OLIGOPEPTIDE HAVING DENGUE VIRUS REPLICATION INHIBITION FUNCTION AND APPLICATION THEREOF

Abstract

The present invention relates to the field of virology, and specifically discloses a short peptide having a dengue virus replication inhibition function and an application thereof. The amino acid sequence of the short peptide provided in the present invention is KHGHHRH, i.e. Lys-His-Gly-His-His-Arg-His (SEQ ID NO. 1). The short peptide has a high specificity affinity with NS5 and has the function of efficiently inhibiting dengue virus replication, the anti-viral effect thereof not been limited to DENV-2, but also having a significant inhibitory effect on the replication of type 1, type 3, and type 4 dengue virus. One cysteine is added to the two ends of the short peptide sequence, the short peptide being cyclised by means of the cysteines at the two ends to form a cyclic peptide. The obtained cyclic peptide strengthens the dengue virus replication inhibition function, and can be used for specific treatment of dengue virus infection.

Claims

1. An oligopeptide having the function of inhibiting dengue virus replication, characterized in that the amino acid sequence of the oligopeptide is KHGHHRH.

2. Use of the oligopeptide according to claim 1 in the manufacture of a medicament for treating dengue virus infection.

3. Use of the oligopeptide according to claim 1 in the manufacture of a medicament for inhibiting dengue virus replication.

4. A pharmaceutical composition, characterized in comprising the oligopeptide according to claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that the oligopeptide may be cychzed to form a cyclic peptide.

6. A oligopeptide having the function of inhibiting dengue virus replication, characterized in that the oligopeptide is a tripeptide, tetrapeptide, pentapeptide or hexapeptide fragment in the oligopeptide according to claim 1.

7. Use of the oligopeptide according to claim 6 in the manufacture of a medicament for treating dengue virus infection.

8. Use of the oligopeptide according to claim 6 in the manufacture of a medicament for inhibiting dengue virus replication.

9. A pharmaceutical composition, characterized in comprising the oligopeptide according to claim 6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is an SDS-PAGE electrophoresis image of the full-length expression of the dengue virus NS5 protein according to the disclosure; the SDS-PAGE electrophoresis image shows that NS5 is mainly expressed in bacterial inclusion bodies, wherein M: protein molecular weight standard; 1: precipitate after bacterial lysis and centrifugation; 2: supernatant after bacterial lysis and centrifugation; 3: effluent after passing the supernatant through a Ni-IDA purification column; 4: 50 inM imidazole eluent after the supernatant is passed through the purification column; 5-6: 100 mM imidazole eluent after the supernatant is passed through the purification column; 7: 500 mM imidazole eluent after the supernatant is passed through the purification column (4-7 are respectively 50 mM, 100 mM, 100 mM, and 500 mM imidazole eluents after the supernatant is passed through the Ni-IDA purification column).

[0043] FIG. 2 shows the Western blotting identification of the recombinant expression product of the dengue virus NSS protein (104 kDa); wherein M: protein molecular weight standard; 1: supernatant of whole strain lysate of expression. strain; 2: precipitate of whole strain lysate of expression strain; 3: inclusion body solution.

[0044] FIG. 3 shows the protective effect of the synthesized cyclic peptide in the example on virus-infected cells; wherein I: cells without addition of the synthesized cyclic peptide; 2: cells with addition of a low concentration of the synthesized peptide; 3: infected cells with addition of a high concentration of the synthesized peptide; 4: non-infected control cells.

DESCRIPTION OF THE EMBODIMENTS

[0045] This disclosure is further explained below with reference to the example. It should be understood that the following example is for illustrative purposes only and is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications and substitutions to the disclosure without departing from the principle and spirit of the disclosure.

[0046] Unless otherwise specified, the experimental methods used in the following example are all conventional methods.

[0047] The materials, reagents and the like used in the following example all can be obtained from commercial sources, unless otherwise specified.

EXAMPLE 1

[0048] This example is used to illustrate the preparation and frictional study of the synthesized peptide of the disclosure.

[0049] 1. The oligopeptide can be synthesized by constructing an expression vector through gene recombination. The codon sequence is AAG AAT ACT CTT CAT ACG TTT or AAG CAT GGT CAT CAT CGT CAT. These are also nucleotide sequences obtained by sequencing during the phage peptide library screening. Alternatively, a nonapeptide with the sequence CKHGHHRHC is synthesized by chemical methods, and the cysteines at both ends are used to cyclize the oligopeptide.

[0050] 2. Dry powder of the synthesized peptide is diluted to a concentration of 1 with a DMEM cell culture medium. The cryopreserved virus is expanded and cultured, and a 500-fold TCID 50/mL of dengue virus suspension is prepared.

[0051] 3. C6/36 cells are cultured on a microplate at 28 C., and the original culture solution on the microplate is aspirated off when the cell density reaches about 70% 100 L of DMEM cell growth and maintenance solution is added to each well in the first row of the culture plate, which are non-infected and pepetide-free control cells; 25 L of peptide solution is added to each well in. the second row, and 50 82 L of peptide solution is added to each well in the third row, which are non-infected control cells with the addition of pepetides; 25 L of virus solution is added to each well in the fourth to eighth rows, and the cell culture plate is placed in an incubator for 1 h to allow the virus to adsorb cells. The fourth row is free of peptide solution, and each of the fifth to eighth rows is loaded with peptide solution by 5 L/well, 10 L/well, 20 L/well and 40 L/well respectively; the pores with a total liquid amount of less than 100 l in the culture system is supplemented to 100 L, and the culture plate is cultured in a carbon dioxide incubator.

[0052] 4. Cell lesions are observed for 4 days, and the number of cell wells and the degree of lesions of cytopathic cells in each row are recorded. The degree of cell lesion is divided into: 0, no cell lesion; I, 0 to 25% of the cells have lesions; II, 25 to 50% of the cells have lesions; III, 50 to 75% of the cells have lesions; IV, 75 to 100 % of the cells have lesions.

[0053] The results show that the cells in the 36 culture wells in the to 1.sup.st to 3.sup.rd rows grow well; the cells in the 12 wells of the 4.sup.th row without the addition of the synthesized peptide all develop lesions, wherein 9 wells have a lesion degree of IV and 3 wells have a lesion degree of III; cells in 12 wells in the 5.sup.th row with the addition of 5 L/well of peptide solution have lesions, and the degrees of which are II to III; 9 wells of cells in the 6.sup.th row with the addition of 10 L/well of peptide solution have lesions, and the degrees of which are I to II; 6 wells of cells in the 7.sup.th row with the addition of 20 L/well of peptide solution have lesions, and the degrees of which are I to II; only 3 wells of cells in the 8.sup.th row with the addition of 40 L/well of peptide solution have lesions, and the degree of which is I; continued culture reveals the return to normality.

[0054] The above cell experiments show that the synthesized peptide of the disclosure has no adverse effect on cell growth at high concentrations; the synthesized peptide has a significant protective effect on cells against virus infection, and the protective effect exhibits a dose-effect relationship.

[0055] It should be understood that the technical solution obtained after proportionally increasing or reducing the amount of the reagents or raw materials used in the above example is substantially the same as that of the above example.

[0056] Although this disclosure has been described in detail with the general descriptions and specific embodiments, it is obvious to those skilled in the art that modifications or improvements can be made to the present invention on the basis of the this disclosure. Therefore, these modifications or improvements made without departing from the spirit of this disclosure belong to the scope of protection of this disclosure.

INDUSTRIAL APPLICABILITY

[0057] The disclosure provides an oligopeptide having the function of inhibiting dengue virus replication and the application thereof. The amino acid sequence of the oligopeptide provided by the disclosure is KHGHHRH, i.e., Lys-His-Gly-His-His-Arg-His. The oligopeptide has a high specific affinity for NS5, and has a highly effective inhibitory effect on dengue virus replication. The antiviral effect thereof is not limited to DENV 2, and it also has a significant inhibitory effect on the replication of type I, type 3, and type 4 dengue viruses. One cysteine is added to each end of the oligopeptide sequence, and the oligopeptide can be cyclized by the cysteines at both. ends to form a cyclic peptide. The obtained cyclic peptide has enhanced effect of inhibiting dengue virus replication, and can be used for specific treatment of dengue virus infection, and has good economic value and application prospect.