GD+mi3 protein nanoparticles for preventing and treating IBR as well as preparation method and application of gD+mi3 protein nanoparticles

Abstract

The invention is suitable for the technical field of gene engineering, and provides gD + mi3 protein nanoparticles for preventing and treating IBR and a preparation method and application thereof.The gD + mi3 protein nanoparticles are formed by coupling gD-st fusion protein and mi3-sc fusion protein, and gD antigens of infectious bovine rhinotracheitis viruses are displayed on the surfaces of the mi3 protein nanoparticles through isopeptide bonds; the amino acid sequence of the mi3 protein nanoparticle is shown as SEQ ID NO: 6 in a sequence table; the amino acid sequence of the gD-st fusion protein is as shown in a sequence table SEQ ID NO: 2; the amino acid sequence of the mi3-sc fusion protein is shown as SEQ ID NO: 4 in a sequence table. After animals are immunized by the gD + mi3 protein nanoparticles, an effective immune protection effect can be generated, and the gD + mi3 protein nanoparticles have a good application prospect in prevention and control of infectious bovine rhinotracheitis caused by IBRV (infectious bovine rhinotracheitis virus).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a gD+mi3 protein nanoparticle for preventing and treating IBR as well as a preparation method and application thereof. BACKGROUND

[0002] Bovine infectious rhinotracheitis (IBR), also known as red nose disease, is a contagious disease caused by bovine infectious rhinotracheitis virus (IBRV). IBRV is also known as bovine herpes virus (BoHV-1). IBRV has strong infectivity and can be transmitted through droplets, artificial insemination and direct contact. The morbidity and mortality of calves are high, and the disease is accompanied by nervous system symptoms. BoHV-1 can be latent in the body of a cow and carried for a long time. When the cow is stressed, the virus is activated and becomes a persistent source of infection, which increases the difficulty of prevention and control. The widespread transmission and latent characteristics of the disease seriously threaten the healthy development of the cattle industry and cause serious economic losses.

[0003] Although traditional vaccines can control the epidemic to some extent, they are difficult to effectively block the latent infection and reactivation of the virus. Therefore, it is of great significance to develop a new type of efficient vaccine for the prevention and control of IBRV. SUMMARY

[0004] The purpose of the present application is to provide a gD+mi3 protein nanoparticle for preventing and treating IBR, which aims to solve the problems raised in the background art.

[0005] To solve the above problems, the present application is realized as follows: a gD+mi3 protein nanoparticle for preventing and treating IBR is composed of gD-st fusion protein and mi3-sc fusion protein coupling. The gD antigen of bovine infectious rhinotracheitis virus is displayed on the surface of the mi3 protein nanoparticle through isopeptide bond. The amino acid sequence of the mi3 protein nanoparticle is shown in SEQ ID NO: 6. The amino acid sequence of the gD-st fusion protein is shown in SEQ ID NO: 2. The amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO: 4.

[0006] Another purpose of the present application is to provide a gene encoding the gD+mi3 protein nanoparticle for preventing and treating IBR, which includes a gD-st gene and a mi3-sc gene. The nucleotide sequence of the gD-st gene is shown in SEQ ID NO: 1. The nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO: 3.

[0007] Another object of the present application is to provide a recombinant expression vector comprising the above-mentioned genes, specifically including a gD-st expression vector and a mi3-sc expression vector; the gD-st expression vector comprises a gD-st gene; and the mi3-sc expression vector comprises a mi3-sc gene.

[0008] Another object of the present application is to provide a preparation method of the above-mentioned gD+mi3 protein nanoparticle, comprising the following steps: constructing a gD-st expression vector of the IBRV gD gene fused with a SpyTag003 tag, and constructing a mi3-sc expression vector of the mi3 gene fused with a SpyCatcher003; transforming the mi3-sc expression vector into a first host cell for expression to obtain a mi3-sc fusion protein; transfecting the gD-st expression vector into a second host cell for expression to obtain a gD-st fusion protein; mixing the gD-st fusion protein and the mi3-sc fusion protein, and performing overnight standing connection to obtain the gD+mi3 protein nanoparticle.

[0009] Further, the first host cell is an E. coli competent cell.

[0010] Further, the second host cell is a CHO cell.

[0011] Further, the molar ratio of the gD-st fusion protein to the mi3-sc fusion protein is (7-9):1.

[0012] Another object of the present application is to provide an application of the above-mentioned gD+mi3 protein nanoparticle, or the above-mentioned gene, or the above-mentioned recombinant expression vector in the preparation of a medicine or vaccine for preventing and treating bovine contagious rhinotracheitis.

[0013] Another object of the present application is to provide a medicine or vaccine for preventing and treating bovine contagious rhinotracheitis, comprising a pharmaceutically acceptable carrier and the above-mentioned gD+mi3 protein nanoparticle.

[0014] The gD+mi3 protein nanoparticle for preventing and treating IBR provided by the present application can form a highly symmetrical, closed virus-like structure by introducing mi3, and has excellent thermal stability and pH tolerance; the gD+mi3 protein nanoparticle can be made into a nanoparticle vaccine, which is non-toxic to mammals, has no biological safety threat, and can induce a more rapid and strong humoral immune response, including a more efficient germinal center reaction and high-titer neutralizing antibody production. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1A schematic diagram of construction of a recombinant expression vector provided for an embodiment of the present application.

[0016] Figure 2 SDS-PAGE and Western Blot identification diagrams of mi3-sc fusion protein (A) and gD-st fusion protein (B) expression and purification provided for an embodiment of the present application.

[0017] Figure 3 Electron microscope diagram of gD+mi3 protein nanoparticles provided for an embodiment of the present application.

[0018] Figure 4 Serum IgG detection result diagram of mice immunized with gD+mi3 protein nanoparticles provided for an embodiment of the present application.

[0019] Figure 5 Spleen lymphocyte stimulation index detection result diagram of mice immunized with gD+mi3 protein nanoparticles provided for an embodiment of the present application.

[0020] Figure 6 Spleen cell supernatant cytokine TNF-α expression level analysis result diagram of mice immunized with gD+mi3 protein nanoparticles provided for an embodiment of the present application. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0022] The Chinese hamster ovary (CHO) cells employed in the embodiments of the present application are one of the most widely used mammalian expression systems for vaccine antigen production. In recent years, a variety of important vaccines (such as respiratory syncytial virus vaccine, herpes zoster vaccine, and new coronavirus subunit vaccine, etc.) have adopted CHO cells to produce their recombinant protein antigens. CHO cells have excellent recombinant protein expression capacity and can achieve high cell density culture in bioreactors, thereby producing high concentrations of target proteins. The monoclonal antibody concentration produced by the CHO cell line after process optimization can reach several grams per liter. CHO cells are easy to suspend culture and can be scaled up for large-scale production of vaccine antigens in large bioreactors while maintaining stable yield. In contrast, many other systems, although they have higher yields at small scale, may face challenges when scaled up. In general, CHO cells provide reliable assurance for large-scale supply of vaccines in terms of yield and scale-up production.

[0023] The mi3 protein nanoparticle is a dodecahedral protein nanocage (60 subunits) with excellent particle uniformity and stability. By linking the SpyCatcher003 sequence at its N-terminus, mi3 becomes a universal base that can spontaneously couple with any antigen carrying the SpyTag003 peptide after mixing. mi3-SpyCatcher003 (mi3-sc) is highly expressed in E. coli, and the prepared nanoparticles have excellent environmental tolerance, and the performance of the coupled mi3 does not decrease after freeze-thawing and freeze-drying treatment. The mi3 platform shows compatibility and high coupling efficiency for different pathogenic antigens.

[0024] To study the mi3 protein nanoparticle vaccine that can prevent IBRV, the present embodiment expresses the IBRV gD antigen fused with SpyTag003 in CHO cells. In addition, mi3-SpyCatcher003 (mi3-sc) nanoparticles are expressed in the prokaryotic system of E. coli, and the purified gD-SpyTag003 (gD-st) is mixed with mi3-SC at a certain ratio, and the co-displaying recombinant nanoparticles are constructed by means of the specific covalent binding between SpyTag003 and SpyCatcher003. This structure can display antigens in a directional and regular manner on the surface of the nanoparticles, thereby maximizing the simulation of the natural virus antigen arrangement, enhancing the activation of B cells and the production of neutralizing antibodies.

[0025] Specifically, in one embodiment of the present application, a gD+mi3 protein nanoparticle for preventing and treating IBR is provided, which is composed of gD-SpyTag003 (gD-st) fusion protein and mi3-SpyChater003 (mi3-sc) fusion protein, and the gD antigen of bovine contagious rhinotracheitis virus is displayed on the surface of the mi3 protein nanoparticle through an isopeptide bond (SpyCatcher003 / SpyTag003). The amino acid sequence of the mi3 protein nanoparticle is shown in SEQ ID NO: 6 of the sequence listing, and the nucleotide sequence is shown in SEQ ID NO: 5 of the sequence listing. The amino acid sequence of the gD-st fusion protein is shown in SEQ ID NO: 2 of the sequence listing, and the amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO: 4 of the sequence listing. The gD-st fusion protein and the mi3-sc fusion protein are added with a signal peptide sequence at the N-terminus and a His6 tag at the C-terminus.

[0026] In another embodiment of the present application, a gene encoding the gD+mi3 protein nanoparticle for preventing and treating IBR is also provided, including a gD-st gene and a mi3-sc gene; the nucleotide sequence of the gD-st gene is shown in SEQ ID NO: 1; and the nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO: 3.

[0027] In another embodiment of the present application, a recombinant expression vector containing the above-mentioned gene is also provided, specifically including a gD-st expression vector and a mi3-sc expression vector; the gD-st expression vector contains the gD-st gene; and the mi3-sc expression vector contains the mi3-sc gene.

[0028] In another embodiment of the present application, a preparation method of the gD+mi3 protein nanoparticle is also provided, including the following steps: S1, constructing a gD-st expression vector of an IBRV gD gene fused with a SpyTag003 tag, and constructing a mi3-sc expression vector of a mi3 gene fused with a SpyCatcher003; S2, transforming the mi3-sc expression vector into a first host cell for expression to obtain a mi3-sc fusion protein; S3, transforming the gD-st expression vector into a second host cell for expression to obtain a gD-st fusion protein; S4, mixing the gD-st fusion protein with the mi3-sc fusion protein, and performing overnight standing connection to obtain the gD+mi3 protein nanoparticle.

[0029] Preferably, the first host cell is an E. coli competent cell; the second host cell is a CHO cell; and the molar ratio of the gD-st fusion protein to the mi3-sc fusion protein is (7-9): 1, more preferably 8:1.

[0030] In another embodiment of the present application, the gD+mi3 protein nanoparticle, the gene, or the recombinant expression vector is also used for preparing a medicine or a vaccine for preventing and treating contagious bovine rhinotracheitis.

[0031] In another embodiment of the present application, a medicine or a vaccine for preventing and treating contagious bovine rhinotracheitis is also provided, including a pharmaceutically acceptable carrier and the gD+mi3 protein nanoparticle.

[0032] Embodiment 1: The embodiment provides a method for obtaining a target gene and constructing an expression vector, specifically as follows: The IBRV-gD (GenBank ID: QB159528.1) gene sequence of bovine contagious nasal tracheitis virus was selected, fused with the SpyTag003 sequence, cloned into the pcDNA3.1(+) expression vector, a signal peptide sequence was introduced at the N-terminal of the gene, a His6 tag was added at the C-terminal, and BamH Iand EcoR Irestriction sites were added at both ends of the constructed sequence; all genes were codon-optimized for mammalian expression systems, and the gD-st expression vector was obtained. The mi3 sequence was fused with the SpyChater003 sequence, a His6 tag was added at the C-terminal, and the mi3-sc expression vector was constructed in pET28a(+) after codon optimization for prokaryotic expression systems. The map of the gene construction is shown in Figure 1 .

[0033] Example 2: This example provides a method for expressing and purifying gD-st fusion protein and mi3-sc fusion protein, as follows: The gD-st expression vector prepared in the above example 1 was introduced into CHO cells by electroporation (the shock cup was 4 mm, the cell number was 1×10 7 cells, and the cell culture density after transfection was 1×10 6 cells / mL), G418 was added 48 h after transfection (the final concentration was 800 μg / mL), and the fresh culture medium containing G418 was replaced 7 days later to obtain a drug-resistant cell population. The supernatant of the cells stably cultured for 4 days was collected, purified using affinity chromatography, and the gD-st fusion protein was obtained; the mi3-sc expression vector prepared in the above example 1 was transformed into E. coli BL21 (DE3) competent cells for expression according to the same process, and the mi3-sc fusion protein was obtained; the above gD-st fusion protein and mi3-sc fusion protein were identified by SDS-PAGE and Western blot, the primary antibody was rabbit His (Abeam), and the secondary antibody was HRP-labeled goat anti-rabbit IgG, and the results are shown in Figure 2 .

[0034] Example 3: This example provides a method for preparing and characterizing gD+mi3 protein nanoparticles, as follows: The gD-st fusion protein and mi3-SC fusion protein prepared in Example 2 above were mixed in a molar ratio of 8:1, mixed uniformly by blowing under sterile operating conditions, and incubated at room temperature overnight. The free antigen was removed by ultrafiltration, and the concentrated solution was the gD+mi3 protein nanoparticle. The sample was filtered through a 0.22 μm filter membrane to remove large particle impurities, and the sample was added dropwise to a carbon film copper grid (300 mesh), and then placed at room temperature for 1 min, and then negatively stained with 2% phosphotungstic acid for 30 s. After removing the excess staining solution, the sample was naturally dried, and the morphology of the nanoparticle was observed under a transmission electron microscope at 80 kV. The results are shown in Figure 3 DLS analysis: 300 μL of the gD+mi3 protein nanoparticle suspension obtained in Example 3 above was measured for average particle size (Z-Average) and polydispersity index (PDI) in a Malvern Zetasizer Nano ZS instrument. Each sample was measured 3 times, and the diameter and uniformity of the nanoparticle were analyzed.

[0035] Example 4: This example is a mouse immunization experiment, and the details are as follows: Forty 6-week-old SPF female BALB / c mice, weighing 20±2 g, were randomly divided into 3 groups, 10 mice in each group. Immunization was performed by subcutaneous multi-point injection at 0 d, 14 d and 28 d. The total dose of gD+mi3 (gD+mi3 protein nanoparticle prepared in Example 3) and gD (gD protein constructed by directly using the IBRV-gD gene without fusing SpyTag003 sequence according to the method of Example 1-2) was 30 μg, and was mixed with M903 adjuvant. The PBS control group was 100 μL of PBS. Tail blood was collected at 7 d, 14 d, 21 d, 28 d, 35 d and 42 d after immunization.

[0036] Serum antibody level detection: ELISA method was used to detect the serum IgG level of mice at different time points (7 d, 14 d, 21 d, 28 d, 35 d, 42 d) after immunization. The gradient-diluted IgG standard and 1:100000-diluted mouse serum sample were incubated at 37°C for 1 h in an ELISA enzyme-labeled plate, and then washed. HRP-labeled goat anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 h. After washing, 90 μL of TMB color developing solution was added and reacted at 37°C for 30 min. After adding 50 μL of 2 M H2SO4 to terminate the reaction, the absorbance value (OD450nm) was measured at 450 nm, and the IgG antibody kinetics curve was plotted, and the results are shown in Figure 4The results showed that the serum IgG levels of gD+mi3 and gD groups gradually increased from 7 d to 42 d after immunization. At 35 d, the serum IgG levels of gD+mi3 and gD groups were significantly increased, and the serum IgG level of gD+mi3 group was significantly higher than that of gD group. At 42 d, the difference between the two groups was enlarged, and the serum IgG levels of the two groups were much higher than that of PBS group. The results showed that gD+mi3 promoted the production of serum IgG in mice.

[0037] Spleen lymphocyte proliferation experiment: On the 35th and 42nd days after immunization, 3 mice were randomly selected from each group and sacrificed by dislocation, and the mouse spleen tissue was collected. The spleen lymphocyte separation medium (Beijing Dakewei Biological Technology Co., Ltd.) was gently ground and prepared into a single cell suspension. Centrifugation at 800xg for 30 min to separate the cells, and the lymphocyte layer was aspirated and washed with RPMI-1640 complete culture medium. According to 1x10 6 cells / mL cell density was inoculated into a 96-well plate, and gD+mi3 protein nanoparticles and gD protein (final concentration 5 μg / mL) were added to each well, and a negative control group (PBS) was set up. After 42 h of culture at 37℃ and 5% CO2, 10 μL of CCK-8 solution was added, and the OD value was measured at 450 nm wavelength after 4 h of incubation at 37℃. The lymphocyte proliferation index (SI) was calculated. Stimulation index (SI) = (experimental group OD value - blank control group OD value) / (negative control group OD value - blank control group OD value). The results are shown in Figure 5 At 35 d, the SI of gD+mi3 group and gD group was about 1.2, and at 42 d, the SI of gD+mi3 group was significantly higher than that of gD group, indicating that gD+mi3 may maintain a high cellular immune response.

[0038] Mouse spleen cell supernatant cytokine level analysis: Commercial ELISA detection kit was used to detect the secretion level of cytokines in the supernatant of mouse spleen cells at different time points (21 d and 42 d) after immunization. First, mouse TNF-α capture antibody was coated in a 96-well ELISA plate, incubated at 4℃ overnight, and then blocked with 5% BSA for 1 h. Then, diluted serum was added, incubated at 37℃ for 2 h, washed, and then biotin-labeled detection antibody was added, incubated at 37℃ for 30 min, and then washed to remove non-specific binding. 90 μL of TMB color developing solution was added, and the reaction was carried out at 37℃ for 15 min. The stop solution was added, and the absorbance value was measured at 450 nm wavelength. The concentration of TNF-α was calculated by standard curve. The results are shown in Figure 6 The expression level of TNF-α in gD+mi3 group was significantly higher than that in PBS and gD groups.

[0039] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A gD+mi3 protein nanoparticle for preventing and treating IBR, characterized in that, The gD+mi3 protein nanoparticles are composed of gD-st fusion protein and mi3-sc fusion protein coupled together, which display the gD antigen of bovine infectious rhinotracheitis virus on the surface of the mi3 protein nanoparticles through isopeptide bonds; the amino acid sequence of the mi3 protein nanoparticles is shown in SEQ ID NO:6 of the sequence listing; the amino acid sequence of the gD-st fusion protein is shown in SEQ ID NO:2 of the sequence listing; and the amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO:4 of the sequence listing.

2. A gene encoding gD+mi3 protein nanoparticles for preventing and treating IBR as described in claim 1, characterized in that, It includes the gD-st gene and the mi3-sc gene; the nucleotide sequence of the gD-st gene is shown in SEQ ID NO:1 of the sequence listing; the nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO:3 of the sequence listing.

3. A recombinant expression vector comprising the gene of claim 2, characterized in that, It includes a gD-st expression vector and a mi3-sc expression vector; the gD-st expression vector contains the gD-st gene; the mi3-sc expression vector contains the mi3-sc gene.

4. A method for preparing gD+mi3 protein nanoparticles as described in claim 1, characterized in that, Includes the following steps: Construct the gD-st expression vector of the IBRV gD gene fused with the SpyTag003 tag, and construct the mi3-sc expression vector of the mi3 gene fused with SpyCatcher003. The mi3-sc expression vector was transformed into the first host cell for expression to obtain the mi3-sc fusion protein; The gD-st expression vector was transfected into a second host cell for expression, yielding the gD-st fusion protein. The gD-st fusion protein was mixed with the mi3-sc fusion protein and allowed to stand overnight for ligation to obtain gD+mi3 protein nanoparticles.

5. The method for preparing gD+mi3 protein nanoparticles according to claim 4, characterized in that, The first host cell is a competent Escherichia coli cell.

6. The method for preparing gD+mi3 protein nanoparticles according to claim 4, characterized in that, The second host cell is a CHO cell.

7. The method for preparing gD+mi3 protein nanoparticles according to claim 4, characterized in that, The molar ratio of the gD-st fusion protein to the mi3-sc fusion protein is (7-9):

1.

8. The use of the gD+mi3 protein nanoparticle as described in claim 1, or the gene as described in claim 2, or the recombinant expression vector as described in claim 3 in the preparation of a drug or vaccine for the prevention and treatment of bovine infectious rhinotracheitis.

9. A drug or vaccine for the prevention and treatment of bovine infectious rhinotracheitis, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes the gD+mi3 protein nanoparticles as described in claim 1.

Images