Rabies virus G protein mutant and its preparation method and application

Abstract

The present invention relates to the field of biomedicine, and more particularly to rabies virus G protein mutants, preparation methods, and applications thereof. The rabies virus G protein mutants of the present invention are obtained by point mutation of the G protein's extracellular domain. These mutants achieve soluble expression of the G protein's extracellular domain and can induce the production of high-titer neutralizing antibodies. Compared to commercial human and canine rabies vaccines, these mutants exhibit superior cellular and antibody immune responses, potentially reducing the number of vaccinations, shortening the immunization schedule, and reducing the immunization dose.

Claims

1. A rabies virus G protein mutant, characterized in that: The amino acid sequence of the G protein mutant is shown in SEQ ID NO:

2. 2 . The G protein mutant according to claim 1 , wherein the N-terminus of the G protein mutant comprises a signal peptide. 3 . The G protein mutant according to claim 2 , wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO:

18. 4 . The G protein mutant according to claim 1 , wherein the C-terminus of the G protein mutant comprises a 6×His tag.

5. A rabies virus G protein multimer, characterized in that: The multimer is formed by self-assembly of the rabies virus G protein mutant according to any one of claims 1 to 4, and the multimer is a hexamer, a nonamer or a dodecamer.

6. The G protein multimer according to claim 5, wherein the multimer is prepared by the following method, comprising the steps of: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 4 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant was harvested, centrifuged and the supernatant was filtered through a membrane. (3) The filtered supernatant was purified by nickel column. (4) Collect samples containing target protein, concentrate by ultrafiltration and then purify by molecular sieve. (5) Diluting the purified target protein with buffer can obtain soluble G protein polymers with uniform particle size; in, The buffer used for molecular sieve purification in step (4) is TBS, and the buffer used in step (5) is TBS or Tween-80, with the concentration of Tween-80 being 0.02-0.1%.

7. The G protein multimer according to claim 6, wherein the step (4) further comprises purifying the product of the first molecular sieve purification by an anion exchange column, and then performing a second molecular sieve purification.

8. The method for preparing the G protein multimer according to any one of claims 5 to 7, comprising the steps of: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 4 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant was harvested, centrifuged and the supernatant was filtered through a membrane. (3) The filtered supernatant was purified by nickel column. (4) Collect samples containing target protein, concentrate by ultrafiltration and then purify by molecular sieve. (5) Diluting the purified target protein with buffer can obtain soluble, uniformly sized G protein trimers or multimers; in, The buffer used for molecular sieve purification in step (4) is TBS, and the buffer used in step (5) is TBS or Tween-80, with the concentration of Tween-80 being 0.02-0.1%.

9. The method for preparing a G protein multimer according to claim 8, wherein step (4) further comprises purifying the product of the first molecular sieve purification by an anion exchange column, and then performing a second molecular sieve purification.

10. An immunogenic complex comprising: (1) An antigen component comprising a rabies virus G protein mutant selected from any one of claims 1 to 4; (2) a granule protein component comprising a nanoparticle protein, wherein the amino acid sequence of the nanoparticle protein is shown in SEQ ID NO: 21; The antigen component and the granule protein component form the immunogenic complex through a covalent binding reaction; The antigen component is formed by fusing the G protein mutant to the binding peptide 1 at the C-terminus via a connecting peptide 1; the particle protein component is formed by fusing the nanoparticle protein to the binding peptide 2 at the N-terminus via a connecting peptide 2; The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO: 19, and the amino acid sequence of the binding peptide 2 is shown in SEQ ID NO: 20; The connecting peptide 1 is GSGGSG; the connecting peptide 2 is (GGS)4. The immunogenic complex according to claim 10 , wherein both the antigen component and the particle protein component comprise a histidine tag.

12. A method for preparing an immunogenic complex: (1) The antigen component and granule protein component encoding genes according to any one of claims 10 to 11 are respectively connected into expression vectors to construct expression recombinant plasmids; (2) constructing a recombinant host cell capable of expressing the antigen component and the granule protein component in the host cell; (3) Using recombinant host cells to express the fusion protein and purifying the recombinant fusion protein; (4) The antigen component and the granule protein component are subjected to a covalent binding reaction to obtain the immunogenic complex.

13. The preparation method according to claim 12, wherein the plasmid for expressing the antigen component in step (1) is selected from pcDNA3.4, and the vector for expressing the granule protein is selected from pET-28a(+) or pET-30a(+).

14. The preparation method according to claim 12 or 13, wherein in step (2), the host cell for expressing the antigen component is CHO, and the host cell for expressing the granule protein is E. coli.

15. An immune composition comprising the rabies virus G protein mutant according to any one of claims 1 to 4, or the rabies virus G protein multimer according to any one of claims 5 to 7, or the immunogenic complex according to any one of claims 10 to 11. The immune composition according to claim 15 , further comprising a pharmaceutically acceptable carrier. The immunogenic composition according to claim 16 , wherein the pharmaceutically acceptable carrier is an excipient.

18. An immune composition according to claim 16, characterized in that The pharmaceutically acceptable carrier is selected from stabilizers, surfactants, buffers, and pH regulators.

19. A rabies vaccine, characterized in that: It contains the immune composition according to any one of claims 15 to 18 and an adjuvant, wherein the adjuvant enhances the immune response through multiple mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages, and the vaccine includes a human vaccine or a veterinary vaccine.

20. The vaccine according to claim 19, wherein the adjuvant is selected from aqueous adjuvants and oil emulsion adjuvants.

21. The vaccine according to claim 19, wherein the adjuvant is selected from the group consisting of mineral adjuvants, liposome adjuvants and saponin adjuvants.

22. The vaccine according to claim 19 or 20, wherein the component contents (w / w) of the adjuvant are as follows: Squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, Citric Acid 0.264%, Sodium Citrate 0.016%.

23. A complete kit, characterized in that: Comprising the vaccine according to any one of claims 19 to 22, and a container required for vaccination with the vaccine.

24. The kit according to claim 23, wherein the container is selected from a needle, a syringe, a powder-carrying container, and a solvent-carrying container.

25. Use of the rabies virus G protein mutant according to any one of claims 1-4, the rabies virus G protein multimer according to any one of claims 5-7, the immunogenic complex according to any one of claims 10-11, the immune composition according to any one of claims 15-18, the vaccine according to any one of claims 19-22, and the kit according to any one of claims 23-24 for the preparation of drugs for preventing or treating diseases caused by rabies virus infection.

26. Use of the rabies virus G protein mutant according to any one of claims 1-4, the rabies virus G protein multimer according to any one of claims 5-7, the immunogenic complex according to any one of claims 10-11, the immune composition according to any one of claims 15-18, the vaccine according to any one of claims 19-22, and the kit according to any one of claims 23-24 for the preparation of drugs for pre-exposure prophylaxis and post-exposure prophylaxis of rabies virus infection.

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