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B cell epitope polypeptide of serine protease inhibitor (Ts-WM5) in larvae phase of trichina muscle, hybridoma cell strain, monoclonal antibody and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunology, can solve problems such as hindering practical application, cumbersome preparation, and uneven batch quality

Pending Publication Date: 2020-06-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the complex composition of ES antigens, cumbersome preparation, long production cycle, uneven batch quality and cross-reactivity hinder its practical application.

Method used

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  • B cell epitope polypeptide of serine protease inhibitor (Ts-WM5) in larvae phase of trichina muscle, hybridoma cell strain, monoclonal antibody and application thereof
  • B cell epitope polypeptide of serine protease inhibitor (Ts-WM5) in larvae phase of trichina muscle, hybridoma cell strain, monoclonal antibody and application thereof
  • B cell epitope polypeptide of serine protease inhibitor (Ts-WM5) in larvae phase of trichina muscle, hybridoma cell strain, monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Prokaryotic expression and purification of Ts-WM5 protein

[0064] 1. Primer Design

[0065] According to the registered WM5 gene sequence in Genbank (accession number: DQ864973.2), design PCR amplification primers, the sequence is as follows:

[0066] WM5-HindIII-atg:5'-ATAC AAGCTT ATGGAAACAGAAATTGCAAA-3' (as shown in SEQ ID No.3)

[0067] WM5-XhoI-tta:5'-ATTAA CTCGAG AATTACCAGAAAAACGTCCAA-3' (as shown in SEQ ID No.4)

[0068] The underlined part is the introduced HindIII and XhoI restriction sites, and the expected length of the amplified product is 1238bp.

[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 ...

Embodiment 2

[0089] Example 2 Preparation of anti-Ts-WM5 protein monoclonal antibody

[0090] 1. Mice Immunization

[0091] Five 6-week-old female BALB / c mice were immunized with recombinant Ts-WM5 protein purified from gel cutting, and immunized five times with a two-week interval between each immunization. The immunization dose was 50 μg / mouse, and the immunization route was intraperitoneal immunization.

[0092] One week after the 4th immunization and the 5th immunization, blood was collected from the tail of the mice, and the serum was separated (4°C, centrifuged at 3000rpm for 30min), and the antibody level was detected by Ts-WM5 indirect ELISA method. The Ts-WM5 indirect ELISA method is operated as follows: the Ts-WM5 protein purified on the column is diluted with a coating solution (0.01M NaOH PH12), and 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...

Embodiment 3

[0100] Identification of embodiment 3 monoclonal antibody

[0101] 1. Subclass identification of monoclonal antibodies

[0102] Subclass identification of the monoclonal antibody obtained in Example 2 was carried out according to the operating instructions of the antibody subclass identification kit. The results show that the heavy chain of the monoclonal antibody of the present invention is IgG1 and the light chain is κ chain, see Table 1 for details.

[0103] Table 1 Identification of monoclonal antibody subclasses

[0104]

[0105]

[0106] 2. Ts-WM5 competition ELISA test

[0107] Coating and blocking are the same as Ts-WM5 indirect ELISA, starting from 1:1, diluting the positive and negative sera of Trichinella spiralis infection by 2 times from 1:1, taking 50 μl of the diluted serum to be tested and mixing it with 50 μl of the monoclonal antibody supernatant respectively, and taking After mixing, 100 μl of the solution was added to a microwell plate and reacted ...

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Abstract

The invention relates to the field of immunology, and particularly relates to a B cell epitope polypeptide of a serine protease inhibitor (Ts-WM5) with high expression in the larvae phase of trichinamuscle, a hybridoma cell strain, a monoclonal antibody and application thereof. The hybridoma cell strain capable of secreting an anti-Ts-WM5 protein specific antibody is obtained, and the Ts-WM5 protein specific B cell epitope polypeptide recognized by the monoclonal antibody secreted by the hybridoma cell strain is identified, so that the B cell epitope polypeptide has important significance indiagnosis of trichinosis and has important significance in establishing sandwich ELSIA for competitive ELISA and detection of circulating antigens for detection of multiple hosts of trichina and 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 of a highly expressed serine protease inhibitor in the muscle larval stage of Trichinella spiralis. Background technique [0002] Trichinellosis is a very serious food-borne zoonotic parasitic disease. Humans are mainly caused by eating raw or undercooked meat containing Trichinella spiralis. Pork is the main source of human infection. The parasite not only causes huge economic losses to animal husbandry production, but also poses a huge threat to human health. 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 as a mandatory inspection of slaughtered animals and a must-check disease for the first inspection. [0003] At present, the prevention and control strategy of trichinellos...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N5/20C12N15/70C12N1/21C07K16/18G01N33/68G01N33/577A61K39/00A61P33/10C12R1/91
CPCC07K14/43536C07K16/18C12N15/70G01N33/68G01N33/577A61K39/0003A61P33/10G01N2333/43526Y02A50/30
Inventor 刘明远刘晓雷刘琰杨勇
Owner JILIN UNIV
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