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Human metapneumovirus multi-epitope antigen and application thereof

A human metapneumovirus, multi-epitope technology, applied in antiviral agents, antibody medical ingredients, medical preparations containing active ingredients, etc., can solve the problems of long time, heavy workload, and difficulty in preparing specific antibodies.

Active Publication Date: 2016-01-06
TIANJIN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first three have the disadvantages of cumbersome operation, heavy workload, long time-consuming, high cost, and difficulty in the preparation of specific antibodies, and their use is subject to certain restrictions.

Method used

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  • Human metapneumovirus multi-epitope antigen and application thereof
  • Human metapneumovirus multi-epitope antigen and application thereof
  • Human metapneumovirus multi-epitope antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: B cell epitope prediction:

[0067] Using the hMPVA-type reference strain NL / 1 / 00F protein (539aa, GenBank accession number AF371337) as a template, the following software was used for prediction:

[0068] 1) BepiPred (http: / / www.cbs.dtu.dk / services / BepiPred / ), the threshold is set to 0.35, and sites >0.35 may be B cell epitopes;

[0069] 2) ABCpred (http: / / www.imtech.res.in / raghava / abcpred / ), the threshold is set to 0.51;

[0070] 3) Bcepred (http: / / www.imtech.res.in / raghava / bcepred / ), when the threshold is set to 2.38;

[0071] 4) LEPS (http: / / leps.cs.ntou.edu.tw / , comprehensively consider secondary structure, hydrophilicity, surface accessibility, plasticity, polarity and antigenicity and other parameters to predict linear epitopes;

[0072] 5) LBTOPE (http: / / crdd.osdd.net / raghava / lbtope / index.php), the possibility > 60% is the possible epitope, select the fragment with high antigenicity judged by the software, and predict the fragment with other methods...

Embodiment 2

[0077] Example 2. T cell epitope prediction

[0078] The amino acid sequences of 7 proteins of N, M, F, G, SH, M2-1 and M2-2 of the type A reference strain NL / 1 / 00 registered on GenBank (GenBank accession number: AF371337) were used as templates , use the following software to predict, and the binding force value >0.426 is the possible CTL and Th epitope:

[0079] For mouse MHC-I restricted CTL epitopes:

[0080] 1) NetMHCpan2.8server ( http: / / www.cbs.dtu.dk / services / NetMHCpan / );

[0081] 2) NetMHC3.4server ( http: / / www.cbs.dtu.dk / services / NetMHC / );

[0082] For mouse MHC-II restricted Th epitopes:

[0083] 3) NetMHCⅡ2.2server ( http: / / genome.cbs.dtu.dk / services / NetMHCII / ):

[0084] 4) ClustalX compared the screened CTL and Th candidate epitopes with the different types of hMPV registered on GenBank for amino acid conservation, and selected the conserved epitopes between A and B types as CTL and Th candidate epitopes, Table 2 , 3.

[0085] Table 2 HMPV mouse CTL...

Embodiment 3

[0092] The hMPV-mea nucleotide sequence was synthesized by Invitrogen Germany. The specific method is to synthesize Oligo by chemical method and splicing it by PCR. Enzyme cutting sites are added to both ends of the full-length hMPV-mea fragment, and the fragment is connected to the T vector. Transform E. coli host cells for massive expansion.

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Abstract

The invention relates to a human metapneumovirus multi-epitope antigen and an application thereof. The human metapneumovirus multi-epitope antigen is formed by combining B cell epitope sequences, Th cell epitope sequences and CTL cell epitope sequences. The epitope sequences in various types are connected in series through connexon. The invention further provides preparing, series connection and expression of human metapneumovirus B cell epitope gene segments, CTL cell epitope gene segments and Th cell epitope gene segments, the human metapneumovirus multi-epitope antigen is prepared, and the cellular-immunity and humoral-immunity activating response level of the human metapneumovirus multi-epitope antigen is evaluated. An evaluation experiment indicates that the human metapneumovirus multi-epitope antigen can activate CTL immunity and Th immunity in cell immunity and can also activate humoral immunity, the certain neutralization effect on hMPV is achieved during an in-vitro experiment, and the human metapneumovirus multi-epitope antigen can be used for developing epitope vaccine, establishing an immunology diagnosis method, researching disease epidemiology, carrying out hMPV infection immunity treatment and the like.

Description

technical field [0001] The invention relates to human metapneumovirus B cell and T cell multi-epitope antigens, belonging to the field of genetic engineering, based on the preparation, series connection and expression of human metapneumovirus B cell epitopes, CTL cell epitopes and Th cell epitope gene fragments, Prepare the multi-epitope antigen of human metapneumovirus, and evaluate its activation of cellular immunity and humoral immune response. Background technique [0002] Human metapneumovirus (Humanmetapneumovirus, hMPV) is a new virus isolated from infant specimens with respiratory tract infection by Dutch scholars in 2001. hMPV is an enveloped single-negative-strand RNA virus, and its genome contains 8 genes encoding 9 different proteins, namely nucleocapsid protein (N), phosphorylated protein (P), matrix protein (M), fusion protein (F ), transcription elongation factor (M2-1), RNA synthesis regulator (M2-2), small hydrophobin (SH), major adhesion protein (G), and R...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/155A61P31/14G01N33/68G01N33/569
Inventor 李晓燕孔梅郭丽茹苏旭杨东靖邹明
Owner TIANJIN CENT FOR DISEASE CONTROL & PREVENTION
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