Antibody preparation method, and antibody and antibody library thus prepared
Provided is a method for preparing antibodies against a protein of interest, through which highly specific antibodies against all proteins can be effectively and rapidly prepared with low cost, and the epitope to which the antibody is directed can be determined, so that a library covering epitopes on the surface of the proteins of interest and a library of antibodies against all the epitopes can be established. The antibodies are proved to be useful in detection, protein function investigation and antibody pharmaceuticals.
1. A method for preparing an antibody against a protein of interest, comprising:
(a) predicting and/or selecting peptide fragment located on the surface of the protein of interest, wherein said peptide fragment is a linear surface signature peptide and/or a conformational surface signature domain of the protein of interest;
(b) synthesizing or expressing one or more of said peptide fragments;
(c) using the product of step (b) to immunize an animal, optionally in combination with an adjuvant;
(d) using lymphocyte from the immunized animal of step (c) to obtain antibodies;
(e) using the peptide fragment of step (a) or said protein of interest in native conformation thereof to screen the antibodies obtained in step (d), so as to obtain an antibody library against said protein of interest,
wherein said peptide fragment of step (a) is predicted or selected through the following process:
(i) determining a surface peptide by calculating a parameter according to the sequence of the protein of interest, said parameter is selected from: solvent accessibility, disorder index, protein-protein interaction domain prediction, or any combination thereof;
(ii) aligning the surface peptide determined in step (i) with the proteome of the species that the protein of interest is originated from, so as to select a specific peptide fragment of said protein of interest;
(iii) aligning the surface peptide determined in step (i) with homologous proteins from other species, so as to screen a conservative sequence of said protein of interest.
2. The method of claim 1 , wherein said protein of interest is a native protein, and/or an alternative splicing isoform thereof, and/or a mutant thereof.
3. The method of claim 1 , wherein said signature peptide is a peptide, which is 6-12 amino acids in length, which is high hydrophilic, which has high antigenicity, which is not signal peptide, which is not in the trans-membrane region, and which is located in disordered region.
4. The method of claim 1 , wherein said signature domain is a sequence specific protein fragment which is 100-500 amino acids in length, and which is expected to have 3 dimensional structure.
5. The method of claim 1 , which is used for producing an antibody library against all the proteins of a species.
6. The method of claim 1 , wherein the antibody library produced in step (e) comprises antibodies against all the epitopes of the protein of interest.
7. The method of claim 1 , wherein the antibody obtained in step (d) is obtained through at least one process selected from:
(1) fusing lymphocyte from the immunized animal of step (c) with amyeloma cell, so that a hybridoma is generated and then expressed to obtain the antibody;
(2) isolating antigen specific B cell from lymphocyte of the immunized animal of step (c), and then using PCR to clone and express the gene of the antibody so as to obtain the antibody;
(3) isolating mRNA from lymphocyte of the immunized animal of step (c), and then obtaining the antibody through phage display, or ribosome display, or yeast display, or bacteria display, or Baculovirus display, or mammal cell display, or mRNA display.
8. The method of claim 1 , wherein one or more of the peptide fragments of step (b) are recombinantly expressed in the form of a fused protein with a protein which enhances the immunogenicity and/or increases the copy number.
9. The method of claim 8 , wherein the protein which enhances the immunogenicity and/or increases the copy number is a virus-like particle protein carrier.
10. The method of claim 9 , wherein the protein which enhances the immunogenicity and/or increases the copy number is Hepatitis B virus nucleocapsid (HBC) protein.
11. The method of claim 10 , wherein said one or more peptide fragments are inserted into loop, N-terminus, or C-terminus of the HBC protein.
12. The method of claim 11 , wherein the position into which said one or more peptide fragments are inserted is located between the amino acid residue at position 77 and the amino acid residue at position 82 of the HBC protein.
13. The method of claim 10 , wherein 2-10 of the peptide fragments are linked by a linker and inserted into the HBC protein.
14. The method of claim 13 , wherein said linker is (GGGGS) n , (SEQ ID NO:158) n , and wherein n=1, 2, 3 or 4.
15. The method of claim 14 , wherein n=1 or 2.
16. The method of claim 1 , wherein the one or more peptide fragments expressed in step (b) are further coupled with an immune-enhancing protein carrier.
17. The method of claim 16 , wherein said immune-enhancing protein carrier is keyhole limpet hemocyanin (KLH).
18. The method of claim 1 , wherein the one or more peptide fragments of step (b) are chemically synthesized.
19. The method of claim 1 , wherein the adjuvant is selected from: Freund's complete adjuvant, aluminum, CpG, or any combination thereof.
20. The method of claim 1 , wherein in said step (c) the animal is immunized at multiple sites.
21. The method of claim 20 , wherein the immunization at multiple sites is performed at at least 2 sites selected from: neck and back, tail end, hind foot palm, hind leg inguen, front leg armpit, hind leg muscle.
22. The method of claim 20 , wherein multiple immunizations are performed, with the time interval of 2-14 days.
23. The method of claim 22 , wherein the immunization protocol used in step (C) comprises the following steps:
(A). the first immunization: the expression product of step (b) together with Freund's complete adjuvant are used to immunize the animal at neck and back, tail end, hind foot palm, hind leg inguen, and front leg armpit; and the expression product of step (b) together with the adjuvant of aluminum+CpG are used to immunize the animal at hind leg muscle;
(B). the second immunization: the expression product of step (b) together with Freund's complete adjuvant are used to immunize the animal at neck and back, hind leg inguen, and front leg armpit; and the expression product of step (b) together with the adjuvant of aluminum+CpG are used to immunize the animal at hind leg muscle;
(C). the third immunization: the expression product of step (b) together with the adjuvant of aluminum+CpG are used to immunize the animal at hind leg muscle, tail end, and front leg armpit;
(D). the fourth immunization: the expression product of step (b) together with the adjuvant of aluminum+CpG are used to immunize the animal at hind leg muscle, tail end, and front leg armpit.
24. The method of claim 23 , wherein the first immunization is performed on the first day, the second immunization is performed on the fifth day, the third immunization is performed on the eighth day, the fourth immunization is performed on the eleventh day.
25. The method of claim 1 , wherein the produced antibody is singly IgG subtype.
26. The method of claim 25 , wherein the produced antibody is monoclonal antibody.
27. The method of claim 25 , wherein the produced antibody is polyclonal antibody.
28. The method of claim 1 , wherein the antibodies produced in step (d) is screened through affinity sorting in said step (e).
29. The method of claim 1 , further comprising a step (f) of screening functional antibody and detection antibody.
30. The method of claim 29 , wherein said detection antibody is screened through Western blotting, IP, IF, IHC, flow cytometry, ELISA, or any combination thereof.
31. The method of claim 29 , wherein said functional antibody is screened through blocking or neutralizing assay.
32. The method of claim 1 , wherein said method can be used to produce detection antibody and/or functional antibody against more than 90% of proteins of interest.
33. The method of claim 22 , wherein multiple immunizations are performed with the time interval of 3-4 days.