Protein binders for irhom2
A conjugate and protein technology, applied in the field of protein conjugates, can solve problems such as the success of inhibitors that have not yet been proven
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Embodiment 1
[0192] Example 1: Generation of Peptides for Immunization and Peptide Binding ELISA Analysis
[0193] Using fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis, on a parallel peptide synthesizer (peptides 1-5, 7-9 and 1b-3b; MultiPep RSi, Intavis AG, Germany), a microwave peptide synthesizer (peptide 6 ; Liberty Blue, CEM, USA) or a custom continuous flow peptide synthesizer (peptides 10, 11 and 4b) [Chan, W.C., White, P.D. Solid Phase Peptide Synthesis, APractical Approach (Oxford University Press Inc., New York, 2000]. Using Fmoc-protected L-amino acids with side-chain protecting groups, the sequence was assembled from C to N-terminus in a step-by-step manner. After the chain assembly was completed, 95% TFA, 4% triethylsilane and 1% Water cleaves the peptide from the resin. The crude product is dissolved in 15% acetonitrile in 0.1% TFA in water and analyzed by reverse phase HPLC using Orbit C18, 10 μm, Column (MZ Analysentechnik, Germany) was used for purif...
Embodiment 2
[0198] Example 2: Propagation of iRhom2 knockout mice for immunization
[0199] Due to the high sequence homology of human to mouse iRhom2 proteins (see NCBI Reference Sequence NP_078875.4. for human iRhom2, NCBI Reference Sequence NP_766160.2. for mouse iRhom2, the extracellular loop 1 of human to mouse iRhom2 The amino acid sequence identities of the , 2, 3 and C-terminal tails were calculated to be 89.96%, 100.00%, 100.00% and 96.97%, respectively), and bred iRhom2 knockout mice instead of wild-type mice for immunization.
[0200] Briefly, the KOMP mouse biology program at the University of California, Davis ordered the Rhbdf2tm1b (KOMP) Wtsi mouse line (Rhbdf2 is an alias for iRhom2) for resuscitation and resulted in the availability of three heterozygous male mice. These three animals on the C57BL / 6N background (C57BL / 6N-Rhbdf2tm1b(KOMP)Wtsi) were mated with wild-type female mice of the 129Sv / J genetic background to generate heterozygous offspring. These heterozygous mic...
Embodiment 3
[0201] Example 3: Immunity and Serum Titer Analysis of Mice
[0202] Three groups of 8- to 10-week-old male and female iRhom2 knockout mice (as described in Example 2) were immunized with peptide mixtures A, B and C, respectively. Mixture A consisted of equal amounts of the four keyhole limpet hemocyanin (KLH)-coupled peptides 1, 2, 3 and 4. Mixture B consisted of equal amounts of the three KLH-conjugated peptides 5, 6 and 7 and mixture C consisted of equal amounts of the four KLH-conjugated peptides 8, 9, 10 and 11. Dissolve 50 μg of the peptide mixture in 20 μl of GERBU Adjuvant MM TM (GERBU Biotechnik, Germany) and adjusted with 10 mM HEPES buffer (PH 7,6) for intraperitoneal (IP) administration in a final volume of 100 μl per injection per mouse. Ten mice per group were injected five times every 10 days. Ten days after the fifth injection, blood (serum) was collected and tested for antibody titers.
[0203] The immune response was assessed by serum antibody titer analy...
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