Nano antibody with PEDV (porcine epidemic diarrhea virus) neutralizing activity and application thereof

Abstract

The invention discloses a nano antibody with PEDV (porcine epidemic diarrhea virus) neutralizing activity and application of the nano antibody. The invention relates to the technical field of biological medicines, and discloses a nano antibody NbH2 with PEDV (porcine epidemic diarrhea virus) neutralizing activity, which is obtained from a natural nano antibody library of camelidae animals through an escherichia coli prokaryotic expression system and a phage display technology on the basis of S1 protein of the PEDV, is a VHH antibody, has an amino acid sequence shown as SEQ ID NO.1, and has a nucleotide sequence shown as SEQ ID NO.2. A nucleotide sequence for coding the nano-antibody is shown as SEQ ID NO.2. The nano-antibody has good PEDV neutralizing activity, has a 100% neutralizing effect on PEDV epidemic strains at a dilution degree of 1: 2, can be used for PEDV detection, can also be used as a therapeutic antibody, can be used for preparing PED therapeutic drugs, has important significance in development of antiviral drugs, and has wide application prospects. Meanwhile, a direction is pointed out for exploration of PEDV vaccine targets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a nanobody with PEDV neutralizing activity and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV). PEDV is a member of the coronavirus genus of the Coronaviridae family, which is a single-stranded RNA virus with an envelope. The genome of PEDV is about 28 kb in length and encodes at least 7 open reading frames, including three non-structural proteins and four structural proteins. The S gene encodes the spike glycoprotein on the surface of the virion, which contains two parts, S1 (1-789 aa) and S2 (790-1383 aa). The S1 region contains multiple neutralizing epitopes and receptor binding domains, which are closely related to viral antigenicity and invasion. The S2 region plays an important role in the signal transduction process of virus and host cell membrane fusion. PED occurs in pigs of all ages, and the mortality rate of piglet is as high as 100%. The main symptoms are vomiting, watery diarrhea, dehydration, and high mortality rate of piglets. Sick pigs have a large amount of yellow, foul-smelling feces around the anus, and the small intestine is distended with yellow liquid, the mesentery is congested, the lymph nodes are edematous, and the villi are short. It can be seen that PED has a high morbidity and mortality rate in lactation piglets, causing significant economic losses.

[0003] In order to detect and prevent PED early, ELISA detection kits are commonly used to detect PEDV or antiserum. At present, most of the core antibody reagents of commercial ELISA kits use polyclonal antibodies or monoclonal antibodies, which have high technical requirements and are expensive. In addition, when PED epidemic occurs in pig farms, antibiotics and other drugs that inhibit bacteria are used for prevention and control to prevent secondary infection of other enteric bacteria. This method has a certain effect in controlling secondary infection of enteric diseases, but it mainly targets bacterial pathogens and does not have effect on the main pathogen, porcine epidemic diarrhea virus.

[0004] The heavy chain antibody (HCAbs) with natural deletion of light chain existing in the peripheral blood of Camelidae has biological characteristics that other terrestrial organisms do not have. The antigen binding site of the heavy chain variable domain of the heavy chain of HCAbs (VHH) is composed of a single domain of the heavy chain variable region, and has good and specific antigen binding capacity like ordinary IgG antibodies. The antigen binding site of the VHH antibody has an extended antigen complementary binding region and tissue penetration, and can bind to antigen epitopes that cannot be contacted by ordinary IgG antibodies. In addition, the recombinant VHH in vitro has unique properties such as easy expression and good water solubility, and has broad application prospects in the fields of miniaturized genetically engineered antibodies, small molecule antibody drugs and high-sensitivity diagnostic reagents. At present, there are still few nano-antibodies acting on PEDV virus. The existing reported nano-antibodies are mostly obtained by screening using an immune nano-antibody library, which usually needs to be immunized multiple times and takes about 6-8 weeks, and the preparation time period is relatively long. For example, the nano-antibody against the S1 protein of porcine epidemic diarrhea virus disclosed in Chinese Patent CN118126167A has specific recognition and binding capacity. However, at present, there is still a lack of therapeutic products for detecting or diagnosing PEDV or PEDV S1 protein, and for the prevention and treatment of porcine epidemic diarrhea virus. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies of the existing therapeutic products for detecting or diagnosing PEDV or PEDV S1 protein, and to provide a nano-antibody with PEDV neutralizing activity and applications thereof.

[0006] The first object of the present application is to provide a nano-antibody with PEDV neutralizing activity.

[0007] The second object of the present application is to provide a gene encoding the nano-antibody.

[0008] The third object of the present application is to provide a recombinant vector.

[0009] The fourth object of the present application is to provide a recombinant engineering bacterium.

[0010] The fifth object of the present application is to provide a product for detecting PEDV virus.

[0011] The sixth object of the present application is to provide a porcine epidemic diarrhea diagnostic reagent or kit.

[0012] The seventh object of the present application is to provide an anti-PEDV virus drug.

[0013] The above objects of the present application are achieved by the following technical solutions:

[0014] The application provides a nanobody with neutralizing activity of PEDV, which is a VHH antibody, and the amino acid sequence of the nanobody is shown as SEQ ID NO. 1.

[0015] The application is based on a porcine epidemic diarrhea virus (PEDV) S1 protein, and a truncated S1 protein of PEDV is obtained by inducing expression through an E. coli prokaryotic expression system. A nanobody NbH2 capable of binding to the truncated S1 and neutralizing PEDV is screened from a camelid natural nanobody library by using phage display technology, and the amino acid sequence of the nanobody gene is shown as SEQ ID NO. 1. The nanobody provided by the application has the activity of neutralizing PEDV, and the dilution degree of the nanobody with 100% neutralizing effect on a PEDV epidemic strain is 1:2. The nanobody can be used not only as a detection antibody for PEDV, but also as a therapeutic antibody, and can be used for preparing a PED treatment drug. In addition, the expression vector of the nanobody provided by the application can use an E. coli expression system, and the culture is convenient, the operation is simple, the cost is low, and the batch production is easy. The nanobody has important guiding significance for the development of antiviral drugs, and also indicates a direction for the exploration of a PEDV vaccine target.

[0016] The application provides a gene encoding the nanobody, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

[0017] The application provides a recombinant vector containing the gene encoding the nanobody.

[0018] The application provides a recombinant engineering bacterium containing the recombinant vector or being the recombinant engineering bacterium for expressing the nanobody with the activity of neutralizing PEDV.

[0019] The application provides application of the nanobody, the gene, the recombinant vector or the recombinant engineering bacterium in preparation of a product for detecting PEDV.

[0020] Further, the product is used for detecting PEDV and / or PEDV S1 protein and / or antiserum.

[0021] Preferably, the product for detecting PEDV is a detection kit for PEDV.

[0022] The application provides a product for detecting PEDV, containing the nanobody, the gene, the recombinant vector or the recombinant engineering bacterium.

[0023] The application provides application of the nanobody, the gene, the recombinant vector or the recombinant engineering bacterium in preparation of a PEDV diagnostic reagent or kit.

[0024] Further, the nanobody having neutralizing activity to PEDV or the coding gene thereof is used to construct a nanobody prokaryotic expression vector, which is introduced into a host bacterium, expressed and purified, and the purified nanobody can specifically bind to PEDV or PEDV S1 protein and effectively neutralize PEDV virus.

[0025] The application provides a pig epidemic diarrhea diagnostic reagent or kit, which contains the nanobody, gene, recombinant vector or recombinant engineering bacterium.

[0026] The application provides application of the nanobody, gene, recombinant vector or recombinant engineering bacterium in preparation of a medicine for treating and / or preventing pig epidemic diarrhea.

[0027] The application provides an anti-PEDV virus medicine, which contains the nanobody, gene, recombinant vector or recombinant engineering bacterium.

[0028] The application has the following beneficial effects:

[0029] The application obtains a nanobody NbH2 having neutralizing activity to porcine epidemic diarrhea virus (PEDV), the amino acid sequence of the coding nanobody gene is shown as SEQ ID NO. 1, the PEDV specific nanobody NbH2 is screened from a natural nanobody library and directed to a PEDV truncated S1 protein, has good neutralizing activity, and the dilution degree of 100 % neutralization to a PEDV epidemic strain is 1:2, can be used as a detection antibody of PEDV and also can be used as a therapeutic antibody, and is used for preparing a PED treatment medicine; and the expression vector of the nanobody NbH2 can adopt an E. coli expression system, is convenient to culture, simple to operate, low in cost and easy to mass produce. The application has important guiding significance for development of an antiviral medicine, and simultaneously indicates a direction for exploration of a PEDV vaccine target. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a schematic diagram of amino acid sequence and domain division of the nanobody NbH2.

[0031] Figure 2 It is an SDS-PAGE electrophoresis result diagram of the nanobody NbH2.

[0032] Figure 3 It is determination of neutralizing titer of the nanobody NbH2 to a PEDV epidemic strain. DETAILED DESCRIPTION

[0033] The application is further illustrated below by combining the description, the accompanying drawings and specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

[0034] Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0035] Example 1 Affinity panning of nanobodies and their identification

[0036] Firstly, the truncated expressed porcine epidemic diarrhea virus (PEDV) S1 protein was used as the coating agent, and the PEDV S1 coating agent was diluted with 50 mM phosphate buffer to a final concentration of 100 μg / mL, and coated at 4°C overnight. The next day, after washing twice with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)), 5% skimmed milk powder was added and incubated at 4°C for 2 h. The liquid in the well was shaken off and dried, 100 μL of phage library was added to each well, and incubated at room temperature for 1 h. The unbound phages were discarded, and the wells were washed 10 times with PBST (0.01 M PBS, 0.01% Tween-20 (v / v)), 5 times with PBS (pH 7.0), and then 50 μL of Gly-HCl (0.2 M, pH 2.2) was added and incubated at room temperature for 15 min, and then immediately neutralized with 50 μL of Tris-HCl (1 M, pH 8.8). 10 μL of the eluted product was used to calculate the titer by plating the bacteria, and the rest of the eluted product was used for the next round of panning after being rescued and amplified by helper phage.

[0037] In the following second, third and fourth rounds of panning, the PEDV S1 coating agent was diluted to a final concentration of 50 μg / mL, 25 μg / mL and 12.5 μg / mL, respectively, and the phages were incubated for 1 h. After washing with PBST (0.01 M PBS containing 0.02%, 0.03% and 0.04% Tween-20 (v / v), respectively) and PBS, the above-mentioned acid elution method was used for elution, and the phages were incubated at room temperature for 1 h. The liquid in the well was then aspirated, and the phages eluted were obtained. The remaining steps were performed in turn as described above.

[0038] After the fourth round of panning, 96 single colonies were randomly picked from the plates of the third and fourth rounds of nanobody natural library elution products, and inoculated into a deep well plate containing 500 μL of LB-Amp medium per well. The plate was incubated at 37°C, 180 rpm overnight. The next day, 10 μL of the overnight culture was inoculated into a deep well plate containing 2 mL of LB-Amp medium per well, and incubated at 37°C, 180 rpm for 3 h. Then 10 μL of IPTG (final concentration 1 mM) was added to each well, and the plate was induced to express at 37°C, 180 rpm for 12 h. The next day, the plate was centrifuged at 4500 rpm for 15 min, and the supernatant was discarded. The plate was then frozen at -80°C for 3 h, and then thawed naturally. 500 μL of PBS was added and incubated at 16°C, 220 rpm for 1 h. The plate was then centrifuged at 4500 rpm for 15 min, and the supernatant was the periplasmic cavity extract of each monoclonal.

[0039] The periplasmic cavity extract of the above monoclonal is subjected to enzyme-linked immunosorbent assay detection, and the specific steps are as follows:

[0040] 1) Plate coating: dilute PEDV S1 to 10 μg / mL with 50 mM phosphate buffer, add 100 μL of the dilution to each well, and coat in a 4°C refrigerator overnight.

[0041] 2) Blocking: wash each well with PBST twice, pat dry the liquid in the well, add 200 μL of blocking solution to each well, and block at 4°C for 3 h. After blocking, discard the blocking solution and pat dry, and place in a 4°C refrigerator for use.

[0042] 3) Sample addition: add 100 μL / well of the periplasmic cavity extract of the monoclonal to the enzyme-labeled plate, and add 100 μL / well of PBS to the control wells, incubate at room temperature for 1 h, and then wash 5 times with PBST and pat dry.

[0043] 4) Secondary antibody: add 100 μL of 5000-fold diluted rabbit anti-VHH secondary antibody to each well, incubate at room temperature for 1 h, wash 5 times with PBST, and pat dry.

[0044] 5) Color development and reading: add 100 μL of TMB two-component color developing solution to each well, incubate in a 37°C incubator for 10 min, then add 50 μL of 10% H2SO4 to each well to stop the reaction, and measure the absorbance at 450 nm with an enzyme-labeled instrument.

[0045] Calculate the ratio of the reading of the well to which the sample to be tested is added to the reading of the control well. Clones with a value greater than 2 are identified as positive clones, and the positive monoclonal is sent to a sequencing company for sequencing, followed by sequence analysis with software for comparative analysis of the amino acid sequence.

[0046] Through ELISA detection of the screened nanobodies, the nanobody NbH2 against PEDV S1 was finally obtained, the amino acid sequence of which is shown in SEQ ID NO. 1, the nucleotide sequence of the gene encoding the nanobody NbH2 against PEDV S1 is shown in SEQ ID NO. 2, and the amino acid sequence and domain division of the nanobody NbH2 are shown in Figure 1 As can be seen, the nanobody NbH2 includes four framework regions and three complementarity determining regions, the framework regions (FR1-FR4) are selected from SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, and SEQ ID NO. 9, respectively; and the complementarity determining regions (CDR1-CDR3) are selected from SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8, respectively.

[0047] Example 2 Expression and purification of specific nanobodies

[0048] Using the nanobody NbH2 sequence obtained in Example 1 as a template, primers NbH2-F and NbH2-R were designed and synthesized. The specific sequences are shown in Table 1 below. The target fragment of the nanobody NbH2 was amplified by PCR under the following conditions: 95℃, 3 min; 95℃, 15 s, 68℃, 15 s, 72℃, 30 s, 30 cycles; 72℃, 5 min. Primers pComb-F and pComb-R were then designed, and the pComb3xss vector fragment was amplified by PCR under the following conditions: 95℃, 30 s, 95℃, 15 s, 60℃, 15 s, 72℃, 4 min, 30 cycles; 72℃, 5 min.

[0049] Table 1 Primer list for constructing the pComb3xss-NbH2 expression vector

[0050]

[0051] A small amount of the PCR amplification product was detected by agarose gel electrophoresis. If the target band was correct and single, enzyme DpnⅠ was added to the reaction product for digestion. After digestion, homologous recombination was performed, and the ligation product was transformed into E. coli BL21. Single clones were picked for sequencing.

[0052] The successfully sequenced overnight bacterial culture was inoculated into 10 mL of LB-Amp liquid medium and incubated overnight at 37°C with shaking at 250 rpm. The next day, the overnight bacteria were added to 750 mL of LB-Amp medium at a ratio of 1:100 and cultured at 37°C with shaking at 250 rpm until the logarithmic growth phase (OD200). 600 (nm = 0.6–0.8) Bacterial culture was inducing expression overnight by adding 1000×IPTG solution to a final concentration of 1 mM. The next day, the bacterial culture was aliquoted into 250 mL centrifuge tubes and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in 5 mL of TES solution. After thorough mixing until no particulate matter remained, the culture was frozen at -80 °C for 3 h. After thawing at room temperature, 15 mL of 5-fold diluted TES solution was added, and the culture was shaken at 4 °C for 1 h. Finally, the culture was centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was collected as the soluble protein solution. Soluble nanobodies were obtained after purification by nickel column affinity chromatography and identification by SDS-PAGE.

[0053] The pComb3xss-NbH2 recombinant vector was successfully constructed via homologous recombination. The nanobody NbH2 was obtained by E. coli BL21 induction expression, and its SDS-PAGE electrophoresis image is shown below. Figure 2 As shown, the expression level of NbH2 is 4.6 mg / L.

[0054] Example 3 Neutralization of PEDV by Nanobody NbH2

[0055] The neutralization activity of the nanobody NbH2 obtained in Example 2 against PEDV was detected according to the following steps:

[0056] ① Vero cell plating: A full T25 bottle of cells was digested and then added to 10 mL of 10% serum-containing DMEM cell culture medium for subculture plating of a 96-well cell culture plate; the next day, when the cells grew to 95%, the antibody neutralization activity was determined;

[0057] ② Virus half dilution: The known TCID 50 of PED virus liquid was diluted to 200 TCID 50 / 0.1 mL with DMEM / High Glucose maintenance solution containing 10 μg / mL of non-EDTA trypsin;

[0058] ③ Nanobody dilution: The nanobody was diluted by two times with 4 mg / mL as the starting concentration, a total of 4 gradients, and each gradient was made in 4 replicates;

[0059] ④ Virus and nanobody co-incubation: The same amount of 200 TCID 50 of virus liquid was added to the diluted nanobody of different gradients, and after thorough mixing, it was placed in a 37°C incubator for 1 hour;

[0060] ⑤ Control group setting: The virus liquid was prepared to contain 0.2, 200 TCID 50 in each inoculum, and then the same amount of low dilution normal control negative PBS was added, and thoroughly mixed; at this time, the virus concentration became 0.1 and 100 TCID 50 , respectively; in addition, a normal cell negative control and a positive control containing only 100 TCID 50 of virus liquid without antibody were set, and they were all thoroughly mixed and then placed in a 37°C CO2 incubator for 1 hour;

[0061] ⑥ Inoculation into Vero cells: The Vero cells in the 96-well cell culture plate were cultured to a density of about 95%, and the culture solution was discarded, and washed 3 times with PBS to remove the serum components in the culture medium. After the virus and nanobody were incubated, the virus and nanobody mixture and the control group were quickly inoculated into the Vero cells, 4 wells of cells were inoculated as replicates for each sample, 100 μL / well. After being placed in a 37°C incubator for 2 h, the liquid in the wells was discarded, washed 2 times with PBS, 100 μL / well of maintenance solution was added, and the culture was continued in a 37°C CO2 incubator;

[0062] ⑦Observation and result determination: after the above treatment, observe the cells daily to see if they are diseased, and finally take the highest dilution of serum that can cause cytopathic effect (CPE) as the standard, and calculate the neutralizing antibody titer according to the Reed-Muench method.

[0063] The statistical results of the neutralizing activity of the nanobody NbH2 are shown in Table 2, showing that the nanobody NbH2 has neutralizing activity on PEDV, and the dilution of 1:2 has 100% neutralizing effect on the epidemic strain of PEDV, as shown in Figure 3 which can be used as a therapeutic antibody and prepared into a PED treatment drug.

[0064] Table 2 Determination of neutralizing titer of nanobody NbH2

[0065]

[0066] In summary, the present application obtains a nanobody NbH2 with neutralizing activity on porcine epidemic diarrhea virus (PEDV), which is a PEDV-specific nanobody NbH2 screened from a natural nanobody library against truncated S1 protein of PEDV, has good neutralizing activity, and the dilution of 1:2 has 100% neutralizing effect on the epidemic strain of PEDV, can not only be used as a detection antibody for PEDV, but also as a therapeutic antibody, and can be used for preparing a PED treatment drug; and the expression vector of the nanobody NbH2 can use an E. coli expression system, which is convenient to culture, simple to operate, low in cost, and easy to mass-produce.

[0067] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. A nanobody, characterized in that, The nanobody is a VHH antibody, and its amino acid sequence is shown as SEQ ID NO.

1.

2. A gene encoding the Nanobody of claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The gene of claim 2.

4. A recombinant engineered bacterium, characterized in that, The recombinant carrier of claim 3, or the recombinant engineering bacteria expressing the nanobody of claim 1.

5. The nanobody of claim 1, the gene of claim 2, the recombinant carrier of claim 3, or the recombinant engineering bacteria of claim 4 for use in the preparation of a product for detecting PEDV virus.

6. The nanobody of claim 1, the gene of claim 2, the recombinant carrier of claim 3, or the recombinant engineering bacteria of claim 4 for use in the preparation of a diagnostic reagent or kit for porcine epidemic diarrhea.

7. A product for detecting PEDV virus, characterized in that, The nanobody of claim 1, the gene of claim 2, the recombinant carrier of claim 3, or the recombinant engineering bacteria of claim 4.

8. A diagnostic reagent or kit for porcine epidemic diarrhea, characterized by, The nanobody of claim 1, the gene of claim 2, the recombinant carrier of claim 3, or the recombinant engineering bacteria of claim 4.

9. The nanobody of claim 1, the gene of claim 2, the recombinant carrier of claim 3, or the recombinant engineering bacteria of claim 4 for use in the preparation of a medicament for treating and / or preventing porcine epidemic diarrhea.

10. A medicine against PEDV virus, characterized in that, The nanobody of claim 1.

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