MERS-CoV specific polypeptides and application thereof

A specific, MHC-I technology, applied in the field of immunoassay

Active Publication Date: 2017-02-15
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the evaluation of T cells in mouse models after MERS-CoV infection or vaccination using the tetramer technique.

Method used

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  • MERS-CoV specific polypeptides and application thereof
  • MERS-CoV specific polypeptides and application thereof
  • MERS-CoV specific polypeptides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Screening of MERS-CoV-specific polypeptides

[0027] Use the peptide prediction software to predict the S protein of MERS-CoV, and then synthesize the peptide, immunize mice with the strain H-2d (BALB / c) with adenovirus carrying the S protein of MERS-CoV virus, and harvest after the immunization For splenocytes, number the synthesized polypeptides and arrange them in an 8×8 matrix, with 8 groups horizontally and 8 groups vertically, the horizontal group and the vertical group have intersections, and the polypeptides with intersections are mixed together. The production of IFN-γ by the mixed polypeptide was detected by Elispot technology, and three functional peptides (as shown in Table 1) were screened from the mixed polypeptides producing IFN-γ in the horizontal and vertical groups, which had strong specificity. It can be used as an effective tool for the evaluation of T cell immunology to evaluate the T cell immune response in mouse models after MERS-CoV inf...

Embodiment 2

[0030] Example 2 Preparation of Polypeptide-MHC Tetramer

[0031] (1) Preparation of polypeptide / MHC complex:

[0032] 1) In H-2K d (Genbank accession number XP_017174460.1) The amino acid sequence GGGLNDIFEAQKIEWHE that can be linked to biotin was added to the C-terminus of the gene to construct the recombinant plasmid pET28a-H-2K d -Bio, connect the B2m gene (Genbank accession number is AAA39668.1) to the vector, construct the recombinant vector pET28a-B2m, and transform the plasmids into Escherichia coli BL21 respectively;

[0033] 2) Culture Escherichia coli BL21 carrying the plasmid at 37°C and add 1mmol / L IPTG to induce protein expression, collect the bacteria, sonicate the bacteria, and dissolve the precipitate in the dissolution buffer (6mol / L guanidine hydrochloride after high-speed centrifugation , 10% glycerin, 50mmol / L Tris pH8.0, 100mmol / LNaCl, 10mmol / L EDTA);

[0034] 3) The polypeptide (named as polypeptide 37-1) with the sequence shown in SEQ ID NO.1 and the...

Embodiment 3

[0049] Example 3 Application of Polypeptide / MHC Tetramer in T Cell Analysis

[0050] Using the characteristics of high affinity and high specificity of MERS-CoV-specific peptide / MHC tetramer to detect T cells in mice after immunization with MERS-CoV-related vaccines, evaluate the effect of vaccines on cellular immunity in mice, and isolate T cells With cloning, in situ staining of the corresponding tissue sections of MERS-CoV-infected mice, etc., the following steps were used to evaluate the level of T cells in BABL / c mice immunized with adenoviral vectors carrying the S protein of MERS-CoV:

[0051] 1) Prepare the polypeptide 37-1 obtained by screening with incomplete Freund's adjuvant and GP96 adjuvant according to the ratio of 20 μg polypeptide, 50 μL incomplete Freund's adjuvant, 50 μL GP96 to prepare an emulsifier, and immunize mice three times, each time Two weeks apart; unimmunized mice were used as negative controls;

[0052] 2) Obtain splenocytes from the immunized m...

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Abstract

The invention discloses MERS-CoV specific polypeptides and application thereof, belongs to the field of immunodetection and provides three MERS-CoV specific polypeptides aiming at BABL / c mice. An MHC-tetramer is prepared by using corresponding polypeptides, affinity between polypeptide-MHC and specific T cell surface TCR is improved effectively, and the MHC-tetramer can serve as an effective tool for T cell evaluation. The technology can be used for separating and cloning of specific T cells and performing in-situ dyeing on mouse corresponding tissue slices after MERS-CoV infection or vaccination and has high application value in the studying aspect of T cells of a mouse model after MERS-CoV infection or vaccination.

Description

technical field [0001] The invention relates to a MERS-CoV specific polypeptide and an application thereof, and belongs to the field of immune detection. Background technique [0002] In June 2012, the first case of MERS was found in Saudi Arabia. Subsequently, more cases were reported in the Arabian Peninsula and spread to Asia, Africa, Europe and North America. The mortality rate was as high as 35.7%. In July 2015, There is also an imported case of infection in our country. The development of safe and effective vaccines is one of the important means to control the spread of MERS-CoV. T cells play a very important role in virus clearance. In the evaluation of the immune effect of MERS-CoV vaccine, the evaluation of T cell immune response is particularly important. The establishment of a susceptible mouse model of MERS-CoV provides a good animal model for the development of its vaccine. It is particularly important to develop efficient, rapid, and sensitive detection meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165A61K39/215A61P31/14G01N33/569
CPCA61K39/12C07K14/005C07K14/70539C07K2319/00C12N2770/20022C12N2770/20034G01N33/56983
Inventor 刘军高福刘科芳谭文杰
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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