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A monoclonal antibody with pedv neutralizing activity

A monoclonal antibody, active technology, applied in the field of genetic engineering, can solve the problems of poor immune effect and large side effects

Active Publication Date: 2021-05-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems in the application of PED vaccines at home and abroad, such as poor immune effect and relatively large side effects.

Method used

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  • A monoclonal antibody with pedv neutralizing activity
  • A monoclonal antibody with pedv neutralizing activity
  • A monoclonal antibody with pedv neutralizing activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the preparation of the hybridoma cell line that can secrete the monoclonal antibody with PEDV neutralizing activity

[0062]Using PEDV S protein (product of Kaioudi Co., Ltd.) as the immunogen, Freund’s adjuvant and PEDV S protein were fully mixed to form an oil emulsion, and 4 SPF grade 6-8 week-old Balb / c mice were selected for immunization, including Primary immunization and three booster immunizations. Orbital blood was collected 7 days after the second booster immunization, and the antibody titer in the serum was detected by ELISA method, and the mouse with the highest antibody titer and positive value was selected for the last booster immunization, and the spleen cells of the mice were collected 4 days later Hybridoma cell fusion was performed with SP2 / 0 cells by PEG2000. When the hybridoma cells form clonal colonies, the cell supernatant is detected by ELISA method, and the positive wells secreting anti-PEDV antibodies are screened out, and the cor...

Embodiment 2

[0115] Example 2, In vivo experimental verification of the supernatant of a hybridoma cell line secreting a monoclonal antibody with PEDV neutralizing activity

[0116]A large-scale pig farm located in Ganzhou City, Jiangxi Province had a full-scale outbreak of PEDV virus, and newborn piglets had symptoms such as severe vomiting, watery diarrhea, and dehydration. And died 24-72 hours after the onset. After collecting the small intestine samples of sick pigs and carrying out PCR amplification, it was confirmed that the piglets in this pig farm were infected with PEDV virus.

[0117] 1. Detection method of disease material

[0118] 1. RNA extraction by Trizol method

[0119] (1) For the small intestine tissue frozen at -80°C, add 1ml Trizol for every 30-50mg of tissue, and use a homogenizer for homogenization.

[0120] (2) Place the homogenized sample at 15-30°C for 5 minutes.

[0121] (3) Add 0.2ml chloroform for every 1ml Trizol used, cover the tube cap, shake vigorously f...

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Abstract

The invention discloses a monoclonal antibody with PEDV neutralizing activity. The monoclonal antibody with PEDV neutralizing activity provided by the present invention is secreted and produced by "monoclonal antibody hybridoma cell line SWS-② with PEDV neutralizing activity". The registration number of the "monoclonal antibody hybridoma cell line SWS‑② with PEDV neutralizing activity" in the General Microbiology Center of China Committee for Culture Collection of Microorganisms is CGMCC No.18312. The invention prepares the monoclonal antibody with neutralizing activity of PEDV, and administers the antibody to newborn piglets, which can effectively reduce the probability of infection of the piglets, and the protection rate reaches 100% in cohabitation infection experiments. The results show that the monoclonal antibody with PEDV neutralizing activity can effectively increase the antibody content in newborn piglets and reduce the risk of disease, and the method is simple to prepare, easy to operate, and has great application prospects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a monoclonal antibody with PEDV neutralizing activity. Background technique [0002] Porcine epidemic diarrhea (PED) is a highly contagious porcine intestinal infectious disease with a mortality rate of up to 100% in suckling piglets. The main symptoms are vomiting, watery diarrhoea, dehydration and high mortality of piglets. There is a large amount of yellow feces around the anus of the sick pig, and the necropsy shows that the small intestine is dilated and filled with yellow liquid, the mesentery is congested, the lymph nodes are edema, and the villi are short. Morbidity and mortality are high in lactating piglets, causing significant economic losses. [0003] Porcine epidemic diarrhea virus (PEDV) belongs to coronavirus, and its genome structure is single-stranded positive-strand RNA. The S protein is located on the outer surface of the virion, which is the key to the vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569G01N33/68A61K39/395A61P31/14C12R1/91
CPCA61K2039/505A61P31/14C07K16/10C07K2317/76G01N33/56983G01N33/577G01N33/68G01N2333/17
Inventor 李秋艳史文姝赵兴波
Owner CHINA AGRI UNIV
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