HSPA8 protein for resisting duck tembusu virus and application of HSPA8 protein

Abstract

The invention discloses an HSPA8 protein capable of resisting a duck tembusu virus and application of the HSPA8 protein. According to the invention, a prokaryotic expression vector for expressing HSPA8 is constructed, protein expression conditions are optimized, and soluble endotoxin-removed HSPA8 protein, including HSPA8-His protein and GST-HSPA8 protein, is expressed and purified. An in-vitro antiviral experiment shows that the HSPA8 protein disclosed by the invention has a good antiviral effect; in-vivo antiviral experiments show that the HSPA8 protein can significantly weaken pathogenicity caused by DTMUV infection, prevent in-vivo proliferation of viruses, weaken mouse weight loss caused by virus infection and reduce mouse death caused by virus infection. Therefore, the HSPA8 protein disclosed by the invention has a remarkable characteristic of resisting DTMUV infection, can be applied to preparation of drugs for resisting the duck tembusu virus, and provides a new strategy for prevention and control of the duck tembusu virus.

Description

Technical Field

[0001] The 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 ovary inflammation and duck yellow virus disease, is a new acute infectious disease caused by duck Tembusu virus (DTMUV). The virus mainly causes the diseased ducks to have elevated body temperature, a significant reduction in feed intake, weakness and paralysis of the limbs, and a significant decrease in egg production. The characteristic lesions of infected ducks include bleeding of the follicle membrane, deformation of the follicles, and even rupture.

[0003] The first and necessary step for DTMUV to infect the host is the binding of the virus to the host cell surface molecules. This process requires a variety of membrane proteins distributed on the cell surface. The binding of the viral envelope protein E to the cell membrane protein determines the susceptibility of DTMUV in vivo and in vitro. Therefore, blocking the binding of the virus to the cell membrane can be used as a direct and efficient antiviral strategy.

[0004] The current prevention and control measures for TMUV are mainly carried out through vaccination. Although there are commercial attenuated live vaccines and inactivated vaccines, immunized ducks can still be infected with TMUV. Therefore, based on vaccination, developing methods to directly block pathogen infection may become a new strategy to combat 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 to 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 N0.1.

[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 a gene encoding the HSPA8 protein as claimed in claim 1, and is used to express the HSPA8 protein.

[0010] Preferably, the conditions for inducing the expression of HSPA8 protein by the prokaryotic expression vector are 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 beneficial effects of the present invention are as follows: the present invention prepares a duck-derived soluble endotoxin-free HSPA8 protein, including HSPA8-His protein and GST-HSPA8 protein. In vitro antiviral experiments show that the HSPA8 protein has a good antiviral effect; in vivo antiviral experiments show that the HSPA8 protein can significantly reduce the pathogenicity caused by DTMUV infection, prevent the proliferation of the virus in vivo, reduce the weight loss of mice caused by viral infection, and reduce the death of mice caused by viral infection. Therefore, the HSPA8 protein in the present invention has significant anti-DTMUV infection properties, can be used to prepare drugs against duck Tembusu virus, and provide a new strategy for the prevention and control of duck Tembusu virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 To optimize the temperature for protein expression;

[0015] Figure 2 To optimize protein expression time;

[0016] Figure 3 To optimize protein expression;

[0017] Figure 4 For the purification of HSPA8 protein, M: protein marker; 1: whole bacterial solution; 2: flow-through solution; 3-4: washing solution; 5-6: elution solution.

[0018] Figure 5 To remove endotoxin and analyze the concentration of HSPA8 protein;

[0019] Figure 6 For 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] Fig. 9is the antiviral effect of GST-HSPA8 protein (the level of virus released in the supernatant);

[0023] Fig.10 The in vivo antiviral effect of HSPA8 protein (brain tissue);

[0024] Fig.11 The in vivo antiviral effect of HSPA8 protein (spinal cord tissue);

[0025] Fig.12 The in vivo antiviral effect of HSPA8 protein (body weight changes);

[0026] Fig.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 2022May 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 in conjunction with 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] According to the genome of the mallard duck Anas platyrhynchos (mallard) in the NCBI gene bank, HSPA8 amplification primers for cloning into pET-28a(+) and pGEX-4T-1 vectors were designed. The specific sequences of the primers 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 homology arms was amplified using the primers in Table 1. The amplified products were 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 products were verified by DNA sequencing and transformed into BL21(DE3) 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 SDS-PAGE analysis of the induced expression bacteria, 1 mM IPTG, 30°C, 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 treated with ultrasound and then repeatedly centrifuged to remove insoluble cell components. Then, referring to the instructions of Ni NTABeads (Changzhou Tiandi Renhe Biotechnology Co., Ltd.), HSPA8-His protein was purified, and the whole bacterial sample, flow-through sample, washing liquid and eluate were retained for SDS-PAGE analysis to determine the protein purification effect. The results showed that the purified HSPA8 protein was of high purity and had no obvious impurities ( Figure 4 ).

[0039] Since the prokaryotic purified protein contains a large amount of endotoxin, in order to prevent endotoxin from causing unnecessary immune response in vivo and in vitro, the purified protein was treated with Protein Endotoxin Removal Kit (Shanghai Biotech Co., Ltd.), and then the protein concentration and purity were analyzed by SDS-PAGE, in which the standard concentration of BSA was used as the concentration reference. The results showed that the purity of the endotoxin-free HSPA8 protein was high, and the protein concentrations of different batches were 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 analyzed by SDS-PAGE. 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. In order to verify whether HSPA8 protein can prevent the infection of DTMUV, this example carried out 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 intracellular and extracellular virus levels, including the genome replication level ( Figure 7 ), viral NS3, NS1 protein accumulation levels ( Figure 8 ) and the level of virus released in the supernatant ( Fig. 9 ).

[0044] The results showed that HSPA8 protein could significantly prevent the infection of DTMUV, and a high dose 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 good antiviral effect in vitro, in order to verify whether HSPA8 protein can prevent DTMUV infection in vivo, this example further carried out in vivo protein blocking experiments. 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 ( Fig.10 ) and spinal cord ( Fig.11 ) and monitored the mouse body weight on days 0-10 of infection ( Fig.12 ) and survival rate ( Fig.13 )Condition.

[0047] The results showed that the viral load in the brain and skull 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 specification 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 labor, all of which are within the protection scope of the present invention.

Claims

1. An HSPA8 protein against duck Tembusu virus, characterized in that: The amino acid sequence of the HSPA8 protein is shown in SEQ ID NO.

1.

2. The HSPA8 protein according to claim 1, characterized in that The HSPA8 protein is a soluble endotoxin-free HSPA8 protein.

3. The HSPA8 protein according to claim 1, characterized in that The HSPA8 protein is HSPA8-His protein and GST-HSPA8 protein.

4. A prokaryotic expression vector, characterized in that: The prokaryotic expression vector contains the gene encoding the HSPA8 protein as claimed in claim 1, and the prokaryotic expression vector is used to express the HSPA8 protein.

5. The prokaryotic expression vector according to claim 4, characterized in that The conditions for inducing the expression of HSPA8 protein by the prokaryotic expression vector are 1 mM IPTG, temperature 30° C., and time 8 h.

6. Use of the HSPA8 protein as claimed in claim 1 in the preparation of a drug against duck Tembusu virus.

7. The use according to claim 6, characterized in that: The application concentration of the HSPA8 protein is 100-150 μg / μl.

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