Method for identifying MHC I binding peptide motif
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An identification method and a technology for binding peptides, applied in the field of biomedicine, can solve the problems of long time and low accuracy of peptide binding motif determination, and achieve the effects of efficient screening, short time-consuming and reliable results.
Active Publication Date: 2019-04-23
CHINA AGRI UNIV +1
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[0004] Aiming at the problems of long time and low accuracy in the determination of polypeptide binding motifs, the present invention provides a method for identifying MHC I binding polypeptide motifs based on a random peptide library, the biggest advantage of which is short detection time and low cost
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Embodiment 1
[0045]Example 1 Porcine SLA I polypeptide motif identification and synthesis of binding polypeptide
[0046] 1.1 Synthesis of random 9-peptide
[0047] Using the Fmoc method to synthesize a 9-peptide library with random sequences, the specific steps are as follows:
[0048] (1) Polypeptide-solid phase carrier cross-linking: 19 kinds of α-amino protecting group amino acids (except cysteine) were prepared by Fmoc (9-fluorenylmethoxycarbonyl), in DMSO at 20-25°C Reaction with alkoxybenzyl alcohol resin;
[0049] (2) Combined deprotection: 19 kinds of cross-linked polypeptide-solid phase carriers were thoroughly mixed, and the amino groups were deprotected by TFA (trifluoroacetic acid);
[0050] (3) Neutralization and washing: neutralize the free amino terminal with triethylamine and fully wash;
[0051] (4) Grouping and condensation: Divide the washed mixture into 19 equal parts, activate the carboxyl group of a new round of amino acids through DCC, and add 19 kinds of amino a...
Embodiment 2
[0105] Example 2 Duck Anpl - MHC I peptide motif identification
[0106] According to the method of Example 1, random sampling was performed on ducks from a duck breeding farm, and lymphocytes were separated by lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd.), and the separation method was carried out with reference to the instructions. The obtained cDNA was reverse-transcribed, and the primer sequences are shown in Table 2:
[0107] Table 2 Anpl -MHC I α chain and β chain primers
[0108] .
[0109] According to the method of Example 1, using cDNA as a template to obtain two Anpl -MHC I, and eventually two Anpl - MHC I binding peptides, and perform LC-MS / MS mass spectrometry sequencing and analysis and data analysis. The result is as Figure 7-13 As shown, the analysis obtained two Anpl - MHC I preference for binding to different sites of the polypeptide. Depend on Figure 13 and Figure 14 It can be seen that Anpl -...
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Abstract
The invention provides a method for identifying an MHC I binding peptide motif. The method includes the following steps that (1) a random peptide with the random sequence and the length of 8-12 is synthesized; (2) amino acid distribution is obtained through DeNovo analysis of the random peptide; (3) an alpha chain and a beta<2m> chain of MHC I are expressed; (4) the random peptide, the alpha chainand the beta<2m> chain form a MHC I-peptide compound in a solution; (5) the MHC I-peptide compound is subjected to thermal denaturation, and an MHC I binding peptide is obtained after separation andpurification; and (6) amino acid distribution of the MHC I binding peptide is obtained through DeNovo analysis of the MHC I binding peptide, and by comparing with amino acid distribution in the step (2), the preference of amino acids at different sites of the MHC I binding peptide and main anchor positioning are obtained. According to the method for identifying the MHC I binding peptide motif, operation is simple, the result is reliable, and the consumed time is short, efficient screening of potential pathogen specific T cell epitopes is achieved, and the method can be widely used in preliminary screening of animal or human body peptide vaccines.
Description
technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for identifying MHCI binding polypeptide motifs by using a random peptide library. Background technique [0002] Major histocompatibility complex class I (Major histocompatibility complex class I, MHCI) molecule-mediated cytotoxic T lymphocyte (Cytotoxic T lymphocyte, CTL) response is an important means for the body to clear diseased cells in the body and resist virus infection. MHC I molecules are expressed on the surface of all nucleated cells and platelets, and bind and present intracellular antigen polypeptides (such as viral polypeptides degraded by proteasomes) for CD8 + The T cell receptor (TCR) on the surface of T cells specifically recognizes and activates CD8 under the cooperation of co-receptor molecules (CD8 molecules, etc.) + T cells specifically kill target cells. This process is called CTL response, and the polypeptides presented by MHC I molecule...
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