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Hybridoma cell strain, monoclonal antibody with resistance of serine protease of trichinella spiralis in intestinal stage generated from hybridoma cell strain and application thereof

A technology of hybridoma cell line and serine protease, which is applied in the direction of anti-enzyme immunoglobulin, anti-animal/human immunoglobulin, antibody, etc. It can solve the problems of cross-reaction in diagnostic blind spots, cumbersome preparation, uneven batch quality, etc.

Active Publication Date: 2020-05-22
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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  • Hybridoma cell strain, monoclonal antibody with resistance of serine protease of trichinella spiralis in intestinal stage generated from hybridoma cell strain and application thereof
  • Hybridoma cell strain, monoclonal antibody with resistance of serine protease of trichinella spiralis in intestinal stage generated from hybridoma cell strain and application thereof
  • Hybridoma cell strain, monoclonal antibody with resistance of serine protease of trichinella spiralis in intestinal stage generated from hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Prokaryotic expression and purification of Ts-ZH68 protein.

[0026] 1. Primer design: According to the registered ZH68 gene sequence (accession number: EM263332) in Genbank, design PCR amplification primers, the sequence is as follows:

[0027] The template used for Ts-ZH68 gene amplification is AD3 stage adult cDNA, and the amplification primers are as follows:

[0028] TsZH68-F:5'-TAAC GAATTC GAAAATTCTCCTGAAG-3';

[0029] TsZH68-R:5'-GACG CTCGAG TTACTTAGAAAAGTG-3'.

[0030] The underlined part is the introduced EcoRI, XhoI restriction site.

[0031] 2. Extraction and reverse transcription of adult RNA of Trichinella spiralis T1 (T.spiralis) AD3

[0032] Dissect the rats infected with Trichinella spiralis muscle larvae for 3 days, dissect the small intestine, wash it with sterilized normal saline, put it on the separation filter cloth, preheat the normal saline to 37°C, submerge the small intestine, and incubate at 37°C for 4 hours, containing 2% ...

Embodiment 2

[0048] Example 2. Preparation method of hybridoma cell line.

[0049] 1. Mice Immunization

[0050] Immunize five 6-week-old female BALB / c mice with the recombinant Ts-ZH68 protein purified by gel cutting in Example 1, and immunize 4 times in total, with an interval of two weeks between each immunization, and the immunization dose is 50 μg / mouse with an equal volume of adjuvant , the first immunization was with Freund's complete adjuvant, the other three were with Freund's incomplete adjuvant, and the immunization route was intraperitoneal immunization.

[0051] One week after the third immunization and the fourth 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 the Ts-ZH68-indirect ELISA method after affinity purification. The Ts-ZH68 indirect ELISA method is operated as follows: the recombinant Ts-ZH68 protein is coated with a coating solution (0.1M carbon...

Embodiment 3

[0057] Example 3. Preparation of anti-Ts-ZH68 protein monoclonal antibody ascites.

[0058] Give 12-week-old healthy BALB / c mice intraperitoneal injection of paraffin oil, 0.5ml / mouse, and 1 week later, intraperitoneal injection of 1×10 6 For the hybridoma cells prepared in Example 2, the ascites was extracted 7 to 10 days later when the abdominal cavity of the mouse was extremely distended, and the ascites was collected once every 2 days. Ascitic fluid was purified using protein G affinity chromatography medium, and before purification, it was centrifuged at 10,000 rpm for 10 min to remove red blood cells and fat, and the supernatant was absorbed for purification. First, connect the chromatography column to the syringe with an adapter, draw 5-10ml of binding buffer with the syringe, inject it into the chromatography column at a flow rate of 1mL / min, draw the prepared ascites and inject it into the chromatography column, use 10-15mL of Rinse the column with washing buffer, el...

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Abstract

The invention provides a hybridoma cell strain, a monoclonal antibody secreted from the hybridoma cell strain and application thereof and belongs to the technical field of prevention and control of trichinella spiralis (T.spiralis). Against the technical problem of specific diagnosis on the T.spiralis, the hybridoma cell strain has a preservation number of CGMCC No.18317. Tests prove that the monoclonal antibody Ts-ZH68-2A40Ab secreted from the hybridoma cell strain can compete against T.spiralis infected positive porcine serum to combine with Ts-ZH68 antigen, and has a recognition peptide being <222>GVDRSATCQGDSGGP236. The monoclonal antibody and the Ts-ZH68 protein B cell epitope peptide recognized by the monoclonal antibody can be used for preparing reagents or vaccines for diagnosing or preventing T.spiralis infection, and lays foundation to establishment of a T.spiralis serological diagnosing method.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of trichinella spiralis (T.spiralis), and specifically relates to a hybridoma cell line and the monoclonal antibody against trichinella spiralis intestinal stage serine protease produced by it and its application. Background technique [0002] Trichinellosis is mainly caused by the host eating raw or semi-raw meat containing trichinella. 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 human health, the World Organization for Animal Health (OIE) has listed trichinel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40C07K7/08C07K7/06G01N33/68G01N33/577A61K39/395A61P33/10C12R1/91
CPCC07K16/40C07K7/08C07K7/06G01N33/68G01N33/577A61P33/10G01N2410/00A61K2039/505A61K39/00C07K16/18C07K2317/34A61K39/0003A61K2039/55566G01N2333/4353G01N33/5308C07K14/4354C12N9/6408C07K2317/14G01N2333/95G01N2800/26
Inventor 刘明远刘晓雷刘琰杨勇
Owner JILIN UNIV
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