Application of bovine lactoferrin in inhibiting tembusu virus infected cells

Abstract

The invention discloses an application of bovine lactoferrin in inhibiting tembusu virus infected cells. The bovine lactoferrin is competitively combined with the tembusu virus receptor on the surface of the host cell to interfere with the combination of the tembusu virus and the specific receptor of the target cell, thereby inhibiting the tembusu virus from infecting the target cell. The result shows that the bovine lactoferrin has the effect of interfering the combination of the tembusu virus and a target cell specific receptor, and shows the effect of inhibiting the tembusu virus from infecting the cells.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to application of bovine lactoferrin in inhibiting Tembusu virus infection of cells. Background Art

[0002] Tembusu virus is a new type of flavivirus that mainly harms ducks and geese. It has the characteristics of fast transmission speed and high infection rate. The morbidity rate in the flock is almost 100%, and the mortality rate is 5%-28%. Sick ducks and geese often show typical symptoms such as fever, loss of appetite, encephalitis and paralysis. Egg laying ducks will show a sharp drop in egg production or even cessation after infection, from 90%-95% at the peak to 5%-10%. Moreover, after the infection, it generally cannot return to the pre-infection level, causing significant economic losses to the duck and goose farming industry. At present, the host spectrum of Tembusu virus infection is still expanding. Chickens, pigeons and sparrows can be infected. Viral nucleic acid and antibodies have also been detected in duck farm workers. Therefore, this disease not only seriously threatens the healthy development of the poultry farming industry, but also poses a potential threat to human health.

[0003] Currently, there are commercial vaccines approved by the state for the prevention of Tembusu virus disease on the market, but there may still be cases of immunization failure or non-immunization in duck and goose farms, resulting in Tembusu virus infection. There is currently no effective treatment for this disease. Therefore, after the onset of the disease, broad-spectrum antiviral drugs or traditional Chinese medicine are generally used for symptomatic treatment, but the treatment effect is difficult to guarantee. Therefore, the development of specific therapeutic agents for the treatment of Tembusu virus infection can serve as a beneficial supplement to vaccine prevention and immunity, which is conducive to the comprehensive prevention and control of Tembusu virus disease.

[0004] Tembusu virus belongs to the Flaviviridae family, which also includes several important human pathogens, including dengue virus, Zika virus, West Nile virus, Japanese encephalitis virus, etc. So far, there is no specific treatment for infection with this genus of virus, and it is difficult to control it through vaccination. Tembusu virus has a high homology with these viruses and uses a similar mechanism to infect cells. Therefore, the development of Tembusu virus-specific therapeutic agents can not only fill the gap in related research, but also provide important references for the prevention and control of other Flaviviridae viruses and the design of antiviral drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inhibiting Tembusu virus infection in cells by using bovine lactoferrin in view of the above-mentioned deficiencies in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The invention discloses an application of the bovine lactoferrin in preparing a Tembusu virus-specific therapeutic agent.

[0008] A Tembusu virus-specific therapeutic composition comprises the bovine lactoferrin described in the present invention.

[0009] The method for preparing bovine lactoferrin of the present invention is obtained by isolating it from bovine milk or by in vitro expression; commercial bovine lactoferrin products can also be purchased. For example, the protein is expressed by a microorganism containing a recombinant nucleic acid molecule encoding the desired peptide under the control of an appropriate transcription promoter, and the desired protein is collected from the microorganism.

[0010] Beneficial effects:

[0011] In order to achieve the above purpose, bovine lactoferrin in the present invention competitively binds to the Tembusu virus receptor on the surface of the host cell, interferes with the binding of the Tembusu virus to the specific receptor of the target cell, and thus inhibits the infection of the target cell by the Tembusu virus. The results show that bovine lactoferrin has the effect of interfering with the binding of the Tembusu virus to the specific receptor of the target cell, and exhibits the effect of inhibiting the infection of the target cell by the Tembusu virus.

[0012] The bovine lactoferrin in the present invention is a natural component in milk, which is non-toxic and harmless. It inhibits the infection of Tembusu virus by inhibiting the binding of the virus to the specific receptor of the target cell, and has no influence or side effects on the host itself, so that its antiviral effect has specificity and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The inhibitory effect of bovine lactoferrin on Tembusu virus infected cells. Bovine lactoferrin with a final concentration of 100 μg / ml can effectively inhibit Tembusu virus infection with an inhibition efficiency of 50%. However, the control bovine serum albumin group did not have this inhibitory effect.

[0014] Figure 2 The results show that the bovine lactoferrin of the present invention can bind to the surface of the target cell membrane, interfere with the binding of Tembusu virus to the target cell specific receptor, and thus inhibit Tembusu virus from infecting the target cell. However, the control bovine serum albumin histone cannot bind to the target cell. DETAILED DESCRIPTION

[0015] Example 1 Inhibitory effect of bovine lactoferrin on Tembusu virus infected cells

[0016] 1. Culture BHK-21 cells to 80% saturation level according to normal cell culture methods.

[0017] 2. Incubate BHK-21 with 100 μg / ml bovine lactoferrin (Sigma-Aldrich, Lactoferrin, L9507-10MG) at 37°C for 1 hour; in the control group, incubate BHK-21 with 100 μg / ml bovine serum albumin at 37°C for 1 hour. After incubation, remove the incubation protein and wash three times with pre-cooled PBS.

[0018] 3. The cells in the experimental and control groups were inoculated with 200 PFU of Tembusu virus, infected at 37°C for 1 hour, and the culture medium was removed. The cells were washed three times with pre-cooled PBS, and cell culture medium containing 100 μg / ml bovine lactoferrin and 100 μg / ml bovine serum albumin was added to the experimental and control groups, respectively. The cells were cultured for another 24 hours, and the viral nucleic acid content was detected by fluorescent quantitative RT-PCR.

[0019] The experimental results are shown in Figure 1 The relative content of viral nucleic acid in the control group (final concentration of bovine serum albumin was 100 μg / ml) was 97%, and the relative content of viral nucleic acid in the test group (final concentration of bovine lactoferrin was 100 μg / ml) was 51.2%. By comparing and analyzing with the control group, bovine lactoferrin can reduce the content of Tembusu virus in BHK-21 cells by nearly 50%, indicating that the use of bovine lactoferrin can inhibit Tembusu virus infection and protect the cells well.

[0020] Example 2 Analysis of the mechanism by which bovine lactoferrin inhibits Tembusu virus infection of cells

[0021] To determine the mechanism by which bovine lactoferrin inhibits Tembusu virus infection of cells, BHK-21 cells were incubated with bovine lactoferrin at a concentration of 100 μg / ml at 37°C for 1 h, washed with PBS, and fixed at room temperature with -20°C precooled acetone: ethanol solution (2:3), and immunofluorescence detection was performed using positive serum prepared from mice immunized with bovine lactoferrin as the primary antibody and goat anti-mouse IgG labeled with Alexa Fluor 594 as the secondary antibody. The control group was incubated with cells with bovine serum albumin at a concentration of 100 μg / ml.

[0022] Experimental results: The bovine lactoferrin of the present invention can bind to BHK-21 cells, competitively preventing the virus from adsorbing to the cells, thereby inhibiting viral infection ( Figure 2 ).

Claims

1. Application of bovine lactoferrin in the preparation of drugs for inhibiting Tembusu virus infection.

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