CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide

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: 2014-06-18
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 cattl

Method used

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  • CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide
  • CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide
  • CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of six strains of pig SAL-I protein

[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 induce for...

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) and Asia1 type foot-and-mouth disease virus VP1 amino acid sequence (EF149009), select Nonapeptide, a CTL mimic epitope peptide that can bind to SLA-I protein and be presented to the cell surface to cause cytotoxic immune response is predicted under the SLA gene. A total of three 9 peptides derived from the VP1 protein of the foot-and-mouth disease virus were designed. The peptides were named Q01, Q02, and AS3 respectively, and their amino acid sequences and other basic information are shown in Table 1. Among them, both Q01 and Q02 are derived from Tibet / CHA / 99, the current epidemic strain of O-type foot-and-mouth disease at home and abroad; AS3 is derived from Asia 1 / Jiangsu / China / 2005, an epidemic strain of As...

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 (Q01, Q02, AS3 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 a CTL (Cytotoxic T Lymphocyte) epitope peptide of a foot-and-mouth disease virus type O as well as a screening method and application of the CTL epitope peptide. The CTL epitope peptide is composed of nine amino acid residues, and the amino acid sequence of the CTL epitope peptide is as follows: Ala-Thr-Arg-Val-Thr-Glu-Leu-Leu-Tyr. The epitope peptide has relatively strong combining capacity with SLA (Swineleukocyteantigen)-I proteins from various strains of swine and can induce cytotoxic immune response so as to be suitable for preparing vaccines for preventing and controlling foot-and-mouth disease viruses of various strains of swine and wide in application range. According to the invention, a CTL simulated epitope peptide of a foot-and-mouth disease virus is combined with a single-chain molecule of SLA-I of six strains of constructed swine in vitro, thus a polypeptide which can be combined with a complex can be screened through mass spectrum measurement; in addition, a simulated epitope peptide which can be induced to generate the immune response capacity of T cells is determined through ELISPOT (Enzyme-Linked Immunospot Assay) detection. The invention provides a method for screening and authenticating the CTL epitope of the foot-and-mouth disease virus in a large scale, and lays the foundation for researching and preparing a multi-epitope vaccine of a foot-and-mouth disease.

Description

technical field [0001] The invention belongs to the field of molecular immunology, and in particular relates to an O-type 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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