阅读说明：本技术 蜂王浆主蛋白的抗体及其用途 (Antibody of royal jelly major protein and application thereof ) 是由 杨柏宏 王俊凯 温国光 于 2018-04-11 设计创作，主要内容包括：特异性针对蜂王浆主蛋白的抗体可被用于纯化包括天然蜂王浆主蛋白的蜂王浆主蛋白的方法中。这些方法可产生富集天然蜂王浆主蛋白的组合物。纯化的蜂王浆主蛋白可被用于产生包含天然蜂王浆主蛋白的美容产品。特异性针对蜂王浆主蛋白的抗体可被用于纯化包括天然蜂王浆主蛋白的蜂王浆主蛋白的方法中。这些方法可产生富集天然蜂王浆主蛋白的组合物。纯化的蜂王浆主蛋白可被用于产生包含天然蜂王浆主蛋白的美容产品。分离的核酸编码与蜂王浆主蛋白特异性结合的单克隆抗体。(antibodies specific for the major protein of royal jelly can be used in a method for purifying the major protein of royal jelly including the major protein of natural royal jelly. These methods can produce compositions enriched in the major proteins of natural royal jelly. The purified royal jelly major protein can be used to produce a cosmetic product comprising a natural royal jelly major protein. Antibodies specific for the major protein of royal jelly can be used in a method for purifying the major protein of royal jelly including the major protein of natural royal jelly. These methods can produce compositions enriched in the major proteins of natural royal jelly. The purified royal jelly major protein can be used to produce a cosmetic product comprising a natural royal jelly major protein. The isolated nucleic acid encodes a monoclonal antibody that specifically binds to a major protein of royal jelly.)

1. A method of purifying a native royal jelly major protein, the method comprising:

Dissolving biological substance containing natural Lac Regis Apis major protein in aqueous solution,

Contacting an aqueous solution comprising dissolved biological substances with a monoclonal antibody immobilized on a substrate, wherein the monoclonal antibody specifically binds to the amino acid sequence of SEQ ID No. 2, thereby binding the monoclonal antibody to the native royal jelly major protein;

Isolating monoclonal antibodies that bind to the major protein of natural royal jelly from the aqueous solution; and

Removing the bound native royal jelly major protein from the monoclonal antibody, e.g., by elution, thereby purifying the native royal jelly major protein.

2. The method of claim 1, wherein the monoclonal antibody comprises:

A Heavy Chain Variable Region (HCVR) comprising:

CDR3 domain of CDR3 domain of SEQ NO 6;

CDR2 domain of CDR2 domain of SEQ NO 6; and

CDR1 domain of CDR1 domain of SEQ NO 6; and

a Light Chain Variable Region (LCVR) comprising:

CDR3 domain of CDR3 domain of SEQ NO 8;

CDR2 domain of CDR2 domain of SEQ NO 8; and

CDR1 domain of CDR1 domain of SEQ NO 8.

3. The method of claim 1, wherein the monoclonal antibody comprises:

HCVR of SEQ ID NO 6; and

LCVR of SEQ ID NO 8.

4. The method of claim 1, wherein the monoclonal antibody comprises:

(a) A Heavy Chain Variable Region (HCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C5, and a Light Chain Variable Region (LCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C 5;

(b) An HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C9, and an LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C 9;

(c) An HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D3, and an LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D 3;

(d) An HCVR having a CDR3 domain of the CDR3 domain of antibody 4G6E2, a CDR2 domain of the CDR2 domain of antibody 4G6E2 and a CDR1 domain of the CDR1 domain of antibody 4G6E2, and an LCVR having a CDR3 domain of the CDR3 domain of antibody 4G6E2, a CDR2 domain of the CDR2 domain of antibody 4G6E2 and a CDR1 domain of the CDR1 domain of antibody 4G6E 2; or

(e) An HCVR having a CDR3 domain of the CDR3 domain of antibody 9G6a2, a CDR2 domain of the CDR2 domain of antibody 9G6a2, and a CDR1 domain of the CDR1 domain of antibody 9G6a2, and an LCVR having a CDR3 domain of the CDR3 domain of antibody 9G6a2, a CDR2 domain of the CDR2 domain of antibody 9G6a2, and a CDR1 domain of the CDR1 domain of antibody 9G6a 2.

5. The method of claim 4, wherein the monoclonal antibody comprises:

(a) The Heavy Chain Variable Region (HCVR) of antibody 4G6C5 and the Light Chain Variable Region (LCVR) of antibody 4G6C 5;

(b) HCVR of antibody 8C5C9 and LCVR of antibody 8C5C 9;

(c) HCVR of antibody 8C5D3 and LCVR of antibody 8C5D 3;

(d) HCVR of antibody 4G6E2 and LCVR of antibody 4G6E 2; or

(e) HCVR of antibody 9G6a2 and LCVR of antibody 9G6a 2.

6. the method of claim 1, wherein the monoclonal antibody competes for binding with at least one of the following antibodies:

(a) other monoclonal antibodies comprising a Heavy Chain Variable Region (HCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C5 and a Light Chain Variable Region (LCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C 5;

(b) additional monoclonal antibodies comprising a HCVR with the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C9 and a LCVR with the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C 9;

(c) Additional monoclonal antibodies comprising a HCVR with the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D3 and a LCVR with the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D 3;

(d) Additional monoclonal antibodies comprising an HCVR with the CDR3 domain of the CDR3 domain of antibody 4G6E2, the CDR2 domain of the CDR2 domain of antibody 4G6E2 and the CDR1 domain of the CDR1 domain of antibody 4G6E2 and an LCVR with the CDR3 domain of the CDR3 domain of antibody 4G6E2, the CDR2 domain of the CDR2 domain of antibody 4G6E2 and the CDR1 domain of the CDR1 domain of antibody 4G6E 2; or

(e) Additional monoclonal antibodies comprising an HCVR with the CDR3 domain of the CDR3 domain of antibody 9G6a2, the CDR2 domain of the CDR2 domain of antibody 9G6a2 and the CDR1 domain of the CDR1 domain of antibody 9G6a2 and an LCVR with the CDR3 domain of the CDR3 domain of antibody 9G6a2, the CDR2 domain of the CDR2 domain of antibody 9G6a2 and the CDR1 domain of the CDR1 domain of antibody 9G6a 2.

7. the method of claim 1, wherein the monoclonal antibody is selected from the group consisting of: antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a2, or an antibody that competes for binding to the major protein of royal jelly with at least one of the following antibodies: antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a 2.

8. The method of any one of claims 1-7, further comprising preparing a composition comprising a purified native royal jelly major protein, wherein the composition comprises a lyophilized native royal jelly major protein, or wherein the composition is comprised in a topical cosmetic product comprising the purified native royal jelly major protein.

9. The method of any one of claims 1-8, wherein the biological substance comprises royal jelly of an insect.

10. the method of any one of claims 1-9, wherein the biological substance comprises a cellular extract genetically engineered to produce a native royal jelly major protein.

11. The method of claim 10, wherein said insect belongs to the genus Apis (Apis).

12. the method of any one of claims 1-11, wherein the matrix comprises agarose.

13. The method of any one of claims 1-12, wherein said contacting, isolating, and removing comprises immunoprecipitation.

14. The method of any one of claims 1-13, wherein the contacting, separating, and removing are performed using an affinity column.

15. The method of any one of claims 8-14, wherein the composition comprises a lyophilized native royal jelly major protein.

16. The method of any one of claims 8-15, wherein the composition comprises at least 30% (w/w) of lyophilized native royal jelly major protein.

17. the method of any one of claims 8-14, wherein the topical cosmetic product comprises a topical lotion, cream, paste, gel, spray, powder, or eyebrow pencil.

18. the method of claim 17, wherein the topical cosmetic product comprises at least 10% royal jelly major protein (w/w).

19. An isolated monoclonal antibody that specifically binds to a major protein of royal jelly, said monoclonal antibody comprising:

(a) A Heavy Chain Variable Region (HCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C5, and a Light Chain Variable Region (LCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C 5;

(b) an HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C9, and an LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C 9;

(c) An HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D3, and an LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D 3;

(d) An HCVR having a CDR3 domain of the CDR3 domain of antibody 4G6E2, a CDR2 domain of the CDR2 domain of antibody 4G6E2 and a CDR1 domain of the CDR1 domain of antibody 4G6E2, and an LCVR having a CDR3 domain of the CDR3 domain of antibody 4G6E2, a CDR2 domain of the CDR2 domain of antibody 4G6E2 and a CDR1 domain of the CDR1 domain of antibody 4G6E 2; or

(e) an HCVR having a CDR3 domain of the CDR3 domain of antibody 9G6a2, a CDR2 domain of the CDR2 domain of antibody 9G6a2, and a CDR1 domain of the CDR1 domain of antibody 9G6a2, and an LCVR having a CDR3 domain of the CDR3 domain of antibody 9G6a2, a CDR2 domain of the CDR2 domain of antibody 9G6a2, and a CDR1 domain of the CDR1 domain of antibody 9G6a 2.

20. The isolated monoclonal antibody of claim 19, comprising:

a Heavy Chain Variable Region (HCVR) comprising:

CDR3 domain of CDR3 domain of SEQ NO 6;

CDR2 domain of CDR2 domain of SEQ NO 6; and

CDR1 domain of CDR1 domain of SEQ NO 6; and

A Light Chain Variable Region (LCVR) comprising:

CDR3 domain of CDR3 domain of SEQ NO 8;

CDR2 domain of CDR2 domain of SEQ NO 8; and

CDR1 domain of CDR1 domain of SEQ NO 8.

21. The isolated monoclonal antibody of claim 19, comprising:

HCVR of SEQ ID NO 6; and

LCVR of SEQ ID NO 8.

22. The isolated monoclonal antibody of claim 19, wherein the monoclonal antibody comprises:

(a) the Heavy Chain Variable Region (HCVR) of antibody 4G6C5 and the Light Chain Variable Region (LCVR) of antibody 4G6C 5;

(b) HCVR of antibody 8C5C9 and LCVR of antibody 8C5C 9;

(c) HCVR of antibody 8C5D3 and LCVR of antibody 8C5D 3;

(d) HCVR of antibody 4G6E2 and LCVR of antibody 4G6E 2; or

(e) HCVR of antibody 9G6a2 and LCVR of antibody 9G6a 2.

23. The isolated monoclonal antibody of claim 19, wherein the monoclonal antibody comprises antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a 2.

24. an isolated monoclonal antibody that competes for binding to a royal jelly major protein having the amino acid sequence of SEQ ID NO:2 with at least one of the antibodies (a) - (e) of claims 19-23.

25. An isolated nucleic acid encoding an isolated monoclonal antibody that specifically binds to a major protein of royal jelly, wherein the antibody comprises:

(HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6; and

A Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8.

26. The isolated nucleic acid of claim 25, wherein the HCVR comprises a HCVR of SEQ ID No. 6, and wherein the LCVR comprises a LCVR of SEQ ID No. 8.

27. the isolated nucleic acid of any one of claims 25-26, wherein the isolated nucleic acid comprises SEQ ID No. 3 and SEQ ID No. 5, wherein SEQ ID No. 3 and SEQ ID No. 5 are contained in the same polynucleotide or are contained in different polynucleotides.

Technical Field

Some embodiments herein relate generally to antibodies specific for a major protein of royal jelly (RA), and methods of using such antibodies, for example, to purify a native royal jelly major protein.

summary of The Invention

some aspects include methods of purifying a native royal jelly major protein. The method may comprise dissolving the biological substance comprising the native royal jelly major protein in, for example, an aqueous solution. The method may comprise contacting a solution comprising dissolved biological material (which may be an aqueous solution, for example, as described above) with a monoclonal antibody immobilized on a substrate, wherein the monoclonal antibody specifically binds to the amino acid sequence of SEQ ID NO:2, and wherein the monoclonal antibody binds to the native royal jelly major protein. The method may comprise isolating monoclonal antibodies that bind to the native royal jelly major protein from the solution, e.g., via washing. The method may further comprise removing the bound native royal jelly major protein from the monoclonal antibody, for example by elution, thereby purifying the native royal jelly major protein. In some embodiments, the method further comprises preparing a composition comprising the purified native royal jelly major protein. The composition may comprise a lyophilized native royal jelly major protein, or may be part of a cosmetic product (e.g., a topical cosmetic product) comprising a native royal jelly major protein. Thus, in some embodiments, the methods result in a composition enriched in the major protein of natural royal jelly. The composition can include at least 1% (w/w) of a native royal jelly major protein, e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a native royal jelly major protein, including ranges between any two of the listed values, e.g., 1% -90%, 1% -50%, 1% -30%, 1% -20%, 1% -10%, 5% -90%, 5% -50%, 5% -30%, 5% -20%, 5% -10%, 10% -90%, 10% -50%, 10% -30%, 10% -20%, 20% -90%, 20% -50% or 20% -30%. In some embodiments, the antibody comprises a mouse antibody. In some embodiments, an antibody of any of the methods for purifying a native royal jelly major protein as described herein comprises a Heavy Chain Variable Region (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, an antibody of any of the methods for purifying a native royal jelly major protein as described herein comprises the HCVR of SEQ ID No. 6 and the LCVR of SEQ ID No. 8. In some embodiments, the antibody comprises antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a2, or a binding fragment thereof. In some embodiments, the antibody comprises: (a) a Heavy Chain Variable Region (HCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C5, and a Light Chain Variable Region (LCVR) having the CDR3 domain of the CDR3 domain of antibody 4G6C5, the CDR2 domain of the CDR2 domain of antibody 4G6C5 and the CDR1 domain of the CDR1 domain of antibody 4G6C 5; or (b) a HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C9 and a LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5C9, the CDR2 domain of the CDR2 domain of antibody 8C5C9 and the CDR1 domain of the CDR1 domain of antibody 8C5C 9; or (C) a HCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D3 and a LCVR having the CDR3 domain of the CDR3 domain of antibody 8C5D3, the CDR2 domain of the CDR2 domain of antibody 8C5D3 and the CDR1 domain of the CDR1 domain of antibody 8C5D 3; or (d) a HCVR having the CDR3 domain of the CDR3 domain of antibody 4G6E2, the CDR2 domain of the CDR2 domain of antibody 4G6E2 and the CDR1 domain of the CDR1 domain of antibody 4G6E2 and a LCVR having the CDR3 domain of the CDR3 domain of antibody 4G6E2, the CDR2 domain of the CDR2 domain of antibody 4G6E2 and the CDR1 domain of the CDR1 domain of antibody 4G6E 2; or (e) a HCVR having the CDR3 domain of the CDR3 domain of antibody 9G6a2, the CDR2 domain of the CDR2 domain of antibody 9G6a2 and the CDR1 domain of the CDR1 domain of antibody 9G6a2 and a LCVR having the CDR3 domain of the CDR3 domain of antibody 9G6a2, the CDR2 domain of the CDR2 domain of antibody 9G6a2 and the CDR1 domain of the CDR1 domain of antibody 9G6a 2. In some embodiments, the antibody competes for binding to the royal jelly major protein with at least one of the antibodies listed above. In some embodiments, the biological substance comprises royal jelly of an insect. In some embodiments, the biological substance comprises a cellular extract genetically engineered to produce a native royal jelly major protein. In some embodiments, the insect belongs to the genus bee (Apis). In some embodiments, the matrix comprises agarose. In some embodiments, the separating and removing comprises immunoprecipitation. In some embodiments, the contacting, separating, and removing are performed using an affinity column. In some embodiments, the composition comprising the purified native royal jelly major protein (and the enriched native royal jelly major protein) comprises at least 30% (w/w) of the lyophilized native royal jelly major protein. In some embodiments, the external cosmetic product comprising the natural royal jelly main protein includes an external lotion, cream, paste, jelly, spray, powder, or eyebrow pencil. In some embodiments, the topical cosmetic product comprises at least 10% royal jelly major protein (w/w). In some embodiments, a kit is provided comprising a composition comprising a purified native royal jelly major protein as described herein. Thus, in some embodiments, the kit comprises a composition enriched in native royal jelly major protein as described herein.

Some aspects include an isolated monoclonal antibody that specifically binds to a major protein of royal jelly. In some embodiments, the antibody comprises antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a2, or the LCVR and HCDR of one of these antibodies, or the HCDR1, HCDR2, HDCR3, LCDR1, LCDR2, and LCDR3 of one of these antibodies. In some embodiments, the isolated monoclonal antibody competes for binding to the royal jelly major protein with one of the antibodies listed above. In some embodiments, the antibody comprises a mouse antibody. Some aspects include kits comprising antibodies specific for a major protein of royal jelly. In some embodiments, an isolated monoclonal antibody comprises a Heavy Chain Variable Region (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, the isolated monoclonal antibody comprises the HCVR of SEQ ID NO. 6 and the LCVR of SEQ ID NO. 8.

Some aspects include an isolated nucleic acid comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding an isolated monoclonal antibody that specifically binds to a major protein of royal jelly. In some embodiments, an isolated monoclonal antibody comprises a (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, the isolated nucleic acid encodes an antibody that specifically binds to a royal jelly major protein comprising, consisting essentially of, or consisting of SEQ ID NO: 2. In some embodiments, the HCVR-encoding nucleic acid and the LCVR-encoding nucleic acid are contained in the same polynucleotide. In some embodiments, the HCVR-encoding nucleic acid and the LCVR-encoding nucleic acid are contained in different polynucleotides (e.g., separate vectors or separate chromosomes). In some embodiments, the nucleic acid comprises, consists essentially of, or consists of the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7. The nucleic acid of SEQ ID NO. 5 can encode the heavy chain variable region and the nucleic acid of SEQ ID NO. 7 can encode the light chain variable region. In some embodiments, the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7 are contained in the same nucleic acid molecule (e.g., a single vector) and are under the control of separate promoters, or under the control of a single promoter, and are separated by an IRES or 2A sequence. In some embodiments, the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7 are comprised in different nucleic acid molecules. In some embodiments, the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7 are contained in different nucleic acid molecules in the same composition. In some embodiments, the isolated nucleic acid encodes an isolated monoclonal antibody that specifically binds to a major protein of royal jelly comprising, consisting essentially of, or consisting of the polypeptide of SEQ ID NO. 2. In some embodiments, the hybridoma cells for expressing the isolated monoclonal antibody that specifically binds to the major protein of royal jelly comprise the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7.

brief Description of Drawings

FIG. 1A is a diagram of the nucleotide sequence (SEQ ID NO:1) (GenBank: AF000633.1) encoding the major protein of royal jelly of honeybees. FIG. 1B is a diagram of the amino acid sequence (SEQ ID NO:2) of the main protein of royal jelly.

FIG. 2A is a diagram of the nucleotide sequence (SEQ ID NO:3) encoding a royal jelly major protein fusion protein that can be used in some embodiments to generate antibodies specific for a royal jelly major protein. FIG. 2B is a diagram of the amino acid sequence (SEQ ID NO:4) of the royal jelly major protein fusion protein. Specific elements of the fusion protein are shown, including a region sharing identity with the native protein, a FLAG tag, and a multiple His tag.

fig. 3A and 3B are images of a dot immunoblot assay measuring the affinity of monoclonal antibodies specific for RA according to some embodiments herein.

Fig. 4A and 4B are images of coomassie stained gels immunoprecipitated by each of five monoclonal antibodies that bind to major proteins of royal jelly according to some embodiments herein. Five antibodies (antibodies 19, 20, 21, 22 and 23) have been identified by immunoblotting as having the highest affinity for the major protein of royal jelly. Figure 4A shows the results of immunoprecipitation of 500ng RA starting material using monoclonal antibodies 19 and 20. Figure 4B shows the results of immunoprecipitation of 500ng RA starting material using monoclonal antibodies 21, 22 and 23.

Fig. 5A is an image of a coomassie-stained SDS-PAGE gel of immunoprecipitated native royal jelly major proteins from royal jelly using monoclonal antibodies that bind to the royal jelly major proteins according to some embodiments herein. Fig. 5B is an image of a western blot of RA based on the input, NB, wash and eluate of immunoprecipitation.

Fig. 6A-D are a series of diagrams illustrating the sequences of the heavy chain variable region and the light chain variable region of antibody 4G6C5 of some embodiments. FIG. 6A shows some embodiments of a nucleic acid encoding the heavy chain variable region of antibody 4G6C5 (SEQ ID NO: 5). FIG. 6B shows a polypeptide comprising the heavy chain variable region of antibody 4G6C5 (SEQ ID NO:6) of some embodiments. FIG. 6C shows nucleic acid encoding the light chain variable region of antibody 4G6C5 (SEQ ID NO:7) of some embodiments. FIG. 6D shows a polypeptide comprising the light chain variable region of antibody 4G6C5 (SEQ ID NO:8) of some embodiments.

Detailed Description

Described herein are methods and compositions for purifying a royal jelly major protein (RA) protein, and compositions comprising the purified royal jelly major protein. In some embodiments, the royal jelly major protein is purified using an antibody specific for the royal jelly major protein (e.g., a monoclonal antibody that binds to a native royal jelly major protein as described herein) so as to produce a composition comprising the purified native royal jelly major protein. The biological substance comprising the main protein of natural royal jelly can be dissolved in an aqueous solution and contacted with an antibody specific for the main protein of royal jelly. In some embodiments, the royal jelly major protein is purified in its native conformation, thereby producing a composition comprising the royal jelly major protein purified in its native conformation. In some embodiments, methods of preparing compositions comprising purified native royal jelly major protein are described. In some embodiments, one or more monoclonal antibodies specific for a major protein of royal jelly and that bind to a native major protein of royal jelly are provided.

the female bee larva can develop into working bee or queen bee. Royal jelly is a substance secreted by worker bees. Royal jelly is a complex mixture of various vitamins, carbohydrates, fatty acids and proteins. Only female larva fed with royal jelly can develop into queen bee. Royal jelly major proteins (e.g., as shown in SEQ ID NO:2) are monomeric 57kDa proteins with NO known family members (i.e., paralogs), which are components of royal jelly. It has been shown that the major protein of royal jelly itself can induce the differentiation of female larvae into queen bees. The royal jelly major protein replicates the action of royal jelly, and has increased growth rate and increased life. It has also been found that the main protein of royal jelly has the same growth and life-increasing effect in another insect, Drosophila melanogaster or common Drosophila melanogaster. It has also been found that the major protein of royal jelly has a life-prolonging effect in the non-insect nematodes, Caenorhabditis elegans. In both insects and helminths, it has been found that the major protein of royal jelly acts mainly through members of the Epidermal Growth Factor Receptor (EGFR). This was demonstrated in Drosophila (Drosophila) and C.elegans by using knock-out mutants. Finally, the main protein of royal jelly has been also shown to have mitogenic effects on mammalian cells. In view of the high degree of conservation of the major royal jelly protein in bees, the major royal jelly protein of honeybees, or more precisely, the major royal jelly protein of insects of the genus Apis, is expected to have biological activity as described herein (such as mitogenic activity on mammalian cells and activity in wound healing) and can be purified using the antibodies and methods as described herein. Thus, "major royal jelly protein" as used herein refers to the major royal jelly protein of the genus Apis. Such royal jelly major proteins can be found in royal jelly of insects of the genus Apis. In some embodiments, the royal jelly major protein comprises or consists of a protein having the amino acid sequence of SEQ ID NO. 2. In some embodiments, the royal jelly major protein is encoded by the nucleic acid of SEQ ID NO. 1. In some embodiments, the major royal jelly protein comprises a major royal jelly protein of the genus apis. In some embodiments, the major royal jelly protein comprises a major royal jelly protein of Apis mellifera (Apis mellifera). In some embodiments, the royal jelly major protein of the genus apis further comprises an affinity tag, such as a his, FLAG, and/or HA tag.

the royal jelly major protein in its native conformation is expected to have biological activities as shown herein, but other proteins comprising the polypeptide sequence of the royal jelly major protein (e.g., denatured royal jelly major protein or synthetic royal jelly major protein polypeptide or fragment) do not necessarily have these activities, or have less potent activities. Thus, in some embodiments, antibodies are provided that specifically bind to the major protein of royal jelly. The antibody specifically aiming at the main protein of the royal jelly can be combined with the main protein of the natural royal jelly. "native" royal jelly major protein as used herein refers to undenatured royal jelly major protein of the genus Apis, such as may be found in royal jelly, for example, the protein of SEQ ID NO: 2. The antibodies can be used to purify a native royal jelly major protein in order to produce a composition comprising the purified native royal jelly major protein as described herein.

Antibodies

Antibodies are members of the class of immunoglobulin molecules. Full-length antibodies are heterotetrameric glycoproteins having a molecular weight of about 150 kDa. See Janeway et al, immunology, 5 th edition, New York: Garland Science 2001, which is hereby incorporated by reference in its entirety. A typical antibody comprises two light chains, each light chain comprising a variable light chain (VL) domain and a light chain constant region, and two heavy chains, each heavy chain comprising a variable heavy chain (VH) domain and a heavy chain constant region. The chains of the antibody are held together via disulfide bonds. From N-terminus to C-terminus, each heavy chain variable region comprises a heavy chain variable region comprising: a first heavy chain framework region (HFR1), a first heavy chain complementarity determining region (HCDR1), a second heavy chain framework region (HFR2), a second heavy chain complementarity determining region (HCDR2), a third heavy chain framework region (HFR3), a third heavy chain complementarity determining region (HCDR3), and a fourth heavy chain framework region (HFR 4). Downstream (toward the C-terminus) of the heavy chain variable region is a heavy chain constant region comprising (from N-terminus to C-terminus) a constant heavy chain 1(CH1) domain, a constant heavy chain 2(CH2) domain and a constant heavy chain 3(CH3) domain. In human antibodies, the heavy chain constant region may be of the IgG1, IgG2, IgG3, or IgG4 type. From N-terminus to C-terminus, each light chain comprises a light chain variable region comprising: a first light chain framework region (LFR1), a first light chain complementarity determining region (LCDR1), a second light chain framework region (LFR2), a second light chain complementarity determining region (LCDR2), a third light chain framework region (LCDR3), a third light chain complementarity determining region (LCDR3), and a fourth light chain framework region (LFR 4). Downstream (toward the C-terminus) of the light chain variable region is a light chain constant region. In human antibodies, the light chain constant region can be of the kappa or lambda type. The CDRs represent hypervariable loops, and the six CDRs, or a subset thereof, are involved in epitope recognition and binding of the antibody. CDR numbering may be according to any art-recognized definition, such as Kabat's definition (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991)), Chothia's definition (see Chothia and Lesk, J.mol.biol.196: 901. 917(1987)), and AbM's definition (Martin et al, Proc.Natl.Acad.Sci.USA,86: 9268. 9272(1989)), each of which is herein incorporated by reference in its entirety. In some embodiments, the CDRs are defined according to a group selected from the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the IMGT definition. As used herein, the term "antibody" encompasses full-length monoclonal and polyclonal antibodies, as well as functional binding fragments thereof, such as Fab, Fab 'F (ab') 2, Fv, diabodies, and the like. In some embodiments, the antibody to the major protein of royal jelly is a full length antibody. An antibody will be understood to "specifically bind" (or "specifically bind" to …, or "specifically target" including variations of these root terms) if the binding of the antibody determines the presence of a particular antigen in a heterogeneous population of proteins, macromolecules, or other possible antibody binding targets. Thus, an antibody specific for a major protein of royal jelly will be understood to bind to a substance comprising the major protein of royal jelly at a level above background and will be understood to preferentially bind to the major protein of royal jelly without significant binding to other potential antigens in the substance. It should be noted that the antibody specific for the main protein of royal jelly can bind to the main protein of natural royal jelly, and can also bind to other forms of main protein of royal jelly, but the binding activity to the main protein of natural royal jelly will be useful for purifying the main protein of natural royal jelly.

Monoclonal antibodies according to some embodiments herein can be constructed by exposing a host organism to one or more administrations of an antigen comprising a major protein of royal jelly. The antigen comprising the major protein of royal jelly further comprises a carrier protein such as Keyhole Limpet Hemocyanin (KLH). According to some embodiments herein, a variety of hosts are suitable, such as mice, rats, rabbits, guinea pigs, hamsters, donkeys, goats, horses, and the like. Thus, it is understood that in some embodiments, antibodies of any desired host type may be produced. In some embodiments, the host comprises a non-human mammal. In some embodiments, the host is a rodent, such as a mouse or rat. Antibody-producing cells, typically B cells, can be isolated from a host. In some embodiments, one or more boosts are performed subsequently to the initial administration of the antigen comprising the major protein of royal jelly to the host. In some embodiments, hybridomas are constructed from the antibody-producing cells and screened to produce antibodies to the royal jelly major protein having desired properties (e.g., affinity for the native royal jelly major protein). In some embodiments, phage display libraries comprising nucleic acids encoding antibodies or binding fragments thereof are generated (see Clackson and Wells, Trends Biotech.12:173-184 (1994)). Phage display libraries of antibodies or binding fragments can be screened for affinity for a desired target (e.g., native royal jelly major protein). Thus, the phage display library can be used for the primary screening of antibodies with suitable affinity, and can also be used for screening variants with suitable affinity for the native royal jelly major protein, for example, for screening higher affinity variants of the lead monoclonal antibody. In some embodiments, the phage display library is screened to identify nucleic acids encoding antibodies with suitable binding properties to the native royal jelly major protein. In some embodiments, the phage display library is used to screen for high affinity variants of monoclonal antibodies that bind to the native royal jelly major protein. The phage display library can be screened against an aqueous solution comprising solubilized native royal jelly major protein (e.g., native royal jelly major protein). Techniques for generating monoclonal Antibodies are discussed, for example, in Greenfield (2014), Antibodies: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, N.Y., which is incorporated herein by reference in its entirety.

in some embodiments, two or more monoclonal antibodies compete for binding to an epitope on a major protein of royal jelly (e.g., a native royal jelly major protein). As used herein, two antibodies "compete" for binding to the major protein of royal jelly when the first antibody inhibits the binding of the second antibody to a common epitope of the major protein of royal jelly. Competition can be determined using a variety of suitable assays (e.g., competition ELISA or competition radioimmunoassay). For example, the binding of a labeled first antibody specific for a major protein of royal jelly can be determined in the presence or absence of an unlabeled (or differently labeled) second antibody specific for a major protein of royal jelly to determine whether the binding of the labeled first antibody is reduced in the presence of the second antibody. The primary antibody may be labeled directly or indirectly. Some epitopes may be linear epitopes that represent a particular set of residues on the royal jelly major protein and may be confirmed, for example, by mapping antibodies that bind to the royal jelly major protein peptide and/or deletion peptide. For example, the linear epitope of the main protein of royal jelly according to some embodiments may comprise residues 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210, 211-220, 221-230, 231-240, 241-250, 251-260, 261-270, 271-280, 281-290, 291-300, 301-310, 311-320, 321-330, 340, 341-350, 351-360, 370-, 371-380, 381-390, 391-400, 401-410, 411-420 or 421-432. Some epitopes may be conformational and, due to the three-dimensional structure of the native royal jelly major protein, may thus be present on the native royal jelly major protein, but do not necessarily comprise contiguous residues in the primary amino acid sequence of the royal jelly major protein. Thus, in some embodiments, a monoclonal antibody specific for a royal jelly major protein as described herein specifically binds to a conformational epitope on the native royal jelly major protein, but binds with lower affinity to the denatured royal jelly major protein, or does not bind significantly to the denatured royal jelly major protein. In some embodiments, a monoclonal antibody specific for a major protein of royal jelly as described herein specifically binds to a linear epitope on the major protein of royal jelly. In some embodiments, the antibody competes for binding to the royal jelly major protein with at least one of the following antibodies: antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a2, or an antibody comprising the six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) of any of the listed antibodies. In some embodiments, the antibody to the royal jelly major protein competes for binding to the royal jelly major protein with two or more of antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a 2. The HCVR and LCVR sequences of antibody 4G6C5 are shown in fig. 6B and 6D, respectively. Nucleic acids encoding the indicated HCVR and LCVR are shown in fig. 6A and 6C, respectively. It will be understood that wherever the antibody "4G 6C 5" (or this root term variant, such as "mAb 4G6C 5", "clone 4G6C 5", etc.) is referred to herein, antibodies comprising a HCVR comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said LCVR comprising the CDR3 domain of the CDR3 domain of SEQ NO. 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. It will be understood that wherever the antibody "4G 6C 5" (or variations of this root term) is referred to herein, antibodies comprising a HCVR which is the HCVR in SEQ No. 6 and a LCVR which is the LCVR of SEQ No. 8 are also expressly contemplated.

in some embodiments, an isolated monoclonal antibody (which may be used as an antibody for any method for purifying a native royal jelly major protein as described herein) comprises a Heavy Chain Variable Region (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, an isolated monoclonal antibody (which may be used as an antibody for any method for purifying a native royal jelly major protein as described herein) comprises a Heavy Chain Variable Region (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8, and said HCVR comprises a polypeptide at least 85% identical to SEQ ID NO. 6 and said LCVR comprises a polypeptide at least 85% identical to SEQ ID NO. 8. For example, the HCVR may comprise a polypeptide at least 90% identical to SEQ ID No. 6 and the LCVR may comprise a polypeptide at least 90% identical to SEQ ID No. 8. For example, the HCVR may comprise a polypeptide at least 95% identical to SEQ ID No. 6 and the LCVR may comprise a polypeptide at least 95% identical to SEQ ID No. 8. For example, the HCVR may comprise a polypeptide at least 97% identical to SEQ ID No. 6 and the LCVR may comprise a polypeptide at least 97% identical to SEQ ID No. 8. For example, the HCVR may comprise a polypeptide at least 99% identical to SEQ ID No. 6 and the LCVR may comprise a polypeptide at least 99% identical to SEQ ID No. 8. In some embodiments, the isolated monoclonal antibody (which can be used as an antibody for any method for purifying a native royal jelly major protein as described herein) comprises the HCVR of SEQ ID NO:6 and the LCVR of SEQ ID NO: 8.

some embodiments include nucleic acids encoding an isolated monoclonal antibody that specifically binds to a major protein of royal jelly. In some embodiments, the nucleic acid can encode an antibody comprising a Heavy Chain Variable Region (HCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6, said Light Chain Variable Region (LCVR) comprising: CDR3 domain of CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, the nucleic acid comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO. 5 and the nucleic acid sequence of SEQ ID NO. 7. The nucleic acid of SEQ ID NO. 5 is capable of encoding HCVR for an antibody that specifically binds to the major protein of royal jelly, and the nucleic acid of SEQ ID NO. 7 is capable of encoding LCVR for an antibody that specifically binds to the major protein of royal jelly. In some embodiments, the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7 are part of the same polynucleotide (e.g., located on the same vector or on the same chromosome). In some embodiments, the nucleic acid of SEQ ID NO. 5 and the nucleic acid of SEQ ID NO. 7 are part of different polynucleotides (e.g., on different vectors or different chromosomes).

In some embodiments, polyclonal antibodies to the native royal jelly major protein are provided. The polyclonal antibodies do not necessarily bind to the same epitope on the main protein of natural royal jelly, and therefore, do not necessarily compete with each other for binding. Polyclonal antibodies (which may also be referred to as "antisera") may be obtained by immunizing a host with an antigen comprising a major protein of royal jelly as described herein. Immune sera from the host can be obtained and polyclonal antibodies having affinity for the main protein of royal jelly can be obtained by affinity purification (e.g., affinity chromatography or immunoprecipitation). For example, a main protein of royal jelly (a main protein of royal jelly in its native conformation) may be immobilized, for example, on a solid phase, and contacted with a polyclonal serum, so that a polyclonal antibody specific for the main protein of royal jelly is bound to the main protein of native royal jelly immobilized on the solid phase. The solid phase can then be washed to remove non-specific antibodies to the major proteins of royal jelly as well as any undesired materials. The antibodies specific for the main protein of royal jelly can then be removed from the solid phase, for example by elution.

Method for purifying main protein of royal jelly

Some embodiments include methods of purifying a major protein of royal jelly. It should be noted that "purifying" a desired substance (such as native royal jelly major protein) as used herein refers to separating the substance from other substances in the heterologous composition, for example, by removing and retaining the desired substance from the heterologous composition, and/or by substantially removing other substances from the heterologous composition. Thus, a "purified" royal jelly major protein (including variations of this term) need not consist entirely of a royal jelly major protein, but also includes compositions in which the royal jelly major protein is substantially enriched compared to the starting heterologous composition. Thus, a method of purifying a royal jelly major protein as discussed according to some embodiments herein will be understood to result in a composition enriched in a royal jelly major protein, e.g., comprising, consisting essentially of, or consisting of a royal jelly major protein (e.g., a natural royal jelly major protein). Products of such compositions in which the major royal jelly proteins are substantially enriched are also understood to comprise purified major royal jelly proteins. For similar reasons, a crude biological extract (comprising the major royal jelly protein and other substances) should be understood as not comprising "purified" major royal jelly protein as used herein. The method may comprise dissolving a biological substance comprising a major protein of natural royal jelly in an aqueous solution. The method may comprise contacting an aqueous solution comprising the dissolved biological substance with an antibody immobilized on a substrate, wherein the monoclonal antibody specifically binds to the major protein of royal jelly. The antibody can be combined with natural royal jelly major protein. The method can include isolating the antibody-bound native royal jelly major protein from the aqueous solution (e.g., by removing the aqueous solution, by washing the antibody and matrix, and/or by removing the matrix from the aqueous solution). The bound native royal jelly major proteins can then be removed from the matrix, for example by elution. The thus obtained natural royal jelly major protein represents a composition comprising the purified natural royal jelly major protein. In some embodiments, a composition comprising a purified royal jelly major protein comprises at least 1% (w/w) of a natural royal jelly major protein, e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a natural royal jelly major protein, including ranges between any two of the listed values, e.g., 1% -90%, 1% -50%, 1% -30%, 1% -20%, 1% -10%, 5% -90%, 5% -50%, 5% -30%, 5% -20%, 5% -10%, 10% -90%, 10% -50%, 10% -30%, 10% -20%, 20% -90%, 20% -50% or 20% -30%. In some embodiments, the purified royal jelly major protein undergoes further purification, such as filtration, centrifugation, size exclusion chromatography, or one or more additional rounds of affinity purification using antibodies specific for the royal jelly major protein. In some embodiments, the purified native royal jelly major protein is used to produce a cosmetic product comprising the native royal jelly major protein as described herein. In some embodiments, the purified native royal jelly major protein is contained in a kit for use in producing a cosmetic product as described herein. Furthermore, it is to be understood that wherever a method of purifying a royal jelly major protein is disclosed herein and includes an antibody, an antibody for purifying a royal jelly major protein is also specifically contemplated.

in some embodiments, antibodies against a major protein of royal jelly can be used to enrich or purify a native royal jelly major protein by: immobilizing an antibody on a substrate, binding the native royal jelly major protein in an aqueous solution to the antibody, separating the bound royal jelly major protein from the remaining aqueous solution (e.g., by washing and/or physically separating the substrate from the aqueous solution), and eluting (or otherwise removing) the bound native royal jelly major protein. In some embodiments, the antibody comprises, consists essentially of, or consists of the following antibodies: a CDR3 domain comprising the CDR3 domain of SEQ NO 6; a CDR2 domain of the CDR2 domain of SEQ NO. 6 and a CDR1 domain of the CDR1 domain of SEQ NO. 6; and a Light Chain Variable Region (LCVR) comprising the CDR3 domain of the CDR3 domain of SEQ NO 8; the CDR2 domain of the CDR2 domain of SEQ NO. 8 and the CDR1 domain of the CDR1 domain of SEQ NO. 8. In some embodiments, the antibody comprises, consists essentially of, or consists of the following antibodies: an antibody comprising the HCVR of SEQ ID NO. 6 and the LCVR of SEQ ID NO. 8. In some embodiments, the antibody comprises one or more of antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a 2. In some embodiments, the antibody competes with one or more of antibody 4G6C5, antibody 8C5C9, antibody 8C5D3, antibody 4G6E2, or antibody 9G6a2 for binding to the major protein of royal jelly. According to some embodiments herein, a variety of suitable techniques may be used to immobilize the antibody on the substrate. For example, the antibody may be immobilized on the matrix by covalent coupling to activated beads (examples include Affi-gel 10, Biorad Laboratories, Richmond, Calif.) or by contacting the antibody with beads that have been coupled to a Protein with high affinity for IgG, such as Protein A (examples include Pierce Protein A Plus Agarose, ThermoFisher scientific, Waltham, Mass.). In some embodiments, antibodies specific for the main protein of royal jelly are used to purify the main protein of natural royal jelly via immunoprecipitation. In some embodiments, antibodies specific for the major protein of royal jelly are used to purify the native royal jelly major protein via affinity chromatography. In some embodiments, after the natural royal jelly major protein binds to the antibody on the matrix, one or more washes are performed to separate the matrix and antibody from the initial solution already containing the royal jelly major protein before eluting or otherwise removing the bound natural royal jelly major protein.

In some embodiments, antibodies specific for the major protein of royal jelly are immobilized directly on the matrix, for example, by covalent attachment (e.g., cross-linking). In some embodiments, the antibody specific for the major protein of royal jelly is indirectly immobilized on the matrix, for example, by contacting the antibody specific for the major protein of royal jelly with a second antibody immobilized on the matrix, wherein the second antibody is specific for the host of the antibody specific for the major protein of royal jelly.

In some embodiments, the main protein of royal jelly comprising an affinity tag (e.g., his or FLAG) or Hemagglutinin (HA) tag is affinity purified. Such affinity-tagged royal jelly major protein proteins can be produced in cell culture (e.g., insect or bacterial cells comprising nucleic acids encoding affinity-tagged royal jelly major proteins). Can make the main protein of royal jelly contact with solid phase with affinity to affinity label. For example, heavy metal ions in the solid phase (e.g., Ni or Co) may have an affinity for his tags. For example, an antibody immobilized on a solid phase may have affinity for FLAG or HA tag. For example, the major protein of royal jelly comprising his and FLAG tags is shown in SEQ ID NO. 4. The affinity-labeled royal jelly major protein was affinity-purified as shown in example 1, and was shown to have biological activity.

In some embodiments, the matrix comprises paramagnetic Beads to aid in the enrichment or purification process (examples include Dynabeads Protein a Magnetic Beads, thermo fisher scientific, waltham, MA). The process is similar to the batch purification method shown above, except that the beads are collected using a magnet rather than centrifugation. In some embodiments, the matrix comprises agarose, such as agarose beads or agarose resin.

In some embodiments, a crude solution of a biological substance (e.g., royal jelly, cell culture, or cell culture extract) comprising a major protein of royal jelly is contacted with an antibody specific for the major protein of royal jelly. In some embodiments, the solution is an aqueous solution. In some embodiments, the biological substance comprising a major protein of royal jelly comprises, consists of, or consists essentially of royal jelly. In some embodiments, the biological substance comprising a major protein of royal jelly comprises, consists of, or consists essentially of a cell culture or extract thereof (e.g., a culture of transgenic cells expressing a major protein of royal jelly). In some embodiments, the cell comprises a bacterial cell or an insect cell. In some embodiments, the solution of biological material comprising a native royal jelly major protein is subjected to one or more initial purification steps prior to contact with the antibody specific for the royal jelly major protein. For example, in some embodiments, the solution of biological matter comprising the major royal jelly protein may undergo an initial round of filtration, centrifugation, size exclusion chromatography, etc., to enrich it with the major royal jelly protein and/or to remove undesired matter. The solution subjected to the initial purification may then be further subjected to a method of purifying a royal jelly major protein using an antibody against the royal jelly major protein as described herein.

In some embodiments, an antibody specific for a major protein of royal jelly immobilized on a substrate (e.g., beads) is placed in a column, and a solution comprising a biological substance containing a major protein of natural royal jelly is passed through the column. After several washes, the native royal jelly major proteins were eluted using a specific buffer that separated the antigen and antibody and collected in the overflow (flowthrough) from the column. Thus, a purified main protein of natural royal jelly can be obtained, resulting in a composition comprising the purified main protein of natural royal jelly. In some embodiments, purification may be performed in a batch process. Instead of using a column, beads were added to the crude solution. The beads may be agglomerated using centrifugation and washed using the same method. The final elution can also be the same as with the column, except that the final elution sucks the beads after they have been centrifuged down. Thus, a purified main protein of natural royal jelly can be obtained, resulting in a composition comprising the purified main protein of natural royal jelly.

composition comprising purified main protein of natural royal jelly

In some embodiments, the methods of purifying a royal jelly major protein as described herein result in a composition that is enriched in, consists of, or consists essentially of a purified natural royal jelly major protein. In some embodiments, the composition comprises a native royal jelly major protein in a solvent (such as an aqueous solution). In some embodiments, the composition comprises a purified native royal jelly major protein in a dried form (e.g., lyophilized or spray dried). It is contemplated that compositions comprising purified native royal jelly major protein may be used in "cosmetic products" (which may also be referred to herein as "cosmetic compositions," including variations of any of the root terms). In some embodiments, the cosmetic product comprises a topical cosmetic product, such as a topical lotion, cream, paste, gel, spray, powder, eyebrow pencil, or the like. Thus, in some embodiments, the cosmetic product comprises a purified native royal jelly major protein as described herein. In some embodiments, the composition comprising the purified native royal jelly major protein is used to prepare a cosmetic product comprising the native royal jelly major protein. In some embodiments, a composition comprising a purified native royal jelly major protein is produced using a purification method as described herein and then used to prepare a cosmetic product comprising a native royal jelly major protein without an intermediate step. In some embodiments, the composition comprising the purified native royal jelly major protein is dried, for example by lyophilization or spray drying, and can subsequently be used to prepare a cosmetic product comprising the native royal jelly major protein. In some embodiments, a composition comprising a purified native royal jelly major protein is included in a kit for preparing a cosmetic product comprising a native royal jelly major protein. In some embodiments, the kit further comprises instructions instructing the user how the composition comprising the purified native royal jelly major protein can be incorporated into a cosmetic product. In some embodiments, the kit further comprises instructions directing the user how to reconstitute the composition comprising the purified native royal jelly major protein. In some embodiments, a composition comprising a purified natural royal jelly major protein comprises at least 1% (w/w) of a natural royal jelly major protein, e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a natural royal jelly major protein, including ranges between any two of the listed values, e.g., 1% -90%, 1% -50%, 1% -30%, 1% -20%, 1% -10%, 5% -90%, 5% -50%, 5% -30%, 5% -20%, 5% -10%, 10% -90%, 10% -50%, 10% -30%, 10% -20%, 20% -90%, 20% -50% or 20% -30%.

In some embodiments, a cosmetic product comprising a native royal jelly major protein comprises at least 1% (w/w) native royal jelly major protein, e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% of a native royal jelly major protein, including ranges between any two of the listed values, e.g., 1% -80%, 1% -50%, 1% -30%, 1% -20%, 1% -10%, 5% -80%, 5% -50%, 5% -30%, 5% -20%, 5% -10%, 10% -80%, 10% -50% > 10-30%, 10-20%, 20-80%, 20-50% or 20-30% of natural royal jelly main protein. Such cosmetic products comprising purified royal jelly major protein as described herein may be topical cosmetic products as described herein. As described herein, it is contemplated that such cosmetic products comprising purified native royal jelly major protein according to some embodiments herein may have biological activity, such as stimulating colony formation and/or stimulating wound healing.

Examples