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Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof

A technology of epitope peptide and foot-and-mouth disease, applied in the field of molecular immunology, to achieve a wide range of applications

Active Publication Date: 2014-04-16
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, all reported FMD virus CTL epitopes have been verified by animals such as mice and cattle, and no FMD virus CTL epitopes directly presented by porcine SLA-I molecules have been reported so far.

Method used

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  • Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof
  • Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof
  • Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of SAL-Ⅰ Protein from Six Strains of Pigs

[0030] (1) Preparation of six strains of porcine SLA-I / pMAL-p2X recombinant Escherichia coli

[0031] Six strains of pig SLA-I / pMAL-p2X recombinant Escherichia coli were constructed and preserved in the Laboratory of Molecular Immunology, School of Life Science and Technology, Dalian University. The six strains of pigs include Landrace, Yorkshire, Topek, Hebao, Yantai and Laiwu black pigs. The SLA-I / pMAL-p2X recombinant Escherichia coli was obtained by the method described in Gao Fengshan et al. (Gene, 2012, 502 (2): 147-153).

[0032] (2) Induced expression of SLA-I recombinant Escherichia coli

[0033] Inoculate 5 mL of the SLA-I / pMAL-p2X recombinant Escherichia coli liquid of six strains of pigs into 500 mL of LB medium, shake and culture in a 37-degree incubator at 170 rpm, and detect the OD at intervals 600 , to be grown to OD 600 When it is between 0.6 and 0.8, add IPTG to 0.5mmol / L, and indu...

Embodiment 2

[0041] Embodiment 2 Design of foot-and-mouth disease virus mimic epitope peptide

[0042] Using the biological information software netMHCpan2.4 (http: / / www.cbs.dtu.dk / services / NetMHCpan), input the O-type foot-and-mouth disease virus VP1 amino acid sequence (AJ539138), the A-type foot-and-mouth disease virus VP1 amino acid sequence (GQ406249) and Asia1 Type FMDV VP1 amino acid sequence (EF149009), select nonapeptide, and predict the CTL mimetic epitope peptide that can bind to SLA-?? protein under the SLA gene and present to the cell surface to cause cytotoxic immune response. Three 9-peptides derived from the VP1 protein of foot-and-mouth disease virus were named Q02, AS3 and QA4 respectively, and their amino acid sequences and other basic information are shown in Table 1. Among them, Q02 is derived from Tibet / CHA / 99, an epidemic strain of type O foot-and-mouth disease at home and abroad; AS3 is derived from the epidemic strain Asia 1 / Jiangsu / China / 2005 of type A foot-and-mo...

Embodiment 3

[0044] Example 3 The combination and screening of SLA-I protein and foot-and-mouth disease virus mimic epitope peptide

[0045] Mix 1 mL of each of the six strains of pig SLA-I proteins purified in Example 1 (protein content 1 mg / mL) with 5 mL of PBS solution, centrifuge at 4500 r / min for 20 min, remove the filtrate, add PBS to a total volume of about 5 mL, and repeat centrifugation Three times, take the upper PBS solution (containing SLA-I) and mix it with the peptide solution (Q02, AS3, QA4 or Co) respectively, the molar ratio of peptide to target protein is 10:1, overnight at 37°C. The mixture was added to a 30K ultrafiltration tube and centrifuged at 4500r / min for 45min. Add PBS, 37°C water bath for 2min, centrifuge at 4500r / min, until the final volume is about 100uL. Add 5mL of citric acid-phosphate buffer solution to the mixture and let it stand at room temperature for 2min. Transfer to a 5K ultrafiltration tube, centrifuge at 4500r / min for 5min, and collect about 5mL ...

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Abstract

The invention discloses type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and a screening method thereof. The CTL epitope peptide consists of nine amino acid residues, and has an amino acid sequence of Ala-Met-Leu-Arg-Ala-Ala-Thr-Tyr-Tyr. The epitope peptide has stronger combining capability with SLA (swine leukocyte antigen)-?? proteins from pigs with different strains and causees cell toxicity immune response, and is applicable to the preparation of prevention and treatment vaccines for foot-and-mouth disease virus of pigs with various strains and wide in application range. According to the type A foot-and-mouth disease CTL epitope peptide and the screening method thereof, constructed SLA-I single-stranded molecules of pigs with six strains are utilized for in vitro combination with CTL simulated epitope peptide of foot-and-mouth disease virus, polypeptide which can be combined with complex is determined and screened by using a mass spectrum, and simulated epitope peptide which can induce the production of T cell immune response capability is determined through ELISPOT (enzyme linked immunospot assay) detection. The invention provides a method for screening and identifying a large number of foot-and-mouth disease virus CTL epitopes in the future, and lays a foundation for the development of multi-epitope vaccines for the foot-and-mouth disease of pigs.

Description

technical field [0001] The invention belongs to the field of molecular immunology, and in particular relates to a type A foot-and-mouth disease CTL epitope peptide capable of inducing cytotoxic immune response combined with SLA-I and a method for screening and identifying the CTL epitope peptide using SLA-I complex. Background technique [0002] Porcine major histocompatibility complex (MHC), also known as swine leukocyte antigen (SLA), is an important immune response molecule in pigs. SLA is divided into three categories, namely SLA-I, SLA-II, and SLA-III. Among them, the SLA-I class molecules include heavy chain and light chain, the heavy chain has polymorphism, and the functional genes are mainly SLA-1, -2, -3. The light chain is encoded by the Beta 2 microglobulin (β2m) gene. In the endoplasmic reticulum, the SLA-I heavy chain, antigen polypeptide and light chain are non-covalently bonded to form a SLA-I-peptides complex, which is processed by the Golgi apparatus and ...

Claims

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Application Information

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IPC IPC(8): C07K14/09A61K39/135A61P31/14
CPCA61K39/00C07K14/005C12N2770/32122C12N2770/32134
Inventor 高凤山
Owner DALIAN UNIV
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