A kind of O-type foot-and-mouth disease ctl epitope peptide and screening method

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

Active Publication Date: 2016-02-17
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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  • A kind of O-type foot-and-mouth disease ctl epitope peptide and screening method
  • A kind of O-type foot-and-mouth disease ctl epitope peptide and screening method
  • A kind of O-type foot-and-mouth disease ctl epitope peptide and screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 6

[0028] Example 1 Preparation of six strains of pig SAL-I protein

[0029] (1) Preparation of six strains of porcine SLA-I / pMAL-p2X recombinant Escherichia coli

[0030] 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).

[0031] (2) Induced expression of SLA-I recombinant Escherichia coli

[0032] Inoculate 5 mL of SLA-I / pMAL-p2X recombinant Escherichia coli liquid from 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 Between 0.6 and 0.8, add IPTG to 0.5mmol / L, and induce for 5 hours at 37 d...

Embodiment 2

[0040] The design of embodiment 2 foot-and-mouth disease virus mimic epitope peptides

[0041] 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 the epidemic strain of Asia1-type foot-and-mou...

Embodiment 3

[0043] The binding and screening of embodiment 3SLA-I protein and foot-and-mouth disease virus mimic epitope peptide

[0044] 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 for 3 The second time, 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, and 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 collec...

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Abstract

The invention discloses an O-type foot-and-mouth disease CTL epitope peptide, a screening method and application thereof. The CTL epitope peptide is composed of nine amino acid residues, and its amino acid sequence is: Ala-Thr-Arg-Val-Thr-Glu-? Leu-Leu-Tyr. The epitope peptide can have strong binding ability with SLA-I proteins derived from different strains of pigs and can cause cytotoxic immune response, and is suitable for the preparation of vaccines for the prevention and treatment of various strains of porcine foot-and-mouth disease viruses, and has a wide range of applications. The invention uses the constructed six-strain pig SLA-I single-chain molecule to bind the CTL mimetic epitope peptide of foot-and-mouth disease virus in vitro, mass spectrometry to screen the polypeptide that can bind to the complex, and through ELISPOT detection, it is determined that it can induce T cell immune response Capable mimotope peptides. The invention provides a method for screening and identifying a large number of CTL epitopes of foot-and-mouth disease virus in the future, and lays a foundation for developing a porcine foot-and-mouth disease multi-epitope vaccine.

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 (majorhistocompatibility complex, MHC), that is, porcine leukocyte antigen (swineleukocyteantigen, 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, 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 Beta2 microglobulin (Beta2microglobulin, β2m) gene. In the endoplasmic reticulum, the SLA-I heavy chain, antigenic polypeptide and light chain are non-covalently bonded to form a ...

Claims

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Application Information

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