KR-20260064853-A - Vaccinia virus recombinant DAL protein and orthopoxvirus vaccine composition comprising the same

Abstract

The present invention relates to a vaccinia virus recombinant DAL protein and an orthophoxvirus vaccine composition containing the same, and more specifically, to a recombinant DAL protein derived from vaccinia virus antigens, a recombinant vector for efficiently expressing said protein, a transformant, a method for producing said recombinant DAL protein, and an orthophoxvirus vaccine composition containing said recombinant DAL protein.

Inventors

• 손은주
• 강향주
• 박소윤
• 김진
• 전혜영
• 송영조
• 유치호
• 김정은

Assignees

• 대한민국(방위사업청장)

Dates

Publication Date

20260508

Application Date

20241029

Claims (17)

1. A gene construct comprising a polynucleotide encoding a vaccinia virus recombinant DAL protein, comprising the amino acid sequence from position 90 to 181 in the amino acid sequence of SEQ ID NO. 1, the amino acid sequence from 3 to 181 in the amino acid sequence of SEQ ID NO. 2, the amino acid sequence from position 64 to 181 in the amino acid sequence of SEQ ID NO. 1, and the amino acid sequence from 3 to 181 in the amino acid sequence of SEQ ID NO. 2.
2. In paragraph 1, The above recombinant DAL protein is a gene construct comprising the amino acid sequence of SEQ ID NO. 3.
3. In paragraph 1, A gene construct further comprising a polynucleotide encoding an endoplasmic reticulum signal peptide and/or a polynucleotide encoding an endoplasmic reticulum retention signal peptide.
4. In paragraph 3, A gene construct further comprising a polynucleotide encoding a His-tag.
5. In paragraph 3, The polynucleotide encoding the above endoplasmic reticulum signal peptide (ER signal peptide) comprises the nucleotide sequence of SEQ ID NO. 5 encoding NB (new chaperone binding protein), and is a gene construct.
6. In paragraph 3, The above-mentioned endoplasmic reticulum residual signal peptide is a gene construct comprising the amino acid sequence of HDEL (His-Asp-Glu-Leu), HEEL (His-Glu-Glu-Leu), KDEL (Lys-Asp-Glu-Leu), KEEL (Lys-Glu-Glu-Leu), RDEL (Arg-Asp-Glu-Leu), or REEL (Arg-Glu-Glu-Leu).
7. In paragraph 3, A gene construct in which the following (i) through (iv) are connected sequentially: (i) Polynucleotide encoding an endoplasmic reticulum signal peptide, (ii) a polynucleotide encoding the recombinant DAL protein, (iii) Polynucleotide encoding the His-tag and (iv) Polynucleotide encoding an ER retention signal peptide.
8. In Paragraph 7, The above gene construct is a gene construct comprising the nucleotide sequence of SEQ ID NO. 13.
9. A recombinant vector comprising a gene construct of any one of claims 1 to 8.
10. Transformed body transformed with the recombinant vector of claim 9.
11. In Paragraph 10, The above-mentioned transformant is a transformant that is a plant body.
12. A method for preparing a vaccinia virus recombinant antigen protein comprising the following (a) and (b): (a) a step of transforming a plant body with the recombinant vector of claim 9; and (b) A step of isolating and purifying vaccinia virus recombinant DAL protein from the above plant.
13. In Paragraph 12, A method for producing a vaccinia virus recombinant antigen protein, wherein the above-mentioned vaccinia virus recombinant DAL protein is expressed in a water-soluble form in the above-mentioned plant.
14. Vaccinia virus recombinant antigen protein containing the amino acid sequence of SEQ ID NO. 3.
15. In Paragraph 14, Vaccinia virus recombinant antigen protein produced using the recombinant vector of claim 9.
16. An orthofoxvirus vaccine composition comprising the vaccinia virus recombinant antigen protein of claim 14.
17. In Paragraph 16, A composition in which the above-mentioned orthopoxvirus is a smallpox virus, a vaccinia virus, or a monkeypox virus.

Description

Vaccinia virus recombinant DAL protein and orthopoxvirus vaccine composition comprising the same The present invention relates to a vaccinia virus recombinant protein and an orthofox virus vaccine composition containing the same. Smallpox is an acute disease characterized by fever, blisters, and pustular pathological skin changes, caused by the smallpox virus. It has a very high mortality rate and once accounted for 10% of all causes of death worldwide, including in Korea; however, in 1979, smallpox was declared eradicated globally. Nevertheless, following the report of a single case of monkeypox in the UK on May 7, 2022, cases of infection have been reported in numerous countries including the United States, Brazil, Spain, France, and Colombia, and interest has recently been growing again as the possibility of the smallpox virus being used as a biological terror weapon has become known. In Korea, monkeypox was designated as a Class 2 infectious disease on June 8, 2022, and the crisis level was raised from 'concern' to 'caution' after a Korean national who had visited Germany was confirmed to have monkeypox in June of the same year. Consequently, the need for the development of vaccines to prevent orthofoxviruses, including monkeypox, is emerging. Meanwhile, A17L, A27L, A28L, A33R, B5R, D8L, L1R, and H3 have been reported as vaccinia virus proteins identified as targets of neutralizing antibodies in humans (Non-patent Literature 1). Against this background, the inventors developed a gene construct derived from major antigens of vaccinia virus and a recombinant vector containing the same for high-efficiency production in plants, and completed the present invention by demonstrating the efficacy of a recombinant protein produced using the said recombinant vector as a vaccine. FIG. 1 shows a gene construct [DAL:6H] encoding a vaccinia virus recombinant DAL protein according to one embodiment of the present invention, the base sequence thereof, and the amino acid sequence of the expressed recombinant DAL protein. FIG. 2 is a band image obtained by performing a Western blot to confirm the expression of vaccinia virus recombinant DAL protein according to one embodiment of the present invention (T, Total extract; S, Soluble; P, Pellet). Figure 3 shows the results of confirming a protein sample obtained during the process of isolating and purifying vaccinia virus recombinant DAL protein according to one embodiment of the present invention by electrophoresis followed by Western blot (A) and Coomassie blue staining (B). Figure 4 is a graph showing the change in body weight (A) and survival rate (B) of mice following vaccinia virus challenge inoculation after administration of vaccinia virus recombinant DAL protein vaccine according to one embodiment of the present invention. Figure 5 is a PRNT result (A) and a graph (B) showing the reduction rate of vaccinia virus plaques compared to a control group (cultured only vaccinia virus) according to the dilution ratio of mouse serum after vaccination with vaccinia virus recombinant DAL protein vaccine according to one embodiment of the present invention. It should be noted that in the following description, only the parts necessary for understanding the embodiments of the present invention are described, and the description of other parts will be omitted to the extent that it does not detract from the gist of the present invention. The terms and words used in this specification and claims described below shall not be interpreted as being limited to their ordinary or dictionary meanings, but shall be interpreted in a meaning and concept consistent with the technical spirit of the invention, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention. Therefore, the embodiments described in this specification and the configurations illustrated in the drawings are merely preferred embodiments of the present invention and do not represent all technical concepts of the present invention; thus, it should be understood that various equivalents and modifications that can replace them may exist at the time of filing this application. The present invention will be described in detail below. As mentioned above, with monkeypox infection cases occurring simultaneously worldwide since 2022, the possibility of the smallpox virus being used as a biological weapon is emerging, and thus, in preparation for this, there is a need to develop a vaccine to prevent orthofoxviruses, including monkeypox. Accordingly, in the present invention, a gene construct encoding a recombinant DAL protein was constructed by fusing A33_1 and A33_2, which are parts of A33, one of the major antigens of vaccinia virus, with L1_1, which is a part of the L1 protein. By confirming the protective efficacy against orthofoxovirus and the ability to generate neutralizing antibodies against it of the recombinant DAL protein produced therefrom, the efficacy of the