CN114146172B

CN114146172B - Nano antibody vaccine capable of preventing porcine pseudorabies virus infection, preparation method and application

Info

Publication number: CN114146172B
Application number: CN202111484962.5A
Authority: CN
Inventors: 曾政; 谢建华; 费磊; 董春霞; 张利; 凌洪权; 蔺露; 欧阳吴莉; 徐斌; 唐颜林
Original assignee: CHONGQING ANIMAL DISEASE PREVENTION AND CONTROL CENTER
Current assignee: CHONGQING ANIMAL DISEASE PREVENTION AND CONTROL CENTER
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-09-16
Anticipated expiration: 2041-12-07
Also published as: CN114146172A

Abstract

The invention provides a nano antibody vaccine capable of preventing porcine pseudorabies virus infection, a preparation method and application thereof. The vaccine has strong neutralizing activity, can be used for preparing a vaccine for preventing and/or treating the pseudorabies, and makes up the defects of the conventional vaccine; the vaccine can effectively neutralize the classical pseudorabies virus strain and the variant pseudorabies virus strain, and can prevent and treat the pseudorabies caused by the classical pseudorabies virus strain and the variant pseudorabies virus strain. And because the sequence background is clear and clear, the vaccine can be produced in a large scale by using a commercial mature means, and is safer and lower in cost.

Description

Nano antibody vaccine capable of preventing porcine pseudorabies virus infection, preparation method and application

Technical Field

The invention relates to the field of animal virology in the field of biotechnology. In particular to a nano antibody vaccine capable of preventing Porcine pseudorabies Virus (PRV) infection, 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 is currently known as a linear double-stranded DNA virus of only one serotype, approximately 145KDa in size, with an average GC content of 73.59%. PRV encodes 70 proteins in total, and the main proteins (gB, gC, gD, gE, gH, gI, gK, gL, gM, gN) are envelope proteins except gG protein. Among them, glycoprotein b (gb), glycoprotein c (gc), glycoprotein d (gd) and glycoprotein h (gh) induce protective immune responses in susceptible animals, producing neutralizing antibodies and antiviral cellular immunity, which are the major protective antigens.

Research shows that the subunit vaccine can provide corresponding protection for immune animals, and the subunit vaccine is prepared by cloning pathogen protective antigen genes into a prokaryotic or eukaryotic expression system by using a genetic engineering method and efficiently expressing the pathogen protective antigen genes. It has now been found that gB, gC, gD in porcine pseudorabies virus glycoprotein all enable the body to produce neutralizing antibodies which have the ability to neutralize PRV, whether in vivo, in vitro or in the presence or absence of a complement. The development of the pseudorabies subunit vaccine (Yancyi, Rougao Gaoming, Zan Nanhui-Jiangxi animal husbandry veterinary journal, 2004 stage 3) discloses that among the 11 glycoproteins currently found in pseudorabies virus, gB, gC and gD all can induce the organism to produce neutralizing antibodies. Monoclonal antibodies to gB, gC, gD neutralize PRV without the involvement of complement. The monoclonal antibodies of gB, gC and gD can resist the attack of PRV virulent virus. Therefore, gB, gC, gD are the first proteins to develop PRV subunit vaccines. Glycoprotein gD is an important neutralizing antigen and also a main target for protective antibodies, and induces a better protective response. US6858385 and US6521231 disclose that a vaccine can be prepared for the prevention of porcine pseudorabies by using porcine pseudorabies virus gD protein. No vaccine in the prior art can solve the problem of the pseudorabies caused by the porcine pseudorabies variant strain.

Monoclonal antibodies (mabs) have since their advent been widely used in medical diagnostics, pharmaceuticals, therapeutics, and scientific research. The nano antibody discovered in recent years not only realizes specific binding force equivalent to that of the traditional antibody, but also has the advantages of a plurality of traditional antibodies as a new generation of antibody derivatives. Therefore, recently, nanobodies are considered as high-value proteins and are applied to various fields including basic research, diagnosis, treatment, food science, and the like. However, no relevant research report is found about the application of the nano antibody in the PRV disease prevention method at present, and no commercial product exists.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide a nano antibody vaccine capable of preventing porcine pseudorabies virus infection, so as to solve the problems in the prior art.

In order to achieve the purpose, the invention provides a nano antibody vaccine capable of preventing porcine pseudorabies virus infection, which comprises three effective components, including gB protein of PRV, gC protein of PRV and nano antibody for specifically recognizing PRVgD protein.

Further, the protein sequence of gB protein of PRV is shown in SEQ ID NO. 4;

further, the protein sequence of the gC protein of the PRV is shown as SEQ ID NO. 5;

further, the protein sequence of the nano antibody for specifically recognizing the PRVgD protein is shown as SEQ ID NO. 3;

furthermore, the invention also provides a nucleotide molecule which codes the amino acid sequence of the nano antibody vaccine.

Furthermore, the invention also provides an expression cassette, a vector, a host cell and an expression system containing the nano antibody vaccine.

Furthermore, the expression cassette, the vector, the host cell and the expression system containing the nano antibody vaccine comprise nucleotide sequences capable of coding the nano antibody vaccine.

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

(1) construction of recombinant eukaryotic expression vectors: connecting DNA sequences of the gB protein including PRV, the gC protein including PRV and the nano antibody specifically recognizing PRVgD protein to an expression vector to obtain a positive plasmid through PCR amplification and enzyme digestion;

(2) and (2) transforming the positive plasmid in the step (1) into a host cell, and inducing and expressing the nano antibody vaccine.

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 also provides application of the nano antibody vaccine in preparation of PRV vaccines.

Further, the vaccine is used for treating and/or preventing PRV, wherein the gB protein of PRV, the gC protein of PRV and the nano-antibody specifically recognizing the PRVgD protein in the vaccine can be used as the only active ingredients and can also be used as one of the active ingredients, and preferably, the active ingredient is the combination of the three ingredients.

Further, the invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of the nanobody vaccine and a pharmaceutically acceptable carrier.

Advantageous effects

The nano antibody vaccine capable of preventing the porcine pseudorabies virus infection comprises three effective components, including gB protein of PRV, gC protein of PRV and nano antibody for specifically recognizing PRVgD protein. Because the vaccine has three proteins which have important functions in PRV infection as antigens, belongs to a multivalent vaccine, covers all common serotype antigens, and can induce more comprehensive neutralizing antibodies than the PRV vaccine reported in the past, the vaccine has stronger neutralizing activity, can be used for preparing the vaccine for preventing and/or treating the pseudorabies, and makes up the defects of the existing vaccine; the vaccine can effectively neutralize the classical pseudorabies virus strain and the variant pseudorabies virus strain, and can prevent and treat the pseudorabies caused by the classical pseudorabies virus strain and the variant pseudorabies virus strain.

Sequencing results of the specific nano antibody provided by the invention show that the nano antibody is highly similar to human VH, and ELISA results show that the specific nano antibody has high binding force and is presumed to be VH-HcAbs naturally existing in camels. The antibody not only has the characteristics of a nano antibody, but also has high similarity with human VH, possibly has great application prospect, not only can be used as an antigen, but also can be used as an antigen carrier, improves the body response, and realizes better immune effect.

The vaccine has the characteristic of broad spectrum, the application object is not limited by animal species, the prevention and treatment efficacy of the vaccine is shown in animal experiments of pigs and dogs, and the effect is excellent. And because the sequence background is clear and clear, the vaccine can be produced in a large scale by using a commercial mature means, and is safer and lower in cost.

Drawings

FIG. 1 shows the expression and purification of gD protein of PRV, wherein M is marker, 1 is the SDS-PAGE identification picture of PET-28a-gD recombinant positive plasmid, and 2 is the SDS-PAGE identification picture of soluble PRV-gD protein obtained by nickel column chromatography purification;

FIG. 2 shows that the ELISA method detects bactrian camel serum titer, wherein the negative control is pre-immune camel serum;

FIG. 3 shows the results of gD protein-specific nanobody ELISA after 3 rounds of panning;

FIG. 4 is a three-dimensional structural view of a fusion protein;

fig. 5 is a temperature curve of the piglets after challenge, wherein the abscissa is days (d) after challenge and the ordinate is temperature (deg.c).

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 soluble expression of porcine pseudorabies gD protein and purification thereof

PRV HN1201 virus (HN1201 strain (Pseudorabisvirus, StrainHN1201) is inoculated on well-grown PK15 cells, the preservation number is CCTCC NO. V201311, the PRV HN1201 virus is preserved in China center for type culture collection, the preservation address is Wuhan university of Wuhan, Hubei province, the preservation date is 2013, 5, 20 days), porcine pseudorabies virus genome DNA is separated by using a conventional method in the field as a template, PCR amplification is carried out by using primers, high fidelity enzyme HS DNA Polymerase with GC Buffer of TAKARA, the amplification conditions are as follows: 3min at 94 ℃; 30cycles at 94 ℃ for 30s, 68 ℃ for 90 s; 5min at 72 ℃. The PCR product was named PRV-gD

gD upstream amplification primer: GGATCCATGCACGTCGCA (SEQ ID NO.1)

gD downstream amplification primers: GCGGCCGCCTAGCGACGCGG (SEQ ID NO.2)

The correctly sequenced PCR product PRV-gD was cloned into PET-28a (purchased from Invitrogen, Cat. A11499), a single clone was picked to identify the insertion direction, the correctly inserted plasmid was sent to Invitrogen for sequencing, and the correctly sequenced plasmid was named PET-28 a-gD. The PET-28a-gD recombinant positive plasmid is expressed, and after being verified by SDS-PAGE (shown in figure 1), the soluble PRV-gD protein is obtained by purification through a nickel column chromatography.

Example 2 screening and characterization of Nanobodies specific for PRV-resistant gD protein

After the purified soluble PRV-gD protein is fully emulsified with the Freund adjuvant, the immune bactrian camel is injected subcutaneously 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, gD protein is used as antigen to measure the antibody titer, and the immunization 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 2), 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 results of screening the specific nano-antibody against gD protein were raised from 0.28 to 3.0 by 3 rounds of panning using phage display technology, indicating that the specific nano-antibody against gE recombinant protein was significantly enriched (FIG. 3). The monoclonal bacteria liquid which is determined to be positive (more than 3 times of negative value) by ELISA detection result,and (5) 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, three nano antibodies aiming at PRV gD protein are finally screened, the nano antibodies are sequenced and then subjected to antigen-antibody neutralization experiment to determine the immune response activity, meanwhile, the nano antibody of the gD protein capable of inducing organism immunity is finally determined by computer analysis, the amino acid sequence of the nano antibody is shown as SEQ ID NO.3, and the nano antibody is named as ND 351.

Example 3 preparation of fusion Nanobody proteins

Optimized design of porcine pseudorabies virus gB protein epitope polypeptide fragment

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 polypeptide are replaced or optimized, and finally the amino acid sequence of the gB epitope polypeptide is determined to be shown as SEQ ID NO. 4.

Optimized design of porcine pseudorabies virus gC protein epitope polypeptide fragment

As mentioned above, identity analysis and computer simulation are carried out on the known porcine pseudorabies virus gC protein, the screened gC protein epitope polypeptide is in tandem connection with the positions 52-113 and 401-458, corresponding optimization is carried out, and finally the amino acid sequence of the gC protein epitope polypeptide is determined to be shown in SEQ ID NO. 5.

Construction of tandem expression cassettes

Amplifying the three polypeptide sequences by using an overlap PCR method, connecting the gB, gC and ND351 sequences in series to construct a gB-gC-ND351 expression cassette, connecting the gB-gC-ND351 expression cassette to a eukaryotic expression vector pcDNA3.1(Invitrogen, San Diego, CA) by using double enzyme digestion to construct pcDNA-gB-gC-ND351, preserving after enzyme digestion sequencing verification (SEQ ID NO.6) is correct, and performing biological analysis on a high-level structure of the pcDNA-gB-gC-ND351 to determine the exposure of an epitope (FIG. 4). Meanwhile, the gB protein antigen epitope polypeptide fragment, the gC protein antigen epitope polypeptide fragment and the two tandem proteins are subjected to the same operation to respectively prepare pcDNA-gB, pcDNA-gC and pcDNA-gB-gC expression plasmids, and the pcDNA-gB-gC expression plasmids are preserved after verification.

Spreading CHO cells in good state in 6-well plate at density of 2-3 × 10 ⁵ Culturing each cell/ml, 2 ml/hole, and culturing at 37 deg.C; when the cells reach 80% confluence degree under the observation of an endoscope, pcDNA-gB-gC-ND351 is transferred into HEK-293T cells (Roche X-tremeGENE HP DNAstrafection Reagent) by a lipid method, and the specific operation is as follows:

a: 2h before transfection, discarding the old culture medium in a 6-well plate, washing 1 time with 1ml PBS/well; discarding PBS, adding 1.8ml of Opti-MEM into each hole, and placing in an incubator at 37 ℃ for continuous culture;

b: taking a 1.5ml EP tube in a clean bench, adding Opti-MEM according to the volume of 200 mul/hole, then adding plasmid, 2 mug/hole, gently blowing, beating and mixing evenly;

c: adding a transfection reagent into the diluted plasmid according to the amount of 6 mul/hole; lightly blowing, beating and mixing uniformly, and standing at room temperature for 20 min;

d: adding the transfection complex into a cell culture hole with 200 mu l/hole, shaking gently and mixing evenly, and putting the cell culture hole into a cell culture box for continuous culture for 48 h.

After the cells are full, collecting cell supernatant in a super clean workbench, centrifuging for 10min at 4 ℃ for 12,000 g, removing cell debris, transferring the supernatant to a new 1.5ml EP tube, subpackaging and storing to-80 ℃ for later use;

collecting cell culture solution, centrifuging at 8000g for 30min, collecting supernatant, filtering with 0.45um filter membrane, loading on Ni-NTA column, washing the column with 10 times column volume of binding buffer solution (20mmol/L Tris-HCl, 0.5mol/L NaCl, 5mmol/L imidazole, pH8.0), and collecting effluent; washing the column with 6 column volumes of washing buffer (20mmol/L Tris-HCl, 0.5mol/L NaCl, 20mmol/L imidazole, pH8.0) and collecting the effluent; finally, the target protein is eluted by 10 column volumes of elution buffer (20mmol/L of LTris-HCl, 0.5mol/L of NaCl, 500mmol/L of imidazole, pH8.0), and the eluate is collected and eluted until no protein is detected. The imidazole eluent containing the target protein is poured into a dialysis bag, dialyzed by 1 XPBS for at least 10 times, and 90ul of reserved sample is taken for detection. In a biological safety cabinet, a 0.22um vacuum filter is used, and a filtered protein solution sample is stored in a refrigerator at minus 80 ℃. Protein concentration and purity determination: determining the protein concentration by using a BCA method; the purity can reach more than 90 percent by adopting an HPLC method to detect the purity.

And performing similar operation on the pcDNA-gB, the pcDNA-gC and the pcDNA-gB-gC to respectively prepare corresponding purified proteins, and performing a vaccine protection experiment after purification and preservation.

Example 4 preparation of Nanobody vaccine and clinical trials thereof

After quantifying the purified nano antibody fusion protein, taking 1128 ml of No. 7 white oil, adding span-80720 ml, and mixing uniformly; then 24 g of aluminum stearate is weighed and added into a mixed sample of No. 7 white oil and span-80; and (3) fully dissolving the mixed sample of the reagent 3 by using a colloid mill, uniformly mixing, and carrying out high-pressure sterilization to obtain the oil phase. Sterile filtering the purified fusion protein, and diluting with sterile water to 1mg/ml to obtain a water phase; mixing 1000mL of oil phase and 500mL of water phase, and stirring at 10000r/min for 2-5min to obtain the nano antibody fusion vaccine.

27 PRV negative pigs were divided into 9 groups for immunization (8 groups in total, 3 in each group; 3 in the blank control group: 1 group), intramuscular injection was performed on the retroauricular neck, and the prepared nanobody fusion vaccines were injected on the left and right ears, respectively, specifically, as shown in Table 1; the two immunizations were performed at intervals of 14 days (first immunization and second immunization on day 1 and day 15), and the blood was collected via the anterior vena cava on

days

7, 14, 21, and 28, respectively, and the neutralizing antibody titers of the vaccine groups were determined by reference to the method of the serum neutralization assay of GB/T18641-2002, and the results are shown in Table 2.

TABLE 1 vaccine grouping

TABLE 2 antibody profiles at different times after immunization of piglets with various porcine pseudorabies vaccine candidate strains

Group of	7d neutralizing antibody titer	14d neutralizing antibody titer	21d neutralizing antibody titer	28d neutralizing antibody titer
					1	1:4.2	1:6.8	1:16.7	1:32.4
2	1:6.1	1:15.7	1:20.4	1:45.2
					3	1:6.3	1:15.7	1:21.2	1:44.8
4	1:3.5	1:6.0	1:11.9	1:21.5
					5	1:3.4	1:5.9	1:12.0	1:23.4
6	1:4.1	1:6.2	1:12.4	1:20.7
					7	1:3.8	1:4.2	1:11	1:15
8	1:4.0	1:4.9	1:12.8	1:22.1
					9	-	-	-	-

From the above results, it can be seen that the first selected strains of the vaccines and the known vaccines thereof can produce high neutralizing antibodies after the piglets are immunized, and the neutralizing antibodies are gradually increased along with the immunization time. The gB and gC proteins can also realize immune response as separate subunit vaccines, meanwhile, in the composition of the vaccine, the increase of the antigen epitope can obviously improve the effect of the vaccine, wherein gB-gC-ND351> gB-gC > gB/gC, which is consistent with the previous research, and similar vaccines exist in the field, and further intensive research can be carried out on the antigen epitope and the optimal arrangement of the fusion protein thereof in the future test. On the basis of neutralizing epitope peptides of gB and gC proteins, a nano antibody ND351 aiming at the gD proteins is added, so that the antibody titer in immune serum can be effectively improved (the titer detected in groups 1-3 is obviously higher than that detected in groups 4-6, and the highest titer is about 2 times of the lower titer), and the difference is obvious. For the gB-gC-ND351 vaccine candidate strain, the immune effect and the immune measure are positively correlated to a certain extent, but the marginal benefit is obvious, the effect of injecting 2ml is not obviously different from the effect of injecting 4ml, side effects appear in the later period, and the specific dosage needs further experimental verification and optimization.

The virus is attacked 28 days after the immunization, and the attacking dose is 2 multiplied by 10 of HN1201 porcine pseudorabies virus ^8.0 TCID 50/head, piglet body temperature was measured daily after challenge (fig. 5), with group 9 negative controls recorded only at

day

2 and 3 experimental pigs all died at day 3. As can be seen from the figure, various vaccine candidate strains can realize certain protection on the porcine pseudorabies virus, although clinical symptoms such as fever and the like appear after challenge, 100 percent (3/3) protection can be provided for piglets, and all the vaccine candidate strains die after 3 days of the control piglets after the challenge, so that the vaccine candidate strains have good protection. Moreover, as can be seen from the body temperature change curve, compared with the enzyme-activated vaccine, the subunit vaccine and the commercialized Bartha-K61 of the application can reduce the temperature rise amplitude and time of piglets to a certain extent, and particularly the gB-gC-ND351 candidate strain of the application can reduce the temperature change amplitude of the piglets after being attacked by virus, shorten the change time and basically maintain the temperature change amplitude to be positiveIs usually horizontal.

The other clinical symptoms of the test pigs are recorded while the temperature is recorded, and observation shows that each vaccine group has the condition of body temperature rise but has no other obvious clinical symptoms, the test pigs of the live vaccine group have the condition of individual initial appetite decline but gradually get better along with time to recover the appetite, no obvious cachexia occurs but the negative control group has obvious conditions of appetite reduction and lassitude, and death occurs, which indicates that each vaccine candidate group has strong protective effect on the porcine pseudorabies virus.

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 vaccine capable of preventing porcine pseudorabies virus infection, preparation method and application

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> gD upstream amplification primer

<400> 1

GGATCCATGCACGTCGCA

<210> 2

<211> 20

<212> DNA

<213> gD downstream amplification primers

<400> 2

GCGGCCGCCTAGCGACGCGG

<210> 3

<211> 124

<212> PROTEIN

<213> Nanobody ND351

<400> 3

EVQLVEAPRRVWPGASLRLSCASSGFTFNDYRYSWIRQTPEKRLEWVQLRYTCKARYAWGSSNTWYQQKPGQSYKLAARQSARHTRYPDWGSGEDLRHTGVQQYGSYPWYSAGQGTQVTVSSAA

<210> 4

<211> 125

<212> protein

<213> gB epitope polypeptide

<400> 4

ATPDGFSAEESLEEIDGAVSPGPSDAPDGEEGDLDARTAVRAVATERDRFYRLNPSEVATAALGQRVSARVLADVMAISRCVEVRGAVYVQNSMRVPGERGTCYSRPLVTFEHNGTAVIEGQLGD

<210> 5

<211> 120

<212> protein

<213> gC epitope polypeptide

<400> 5

STPEPVSGTTGAEASTPAAVSTPRVPAPSVSRRKPQRNGNRTRVHGDKATSHGRKPIVCRERAEHAGLLNVRSARPLSDLDGPVDYTCRLEGMPSVLPIFEDTQRYDASPTSESWPVVTS

<210> 6

<211> 369

<212> protein

<213> gB-gC-ND351 fusion protein

<400> 6

ATPDGFSAEESLEEIDGAVSPGPSDAPDGEEGDLDARTAVRAVATERDRFYRLNPSEVATAALGQRVSARVLADVMAISRCVEVRGAVYVQNSMRVPGERGTCYSRPLVTFEHNGTAVIEGQLGDSTPEPVSGTTGAEASTPAAVSTPRVPAPSVSRRKPQRNGNRTRVHGDKATSHGRKPIVCRERAEHAGLLNVRSARPLSDLDGPVDYTCRLEGMPSVLPIFEDTQRYDASPTSESWPVVTSEVQLVEAPRRVWPGASLRLSCASSGFTFNDYRYSWIRQTPEKRLEWVQLRYTCKARYAWGSSNTWYQQKPGQSYKLAARQSARHTRYPDWGSGEDLRHTGVQQYGSYPWYSAGQGTQVTVSSAA

Claims

1. A nanometer antibody vaccine capable of preventing porcine pseudorabies virus infection comprises three effective components, including gB protein of PRV, gC protein of PRV and nanometer antibody for specifically recognizing PRVgD protein, wherein the protein sequence of gB protein of PRV is shown in SEQ ID NO. 4; the protein sequence of gC protein of the PRV is shown as SEQ ID NO. 5; the protein sequence of the nano antibody for specifically recognizing the PRVgD protein is shown in SEQ ID NO. 3.

2. A nucleotide molecule encoding the nanobody vaccine amino acid sequence of claim 1.

3. An expression cassette, vector, host cell or expression system comprising the nanobody vaccine of claim 1.

4. Use of the nanobody vaccine of claim 1, the nucleotide molecule of claim 2 or the expression cassette, vector, host cell or expression system of claim 3 for the preparation of a porcine pseudorabies virus vaccine.

5. A vaccine composition for preventing porcine pseudorabies virus infection, comprising the nanobody vaccine of claim 1, the nucleotide molecule of claim 2, or the expression cassette, vector, host cell or expression system of claim 3.