Immunogen preparation of variable peptidic epitopes and preparation method thereof
An immunogen and variable technology, applied in biochemical equipment and methods, medical preparations containing active ingredients, chemical instruments and methods, etc., can solve problems such as difficulties in defining synthetic routes and qualitative work, and the danger of mixed peptides
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[0058]The method of the present invention for preparing an immunogen mixture begins with the analysis of the sequence of a naturally occurring pathogen that has been reported in a scientific database or peer-read scientific journal. This work was used to reduce the number of different peptides produced during the synthesis. The amino acids to be added and at which step of the synthesis reaction are determined in advance according to the specific steps, ie after the first calibration of sequences containing known T-helper and B-cell neutralizing epitopes. The resulting mixture of peptides, termed a hypervariable epitope construct (HEC), embodies the variation in the epitope sequence formed during the synthesis of a single peptide by adding a statistically weighted mixture of amino acids to align with the original chain This is achieved by linking the previously assembled amino acids. Thus, unlike mimetic clusters, peptides in HECs are not completely randomly formed, which can ...
Embodiment 1
[0115] Example 1: HIV-1-based HECs
[0116] Epitope protein sequences are provided by the Human Retrovirus and AIDS database (Los Alamos, 1998). Based on the sequence data, five regions of the HIV-1 envelope glycoprotein (gp120) were identified as hypervariable regions. These five hypervariable regions include antibody neutralizing, CTL, and / or T helper epitopes (HIV Molecular Immunity Database, 1998).
[0117] The probable amino acids at each site on the neutralizing epitope were determined from the sequence composition of in vivo isolates of HIV-1 clade B strains, and the sequence composition of each epitope was calibrated and evaluated. Subsequently, the mixture of suitable amino acids determined from the sequence data obtained above is subjected to an amino acid coupling step for the synthesis of a neutralizing epitope. This step is repeated at each amino acid coupling step in the synthesis. Thus, in one synthesis, a mixture of peptides was prepared representing all mea...
Embodiment 2
[0124] Example 2: HECs based on hepatitis C virus epitopes
[0125] The present invention designs two kinds of HECs against two hypervariable regions of hepatitis C virus (HCV). Sequences of viral in vivo isolates were obtained from databases and peer-read scientific literature, calibrated against these epitopes. According to the principles established by the present invention, when determining the amino acids contained in the variable residue base, the amino acid frequency is rounded to the nearest 25%, and then the proportion of amino acids added in the coupling step is determined. Figure 9 shows the amino acids occurring at different positions in the peptide mixture. The mixture was synthesized using standard Fmoc chemistry.
[0126] In particular, HCV HEC-1 in Figure 9 constitutes a 24-mer with equal amounts of tyrosine (Y) and histidine (H) at residue 4 and an equal amount of leuco at position 17. amino acid (L) and phenylalanine (F), with equal amounts of alanine (A) ...
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