HSPA8 protein resistant to duck Tembusu virus and its application
By preparing and purifying the HSPA8 protein against the Ducktambusu virus, the problem of infection after immunization of existing vaccines was solved, significant antiviral effects in vitro and in vivo were achieved, and new prevention and control strategies were provided.
Patent Information
- Application Number
- CN202411508047.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2044-10-28
AI Technical Summary
Ducks may still be infected with the Tampusu virus after existing vaccines are immunized, and there is a lack of effective methods to directly block the binding of the virus to the cell membrane.
A HSPA8 protein against the tambusu virus, including soluble deendotoxin HSPA8 proteins such as HSPA8-His and GST-HSPA8 proteins, is prepared and purified by prokaryotic expression vectors for the preparation of antiviral drugs.
HSPA8 protein exhibits significant antiviral effects in vitro and in vivo, which can prevent virus proliferation, weaken the pathogenicity caused by viral infection, improve survival rate and reduce viral load.
Smart Images
Figure CN119080953B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to an HSPA8 protein resistant to duck Tembusu virus and an application thereof. Background Art
[0002] Duck Tembusu virus disease, also known as duck hemorrhagic oophoritis and duck yellow virus disease, is a new, acute infectious disease caused by the duck tembusu virus (DTMUV). The virus primarily causes fever in infected ducks, a significant decrease in feed intake, limb weakness and paralysis, and a significant decrease in egg production. Characteristic lesions in infected breeder ducks include bleeding in the ovarian membrane, follicular deformation, and even rupture.
[0003] The first and necessary step for DTMUV to infect a host is the binding of the virus to host cell surface molecules. This process requires multiple membrane proteins distributed on the cell surface. The binding of the viral envelope protein E to cell membrane proteins determines the susceptibility of DTMUV in vivo and in vitro. Therefore, blocking the binding of the virus to the cell membrane can serve as a direct and effective antiviral strategy.
[0004] Current prevention and control measures for TMUV primarily rely on vaccination. Although commercially available live attenuated and inactivated vaccines are available, TMUV infection can still occur in immunized ducks. Therefore, developing methods to directly block pathogen infection, in addition to vaccination, may be a new strategy for combating DTMUV infection. Summary of the Invention
[0005] The purpose of the present invention is to solve the above problems existing in the prior art and provide an HSPA8 protein resistant to duck Tembusu virus and its application.
[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: an HSPA8 protein resistant to duck Tembusu virus, the amino acid sequence of the HSPA8 protein is shown in SEQ ID NO.1.
[0007] Preferably, the HSPA8 protein is a soluble endotoxin-free HSPA8 protein.
[0008] Preferably, the HSPA8 protein is HSPA8-His protein and GST-HSPA8 protein.
[0009] The present invention also provides a prokaryotic expression vector, which contains the gene encoding the HSPA8 protein according to claim 1, and is used to express the HSPA8 protein.
[0010] Preferably, the prokaryotic expression vector induces expression of HSPA8 protein under the following conditions: 1 mM IPTG, temperature 30° C., and time 8 h.
[0011] The present invention also provides the use of the HSPA8 protein in preparing a drug for resisting duck Tembusu virus.
[0012] Preferably, the HSPA8 protein is used at a concentration of 100-150 μg / μl.
[0013] The present invention has the beneficial effects of preparing a duck-derived soluble, endotoxin-free HSPA8 protein, comprising an HSPA8-His protein and a GST-HSPA8 protein. In vitro antiviral experiments demonstrated that the HSPA8 protein exhibited excellent antiviral efficacy. In vivo antiviral experiments also demonstrated that the HSPA8 protein significantly reduced the pathogenicity of DTMUV infection, inhibited viral proliferation in vivo, mitigated viral-induced weight loss in mice, and reduced viral-induced mortality in mice. Therefore, the HSPA8 protein of the present invention exhibits significant resistance to DTMUV infection and can be used to prepare drugs against duck Tembusu virus, providing a new strategy for the prevention and control of duck Tembusu virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 Optimize protein expression temperature;
[0015] Figure 2 To optimize protein expression time;
[0016] Figure 3 To optimize protein expression;
[0017] Figure 4 For the purification of HSPA8 protein; wherein, M: protein marker; 1: whole bacterial solution; 2: flow-through; 3-4: washing solution; 5-6: eluent.
[0018] Figure 5 For endotoxin removal and concentration analysis of HSPA8 protein;
[0019] Figure 6 Purity analysis of GST and GST-HSPA8 proteins;
[0020] Figure 7 is the antiviral effect of GST-HSPA8 protein (genome replication level);
[0021] Figure 8 is the antiviral effect of GST-HSPA8 protein (viral protein accumulation level);
[0022] Figure 9is the antiviral effect of GST-HSPA8 protein (the level of virus released in the supernatant);
[0023] Figure 10 The in vivo antiviral effect of HSPA8 protein (brain tissue);
[0024] Figure 11 The antiviral effect of HSPA8 protein in vivo (spinal cord tissue);
[0025] Figure 12 The in vivo antiviral effect of HSPA8 protein (body weight changes);
[0026] Figure 13 The in vivo antiviral effect (survival rate) of HSPA8 protein.
[0027] DTMUV was provided by the Poultry Disease Control Research Center of Sichuan Agricultural University. The public literature information is as follows: He Y, et al. Assembly-defective Tembusu virus ectopically expressing capsid protein is an approach for live-attenuated flavivirus vaccine development. NPJ Vaccines. 2022; 7(1): 51. Published 2022 May 12. doi: 10.1038 / s41541-022-00468-y
[0028] 17-day-old female Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. DETAILED DESCRIPTION
[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0030] Example 1 Preparation of Soluble Endotoxin-Free HSPA8 Protein
[0031] 1. Construction of HSPA8 prokaryotic expression vector
[0032] Based on the genome of the mallard duck Anas platyrhynchos (mallard) from the NCBI gene bank, HSPA8 amplification primers were designed for cloning into pET-28a(+) and pGEX-4T-1 vectors. The specific primer sequences are shown in Table 1:
[0033] Table 1 Primers for constructing HSPA8 prokaryotic expression vector
[0034]
[0035] Using duck cDNA as a template, the HSPA8 gene carrying the homology arms was amplified using the primers listed in Table 1. The amplified product was purified and ligated into the pET-28a(+) vector digested with BamHI and XhoI or the pGEX-4T-1 vector digested with EcoRI and XhoI. The ligated plasmid product was verified by DNA sequencing and transformed into BL21(DE3) cells for protein expression.
[0036] 2. Expression, Purification, and Endotoxin Removal of Soluble HSPA8 Protein
[0037] The NS1 expression bacteria were inoculated into LB liquid medium containing kana resistance to expand the culture, and pET-28a (+) empty load was used as a control. The expression conditions were optimized in sequence, including the induction temperature (25 ° C, 30 ° C, 37 ° C) ( Figure 1 ), induction time (4h, 6h, 8h, 10h) ( Figure 2 ) and expression forms (supernatant, inclusion bodies) ( Figure 3 After induced expression, the cells were analyzed by SDS-PAGE, and 1 mM IPTG, 30°C, and 8 h were selected as the conditions for large-scale expression.
[0038] According to the optimized prokaryotic expression conditions, HSPA8-His protein was expressed in large quantities. The bacterial precipitate obtained after large-scale expression was sonicated and repeatedly centrifuged to remove insoluble cellular components. Then, the HSPA8-His protein was purified according to the instructions of Ni NTABeads (Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The whole bacterial sample, flow-through sample, washing solution and eluate were retained and analyzed by SDS-PAGE to determine the protein purification effect. The results showed that the purified HSPA8 protein was of high purity and had no obvious impurity bands ( Figure 4 ).
[0039] Since the prokaryotic purified protein contains a large amount of endotoxin, in order to prevent endotoxins from causing unnecessary immune responses in vivo and in vitro, the purified protein was treated with endotoxin removal using the Protein Endotoxin Removal Kit (Shanghai Biyuntian Biotechnology Co., Ltd.), and then the protein concentration and purity were analyzed by SDS-PAGE, in which a standard concentration of BSA was used as a concentration reference. The results showed that the endotoxin-free HSPA8 protein was highly pure, with the protein concentrations of different batches being 10 and 20 mg / ml ( Figure 5 ).
[0040] According to the above process, GST and GST-HSPA8 proteins were expressed and purified, and then 100 μg of protein was subjected to SDS-PAGE analysis. The results showed that the purified GST and GST-HSPA8 proteins were highly pure ( Figure 6 ).
[0041] Example 2 HSPA8 protein has anti-DTMUV activity in vitro
[0042] The HSPA8 proteins (GST-HSPA8 and HSPA8-His) in this example were prepared in Example 1.
[0043] 1. To verify whether HSPA8 protein can prevent DTMUV infection, this example conducted an in vitro protein blocking experiment. Duck embryo fibroblasts (DEF) were passaged into 12-well plates. When the cell density reached 100%, 100 μg GST, 20 μg GST-HSPA8 and 100 μg GST-HSPA8 were added to 5*10 3 TCID 50 The DTMUV was mixed and then infected into DEF cells. After 36 h of infection, the virus levels inside and outside the cells were analyzed, including the genome replication level ( Figure 7 ), viral NS3, NS1 protein accumulation levels ( Figure 8 ) and the level of virus released in the supernatant ( Figure 9 ).
[0044] The results showed that HSPA8 protein could significantly prevent the infection of DTMUV, and high doses of GST-HSPA8 protein almost completely blocked the infection of the virus, indicating that HSPA8 has anti-DTMUV function.
[0045] 2. HSPA8 protein has anti-DTMUV activity in vivo
[0046] Since HSPA8 protein has a good antiviral effect in vitro, in order to verify whether HSPA8 protein can prevent DTMUV infection in vivo, this example further carried out an in vivo protein blocking experiment. 5 TCID 50 / ml) was mixed with 150 μg HSPA8-His protein and then injected intracranially into 17-day-old female Kunming mice. The brain was detected on the second and fifth days after infection. Figure 10 ) and spinal cord ( Figure 11 ) and monitored the mouse body weight ( Figure 12 ) and survival rate ( Figure 13 )Condition.
[0047] The results showed that the viral load in the brain and cranium of mice treated with HSPA8 protein was lower than that in the control group. At the same time, the weight changes and survival rate also indicated that HSPA8-treated mice had lower viral pathogenicity.
[0048] In summary, the soluble endotoxin-free HSPA8 protein of the present invention has high purity and high safety, and exhibits significant anti-DTMUV ability in both in vitro and in vivo animal experiments, and can be used to prepare drugs against duck Tembusu virus.
[0049] The description and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative work, and all of them are within the scope of protection of the present invention.
Claims
1. A use of HSPA8 protein in the preparation of a drug against duck Tembusu virus, characterized in that: The amino acid sequence of the HSPA8 protein is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The application concentration of the HSPA8 protein is 100-150 μg / μl.