B-cell epitope polypeptide, hybridoma cell line, monoclonal antibody, application of trichinella spiralis intestinal stage cysteine ​​protease inhibitor

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunology, can solve the problems of complex ES antigen components, cross-reaction in diagnostic blind areas, and complicated preparation.

Active Publication Date: 2021-08-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ES antigen has complex components, cumbersome preparation, long production cycle, uneven batch quality, and serious diagnostic blind spots (cannot be detected before 19 days after infection) and cross-reactivity, which hinder its practical application.

Method used

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  • B-cell epitope polypeptide, hybridoma cell line, monoclonal antibody, application of trichinella spiralis intestinal stage cysteine ​​protease inhibitor
  • B-cell epitope polypeptide, hybridoma cell line, monoclonal antibody, application of trichinella spiralis intestinal stage cysteine ​​protease inhibitor
  • B-cell epitope polypeptide, hybridoma cell line, monoclonal antibody, application of trichinella spiralis intestinal stage cysteine ​​protease inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Prokaryotic expression and purification of embodiment 1Ts-WN10 protein

[0064] 1. Primer Design

[0065] According to the registered TsWN10 gene sequence in Genbank (accession number: EU263325), design PCR amplification primers, the sequence is as follows:

[0066] TsWN10-EcoRI-atg:5'-TAAC GAATTC ATGCAGATACTTGGTGA-3' (as shown in SEQ ID No.3)

[0067] TsWN10-XhoI-tta:5'-GACG CTCGAG TTAACATTCAACA-3' (as shown in SEQ ID No.4)

[0068] The underlined part is the introduced EcoRI and XhoI restriction sites, and the expected length of the amplified product is 1187bp.

[0069] 2. Extraction and reverse transcription of Trichinella spiralis T1 (T.spiralis) RNA

[0070] Take one Trichinella spiralis T1 (T.spiralis) conservation mouse in this experiment, kill it by neck dislocation, peel off the skin, tail, viscera and claws, wash the carcass and mince it, put it in 300ml containing 1% HCl and 1% stomach The protease digestion solution was stirred and digested in a 37°C...

Embodiment 2

[0091] Example 2 Preparation of anti-Ts-WN10 protein monoclonal antibody

[0092] 1. Mice Immunization

[0093] Five 6-week-old female BALB / c mice were immunized with the purified recombinant Ts-WN10 protein for 5 times with a two-week interval between each immunization.

[0094] One week after the 4th immunization and 5th immunization, blood was collected from the tail of the mice, and the serum was separated (centrifuged at 3000rpm at 4°C for 30min), and the antibody level was detected by Ts-WN10 indirect ELISA method. The Ts-WN10 indirect ELISA method is operated as follows: Ts-WN10 protein refolded and purified on the column is diluted with coating solution (0.01M NaOH PH12), the coating amount is 0.125ug / well, 100ul per well. Coat at 37°C for 2h or overnight at 4°C. After that, the plate was washed 3 times with PBST (containing 0.05% Tween 20). Block with blocking solution (5% skim milk) at 37°C for 2h. After that, the plate was washed 3 times with PBST. Serum to be ...

Embodiment 3

[0102] Identification of embodiment 3 monoclonal antibody

[0103] 1. Subclass identification of monoclonal antibodies

[0104] Subclass identification of the monoclonal antibody obtained in Example 2 was carried out according to the instruction manual of the antibody subclass identification kit. The results show that the heavy chains of the monoclonal antibodies of the present invention are all IgG1 light chains and the κ chains, see Table 1 for details.

[0105] Table 1 Identification of monoclonal antibody subclasses

[0106] Monoclonal antibody name lgG1 lgG2a wxya lgG3 lm w wxya kappa chain lambda chain WN10-1H9 0.837 0.062 0.045 0.048 0.031 0.046 0.757 0.039

[0107] 2. Ts-WN10 competition ELISA test

[0108] Coating and sealing are the same as Ts-WN10 indirect ELISA, starting from 1:1, diluting the positive and negative sera of Trichinella spiralis infection by 2 times from 1:1, taking 50ul of the diluted serum to be tested ...

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Abstract

The invention relates to the field of immunology, in particular to a B cell epitope polypeptide of a trichinella spiralis intestinal stage cysteine ​​protease inhibitor (Ts-WN10), a hybridoma cell line, a monoclonal antibody and applications thereof. The present invention obtains a hybridoma cell line that can secrete anti-Ts-WN10 protein-specific antibody, and identifies the Ts-WN10 protein-specific B cell epitope polypeptide recognized by the monoclonal antibody secreted by it, which is effective against trichinellosis The diagnosis is of great significance, and it is of great significance for the establishment of competitive ELISA and sandwich ELSIA for the detection of circulating antigens for the detection of Trichinella multi-host and the development of subunit vaccines.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a B cell epitope polypeptide, a hybridoma cell line, a monoclonal antibody and an application thereof of a trichinella spiralis intestinal stage cysteine ​​protease inhibitor. Background technique [0002] Trichinellosis is mainly caused by the host eating raw or semi-raw meat containing trichinella, and pork is the main source of human infection. The average incubation period of human trichinellosis is 2 weeks. The main symptoms of patients in the acute stage are fever, severe muscle pain, severe diarrhea, facial edema, and eosinophilia, etc. The symptoms can last for several weeks and lead to body failure, especially severe infection Severe damage to the myocardium and brain may occur, and even death may occur. [0003] In view of the great threat and harm caused by trichinella spiralis to public health and safety, the World Organization for Animal Health (OIE) listed trichinellosis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C07K16/38C12N15/15C12N5/20G01N33/68G01N33/577G01N33/53A61K39/395A61P33/10C12R1/91
CPCC07K14/8139C07K16/38G01N33/6893G01N33/577G01N33/5308A61P33/10C07K2317/76C07K2317/33C07K2317/34G01N2333/8139A61P33/00C07K16/18C07K2317/92G01N2333/4353C07K16/20C07K2317/56C07K2317/565G01N33/56905
Inventor 刘明远刘晓雷刘琰杨勇唐斌
Owner JILIN UNIV
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