CN116063406A - Cat infectious rhinotracheitis vaccine, and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological products, in particular to a cat infectious rhinotracheitis vaccine, a preparation method and application thereof. The feline infectious rhinotracheitis vaccine includes an immunogenic composition comprising: cat infectious rhinotracheitis gB protein, cat infectious rhinotracheitis gC protein, and cat infectious rhinotracheitis gD protein. The invention constructs gB-PA3, gC-PA3 and gD-PA3 genes into plasmids, obtains gB, gC and gD proteins through expression, and obtains cat infectious rhinotracheitis vaccines by matching with an adjuvant. The cat infectious rhinotracheitis provided by the invention can stimulate organisms to generate good humoral and cellular immune responses after immunizing animals, and has the advantages of good immunogenicity, high safety and the like.

Description

Cat infectious rhinotracheitis vaccine, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological products, in particular to a cat infectious rhinotracheitis vaccine, a preparation method and application thereof.

Background

Feline infectious rhinotracheitis (Feline infectious rhinotracheitis, FIR), also known as feline viral rhinotracheitis, is an acute, highly contagious upper respiratory disease caused by feline herpes virus type I (Feline herpesvirus, FHV-1). FHV-1 mainly infects cats, and also infects felines such as tigers and leopards, and constitutes a great threat to the health of cats. It is reported that the disease of respiratory tract infection caused by FHV-1 infection accounts for about 50-75% of all definite viral upper respiratory tract infections in cats; the most common symptoms of FHV-1 infection in cats are conjunctivitis and rhinotracheitis, and the virus may also cause the cats to develop glossitis, pneumonia, abortion, keratitis, systemic infection, or rare dermatitis. FHV-1 infected cats are mainly transmitted into the trigeminal ganglion through sensory neurons, and can cause latent infection of organisms; because the latent infection of the virus is longer, the infected cats carry the virus for a long time to expel the virus, and great difficulties are brought to the diagnosis and prevention work of the infectious rhinotracheitis of the cats.

Vaccination is one of the safest and most effective measures to control and prevent infectious diseases. Currently, FHV-1 attenuated live vaccines and inactivated viral vaccines are widely used. While live attenuated vaccines can provide protection, there are often some side effects on cats such as sneezing, ocular secretions and elevated body temperature. In contrast to live attenuated vaccines, inactivated vaccines may cause adverse reactions at the injection site, especially when combined with aluminium adjuvants, sarcoma may occur at the injection site.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a cat infectious rhinotracheitis vaccine, and a preparation method and application thereof.

In a first aspect, the present invention provides an immunogenic composition comprising: cat infectious rhinotracheitis gB protein, cat infectious rhinotracheitis gC protein, and cat infectious rhinotracheitis gD protein.

Further, the cat infectious rhinotracheitis gB protein is obtained by encoding a nucleotide sequence shown as SEQ ID NO. 1; the cat infectious rhinotracheitis gC protein is obtained by encoding a nucleotide sequence shown as SEQ ID NO. 2; the cat infectious rhinotracheitis gD protein is obtained by encoding a nucleotide sequence shown as SEQ ID NO. 3.

Further, the molar ratio of the cat infectious rhinotracheitis gB protein, the cat infectious rhinotracheitis gC protein and the cat infectious rhinotracheitis gD protein is 1-5: 1 to 5:1 to 5.

In a second aspect, the invention provides a feline infectious rhinotracheitis vaccine comprising the immunogenic composition.

Further, the dosage of the cat infectious rhinotracheitis gB protein in the cat infectious rhinotracheitis vaccine is 0.1-1 mg/mL; and/or the dosage of the cat infectious rhinotracheitis gC protein is 0.1-1 mg/mL; and/or the dosage of the cat infectious rhinotracheitis gD protein is 0.1-1 mg/mL.

Further, an adjuvant is included; the adjuvants include one or more of Gel02, natural source adjuvants (e.g., quilla, saponins, propolis), oil emulsion adjuvants (e.g., freund's adjuvant, white oil adjuvant, ISA206, MF-59, adjuvant-65, SAF series), cytokine adjuvants (e.g., interleukins, interferons, tumor necrosis factors, colony stimulating factors), microbial source adjuvants (e.g., lipopolysaccharide, muramyl dipeptide, cholera toxin, cpG immunomodulating sequences), liposome adjuvants, nanoadjuvants.

Further, the adjuvant is Gel02; the mass ratio of the immunogenic composition to the adjuvant is 20:1-5:1.

In a third aspect, the invention provides a method of preparing the feline infectious rhinotracheitis vaccine comprising:

the nucleotide sequences shown as SEQ ID NO.1-3 are respectively constructed on a carrier, and are expressed by an insect cell-baculovirus expression system to obtain cat infectious rhinotracheitis gB protein, cat infectious rhinotracheitis gC protein and cat infectious rhinotracheitis gD protein, and the cat infectious rhinotracheitis vaccine is prepared by matching with an adjuvant.

Further, the cat infectious rhinotracheitis gB protein, the cat infectious rhinotracheitis gC protein and the cat infectious rhinotracheitis gD protein are obtained through the expression of a GEM-PA surface display system.

Further, the expression cassette for expressing the cat infectious rhinotracheitis gB protein is: form of signal peptide+cat infectious rhinotracheitis gB protein+linker+pa 3 sequence;

the expression cassette for expressing cat infectious rhinotracheitis gC protein is: form of signal peptide+cat infectious rhinotracheitis gC protein+linker+pa 3 sequence;

the expression cassette for expressing cat infectious rhinotracheitis gD protein is: form of signal peptide+cat infectious rhinotracheitis gD protein+linker+PA 3 sequence.

Further, the sequence of PA3 is shown as SEQ ID NO.4, the gp64 signal peptide sequence is shown as SEQ ID NO.5, and the Linker sequence is shown as SEQ ID NO. 6.

Further, the vector used in the insect cell-baculovirus expression system is the pFastBac1 vector.

Further, the cells transfected in the insect cell-baculovirus expression system are Sf9 cells.

The invention further provides the use of said immunogenic composition in the manufacture of a medicament for immunizing cats against infectious rhinotracheitis.

The invention further provides a biological material, which comprises any one or more nucleotide sequences shown in SEQ ID NO. 1-3; the biological material is an expression cassette, a vector or a transgenic cell.

The invention has the following beneficial effects:

the sequences of the gB-PA3, the gC-PA3 and the gD-PA3 genes are optimized and then expressed to obtain three cat infectious rhinotracheitis proteins, and then the three proteins and the adjuvant are combined to obtain the specific cat infectious rhinotracheitis vaccine.

Drawings

FIG. 1 is a schematic diagram of a recombinant plasmid construction strategy according to example 1 of the present invention; wherein A is rpFB1-gp64-gB-PA3; b is rpFB1-gp64-gC-PA3; c is rpFB1-gp64-gD-PA3.

FIG. 2 is a diagram showing the indirect immunofluorescence assay of recombinant baculovirus provided in example 1 of the present invention; wherein A is rBV-gp64-gB-PA3; b is rBV-gp64-gC-PA3; c is rBV-gp64-gD-PA3; d is a cell control.

FIG. 3 is a diagram showing the WB identification of the fusion protein according to example 1 of the present invention; wherein A is a gB-PA3 fusion protein; b is gC-PA3 fusion protein; c is gD-PA3 fusion protein; in the figure, M is a protein molecule Marker,1 is supernatant, 2 is supernatant after ultrasonication, and 3 is Sf9 cell control.

Fig. 4 is a GEM particle electron microscope observation chart provided in embodiment 2 of the present invention; wherein A is gB-GEM; b is gC-GEM; c is gD-GEM; d is GEM control.

FIG. 5 is a graph of indirect immunofluorescence assay of GEM particles provided in example 2 of the invention; wherein A is gB-GEM; b is gC-GEM; c is gD-GEM; d is GEM control.

FIG. 6 is a WB identification map of GEM particles provided in example 2 of the present invention; wherein A is gB-GEM; b is gC-GEM; c is gD-GEM; in the figure, M is a protein molecule Marker;1 is GEM granule; 2 is GEM control.

FIG. 7 is a SDS-PAGE identification of GEM particles according to example 2 of the present invention; a is gB-GEM; b is gC-GEM; c is gD-GEM.

FIG. 8 is a schematic diagram showing the detection results of FHV-1 neutralizing antibodies in mouse serum as provided in example 3 of the present invention.

FIG. 9 is a graph showing the results of ELISPot assay of secreted cytokines IFN-. Gamma.and IL-4 provided in example 3 of the present invention.

FIG. 10 is a graph showing the result of secretion of cytokines by spleen cells according to example 3 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1 expression and characterization of FHV-1gB-PA3, gC-PA3, gD-PA3 fusion proteins

1.1 construction and identification of recombinant donor plasmids and shuttle plasmids

The gene sequences gB, gC and gD of lactococcus lactis MG1363 fluke protein PA3 sequence and FHV CH-B strain are obtained from GenBank, and are optimized and synthesized according to the codon preference of insect cells. The primer is designed for the optimized gB, gC and gD sequences by PrimerPremier5.0 software, and the primer sequences are shown in Table 1. Sequence optimization and synthesis, and primer synthesis are completed by Shanghai bioengineering limited company.

According to the primers shown in Table 1, the gB-PA3, gC-PA3 and gD-PA3 genes are subjected to overlapping PCR amplification and ligation, and the construction strategy is shown in FIG. 1 (the sequence of PA3 is shown as SEQ ID NO.4, the sequence of gp64 signal peptide is shown as SEQ ID NO.5, and the sequence of Linker is shown as SEQ ID NO. 6). After the PCR amplification product is subjected to agarose gel electrophoresis with concentration of 1%, the gene is recovered and purified according to the operation steps of the gel recovery kit instruction book, and the obtained gene fragments are named gB-PA3, gC-PA3 and gD-PA3. The gB-PA3, gC-PA3 and gD-PA3 gene fragments are cloned to eukaryotic expression plasmids pFastBac1-gp64 respectively, double digestion identification is carried out on recombinant plasmids by using Xba I and Kpn I, and the identified correct plasmids are sent to Jilin Kyoto Biotechnology Co.Ltd.

The recombinant plasmid plasmids with correct sequencing were designated pFB1-gp64-gB-PA3, pFB1-gp64-gC-PA3, pFB1-gp64-gD-PA3. Three plasmids were transformed into DH10Bac competent, cultured at 37℃for 48h, after screening for bluish white spots containing tetracycline, gentamicin and kanamycin, white colonies were picked up, recombinant baculovirus plasmids were extracted and subjected to PCR identification, and the identified correct recombinant baculovirus plasmids were designated rBac-gp64-gB-PA3, rBac-gp64-gC-PA3 and rBac-gp64-gD-PA3.

Table 1 amplification primers for gB, gC, gD and PA3 genes

/>

Note that: the lower line mark in the table represents the cleavage site sequence information. The underlined wavy line represents Linker sequence information.

1.2 rescue of recombinant baculoviruses

Sf9 cells were transfected with rBac-gp64-gB-PA3, rBac-gp64-gC-PA3 and rBac-gp64-gD-PA3, respectively, and cytopathic effect was observed after incubation at 27℃for 72 hours. When cytopathy reaches about 80%, collecting the mixture to obtain recombinant baculovirus, extracting genome, and identifying correctly by PCR, and naming rBV-gp64-gB-PA3, rBV-gp64-gC-PA3 and rBV-gp64-gD-PA3. The recombinant baculovirus is continuously passaged downwards, obvious cytopathy is visible after Sf9 cells are infected for 48 hours, and the cells become large, round and fall off.

1.3 Indirect immunofluorescence

The Sf9 adherent cells are inoculated with rBV-gp64-gB-PA3, rBV-gp64-gC-PA3 and rBV-gp64-gD-PA3, and indirect immunofluorescence identification is carried out by using cat-source anti-FHV-1 polyclonal antibody.

As shown in FIG. 2, the recombinant baculoviruses rBV-gp64-gB-PA3, rBV-gp64-g C-PA3 and rBV-gp64-gD-PA3 infected Sf9 cells all showed obvious green fluorescence, and normal Sf9 cells showed no specific fluorescence reaction, but only red cell background. This shows that the gB, gC, gD proteins are expressed in cells, and the expressed target proteins can be specifically recognized by cat-derived anti-FH V-1 polyclonal antibodies.

1.4WB identification of expression and expression forms of the protein of interest

The P3 generation rBV-gp64-gB-PA3, rBV-gp64-gC-PA3 and rBV-gp64-gD-PA3 were inoculated with Sf9 suspension cells at MOI=0.5, respectively, and the mixture was harvested after 96 hours. The mixture was centrifuged at 3000rpm for 10min, the pellet was resuspended in an equal volume of 10mM PBS, and after sonication, the pellet was centrifuged at 6000rpm for 10min, and the pellet was resuspended in an equal volume of 10mM PBS. Adding 5×loading Buffer into the sample, boiling for 10min, performing SDS-PAGE electrophoresis, transferring to NC membrane, and performing WB analysis with cat-derived anti-FHV-1 polyclonal antibody as primary antibody (1:1000 dilution).

The WB identification result is shown in FIG. 3, the specific band of the gB protein is visible at 130KD for the rBV-gp64-gB-PA3 sample, the gB protein is soluble expressed, the specific band of the gC protein is visible at 100KD for the rBV-gp64-gC-P A3 sample, the specific band of the gD protein is visible at 70KD for the rBV-gp64-gD-PA3 sample, and the gD protein is soluble expressed.

Example 2 binding of fusion proteins to GEM particles and identification

1.1 preparation of GEM particles

50. Mu.L of the preserved strain was added to 5mL of M17 medium, and the bacterial liquid was recovered. The resuscitated bacterial liquid is added into M17 culture medium according to the proportion of 1:500, and is cultured for about 16 hours at the temperature of 30 ℃. Centrifuge at 7000rpm for 10min. The supernatant was discarded and the pellet was resuspended in 10mM PBS. Boiling: the precipitate was resuspended in 10% TCA solution, thoroughly mixed by shaking and boiled in water for 30min. Wash thoroughly 5 times with PBS. Finally resuspended in PBS. Optical deviceThe number of GEM particles was observed and recorded under a microscope, as 1U GEM particles = 2.5 x 10 ⁹ And (3) quantifying and split charging the mixture, and preserving the mixture at-80 ℃.

1.2 binding of fusion proteins to GEM particles

Respectively taking 1U GEM particles to combine with 10mL fusion proteins gB-PA3, gC-PA3 and gD-PA3, incubating for 30min at room temperature, centrifuging, discarding supernatant, washing the precipitate with PBS for 4-5 times, and finally re-suspending with PBS to obtain a mixture, namely gB-GEM, gC-GEM and gD-GEM.

1.3 identification by Transmission Electron microscopy

And respectively adding 2.5% glutaraldehyde fixing solution into the obtained gB-GEM, gC-GEM and gD-GEM, fixing at 4 ℃ overnight, finally preparing an electron microscope slice, and observing the morphology and structure of GEM particles of the load fusion protein by using a transmission electron microscope. The results are shown in FIG. 4: compared with GEM control, the gB-GEM, gC-GEM and gD-GEM samples have flocculent conjugates on the surface of GEM particles, which shows that the gB-PA3, gC-PA3 and gD-PA3 fusion proteins are successfully displayed on the surface of GEM particles.

1.4 Indirect immunofluorescence assay

The combined mixture gB-GEM, gC-GEM and gD-GEM are subjected to indirect immunofluorescence identification by using cat-source anti-FHV-1 polyclonal antibody. The results are shown in FIG. 5: the gB-GEM, the gC-GEM and the gD-GEM all have obvious green fluorescence, and the GEM particle control has no green fluorescence. This suggests that the gB-PA3, gC-PA3, and gD-PA3 fusion proteins were successfully displayed on the surface of GEM particles.

1.5WB assay

Mixing the mixture gB-GEM, gC-GEM and gD-GEM with 5×loading Buffer, and boiling for 10min. WB identification gave the results shown in fig. 6: the specific band of the gB-PA3 fusion protein was seen at 130KD for the gB-GEM sample, at 100KD for the gC-PA3 fusion protein, and at 70KD for the gD-GEM sample.

1.6 determination of the amount of fusion protein bound to GEM particles

SDS-PAGE is carried out on gB-GEM, gC-GEM and gD-GEM, BSA with different gradient concentrations is used as a standard protein, and the protein binding amount is determined by gray-scale analysis software. The results shown in fig. 7 were obtained by quantitative analysis: the 1U GEM particles bind about 31.8 μg of the gB-PA3 fusion protein, 86.8 μg of the gC-PA3 fusion protein, and 114 μg of the gD-PA3 fusion protein.

EXAMPLE 3 immunogenicity Studies of FHV GEM vaccine

1.1 immunization of mice

gB-GEM, gC-GEM and gD-GEM are mixed according to the mass ratio of 1:1:1 (16.67. Mu.g each) were mixed to obtain gB & gC & gD-GEM. The gB-GEM, the gC-GEM, the gD-GEM and the gB & gC & gD-GEM are respectively mixed with Gel02 adjuvant in a volume ratio of 5:1 to prepare the vaccine for immunization. The mice were randomly divided into 5 groups, see table 2. Immunization was performed 3 times, with booster immunization at week 3 and week 6, respectively. Serum samples were collected at weeks 0, 7, 9, 11 and 13 after mouse immunization.

Table 2 mice immunization groups

1.2 detection of FHV-1 neutralizing antibodies

Serum samples were collected at weeks 0, 7, 9, 11 and 13 after the first immunization of mice and tested for FHV-1 neutralizing antibodies. The specific method comprises the following steps: in a 96-well cell plate, 50 μl of double culture broth was added per well, serum was diluted 2-fold to column 6, and 4 replicates per sample were performed; diluting calibrated FHV-1 cytotoxicity to 200TCID using double MEM-free ₅₀ Per mL, 50. Mu.L/well in 96-well plates; in the virus control wells, 50 μl of double MEM free was added to each well, and the diluted virus was diluted 10-fold, 50 μl/well, and repeated four times; 37 ℃ and 5% CO ₂ After 1h incubation, digested F81 cells were added, 100. Mu.l/well. 37 ℃ and 5% CO ₂ Culturing in a incubator for 4 days, and observing cytopathy under an optical microscope to judge the titer of the FHV-1 neutralizing antibody in serum.

The results are shown in FIG. 8: neutralizing antibody titers were significantly higher in immunized mice than in PBS. In particular, the neutralizing antibody titres of the gB & gC & gD-GEM groups are higher than those of the gB-GEM, gC-GEM and gD-GEM groups. The results show that after the gB, the gC, the gD-GEM and the Gel02 adjuvant are mixed, the mice can be stimulated to generate FHV neutralizing antibodies, and the mice have good immune effect.

1.3 IFN-gamma and IL-4ELISpot assays

Mice were euthanized 1 week after the last immunization (3 mice per group), spleen cells were isolated and 1.25X10 ⁵ Individual cells were transferred to ELISpot plates and splenocytes were stimulated with FHV-1 cytotoxicity (moi=0.04). Cell plates were incubated in an incubator for 36h, stained for antibody incubation according to the instructions, and finally counted using AID ELISPOT reader iSpot.

The ELISPot assay results are shown in FIG. 9, where the immune group has significantly increased numbers of IFN-gamma and IL-4 secreting cells compared to the PBS group. IL-4 secreting cells were significantly higher in the gB & gC & gD-GEM groups than in the gB-GEM, gC-GEM and gD-GEM groups.

1.4 detection of cytokine secretion from spleen cell supernatant

To detect cytokine levels, 1.25X10 were used ⁵ Individual cells were transferred to 96-well plates and stimulated with FHV-1 cytotoxicity (moi=0.04). After 48h, the supernatant was collected and the secretion level of cytokines in the spleen cell supernatant was evaluated using Meso Scale Discovery kit. The results are shown in FIG. 10: IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha cytokines were significantly higher in the immunized group than in the PBS group. In addition, gB&gC&Th 2-related cytokines (IL-4, IL-6 and IL-10) were significantly higher in the gD-GEM group than in the gB-GEM, gC-GEM and gD-GEM groups. The above data indicate that gB&gC&gD-GEM promotes Th1 and Th2 responses of mouse spleen cells and cytokine secretion.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. An immunogenic composition comprising: cat infectious rhinotracheitis gB protein, cat infectious rhinotracheitis gC protein, and cat infectious rhinotracheitis gD protein.

2. The immunogenic composition of claim 1, wherein the feline infectious rhinotracheitis gB protein is encoded by a nucleotide sequence set forth in SEQ ID No. 1; and/or, the cat infectious rhinotracheitis gC protein is obtained by encoding a nucleotide sequence shown as SEQ ID NO. 2; and/or, the cat infectious rhinotracheitis gD protein is obtained by encoding a nucleotide sequence shown as SEQ ID NO. 3.

3. The immunogenic composition of claim 1 or 2, wherein the molar ratio of feline infectious rhinotracheitis gB protein, feline infectious rhinotracheitis gC protein, and feline infectious rhinotracheitis gD protein is 1-5: 1 to 5:1 to 5.

4. A feline infectious rhinotracheitis vaccine comprising the immunogenic composition of any one of claims 1-3.

5. The feline infectious rhinotracheitis vaccine of claim 4 wherein the feline infectious rhinotracheitis vaccine has an amount of feline infectious rhinotracheitis gB protein of 0.1-1 mg/mL; and/or the dosage of the cat infectious rhinotracheitis gC protein is 0.1-1 mg/mL; and/or the dosage of the cat infectious rhinotracheitis gD protein is 0.1-1 mg/mL.

6. The feline infectious rhinotracheitis vaccine of claim 1, further comprising an adjuvant; the adjuvant comprises one or more of Gel02, natural source adjuvant, oil emulsion adjuvant, cytokine adjuvant, microorganism source adjuvant, liposome adjuvant and nanometer adjuvant.

7. The feline infectious rhinotracheitis vaccine of claim 6, wherein the adjuvant is Gel02; the volume ratio of the immunogenic composition to the adjuvant is 20:1-5:1.

8. A method of preparing a feline infectious rhinotracheitis vaccine as defined in any one of claims 4-7, comprising:

the nucleotide sequences shown as SEQ ID NO.1-3 are respectively constructed on a carrier, and are expressed by an insect cell-baculovirus expression system to obtain cat infectious rhinotracheitis gB protein, cat infectious rhinotracheitis gC protein and cat infectious rhinotracheitis gD protein, and the cat infectious rhinotracheitis vaccine is prepared by matching with an adjuvant.

9. Use of an immunogenic composition according to any one of claims 1-3 in the manufacture of a medicament for immunizing cats against infectious rhinotracheitis.

10. A biological material, characterized in that the biological material comprises any one or more nucleotide sequences as set forth in SEQ ID No. 1-3; the biological material is an expression cassette, a vector or a transgenic cell.

Images