinant phages was evaluated by detecting the titer of recombinant phages in each round of elution. Specifically, the PDCoV-S protein was diluted to 100 μg / mL with carbonate buffer, added to a 96-well ELISA plate at 100 μL / well, and placed at 4°C for overnight coating; the next day, the coating solution in the well was discarded, 200 μL of blocking solution was added to each well, and the plate was blocked at 37°C for 2 hours; the liquid in the well was discarded, the plate was washed 5 times with PBST, and then 100 μL of the diluted phage library (diluted to 5×10 11 pfu / ml), incubate at 37℃ for 1h; discard the liquid in the wells, wash the plate 10 times with PBST, then add 100μL 0.1M triethylamine solution to each well, incubate at room temperature for 10min, aspirate the eluate in the wells and neutralize with an equal volume of 1M Tris-HCl (PH 7.4). Next, take a small amount of eluate for phage titer determination and output calculation, and at the same time, infect the remaining eluted phage for amplification culture and carry out the next round of screening, and repeat 3 rounds of screening.

[0060] The number of phages added before each round of screening was recorded as Input, the amount of phages harvested from the positive enzyme-labeled wells after screening was recorded as P, and the amount of phages harvested from the negative control wells was recorded as N. P / Input represents the recovery rate, and the higher the P / N value, the higher the proportion of specific phages. The results showed that during the three rounds of screening, the phage recovery rate increased round by round. After the third round of screening, the P / N value could reach 7500, indicating that the specific recombinant phages were significantly enriched (Table 1).

[0061] Table 1 Enrichment results of nanoantibodies against PDCoV-S protein-specific phages during the three rounds of screening

[0062]

[0063] Phage ELISA screening:

[0064] 940 single clones were randomly selected from the bacterial plate after the third round of screening. After amplification and induction, they were centrifuged at 5000r / min for 10 minutes. 600μL of the supernatant was carefully aspirated and dispensed into a 1.5mL centrifuge tube for phage enzyme-linked immunosorbent assay. The specific operation is as follows:

[0065] 96-well ELISA plates were coated with PDCoV-S protein (10 ng / well) at 4°C overnight, blocked with 2% skim milk powder at 37°C for 1 h, 100 μL of phage supernatant was added to each well, incubated at 37°C for 1 h, washed 3 times with PBST, 100 μL of HRP-labeled anti-M13 antibody (1:15000) was added to each well, incubated at 37°C for 1 h, washed 3 times with PBST; then 100 μL of TMB colorimetric solution was added to each well, color developed at 37°C for 15 min, then 100 μL of stop solution (2M H2SO4) was added to each well, and the absorbance value (OD) at 450 nm was measured by an ELISA instrument. 450nm A total of 153 positive clones were screened and obtained ( Figure 7 ).

[0066] The above positive clones were selected and verified by enzyme-linked immunosorbent assay with PDCoV-S protein, PDCoV and multi-tag protein (containing HA and His tags), and finally 22 positive clones were obtained ( Figure 8 ).

[0067] Soluble ELISA Screening:

[0068] The 22 positive clones obtained by screening were amplified by PCR and sequenced, and a total of 14 unique sequences were obtained. The 14 unique sequences (in E.coli TG1) were induced by IPTG at 37°C, the cells were collected after centrifugation, the periplasmic soluble proteins were extracted, and the enzyme-linked immunosorbent assay was performed:

[0069] 96-well ELISA plates were coated with PDCoV-S protein, PDCoV or His-tagged protein at 4°C overnight, blocked with 2% skim milk powder at 37°C for 1h, 100 μL periplasmic supernatant was added to each well, incubated at 37°C for 1h, washed 3 times with PBST, 100 μL HRP-labeled Anti-HA Tag Antibody (1:4000) was added to each well, incubated at 37°C for 1h, washed 3 times with PBST; then 100 μL TMB colorimetric solution was added to each well, color developed at 37°C for 15min, then 100 μL stop solution (2M H2SO4) was added to each well, and the absorbance value (OD) at 450nm was measured by an ELISA reader. 450nm Finally, three positive cloned nanoantibodies Nb1, Nb3, and Nb8 were obtained, which bound to both the antigen PDCoV-S and the virus ( Fig. 9 ).

[0070] Example 4: Expression, purification and specificity analysis of Nb3

[0071] Expression and purification of nanobodies:

[0072] The VHH genes of Nb1, Nb3, and Nb8 were subcloned into the pCold II expression vector. After induction of expression, SDS-PAGE showed that the molecular weights of the three purified nanobodies were consistent with the expected molecular weight ( Fig.10 ).

[0073] Transform the nanobodies Nb1, Nb3, and Nb8 into competent E. coli BL21 (DE3) cells:

[0074] Pick a single colony and inoculate it into 5-10mL LB liquid medium containing 100μg / mL ampicillin, and shake and culture it at 37℃, 220r / min overnight. The next day, inoculate it into 500mL LB medium at 1:100, shake and culture it at 37℃, 220r / min for 4-5h until the D600 value reaches 1.0, then take out the culture bottle and put it in ice water for 30min, the purpose is to cool the culture medium to about 15℃. Then add IPTG with a final concentration of 0.5mM to the culture medium, and induce culture in a 15℃ shaker for 24h. After 24h, collect the bacterial precipitate, resuspend the collected bacterial precipitate with PBS (20mL PBS / g bacterial wet weight), and then perform ultrasonic disruption (300W, 3s, 3s, 40min, ice water bath). After the ultrasonication is completed, centrifuge at 1000r / min for 30min and collect the supernatant. The results showed that Nb3 was expressed in a soluble form in the supernatant, while Nb1 and Nb8 were expressed in the form of insoluble inclusion bodies.

[0075] The present invention selected Nb3 expressed in a soluble form for further testing, and Nb3 was purified by Ni column affinity chromatography (the purification steps of Nb3 were consistent with the purification steps of S protein) and SDS-PAGE detection. The results showed that the expression level of Nb3 was high and the purity was good ( Fig.11 ).

[0076] Specificity and antibody titer detection of nanobodies:

[0077] The purified nanobody Nb3 was labeled with HRP using the Biolon (BF06095S) HRP labeling kit, and the specificity and potency of Nb3-HRP were further determined by the indirect ELISA method. The specific operation is as follows:

[0078] Potential cross-reactivity detection was performed with 5 different viral proteins (PDCoV-S, PDCoV-N, PEDV-S, PEDV-N and His-tag protein). In brief, the five proteins were coated in equal amounts on a 96-well ELISA plate and incubated overnight at 4°C. The plate was blocked with 5% BSA. After blocking, the HRP-labeled nanoantibodies were diluted to different concentrations (4000ng / mL, 400ng / mL, 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL) and incubated with the antigen at 37°C for 1h. The plate was washed 5 times with PBST, 100μL of TMB solution was added to each well, and incubated at 37°C for 15min. Then 100μL of 2M H2SO4 was added to each well to terminate the reaction, and the absorbance at 450nm was read on an ELISA reader. The results showed that Nb3 only reacted with PDCoV-S protein, with good specificity and a titer as high as 1:160000 ( Fig.12 ).

[0079] Example 5: Establishment of a competitive ELISA method for detecting PDCoV antibodies

[0080] Establishment of competitive ELISA method:

[0081] The present invention establishes a competitive ELISA method for detecting PDCoV antibodies using Nb3-HRP and optimizes the method. Specifically, the square array titration method was used to screen and determine the optimal conditions for the ELISA method, and the antigen coating concentration (10, 5, 2.5, 2.0, 1.0, 0.5 and 0.2 μg / mL, respectively), antigen coating conditions (37°C 1h, 37°C 2h, 4°C 12h, respectively); blocking conditions (37°C 1h, 37°C 2h, 4°C 12h, respectively); HRP-Nb dilution (1:2500, 1:5000, 1:10000, 1:15000, 1:20000, 1:40000); competition reaction time (37°C for 30min, 60min, 90min, 120min, respectively); TMB color development time (37°C for 5min, 10min and 15min), etc. were optimized, and the reaction conditions with a large blocking rate and a positive and negative value within a reasonable range were selected as the optimal reaction conditions for the ELISA method. Blocking rate PI = (1 – positive serum OD 450nm / Negative serum OD 450nm )×100%.

[0082] The optimal operating procedure for this ELISA method is as follows:

[0083] First, the S protein was diluted to 2 μg / mL with carbonate buffer, and then 100 μL of the diluted S protein solution was added to each well of the ELISA plate, and the plate was placed in a 37°C constant temperature incubator for 2 hours. Then, the plate was washed 3 times with PBST and patted dry; 100 μL / well of ELISA plate stabilizer (protein-free rapid blocking solution) was added to the wells, and the plate was blocked at 37°C for 2 hours, the blocking solution was discarded, the plate was patted dry, and the plate was dried at 37°C for 2 hours. Add 25 μL of the sample to be tested to each well of the serum dilution plate, set up two wells of negative serum and positive serum as controls, 25 μL / well; then add 50 μL of enzyme-labeled antibody working solution (HRP-Nb dilution 1:20000) to each well; shake and mix the serum dilution plate for 5 minutes, then use a spray gun to transfer the mixed solution to the enzyme-labeled reaction plate, and incubate at 37°C for 2 hours; take out the enzyme-labeled reaction plate, spin dry, wash it with 1x PBST 5 times, pat dry on absorbent paper, add 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color developer A solution to each well, and then add 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color developer B solution, and stand at 37°C in the dark for 10 minutes; finally, add 100 μL of stop solution to each well to terminate the reaction, and read the OD with an enzyme reader 450nm value.

[0084] Immunofluorescence assay (IFA):

[0085] Since there is no commercial kit for PDCoV antibody detection, the present invention uses indirect immunofluorescence to determine the positive and negative of pig serum PDCoV antibodies. Specifically, PDCoV (MOI = 0.01) was placed in a 96-well plate filled with 80% LLC-PK1 cells. After 24 hours, the culture medium was discarded and washed three times with PBS. 4% paraformaldehyde was fixed at 4°C for 1 hour, 0.25% Triton-100 was permeabilized at room temperature for 10 minutes, and then blocked with 5% BSA for 1 hour. Pig serum samples and 488-labeled goat anti-pig IgG were used as primary and secondary antibodies, respectively. 4′, 6-diamidino-2-phenylindole was stained in the dark for 5 minutes, and observed under a fluorescence microscope after washing 3 times with PBS.

[0086] The above method was used to test 300 pig serum samples stored in the applicant's laboratory. Fig.13 As shown, a, b and c represent PDCoV strong positive sera, which are generally serum samples 28 days after infection or 14 days after the second immunization; d, e and f represent PDCoV weak positive sera, most of which are serum samples 14 days after infection or 14 days after the first immunization; g, h and i represent PDCoV negative sera, which are basically uninfected and unimmunized serum samples. Finally, a total of 94 PDCoV negative sera and 206 PDCoV positive sera were determined by IFA testing (Table 2).

[0087] Table 2 Results of immunofluorescence test of pig serum

[0088]

[0089] Determination of critical value:

[0090] 94 PDCoV negative sera (negative by IFA test) were detected by optimized competitive ELISA method, and each sample was repeated twice to calculate the PI value. The mean (X) and standard deviation (SD) of the 94 serum PI values ​​were calculated. When the sample PI value ≥X+3SD, it was judged as positive; when the sample PI value ≤X+2SD, it was judged as negative; when X+2SD<sample PI value<X+3SD, it was judged as suspicious. Specifically, it is defined as positive when the sample PI value ≥50%; negative when the sample PI value ≤40%; and suspicious when the sample PI value is between 40%-50%.

[0091] Competitive ELISA method specificity, sensitivity and repeatability verification:

[0092] Specificity verification: The competitive ELISA method established by the present invention was used to detect common swine disease positive sera stored in the applicant's laboratory, including porcine epidemic diarrhea virus (PEDV) positive serum, porcine transmissible gastroenteritis virus (TGEV) positive serum, porcine rotavirus (PoRV) positive serum, porcine pseudorabies virus (PRV) positive serum, classical swine fever virus (CSFV) positive serum, porcine respiratory and reproductive syndrome virus (PRRSV) positive serum and porcine circovirus (PCV2) positive serum. Three replicates were made for each sample, and a PDCoV positive control was set at the same time to verify the specificity of the method.

[0093] The results showed that the above positive serum PI values ​​were all less than 40% ( Fig.14 A), showing PDCoV negative, indicating that the competitive ELISA detection method established by the present invention has good specificity.

[0094] Sensitivity verification: Three PDCoV-positive sera were diluted according to the ratios of 1:3, 1:7, 1:15, 1:31, 1:63, 1:127 and 1:255, and detected by the competitive ELISA method established by the present invention. Three replicate wells and a negative control were set to analyze the sensitivity of the method.

[0095] The results showed that the three positive sera were still positive (PI value ≥ 50%) when diluted 1:15, 1:15 and 1:127 respectively. Fig.14 B), indicating that the competitive ELISA detection method established by the present invention has high sensitivity.

[0096] Intra-batch repeatability test: The purified PDCoV-S protein was coated with carbonate coating buffer, and one ELISA plate was coated. Six pig sera with clear background were randomly selected for testing. Three replicate wells were set for each serum sample, and six repeatability tests were performed; inter-batch repeatability test: Three ELISA plates were coated at different times, and cELISA tests were performed on six serum samples. Three replicate wells were set for each serum sample.

[0097] The results showed that the coefficients of variation within and between batches were less than 10% (Table 3), indicating that the competitive ELISA detection method established in the present invention has good repeatability.

[0098] Table 3 Intra-batch and inter-batch repeatability test results of the competitive ELISA method of the present invention

[0099]

[0100] Example 6: Evaluation of the neutralization activity of nanobodies in vivo and in vitro

[0101] In order to evaluate the neutralizing activity of anti-PDCoV nanoantibody Nb3 on cells in vitro, the nanoantibody Nb3 was serially diluted and the 50% tissue culture infectious dose (TCID 50 / mL). The specific operation is as follows:

[0102] The purified nanobody Nb3 was diluted serially in a 96-well plate with culture medium. Then, an equal volume of virus solution (200 TCID 50 ) and placed in a 37°C 5% CO2 incubator for 1 hour. Then, 100 μl of the serum-virus mixture was added to the 96-well plate covered with 70-80% pig kidney cells, incubated in a 37°C, 5% CO2 incubator for one hour, and then the liquid in the 96-well plate was discarded, washed three times with PBS, and 200 μl of maintenance culture medium (containing 20 μg / ml trypsin) was added to each well. Then, the cytopathic effect (CPE) was observed and recorded for 5-7 days. The neutralizing antibody titer was calculated using the Reed-Muench method. The neutralizing antibody titer is the highest dilution multiple of the serum that can protect 50% of the cells from pathological changes. The results showed that the neutralizing titer of Nb3 can reach 1:128 ( Fig.15 A).

[0103] Furthermore, in order to evaluate the protective efficacy of anti-PDCoV nanoantibody Nb3 on suckling piglets after oral administration, the nanoantibody Nb3 was orally inoculated with milk into 4-day-old piglets before and after the virus attack, and the clinical symptoms and protection rate of 0-7dpi were recorded and observed. The results showed that oral administration of 5mg / ml Nb3 to piglets with milk before and after the virus attack could significantly reduce the incidence of diarrhea in piglets and protect 3 / 5 piglets (Table 4 results show that 2 / 5 piglets in the oral Nb3 group developed the disease after the virus attack, while 5 / 5 piglets in the negative control group (Mock group) developed the disease) from the attack of the strong strain of PDCoV ( Fig.15 B).

[0104] Table 4 Disease status of piglets from 0 to 7 days after infection

[0105]

[0106] Piglet disease determination criteria: When the diarrhea score is ≥1 and the CT value is <30, it is determined to be an illness.

Claims

1. A nanobody with neutralizing activity against porcine delta coronavirus, characterized in that: The nanobody is Nb3, and its amino acid sequence is shown in SEQ ID No.

2.

2. A competitive ELISA method for detecting PDCoV antibodies, characterized in that The following steps are involved: (1) The nanobody Nb3 in claim 1 is labeled with HRP and diluted with serum at a volume ratio of 1:20000 to obtain an enzyme-labeled antibody working solution; (2) Dilute the PDCoV S protein to 2 μg / mL with carbonate buffer, then add 100 μL / well to the ELISA plate and place it in a 37°C constant temperature incubator for 2 h; after coating, wash with PBST buffer and pat dry; add 100 μL / well of ELISA plate stabilizer to each well, block at 37°C for 2 h, discard the blocking solution, gently pat dry, and dry at 37°C for 2 h; (3) Add 25 μL of the serum sample to be tested to each well of the serum dilution plate, and set up two wells of negative serum and positive serum as controls, 25 μL / well; then add 50 μL of the enzyme-labeled antibody working solution in step (1) to each well, shake and mix, then transfer to each well of the enzyme-labeled reaction plate with a dispenser and incubate at 37°C for 2 h; (4) Shake dry the ELISA reaction plate, wash it five times with 1x PBST, pat it dry, add 50 μL of TMB colorimetric reagent A solution to each well, then add 50 μL of TMB colorimetric reagent B solution, and incubate at 37°C in the dark for 10 min. Finally, add 100 μL of stop solution to each well to terminate the reaction, and read the OD value using an ELISA reader. 450nm Value, according to the formula PI=(1-positive serum OD 450nm / Negative serum OD 450nm )×100% was used to calculate the blocking rate PI. When the sample PI value was ≥50.02%, it was PDCoV positive; when the sample PI value was ≤40.29%, it was PDCoV negative; and when the sample PI value was between 40.29% and 50.02%, it was suspicious.

3. A competitive ELISA method for detecting PDCoV antibodies as claimed in claim 2, characterized in that In step (2), the ELISA plate stabilizer is a protein-free rapid blocking solution.

4. A competitive ELISA method for detecting PDCoV antibodies as described in claim 2 or 3, characterized in that In step (4), the stop solution is 2M H2SO4.

5. Use of a nano antibody with neutralizing activity against porcine delta coronavirus as claimed in claim 1 in the preparation of a porcine delta coronavirus detection reagent or kit.

6. Use of a nanobody with neutralizing activity against porcine delta coronavirus as claimed in claim 1 in the preparation of a drug for preventing or treating porcine delta coronavirus infection.

7. Use of a nanobody with neutralizing activity against porcine delta coronavirus according to claim 6 in the preparation of a drug for preventing or treating porcine delta coronavirus infection, characterized in that: The medicine is an oral medicine.

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