CN109943592B

CN109943592B - Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus, preparation method and application

Info

Publication number: CN109943592B
Application number: CN201910354213.7A
Authority: CN
Inventors: 钱平; 李祥敏; 李江龙; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2020-11-17
Anticipated expiration: 2039-04-29
Also published as: CN109943592A

Abstract

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD protein gene, a recombinant baculovirus, a preparation method and application thereof, and belongs to the technical field of veterinary vaccines. The recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD protein gene is inserted into a restriction enzyme cutting site between BamHI and EcoRI and a restriction enzyme cutting site between SpeI and HindIII of the baculovirus transfer vector by taking HBM signal peptide-gD-His label or HBM signal peptide-gD-IgGFc-His label as an exogenous gene respectively; the gD is a gene encoding gD protein with a deleted transmembrane region segment. The vector promotes the secretory expression of the gD protein and the gD-IgGFc fusion protein of the porcine pseudorabies virus. The subunit vaccine prepared by adopting the gD protein and the gD-IgGFc fusion protein has the characteristics of good safety, strong specificity and high virus attack protection rate.

Description

Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus, preparation method and application

Technical Field

The invention belongs to the technical field of veterinary vaccines, and particularly relates to a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD protein gene, a recombinant baculovirus, a preparation method and an application.

Background

Porcine Pseudorabies (PR, also known as Aujeszky's disease, AD) is an infectious disease caused by porcine Pseudorabies Virus (PRV). Except that pigs and cattle of various ages are susceptible, animals such as sheep, dogs, cats, rabbits, mice, minks, foxes and the like are infected and attacked under natural conditions, and great economic loss is easily caused to the livestock breeding industry. Porcine Pseudorabies virus (PRV) is also called as porcine herpesvirus type I, infectious bulbar palsy virus, severe itching syndrome virus and Orientszky's disease virus, and is easy to cause symptoms such as fever, severe itching (except for pigs) and encephalomyelitis of various domestic animals such as cows, sheep, pigs, dogs and cats and wild animals. The clinical symptoms of the disease are similar to rabies, so the disease name of pseudorabies is used.

The infection characteristics of the porcine pseudorabies virus comprise two types, one type is latent infection, and the other type is recessive infection. Latent infection is a condition in which an infected pig does not show clinical symptoms, but infectious viruses exist in a latent state for a long period of time in the pig, and the viruses are not isolated, but the presence of viral genomic DNA can be detected by a polymerase probe method, and the latent viruses can be converted into infectious viruses when immunity is weakened due to external adverse environmental stimuli and the like. The recessive infection is that after a pig herd is inoculated with a vaccine or naturally infected with a small amount of virus, the pig is only partially immunized, and recessive infection pigs and pigs with subclinical symptoms can occur, and the virus elimination of the pig with the virus can last for more than one year. In response to the infection characteristics of porcine pseudorabies virus, attempts have been made to control the outbreak of disease in pigs using improved live and inactivated vaccine immunization. Live attenuated vaccines generally have long-term immune effects but risk insufficient attenuation and genetic instability. Inactivated vaccines are not as effective as attenuated vaccines, and repeated vaccination is required, which greatly increases the amount of inactivated vaccine used. Subunit vaccines carrying a single gene of PRV have not been presented.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD protein gene, which promotes the secretory expression of the porcine pseudorabies virus gD protein.

The invention also aims to provide a recombinant baculovirus expressing the gD protein or the gD-IgGFc fusion protein of the porcine pseudorabies virus, and the virus has the characteristic of secreting and expressing the gD protein or the gD-IgGFc fusion protein.

The invention also aims to provide the subunit vaccine for the porcine pseudorabies, which has the characteristics of high safety, strong specificity, high immunity efficiency and the like.

The invention provides a recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, which is characterized in that HBM signal peptide-gD-His label is taken as an exogenous gene and is respectively inserted into a restriction enzyme cutting site between BamHI and EcoRI and a restriction enzyme cutting site between SpeI and HindIII of the baculovirus transfer vector;

the gD is a gene encoding gD protein with a deleted transmembrane region segment; the nucleotide sequence of the gene of the gD protein with the deleted transmembrane region segment is shown as SEQ ID No. 1.

The invention provides a construction method of the recombinant baculovirus transfer vector, which comprises the following steps:

(1) PCR amplifying gD segment with C end transmembrane region segment deletion by using PRV genome as template;

(2) connecting the gD fragment with the deletion of the transmembrane region fragment with a basic vector to obtain a gD-containing recombinant vector;

(3) performing PCR amplification by using a gD-containing recombinant vector as a template and a first primer pair to obtain a first PCR amplification product which is an HBM signal peptide-gD-His tag target fragment;

respectively adding enzyme cutting sites by taking the first PCR amplification product as a template to obtain an HBM signal peptide-gD-His label target fragment with two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and an HBM signal peptide-gD-His label target fragment with two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites;

(4) and connecting the HBM signal peptide-gD-His tag target fragment with the two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and the HBM signal peptide-gD-His tag target fragment with the two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites to a baculovirus transfer vector to obtain the recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD protein gene.

Preferably, the first primer pair comprises a forward primer for adding an HBM signal peptide to the 5 'end of gD and a reverse primer for adding a His tag to the 3' end of gD, and the nucleotide sequences of the forward primers are shown as SEQ ID No.3 and EQ ID No. 4; the nucleotide sequence of the reverse primer is shown as SEQ ID No. 5.

Preferably, the method for obtaining the HBM signal peptide-gD-His tag target fragment with BamHI and EcoRI enzyme cutting sites connected at two ends by adding the enzyme cutting sites comprises the steps of carrying out PCR amplification by adopting SEQ ID No.6 and SEQ ID No. 7;

the method for obtaining the HBM signal peptide-gD-His tag target fragment with two ends respectively connected with SpeI and HindIII enzyme cutting sites by adding the enzyme cutting sites comprises the step of carrying out PCR amplification by adopting SEQ ID No.8 and SEQ ID No. 9.

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD-IgGFc fusion protein gene, wherein HBM signal peptide-gD-IgGFc fusion protein-His label is taken as an exogenous gene and is respectively inserted into a restriction enzyme cutting site between BamHI and EcoRI and a restriction enzyme cutting site between SpeI and HindIII;

the nucleotide sequence of the gD-IgGFc fusion protein is obtained by connecting a gene of gD protein with a coding sequence of IgGFc protein, wherein the gene is deleted by a coding transmembrane region segment, and the coding sequence of the IgGFc protein is connected in sequence; the nucleotide sequence of the gene of the gD protein with the deleted coding transmembrane region segment is shown as SEQ ID No. 1; the nucleotide sequence of the IgGFc protein coding sequence is shown as SEQ ID No. 2.

The invention provides a construction method of a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD-IgGFc fusion protein gene, which comprises the following steps:

(1) using PRV genome as template, PCR amplifying gD segment with C end transmembrane region segment deletion and artificially synthesizing IgGFc-His segment;

(2) connecting the gD segment or the IgGFc-His segment with the transmembrane region segment, which is deleted, with a basic vector respectively to obtain a gD-containing recombinant vector or an IgGFc-His-containing recombinant vector;

(4) performing fusion PCR amplification by using the HBM signal peptide-gD-His tag target fragment and the recombinant vector containing IgGFc-His as templates and using a second primer pair to obtain a second PCR amplification product which is the HBM signal peptide-gD-IgGFc-His tag target fragment;

respectively adding enzyme cutting sites by taking the second PCR amplification product as a template to obtain an HBM signal peptide-gD-IgGFc-His label target fragment with two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and an HBM signal peptide-gD-His label target fragment with two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites;

(5) and connecting the HBM signal peptide-gD-IgGFc-His label target fragment with the two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and the HBM signal peptide-gD-IgGFc-His label target fragment with the two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites to a baculovirus transfer vector to obtain the recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD-IgGFc fusion protein gene.

Preferably, the second primer pair comprises a forward primer of which the 5 'end of gD is added with an HBM signal peptide sequence as shown in SEQ ID No.10, a reverse primer of which the 3' end of gD is added with a homology arm sequence as shown in SEQ ID No.11, a forward primer of which the 5 'end of IgGFc is added with a homology arm sequence as shown in SEQ ID No.12, and a reverse primer of which the 3' end of IgGFc is added with a His tag sequence as shown in SEQ ID No. 13.

The invention provides a recombinant baculovirus expressing porcine pseudorabies virus gD protein, which is obtained by transfecting a recombinant baculovirus transfer vector to insect cells, culturing until the cells have pathological changes and collecting supernatant.

The invention provides a recombinant baculovirus expressing porcine pseudorabies virus gD-IgGFc fusion protein, which is obtained by transfecting a recombinant baculovirus transfer vector to an insect cell, culturing until the cell has pathological changes, and collecting supernatant.

The recombinant baculovirus transfer vector or the recombinant baculovirus obtained by the construction method of the recombinant baculovirus transfer vector or the application of the recombinant baculovirus in producing the vaccine of porcine pseudorabies is provided.

The invention provides a subunit vaccine for preventing and treating porcine pseudorabies, which comprises an adjuvant and one of the following proteins: the porcine pseudorabies virus gD protein expressed by the recombinant baculovirus and the gD-IgGFc fusion protein expressed by the recombinant baculovirus.

Preferably, the concentration of the porcine pseudorabies virus gD protein is 500-700 mug/ml; the concentration of the gD-IgGFc fusion protein is 100-150 mu g/ml.

The invention provides a recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, wherein HBM signal peptide-gD-His label is an exogenous gene which is respectively inserted into BamHI and EcoRI enzyme cutting sites and SpeI and HindIII enzyme cutting sites of the baculovirus transfer vector; the gD is a gene encoding gD protein with a C-terminal transmembrane region segment deleted. The sequence of gD is according to the DNA sequence of the coding gD protein of the deletion of the coding transmembrane region segment in the epidemic dominant strain in China, the gD protein is glycoprotein of PRV, the total length is 993bp, the coding 331aa, the deletion of the transmembrane region is more beneficial to the secretory expression of the gD protein, and meanwhile, the HBM signal peptide-gD-His label is placed under a promoter in a baculovirus transfer vector in a 2-copy form in the recombinant baculovirus transfer vector, so that the expression quantity of a target gene can be improved, and convenience is provided for subsequent separation and purification and in-vitro expression of the protein.

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD-IgGFc fusion protein gene, wherein an HBM signal peptide-gD-IgGFc fusion protein-His label is an exogenous gene which is respectively inserted into a BamHI enzyme digestion site and an EcoRI enzyme digestion site and a SpeI enzyme digestion site and a HindIII enzyme digestion site of the baculovirus transfer vector; the gD-IgGFc fusion protein is obtained by connecting a gene of gD protein with a coding sequence of IgGFc protein, wherein the gene is used for coding the gD protein with a transmembrane region segment deleted, and the coding sequence of the IgGFc protein is connected in sequence. The protein coded by the gD-IgGFc is a gD and murine IgGFc fusion protein of PRV, the total length is 1830bp, the coding is 609aa, the deletion of a gD transmembrane region is more beneficial to the secretory expression of the gD-IgGFc protein, the PRV glycoprotein gD and the IgGFc are fused to enhance the protective immunity to the PRV, and the IgG2a Fc fragment plays a potential molecular adjuvant role. Meanwhile, the HBM signal peptide-gD-IgGFc fusion protein-His label is placed under a promoter of a baculovirus transfer vector in a 2-copy mode, so that the expression quantity of a target gene is improved, and convenience is provided for subsequent separation and purification and in-vitro expression of the protein.

The recombinant baculovirus transfer vector or the recombinant baculovirus obtained by the construction method of the recombinant baculovirus transfer vector or the application of the recombinant baculovirus in producing the vaccine of porcine pseudorabies is provided. Because the gD protein expressed by the porcine pseudorabies virus gD protein recombinant baculovirus and the gD-IgGFc fusion protein expressed by the gD-IgGFc fusion protein recombinant baculovirus are not all viruses, the subunit vaccine prepared by the fusion protein is free from the risk of virus diffusion, and the production process is safer. After the gD protein and the gD-IgGFc fusion protein which are prepared simultaneously immunize organisms, the gD protein and the gD-IgGFc fusion protein have the characteristics of strong antigen specificity diagnosis, high sensitivity and rapid diagnosis; can realize the high-efficiency production of genetic engineering vaccines. In addition, compared with gD protein, the antigen immunogenicity of the obtained gD-IgGFc fusion protein is better, and the protection rate to mice is higher. Therefore, the IgG2a Fc fragment serves as a molecular adjuvant to be fused with an antigen, and plays a role in improving the immunogenicity of the antigen.

Drawings

FIG. 1 shows the restriction enzyme cleavage identification map of recombinant baculovirus transfer vectors PFBD-2-PRVgD and PFBD-2-PRVgD-IgGFc;

FIG. 2 is an electrophoresis diagram of Western blot and SDA-PAGE analysis for recombinant baculovirus AC-2-PRV-gD protein expression;

FIG. 3 is an electrophoresis diagram of Western blot and SDA-PAGE analysis for recombinant baculovirus AC-2-PRV-gD-IgG2aFc protein expression;

FIG. 4 is a graph of antibody level measurements against PRV at different time points after immunization of mice with two subunit vaccines;

FIG. 5 shows the measurement of cytokine levels induced by mice immunized with two subunit vaccines;

FIG. 6 shows the survival rate of mice immunized with the A two subunit vaccines after challenge.

Detailed Description

the gD is a gene for coding a gD protein with a deleted C-terminal transmembrane region segment; the nucleotide sequence of the gene of the gD protein with the deleted transmembrane region segment is shown as SEQ ID No. 1. The complete sequence of gD gene is shown as SEQ ID No.14, and the amino acid sequence coded by the complete sequence of gD gene is shown as SEQ ID No.15

In the invention, the gene of the gD protein which is deleted by the C-terminal transmembrane region segment is not limited to the nucleotide sequence shown in SEQ ID No.1, and also comprises a nucleotide sequence which is complementary with the SEQ ID No.1 and a sequence which is obtained by simply adding, deleting and replacing one or more nucleotides on the basis of the SEQ ID No.1, thereby realizing the purpose of the invention and belonging to the protection scope of the invention. In the embodiment of the invention, the nucleotide sequence of the HBM signal peptide-gD-His tag is shown as SEQ ID No. 16.

The source of the baculovirus transfer vector in the present invention is not particularly limited, and baculovirus transfer vectors known in the art may be used. To illustrate the recombinant baculovirus transfer vector composition, recombinant baculovirus was prepared using the PFBDHmHNM1P10 efp plasmid as a recombinant baculovirus transfer vector in the examples in the art. The PFBDHmHNM1P10 efp plasmid was constructed by the laboratory. The core of the recombinant baculovirus transfer vector is that the HBM signal peptide-gD-His label is an exogenous gene, and the type of the baculovirus transfer vector is not specific, so that the recombinant baculovirus transfer vector only contains two or more multiple cloning sites. The type of the multiple cloning site is not limited, but is not limited to a cleavage site between BamHI and EcoRI and a cleavage site between SpeI and HindIII.

The nucleotide sequence of the HBM signal peptide is not particularly limited in the present invention, and the nucleotide sequence of the HBM signal peptide known in the art may be used. In the embodiment of the invention, the sequence of the HBM signal peptide is shown in SEQ ID No.3 in the construction method. The nucleotide sequence of the His tag is not particularly limited in the present invention, and a nucleotide sequence of a His tag known in the art may be used. In the embodiment of the invention, the sequence of the His tag is shown in SEQ ID No.4 in the construction method.

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD-IgGFc fusion protein gene, wherein an HBM signal peptide-gD-IgGFc fusion protein-His label is an exogenous gene which is respectively inserted into a restriction enzyme cutting site between BamHI and EcoRI and a restriction enzyme cutting site between SpeI and HindIII of the baculovirus transfer vector; the gD-IgGFc fusion protein is obtained by connecting a gene of gD protein with a coding sequence of IgGFc protein, wherein the gene is deleted from a coding transmembrane region segment, and the coding sequence of the IgGFc protein is connected in sequence; the nucleotide sequence of the gene of the gD protein with the deleted coding transmembrane region segment is shown as SEQ ID No. 1; the nucleotide sequence of the IgGFc protein coding sequence is shown as SEQ ID No. 2.

In the present invention, the sequence encoding the gD protein with the deletion of the transmembrane region segment is described above, and thus the description thereof is omitted. The IgGFc protein is selected from mouse-derived IgGFc protein, preferably mouse-derived IgG2aFc protein, but not other IgG subtypes, because IgG2aFc has higher affinity with mouse-derived Fc gamma RI receptor, and the Fc gamma RI receptor is a high-affinity receptor of immunoglobulin, which is favorable for forming fusion protein with stronger specificity. The IgGFc protein coding sequence is not limited to the nucleotide sequence shown in SEQ ID No.2, and also comprises a nucleotide sequence complementary with the SEQ ID No.2 and a sequence obtained by simply adding, deleting and replacing one or more nucleotides on the basis of the SEQ ID No.2, which can achieve the purpose of the invention and belongs to the protection scope of the invention. The connecting sequence is used for connecting the 5 'end of the nucleotide sequence of the IgGFc protein and the 3' end of the gD protein. The linker sequence has high flexibility and can be used as a linker sequence for ligation as is well known in the art. In the embodiment of the present invention, the specific nucleotide sequence of the connecting sequence may be found in the following construction method, wherein the reverse primer is a reverse primer in which a homology arm sequence is added to the 3 'end of gD as shown in SEQ ID No.8 or the forward primer is a forward primer in which a homology arm sequence is added to the 5' end of IgGFc as shown in SEQ ID No. 9. The kind of the baculovirus transfer vector is the same as the recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD protein gene, and the details are not repeated herein.

In the invention, the nucleotide sequence of the coding sequence of the HBM signal peptide-gD-IgGFc fusion protein-His tag is shown as SEQ ID No.17, the nucleotide sequence of the gD-IgG2aFc gene is 1830bp in total, and 609aa is coded; the protein sequence coded by the gD-IgG2aFc gene is shown as SEQ ID No. 18.

The invention artificially synthesizes gD fragments with deleted transmembrane region fragments. The gD segment with the deletion of the transmembrane region segment is obtained by performing PCR amplification by taking the genome of the porcine pseudorabies virus as a template. The forward primer for PCR amplification comprises gtgcccgcgccgaccttccccccgcccgcgtac (SEQ ID No. 19); the reverse primer for PCR amplification comprises gcggtggcgcgagacgcccggcgcggcggtg (SEQ ID No. 20). The procedure for the PCR amplification was as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 60 ℃, 30 s; 72 ℃, 1kb/min) for 30 cycles; 72 ℃ for 5 min; the PCR amplification system is as follows: the amplification system amounted to 50. mu.l: 10X buffer (containing Mg)²⁺) 5 mul; dNTP, 6. mu.l; 10 μ M forward primer 2 μ l; 10 μ M reverse primer 2 μ l; template, 200 ng; taq enzyme 0.5. mu.l; ddH₂And O is used for supplementing the system.

After a gD fragment with a deleted transmembrane region fragment is obtained, the gD fragment with the deleted transmembrane region fragment is connected with a basic vector to obtain a gD-containing recombinant vector.

In the present invention, the kind of the base vector is not particularly limited, and a base vector well known in the art may be used. In the examples of the present invention, the basic vector is pEASYBLUNT vector for illustrating the ligation process. The pEASYBLUNT vector and gD fragment with deletion of the transmembrane region fragment are mixed and reacted for 5min at room temperature, and are connected through a blunt end.

By taking a gD recombinant vector as a template, carrying out PCR amplification by using a first primer pair to obtain a first PCR amplification product which is an HBM signal peptide-gD-His tag target fragment; and respectively adding enzyme cutting sites by taking the first PCR amplification product as a template to obtain an HBM signal peptide-gD-His label target fragment with two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and an HBM signal peptide-gD-His label target fragment with two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites.

In the present invention, the first primer pair preferably comprisesA forward primer for adding HBM signal peptide to the 5 'end of gD and a reverse primer for adding His tag to the 3' end of gD, wherein the nucleotide sequences of the forward primers are shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequence of the reverse primer is shown as SEQ ID No. 5. The reaction system for carrying out PCR amplification on the first primer pair is as follows: 50 μ l: 10 × buffer (containing Mg)²⁺) 5 mul; dNTP, 6. mu.l; 10 μ M forward primer, 2 μ l; 10. mu.M reverse primer, 2. mu.l; template, 200 ng; taq enzyme, 0.5. mu.l; ddH₂O complement system; the reaction program of PCR amplification of the first primer pair is 94 ℃ and 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min) for 30 cycles; 72 ℃ for 5 min.

In the invention, the method for obtaining the target fragment of HBM signal peptide-gD-His label with BamHI and EcoRI enzyme cutting sites connected at two ends by adding the enzyme cutting sites preferably comprises the step of carrying out PCR amplification by adopting SEQ ID SEQ ID No.6 and SEQ ID No. 7. Adding a BamHI enzyme cutting site to the 5' end of the HBM signal peptide-gD-His tag target fragment by using the primer shown in SEQ ID No. 6; the primer shown in SEQ ID No.7 adds an EcoRI cleavage site to the 3' end of the target fragment of HBM signal peptide-gD-His tag. The reaction sequence in PCR amplification of SEQ ID No.6 and SEQ ID No.7 is preferably as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min) for 30 cycles; 72 ℃ for 5 min. The reaction system is the same as above.

In the invention, the method for obtaining the HBM signal peptide-gD-His tag target fragment with SpeI and HindIII enzyme cutting sites connected at two ends respectively by adding the enzyme cutting sites preferably comprises the step of carrying out PCR amplification by adopting SEQ ID No.8 and SEQ ID No. 9. The primer shown in SEQ ID No.8 adds a SpeI enzyme cutting site to the 5' end of the target fragment of HBM signal peptide-gD-His tag. The primer shown in SEQ ID No.9 adds HindIII enzyme cutting site to the 3' end of HBM signal peptide-gD-His label target segment. The reaction sequence in the case of PCR amplification with SEQ ID No.8 and SEQ ID No.9 is preferably as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min) for 30 cycles; 72 ℃ for 5 min. The reaction system is the same as above.

After two fragments are obtained, the HBM signal peptide-gD-His label target fragment with the two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and the HBM signal peptide-gD-His label target fragment with the two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites are connected to a baculovirus transfer vector to obtain the porcine pseudorabies virus gD protein recombinant baculovirus transfer vector.

In the present invention, the baculovirus transfer vector or both target fragments are preferably cleaved with BamHI followed by EcoRI and SpeI and HindIII enzymes before ligation. The ligation method is preferably performed overnight at 16 ℃ using T4DNA ligase.

(4) performing PCR fusion amplification by using the HBM signal peptide-gD-His tag target fragment and the recombinant vector containing IgGFc-His as templates and using a second primer pair to obtain a second PCR amplification product which is the HBM signal peptide-gD-IgGFc-His tag target fragment;

taking the second PCR amplification product as a template to obtain an HBM signal peptide-gD-IgGFc-His label target fragment with two ends respectively connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites and an HBM signal peptide-gD-His label target fragment with two ends respectively connected with SpeI enzyme cutting sites and HindIII enzyme cutting sites;

(5) and connecting the HBM signal peptide-gD-IgGFc-His label target fragment with the BamHI enzyme cutting site and the EcoRI enzyme cutting site connected at the two ends respectively and the HBM signal peptide-gD-IgGFc-His label target fragment with the SpeI enzyme cutting site and the HindIII enzyme cutting site connected at the two ends respectively to a baculovirus transfer vector to obtain the recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD-IgGFc fusion protein gene.

In the present invention, the second primer pair preferably includes a forward primer in which an HBM signal peptide sequence is added to the 5 'end of gD as shown in SEQ ID No.10, a reverse primer in which a homology arm sequence is added to the 3' end of gD as shown in SEQ ID No.11, a forward primer in which a homology arm sequence is added to the 5 'end of IgGFc as shown in SEQ ID No.12, and a reverse primer in which a His tag sequence is added to the 3' end of IgGFc as shown in SEQ ID No. 13. The reaction program of the second primer pair for PCR amplification is as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min) for 30 cycles; 72 ℃ for 5 min; the reaction system during PCR amplification is as follows: 50 μ l: 10X buffer (containing Mg)²⁺) 5 mul; dNTP, 6. mu.l; 10 μ M forward primer, 2 μ l; 10. mu.M reverse primer, 2. mu.l; template (HBM-gD-His or IgGFc-His), 200 ng; taq, 0.5. mu.l; ddH₂O complement system; amplification System 2: 50 μ l: 10X buffer (containing Mg)²⁺) 5 mul; dNTP, 6. mu.l; 10 μ M forward primer, 2 μ l; 10. mu.M reverse primer, 2. mu.l; template 1(HBM signal peptide-gD-His), 200 ng; template 2(IgGFc-His), 200 ng; taq enzyme, 0.5. mu.l; ddH₂O make up the system.

In the invention, the method for obtaining the target fragment of the HBM signal peptide-gD-IgGFc-His tag with BamHI and EcoRI enzyme cutting sites connected at two ends by respectively adding enzyme cutting sites by taking the second PCR amplification product as a template preferably comprises the step of carrying out PCR amplification by adopting SEQ ID No.6 and SEQ ID No. 7. Adding a BamHI enzyme cutting site at the 5' end of the HBM signal peptide-gD-IgGFc-His tag target fragment by using a primer shown in SEQ ID No. to obtain a target fragment; the primer shown in SEQ ID No.7 adds an EcoRI restriction site to the 3' end of the target fragment of HBM signal peptide-gD-IgGFc-His tag. The reaction sequence in the case of PCR amplification with SEQ ID No.6 and SEQ ID No.7 is preferably as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min) x30(30 cycles); 72 ℃ for 5 min. The reaction system is the same as above.

In the invention, the method for obtaining the HBM signal peptide-gD-IgGFc-His tag target fragment with SpeI and HindIII enzyme cutting sites connected at two ends by adding the enzyme cutting sites preferably comprises the step of carrying out PCR amplification by adopting SEQ ID No.8 and SEQ ID No. 9. The primer shown in SEQ ID No.8 adds a SpeI enzyme cutting site to the 5' end of the target fragment of HBM signal peptide-gD-IgGFc-His tag. The primer shown in SEQ ID No.9 adds HindIII enzyme cutting site to the 3' end of target fragment of HBM signal peptide-gD-IgGFc-His tag. The reaction sequence in the case of PCR amplification with SEQ ID No.8 and SEQ ID No.9 is preferably as follows: 94 ℃ for 5 min; (94 ℃, 30 s; 65 ℃, 30 s; 72 ℃, 1kb/min), 30 cycles; 72 ℃ for 5 min. The reaction system is the same as above.

The invention provides a recombinant baculovirus expressing porcine pseudorabies virus gD protein or gD-IgGFc fusion protein, which is obtained by transfecting a recombinant baculovirus transfer vector prepared by the construction method to insect cells, culturing until the cells have pathological changes, and collecting supernatant.

In the present invention, the method for transfecting the recombinant baculovirus transfer vector into the insect cell is preferably transfection using liposome-mediated transfection. The present invention is not particularly limited in kind of the insect cell, and insect cells well known in the art may be used. In the present embodiment, the insect cell is preferably an Sf9 insect cell. The method of culturing transfected cells in the present invention is not particularly limited, and a conventional method of culturing transfected cells in the art may be used. After the cells are diseased, collecting the supernatant by adopting a conventional centrifugation method.

In the present invention, the method for producing a vaccine for porcine pseudorabies preferably comprises the steps of: inoculating the recombinant baculovirus expressing the porcine pseudorabies virus gD protein or the gD-IgGFc fusion protein into transfected insect cells at the dose of 0.1MOI, continuously culturing, collecting supernatant when the cells have pathological changes, and detecting gD and gD-IgGFc fusion protein secretion supernatant through Westernblot analysis.

In the invention, the concentration of the porcine pseudorabies virus gD protein is preferably 500-700 mug/ml, and more preferably 600 mug/ml; the concentration of the gD-IgGFc fusion protein is 100-150 mu g/ml, and more preferably 120 mu g/ml.

In the present invention, the adjuvant is preferably SEPPIC IMS-1313 adjuvant. The volume ratio of the gD or gD-IgGFc protein to the adjuvant is preferably 7: 3. The nasal drop dose of the subunit vaccine is preferably 20 mug/6-week-old mouse, and the variety of the mouse is Babic. The same dose was boosted once 14 days after the first immunization.

The porcine pseudorabies virus gD protein recombinant baculovirus transfer vector, the recombinant baculovirus, the preparation method and the application thereof provided by the invention are explained in detail by combining the following examples, but the vector and the recombinant baculovirus are not understood to limit the protection scope of the invention.

Example 1

Construction method of recombinant baculovirus transfer vector PFBD-2-PRVgD containing porcine pseudorabies virus gD protein gene

And (3) coding gD deleted from the transmembrane region fragment, connecting the gD with a pEASYBLUNT vector to obtain a pEASYBLUNT-gD recombinant vector, and then carrying out sequencing verification to obtain a result which is correct and ready for use after comparison.

Plasmid pEASYBLUNT-gD is taken as a template, PCR amplification is respectively carried out through 2 groups of primer pairs to obtain gD and gD genes connected with GP67 signal peptide sequences, and then BamHI, EcoRI, XbaI and HindIII are used for cloning target genes into a baculovirus transfer vector PFBDHmHNM1P10eEFP for PFBD (see a recombinant baculovirus expressing a synthetic sandwich H1N1 influenza virus HA-NA-M1 gene modified and thrown in China patent 200910063217.6). Specifically, pEASYBLUNT-gD is taken as a template, and a first primer pair is used for carrying out PCR amplification respectively to obtain a GP67-gD-His label target fragment; taking a fragment HBM-gD-His tag target fragment obtained by amplification of the first primer pair as a template, and respectively carrying out PCR amplification by using a second primer pair to obtain an HBM signal peptide-gD-His tag target sequence connected with BamHI and EcoRI enzyme cutting sites; and (3) taking the amplification product of the second primer as a template, and amplifying by using a third primer to obtain an HBM signal peptide-gD-His tag target sequence connected with XbaI and HindIII enzyme cutting sites. Wherein the sequences of the first group of primer pairs are shown as SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; the sequence of the second primer pair is shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 9; the primer sequences are shown in Table 1.

TABLE 1 primer sequence information List

Sequence of	Numbering
		ttttatggtcgtatacatttcttacatctatgcggtgcccgcgccgaccttccccccgc-F	SEQ ID No.3
gccaccatgaaattcttagtcaacgttgcccttgtttttatggtcgtatacatttctta-F	SEQ ID No.4
		ctagtgatggtgatggtgatgagagcccgatccgcggtggcgcgagacgcccggcgcgg-R	SEQ ID No.5
ggatccgccaccatgaaattcttagtcaac-F	SEQ ID No.6
		gaattcctagtgatggtgatggtgatgagagc-R	SEQ ID No.7
actagtgccaccatgaaattcttagtcaacg-F	SEQ ID No.8
		aagcttctagtgatggtgatggtgatgagagcc-R	SEQ ID No.9

The transfer vector obtained was identified by digestion with BamHI and EcoRI, SpeI and HindIII, and the result is shown in A of FIG. 1, where M is 15000bp Marker. The enzyme digestion result shows that the size of the enzyme digestion identification fragment is consistent with the expected result, and the expression transfer vector is successfully constructed. The transfer vector PFBD-2-PRV-gD contained 2 copies of the PRV gD gene sequence.

Example 2

Construction method of recombinant baculovirus AC-2-PRVgD

1 mu g of baculovirus transfer plasmid PFBD-2-PRV-gD was added to the DHI0Bac coliform competent cells, and ice-cooled for 30 minutes. The cells are thermally shocked in water at 42 ℃ for 45 seconds, then are iced for 2 minutes in an ice bath, 900 microliters of SOC liquid culture medium is added, shaking culture is carried out at 37 ℃ for 4 hours, 100 microliters of the SOC liquid culture medium is taken and coated on a three-resistance LB plate (Kan, Gen and Tel), and culture is carried out at 37 ℃ for 24-48 hours. Single white colonies screened by 3 rounds of blue-white screening were picked up and cultured in 10mL LB medium containing kanamycin, gentamicin and tetracycline triple antibodies at 37 ℃ for 16h in a shaker (180 rpm/min). Centrifuging to collect bacteria, adding 0.3mL of Solution I for resuspension, adding 0.3mL of Solution II for gentle mixing, immediately adding 0.3mL of Solution III for mixing, carrying out ice bath for 10min, centrifuging at 4 ℃ and 12000rpm for 10min, transferring supernatant into another sterile blank 1.5mL of LEP tube, adding 0.5mL of isopropanol for mixing, standing for 10min, centrifuging at 4 ℃ and 12000rpm for 10min, discarding supernatant, washing with 75% ethanol, drying, adding 30-50 mu LTE for dissolving, and storing at 4 ℃.

Positive rods were transfected into Sf9 insect cells using liposome-mediated transfection: sf9 cells are inoculated in a six-hole plate, transfection is carried out when the cells grow to 80-90%, and two 1.5mL sterile Ep tubes are taken. And adding 100 mu L of serum-free Grace's culture medium and 8 mu L of Lcellinfection into the tube A, sequentially adding 100 mu L of serum-free Grace's culture medium and 1-2 mu g of recombinant Bacmid into the tube B, standing at room temperature for 5-10min, gradually dripping the solution in the tube B into the tube A, standing at room temperature for 20min, and then supplementing the serum-free Grace's culture medium to 1 mL. And adding the mixture in the 1.5mLEp tube onto the monolayer cells, culturing for 5-6 h in an incubator at 27 ℃, replacing complete Grace's culture solution, continuously culturing for 2-3 d, and collecting culture supernatant after cytopathic effect to obtain the recombinant baculovirus AC-2-PRVgD.

Example 3

Artificially synthesizing a nucleotide sequence gD of a gD protein and an IgG2aFc of a nucleotide sequence of a murine IgG2aFc protein, wherein the nucleotide sequence gD of the gD protein and the IgG2aFc protein are deleted from a C-terminal transmembrane region segment; and mixing the gD or IgG2aFc and the pEASYBLUNT vector according to the volume ratio of 0.5-4: 1, reacting for 5min at room temperature, connecting to obtain pEASYBLUNT-gD and pEASYBLUNT-IgG2aFc recombinant vectors, and carrying out sequencing verification to obtain correct and spare use after the results are compared.

Respectively carrying out PCR amplification by using pEASYBLUNT-gD as a template and the first primer pair in the embodiment 1 to obtain target fragments connected with HBM signal peptide-gD-His; products HBM-gD-His and pEASYBLUNT-IgG2aFc obtained by amplification of the first primer pair are taken as templates, and a second primer pair is respectively used for PCR amplification to obtain HBM signal peptide-gD-IgGFc-His target fragments connected with BamHI enzyme cutting sites and EcoRI enzyme cutting sites; and amplifying by using a third primer pair and connecting HBM signal peptide-gD-IgG 2aFc-His tag target fragments with BamHI and EcoRI enzyme cutting sites by using fragments HBM-gD-IgG2aFc-His and pEASYBLUNT-IgG2aFc obtained by amplifying the first primer pair as templates.

Wherein the sequences of the second group of primer pairs are shown as SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 and SEQ ID No. 13; the primer sequences are shown in Table 2.

TABLE 2 primer sequence information List

Sequence of	Numbering
		gccaccatgaaattcttagtcaacgttgccc-F	SEQ ID No.10
ccacctcctccggacccacccccgcctgatccgcggtggcgcgagacgcccggcgcggc-R	SEQ ID No.11
		atcaggcgggggtgggtccggaggaggtggctcgggatctgagcccagagggcccacaa-F	SEQ ID No.12
ctagtgatggtgatggtgatgagagcccgatcc-R	SEQ ID No.13

The baculovirus transfer vector PFBD-2-PRVgD-IgG2aFc was obtained by ligating the gD-IgG2aFc target fragment ligated to the HBM signal peptide sequence and ligated to the BamHI and EcoRI cleavage sites, and the gD-IgG2aFc target fragment ligated to the HBM signal peptide sequence and ligated to the SpeI and HindIII cleavage sites, to the baculovirus transfer vector PFBDHmHNM1P10 eEFP.

The baculovirus transfer vector PFBD-2-PRVgD-IgG2aFc was identified by double digestion. The results are shown in FIG. 1, B, where M is 15000bp Marker. The enzyme digestion result shows that the size of the enzyme digestion identification fragment is consistent with the expected result, and the expression transfer vector is successfully constructed. The enzyme digestion identification result shows that the transfer vector PFBD-2-PRV-gD contains 2 copies of PRV gD gene sequence.

Recombinant baculovirus AC-2-PRVgD-IgG2aFc expressing the gD-IgG2aFc fusion protein was prepared according to the construction method of example 2.

Example 4

Expression of gD gene and gD-IgG2aFc fusion gene was detected by Westernblot analysis using the recombinant baculovirus AC-2-PRVgD obtained in example 2 and the recombinant baculovirus AC-2-PRVgD-IgG2aFc obtained in example 3 as targets

Sf9 cells were seeded in 6-well plates and the recombinant baculovirus of example 2 or 3 was seeded at 0.1MOI dose when the cells grew to 90%. Cell culture supernatant and cells were collected separately 72 hours after seeding. After SDA-PAGE running, the gel was transferred to PVDF membrane, and APPVE2 protein expression was detected by ECL display using commercial His antibody as primary antibody (MBL, USA) and HRP-labeled goat anti-mouse IgG (Wuhan Boston Biotech) as secondary antibody. The experimental results are shown in fig. 2 and the left panel of fig. 3. Wherein a 41kDa stripe appears in the left diagram of FIG. 2 and a 90kDa stripe appears in the left diagram of FIG. 3. Thus, recombinant virus bacillus AC-2-PRVgD and AC-2-PRVgD-IgG2aFc infect gD protein and gD-IgG2aFc fusion protein secretion supernatant expressed by insect cells.

SDA-PAGE detection of purified gD protein and gD-IgG2aFc fusion protein

The recombinant baculovirus AC-2-PRVgD or AC-2-PRVgD-IgG2aFc obtained in example 2 was inoculated with HF cells (1 x 10) at a dose of 0.0001MOI⁶/ml), harvested 4 days or 5 days after inoculation, and stored at 4 degrees. After harvesting, 10000rpm can be directly used, centrifugation is carried out for 10min, and culture supernatant is obtained. The supernatant was filtered through a 0.45mm or 0.22 μm filter. 20ml of His filler equilibrated with equilibration buffer was added to the filtered supernatant and stirred overnight at 4 ℃. The gD protein or gD-IgG2aFc fusion protein bound overnight to His filler was placed in a column and allowed to drip freely, and the flow-through was bound again to His and allowed to drip freely. Eluting 2 column volumes with 0mm imidazole 10mmTris-200mmNaCl buffer solution, and then eluting 2 column volumes with 20mm imidazole 10mmTris-200mmNaCl buffer solution to wash the hybrid protein. Finally, 200mm imidazole 10mmTris-200mmNaCl buffer solution elutes 2 column volumes, and elution of the target protein is carried out. And (3) carrying out imidazole removal treatment on the eluted protein, and taking 30-50 mu l of sample to carry out SDS-PAGE detection.

SDA-PAGE analysis recombinant baculovirus AC-2-PRV-gD protein expression electrophoresis picture is shown in the right picture in figure 2, wherein a specific band purified by about 41KDa appears. SDA-PAGE analysis recombinant baculovirus AC-2-PRV-gD-IgG2aFc protein expression electrophoresis chart is shown in the right chart in figure 3, wherein a specific band purified from about 71KDa appears. Recombinant virus bacillus AC-2-PRVgD and AC-2-PRVgD-IgG2aFc infect gD protein and gD-IgG2aFc fusion protein secretion supernatant expressed by insect cells.

Example 5

Vaccine for preventing and treating porcine pseudorabies

(I) preparation of recombinant baculovirus AC-2-PRVgD and AC-2-PRVgD-IgG2aFc subunit vaccine

Recombinant baculovirus AC-2-PRVgD obtained in example 2 and AC-2-PRVgD-IgG2aFc prepared in example 3 were used to inoculate HF cells (1 x 10) at a dose of 0.0001MOI⁶Ml), after 5 days of culture, the supernatant was harvested by centrifugation, purified by nickel column, and examined by SDS-PAGE. Calculating the content of gD and gD-IgG2aFc protein, wherein the concentration of the gD protein in the vaccine is 200 mug/ml; the concentration of gD-IgG2aFc protein in the vaccine was 300. mu.g/ml. Subunit vaccines were prepared, each immunized with 20. mu.g protein, by emulsification with SEPPIC adjuvant IMS-1313 at a dose of 200. mu.g/ml. The prepared subunit vaccine is stored at 4 ℃ after sterile inspection.

And (II) safety test of subunit vaccines prepared from the recombinant baculovirus AC-2-PRVgD and AC-2-PRVgD-IgG2 aFc.

The subunit vaccines were prepared to immunize 6 week old Babic mice separately, 12 mice were inoculated per batch separately. The inoculation mode is nasal drop immunization. The test result shows that the body temperature, spirit and appetite are all normal after the vaccination. The vaccine was confirmed to be safe for mice.

(III) immunogenicity test of subunit vaccine prepared from recombinant baculovirus AC-2-PRVgD and AC-2-PRVgD-IgG2aFc on mice

In order to systematically evaluate the effectiveness of the subunit vaccine of the above index. 60 mice, 6-7 weeks old, PCR-tested PRV negative, were purchased and randomly divided into 4 groups. The immunization dose is 20 mug/mouse, and the immunization route is nasal drop immunization. The same dose was boosted once 14 days after the first immunization. Collecting blood under eyelid before immunization and before immunization for 14, 28 and 42 days, separating splenic lymphocyte for immunization for 42 days, separating lymphocyte through gD and gD protein stimulation, harvesting supernatant, and determining cytokine content.

1. Detection of specific antibodies produced by the body against PRV

The PRV antibody is detected by an indirect ELISA method, and the operation steps are as follows: the PRgD protein expressed and purified by the recombinant baculovirus of the invention is used for coating an ELISA plate, matrix titration is carried out, each hole is determined to be coated with 0.5 mu g, and the temperature is kept overnight at 4 ℃. Blocking was then performed with 1% BSA at 37 ℃ for 1 h. After washing, 50. mu.L of 100-fold diluted serum to be detected was added to each well and incubated for 1 h. Washing for 3 times50 μ L of 10000-fold diluted HRP-labeled goat anti-mouse or rabbit IgG was added to each well and incubated at 37 ℃ for 1 h. After washing, TMB substrate solution was added and color development was carried out at room temperature for 10 min. 50 μ L of 2mol/L H per well₂SO₄The reaction was terminated by terminating the solution, and the OD was measured₄₅₀The value is obtained. The results show that immunization of the subunit vaccine prepared in this example, all groups except the PBS group produced PRV-specific IgG antibodies at 14, 28, 42dpi and peaked at 42 dpi. Furthermore, IgG antibody titers of mice vaccinated with gD-IgG2aFc and gD-IgG2aFc were higher than those of mice vaccinated with gD and gD (as shown in fig. 4). The results demonstrate that subunit vaccines prepared from AC-2-PRVgD and AC-2-PRVgD-IgG2aFc are effective in inducing the production of atopic antibodies against APPV. Clearly, the gD-IgGFc immunization group produced significantly higher PRV-specific IgG antibodies than either the gD or gD immunization groups. These results indicate that fusion of antigen to IgG2aFc is effective in promoting humoral immune responses.

2. Detection of specific cellular immune response produced by body

(1) Taking out the spleen of the mouse aseptically, placing the mouse spleen in a dish containing incomplete RPMI-1640, washing, transferring the mouse spleen into an aseptic homogenizer, adding a little incomplete 1640, lightly grinding, adding the incomplete RPMI-1640, uniformly mixing, and filtering by a cell filter to remove tissue fragments. The filtrate was gently pipetted into a 15ml centrifuge tube and centrifuged at 1000rpm for 10 min. The supernatant was discarded and 5ml of sterile 8.3g/LNH was added₄Cl, resting for 5min (red blood cells removed) and centrifuging at 1000rpm for 10 min. The supernatant was discarded, washed with incomplete RPMI-1640 and centrifuged at 1000rpm for 10 min. The supernatant was discarded and the pellet was suspended in 2.5ml of RPMI-1640 (10% FBS). Cell counting, trypan blue staining, requires that the cell activity should be above 95%. Adjusting the cell concentration to 1X 10⁶and/mL. After mixing, add 500. mu.L of the mixture into a 12-well plate. Each sample was stimulated with purified gD protein, concanavalin (positive control) and culture broth (negative control), and 3 replicates of each stimulus were made; 12 well cell culture plates were placed at 37 ℃ in 5% CO₂The cells were incubated in an incubator for 24 hours and then centrifuged to harvest the supernatant. And (3) detecting the expression levels of IFN-gamma and IL-4 proteins in the supernatant by using commercial mouse IFN-gamma and IL-4ELISA detection kits, wherein the kits adopt double-antibody sandwich ELISA.

The results show that the IL-4 concentration of the four subunit vaccine immunization groups is obviously higher than that of IFN-gamma. Furthermore, the IL-4 concentration in the gD-IgG2aFc immunized group was significantly higher than in the gD or gD groups (FIG. 5, in which the left panel is the change in IL-4 concentration in the three treatment groups and the right panel is the change in IFN-. gamma.concentration in the three treatment groups). These data indicate that Fc-linked PRVgD or gD proteins can significantly promote cellular immune responses.

(IV) challenge protection experiment of subunit vaccine prepared from recombinant baculovirus AC-2-PRVgD and AC-2-PRVgD-IgG2aFc on mice

To assess the suitability of mouse IgG2aFc fragments as potential molecular adjuvants, all mice were infected intranasally with 100LD₅₀The PRV GX wild strain of (a). During monitoring, the protection rates of the different groups are shown in fig. 6. After challenge, the protection rate of the gD-IgG2aFc group is obviously higher than that of the gD group. Mice immunized with gD-IgG2aFc were all alive without any clinical symptoms; the survival rate of mice immunized with gD-IgG2aFc is 83%; the survival rate of mice immunized with gD is 50%; the survival of mice immunized with gD-IgG2aFc was 67% (as shown in FIG. 6). These data indicate that PRV gD or gD protein expressed by recombinant baculovirus can effectively protect mice from PRV infection. In addition, the fusion of gD with IgG2aFc fragment subunit vaccine can achieve the best protection.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus, preparation method and application

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 993

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtgcccgcgc cgaccttccc cccgcccgcg tacccgtaca ccgagtcgtg gcagctgacg 60

ctgacgacgg tcccctcgcc cttcgtcggc cccgcggacg tctaccacac gcgcccgctg 120

gaggacccgt gcggggtggt ggcgctgatc tccgacccgc aggtggaccg gctgctgaac 180

gaggcggtgg cccaccggcg gcccacgtac cgcgcccacg tggcctggta ccgcatcgcg 240

gacgggtgcg cgcacctgct gtactttatc gagtacgccg actgcgaccc caggcagatc 300

tttgggcgct gccggcgccg caccacgccg atgtggtgga ccccgtccgc ggactacatg 360

ttccccacgg aggacgagct ggggctgctc atggtggccc cggggcggtt caacgagggc 420

cagtaccggc gcctggtgtc cgtcgacggc gtgaacatcc tcaccgactt catggtggcg 480

ctccccgagg ggcaagagtg cccgttcgcc cgcgtggacc agcaccgcac gtacaagttc 540

ggcgcgtgct ggagcgacga cagcttcaag cggggcgtgg acgtgatgcg attcctgacg 600

ccgttctacc agcagccccc gcaccgggag gtggtgaact actggtaccg caagaacggc 660

cggacgctcc cgcgggccta cgccgccgcc acgccgtacg ccatcgaccc cgcgcggccc 720

tcggcgggct cgccgaggcc caggccccgg ccccggccca ggccccggcc gaagcccgag 780

cccgccccgg cgacgcccgc gccccccggc cgcctgcccg agccggcgac gcgggaccac 840

gccgccgggg ggcgccccac gccgcgaccc ccgaggcccg agacgccgca ccgccccttc 900

gccccgccgg ccgtcgtgcc cagcgggtgg ccgcagcccg cggagccgtt cccgccccgg 960

accaccgccg cgccgggcgt ctcgcgccac cgc 993

<210> 2

<211> 699

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagcccagag ggcccacaat caagccctgt cctccatgca aatgcccagc acctaacctc 60

ttgggtggac catccgtctt catcttccct ccaaagatca aggatgtact catgatctcc 120

ctgagcccca tagtcacatg tgtggtggtg gatgtgagcg aggatgaccc agatgtccag 180

atcagctggt ttgtgaacaa cgtggaagta cacacagctc agacacaaac ccatagagag 240

gattacaaca gtactctccg ggtggtcagt gccctcccca tccagcacca ggactggatg 300

agtggcaagg agttcaaatg caaggtcaac aacaaagacc tgccagcgcc catcgagaga 360

accatctcaa aacccaaagg gtcagtaaga gctccacagg tatatgtctt gcctccacca 420

gaagaagaga tgactaagaa acaggtcact ctgacctgca tggtcacaga cttcatgcct 480

gaagacattt acgtggagtg gaccaacaac gggaaaacag agctaaacta caagaacact 540

gaaccagtcc tggactctga tggttcttac ttcatgtaca gcaagctgag agtggaaaag 600

aagaactggg tggaaagaaa tagctactcc tgttcagtgg tccacgaggg tctgcacaat 660

caccacacga ctaagagctt ctcccggact ccgggtaaa 699

<210> 3

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttttatggtc gtatacattt cttacatcta tgcggtgccc gcgccgacct tccccccgc 59

<210> 4

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gccaccatga aattcttagt caacgttgcc cttgttttta tggtcgtata catttctta 59

<210> 5

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctagtgatgg tgatggtgat gagagcccga tccgcggtgg cgcgagacgc ccggcgcgg 59

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggatccgcca ccatgaaatt cttagtcaac 30

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaattcctag tgatggtgat ggtgatgaga gc 32

<210> 8

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

actagtgcca ccatgaaatt cttagtcaac g 31

<210> 9

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aagcttctag tgatggtgat ggtgatgaga gcc 33

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gccaccatga aattcttagt caacgttgcc c 31

<210> 11

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccacctcctc cggacccacc cccgcctgat ccgcggtggc gcgagacgcc cggcgcggc 59

<210> 12

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atcaggcggg ggtgggtccg gaggaggtgg ctcgggatct gagcccagag ggcccacaa 59

<210> 13

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctagtgatgg tgatggtgat gagagcccga tcc 33

<210> 14

<211> 1209

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgctgctcg cagcgctatt ggcggcgctg gtcgcccgga cgacgctcgg cgcggacgtg 60

gacgccgtgc ccgcgccgac cttccccccg cccgcgtacc cgtacaccga gtcgtggcag 120

ctgacgctga cgacggtccc ctcgcccttc gtcggccccg cggacgtcta ccacacgcgc 180

ccgctggagg acccgtgcgg ggtggtggcg ctgatctccg acccgcaggt ggaccggctg 240

ctgaacgagg cggtggccca ccggcggccc acgtaccgcg cccacgtggc ctggtaccgc 300

atcgcggacg ggtgcgcgca cctgctgtac tttatcgagt acgccgactg cgaccccagg 360

cagatctttg ggcgctgccg gcgccgcacc acgccgatgt ggtggacccc gtccgcggac 420

tacatgttcc ccacggagga cgagctgggg ctgctcatgg tggccccggg gcggttcaac 480

gagggccagt accggcgcct ggtgtccgtc gacggcgtga acatcctcac cgacttcatg 540

gtggcgctcc ccgaggggca agagtgcccg ttcgcccgcg tggaccagca ccgcacgtac 600

aagttcggcg cgtgctggag cgacgacagc ttcaagcggg gcgtggacgt gatgcgattc 660

ctgacgccgt tctaccagca gcccccgcac cgggaggtgg tgaactactg gtaccgcaag 720

aacggccgga cgctcccgcg ggcctacgcc gccgccacgc cgtacgccat cgaccccgcg 780

cggccctcgg cgggctcgcc gaggcccagg ccccggcccc ggcccaggcc ccggccgaag 840

cccgagcccg ccccggcgac gcccgcgccc cccggccgcc tgcccgagcc ggcgacgcgg 900

gaccacgccg ccggggggcg ccccacgccg cgacccccga ggcccgagac gccgcaccgc 960

cccttcgccc cgccggccgt cgtgcccagc gggtggccgc agcccgcgga gccgttcccg 1020

ccccggacca ccgccgcgcc gggcgtctcg cgccaccgct cggtgatcgt cggcacgggc 1080

accgcgatgg gcgcgctcct ggtgggcgtg tgcgtctaca tcttcttccg cctgaggggg 1140

gcgaaggggt atcgcctcct gggcggtccc gcggacgccg acgagctaaa agcgcagccc 1200

ggtccgtag 1209

<210> 15

<211> 402

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu

1 5 10 15

Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala

20 25 30

Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser

35 40 45

Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp

50 55 60

Pro Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu

65 70 75 80

Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val

85 90 95

Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile

100 105 110

Glu Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg

115 120 125

Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro

130 135 140

Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn

145 150 155 160

Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu

165 170 175

Thr Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala

180 185 190

Arg Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp

195 200 205

Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe

210 215 220

Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys

225 230 235 240

Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr Ala

245 250 255

Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg Pro Arg

260 265 270

Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro

275 280 285

Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala

290 295 300

Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg

305 310 315 320

Pro Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala

325 330 335

Glu Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val Ser Arg His

340 345 350

Arg Ser Val Ile Val Gly Thr Gly Thr Ala Met Gly Ala Leu Leu Val

355 360 365

Gly Val Cys Val Tyr Ile Phe Phe Arg Leu Arg Gly Ala Lys Gly Tyr

370 375 380

Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Glu Leu Lys Ala Gln Pro

385 390 395 400

Gly Pro

<210> 16

<211> 1107

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctcgaggcca ccatgaaatt cttagtcaac gttgcccttg tttttatggt cgtatacatt 60

tcttacatct atgcggtgcc cgcgccgacc ttccccccgc ccgcgtaccc gtacaccgag 120

tcgtggcagc tgacgctgac gacggtcccc tcgcccttcg tcggccccgc ggacgtctac 180

cacacgcgcc cgctggagga cccgtgcggg gtggtggcgc tgatctccga cccgcaggtg 240

gaccggctgc tgaacgaggc ggtggcccac cggcggccca cgtaccgcgc ccacgtggcc 300

tggtaccgca tcgcggacgg gtgcgcgcac ctgctgtact ttatcgagta cgccgactgc 360

gaccccaggc agatctttgg gcgctgccgg cgccgcacca cgccgatgtg gtggaccccg 420

tccgcggact acatgttccc cacggaggac gagctggggc tgctcatggt ggccccgggg 480

cggttcaacg agggccagta ccggcgcctg gtgtccgtcg acggcgtgaa catcctcacc 540

gacttcatgg tggcgctccc cgaggggcaa gagtgcccgt tcgcccgcgt ggaccagcac 600

cgcacgtaca agttcggcgc gtgctggagc gacgacagct tcaagcgggg cgtggacgtg 660

atgcgattcc tgacgccgtt ctaccagcag cccccgcacc gggaggtggt gaactactgg 720

taccgcaaga acggccggac gctcccgcgg gcctacgccg ccgccacgcc gtacgccatc 780

gaccccgcgc ggccctcggc gggctcgccg aggcccaggc cccggccccg gcccaggccc 840

cggccgaagc ccgagcccgc cccggcgacg cccgcgcccc ccggccgcct gcccgagccg 900

gcgacgcggg accacgccgc cggggggcgc cccacgccgc gacccccgag gcccgagacg 960

ccgcaccgcc ccttcgcccc gccggccgtc gtgcccagcg ggtggccgca gcccgcggag 1020

ccgttcccgc cccggaccac cgccgcgccg ggcgtctcgc gccaccgcgg atcgggctct 1080

catcaccatc accatcacta ggcatgc 1107

<210> 17

<211> 1830

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat 60

gcggtgcccg cgccgacctt ccccccgccc gcgtacccgt acaccgagtc gtggcagctg 120

acgctgacga cggtcccctc gcccttcgtc ggccccgcgg acgtctacca cacgcgcccg 180

ctggaggacc cgtgcggggt ggtggcgctg atctccgacc cgcaggtgga ccggctgctg 240

aacgaggcgg tggcccaccg gcggcccacg taccgcgccc acgtggcctg gtaccgcatc 300

gcggacgggt gcgcgcacct gctgtacttt atcgagtacg ccgactgcga ccccaggcag 360

atctttgggc gctgccggcg ccgcaccacg ccgatgtggt ggaccccgtc cgcggactac 420

atgttcccca cggaggacga gctggggctg ctcatggtgg ccccggggcg gttcaacgag 480

ggccagtacc ggcgcctggt gtccgtcgac ggcgtgaaca tcctcaccga cttcatggtg 540

gcgctccccg aggggcaaga gtgcccgttc gcccgcgtgg accagcaccg cacgtacaag 600

ttcggcgcgt gctggagcga cgacagcttc aagcggggcg tggacgtgat gcgattcctg 660

acgccgttct accagcagcc cccgcaccgg gaggtggtga actactggta ccgcaagaac 720

ggccggacgc tcccgcgggc ctacgccgcc gccacgccgt acgccatcga ccccgcgcgg 780

ccctcggcgg gctcgccgag gcccaggccc cggccccggc ccaggccccg gccgaagccc 840

gagcccgccc cggcgacgcc cgcgcccccc ggccgcctgc ccgagccggc gacgcgggac 900

cacgccgccg gggggcgccc cacgccgcga cccccgaggc ccgagacgcc gcaccgcccc 960

ttcgccccgc cggccgtcgt gcccagcggg tggccgcagc ccgcggagcc gttcccgccc 1020

cggaccaccg ccgcgccggg cgtctcgcgc caccgcggat caggcggggg tgggtccgga 1080

ggaggtggct cgggatctga gcccagaggg cccacaatca agccctgtcc tccatgcaaa 1140

tgcccagcac ctaacctctt gggtggacca tccgtcttca tcttccctcc aaagatcaag 1200

gatgtactca tgatctccct gagccccata gtcacatgtg tggtggtgga tgtgagcgag 1260

gatgacccag atgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 1320

acacaaaccc atagagagga ttacaacagt actctccggg tggtcagtgc cctccccatc 1380

cagcaccagg actggatgag tggcaaggag ttcaaatgca aggtcaacaa caaagacctg 1440

ccagcgccca tcgagagaac catctcaaaa cccaaagggt cagtaagagc tccacaggta 1500

tatgtcttgc ctccaccaga agaagagatg actaagaaac aggtcactct gacctgcatg 1560

gtcacagact tcatgcctga agacatttac gtggagtgga ccaacaacgg gaaaacagag 1620

ctaaactaca agaacactga accagtcctg gactctgatg gttcttactt catgtacagc 1680

aagctgagag tggaaaagaa gaactgggtg gaaagaaata gctactcctg ttcagtggtc 1740

cacgagggtc tgcacaatca ccacacgact aagagcttct cccggactcc gggtaaagga 1800

tcgggctctc atcaccatca ccatcactag 1830

<210> 18

<211> 609

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala Tyr

20 25 30

Pro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser Pro

35 40 45

Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp Pro

50 55 60

Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu Leu

65 70 75 80

Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val Ala

85 90 95

Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile Glu

100 105 110

Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg Arg

115 120 125

Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr

130 135 140

Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu

145 150 155 160

Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr

165 170 175

Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg

180 185 190

Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp Asp

195 200 205

Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe Tyr

210 215 220

Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys Asn

225 230 235 240

Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr Ala Ile

245 250 255

Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg Pro Arg Pro

260 265 270

Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro Ala

275 280 285

Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala Gly

290 295 300

Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg Pro

305 310 315 320

Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala Glu

325 330 335

Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val Ser Arg His Arg

340 345 350

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Glu Pro

355 360 365

Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro

370 375 380

Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys

385 390 395 400

Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val

405 410 415

Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn

420 425 430

Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr

435 440 445

Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp

450 455 460

Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

465 470 475 480

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg

485 490 495

Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys

500 505 510

Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp

515 520 525

Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys

530 535 540

Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser

545 550 555 560

Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser

565 570 575

Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser

580 585 590

Phe Ser Arg Thr Pro Gly Lys Gly Ser Gly Ser His His His His His

595 600 605

His

<210> 19

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtgcccgcgc cgaccttccc cccgcccgcg tac 33

<210> 20

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gcggtggcgc gagacgcccg gcgcggcggt g 31

Claims

1. The recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD-IgGFc fusion protein gene is characterized in that HBM signal peptide-gD-IgGFc fusion protein-His label is taken as an exogenous gene and is respectively inserted into a restriction enzyme cutting site between BamHI and EcoRI and a restriction enzyme cutting site between SpeI and HindIII of the baculovirus transfer vector;

2. A method for constructing a recombinant baculovirus transfer vector containing the porcine pseudorabies virus gD-IgGFc fusion protein gene as claimed in claim 1, which comprises the following steps:

3. The method for constructing according to claim 2, wherein the second primer pair comprises a forward primer in which an HBM signal peptide sequence is added to the 5 'end of gD as shown in SEQ ID No.10, a reverse primer in which a homology arm sequence is added to the 3' end of gD as shown in SEQ ID No.11, a forward primer in which a homology arm sequence is added to the 5 'end of IgGFc as shown in SEQ ID No.12, and a reverse primer in which a His tag sequence is added to the 3' end of IgGFc as shown in SEQ ID No. 13.

4. A recombinant baculovirus expressing porcine pseudorabies virus gD-IgGFc fusion protein, which is obtained by transfecting the recombinant baculovirus transfer vector of claim 1 into insect cells, culturing until the cells have lesions, and collecting supernatant.

5. Use of the recombinant baculovirus transfer vector of claim 1 or the recombinant baculovirus of claim 4 for the production of a vaccine for porcine pseudorabies.

6. A subunit vaccine for preventing and treating porcine pseudorabies is characterized by comprising an adjuvant and one of the following proteins: the recombinant baculovirus of claim 4 expressing a gD-IgGFc fusion protein.

7. The subunit vaccine of claim 6, wherein the gD-IgGFc fusion protein is present at a concentration of 100-150 μ g/ml.