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Hybridoma cell strain secreting CpHV-1 monoclonal antibodies and application thereof

A monoclonal antibody, hybridoma cell technology, applied in the field of immunization, can solve the problems of long time consumption and easy occurrence of false positives

Active Publication Date: 2018-11-30
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are practical, they also have many disadvantages.
The PCR method is a more commonly used identification method, which is used to detect the nucleic acid of the virus. Although it has certain sensitivity and specificity, it is prone to false positives.
The virus isolation and identification experiment is to obtain the virus strain through cell isolation and culture, and then conduct a series of molecular biological identification. Although classic, this method takes a long time

Method used

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  • Hybridoma cell strain secreting CpHV-1 monoclonal antibodies and application thereof
  • Hybridoma cell strain secreting CpHV-1 monoclonal antibodies and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0070] The establishment of embodiment 1 monoclonal antibody hybridoma cell line

[0071] 1. Preparation of CpHV-1 antigen

[0072] The inventor isolated a new CpHV-1 domestically prevalent and virulent strain JSHA1405 from sheep with severe respiratory infections (Hao Fei et al., Goat herpesvirus type I vaccine strain and its application [P]. Jiangsu: CN107893056A, 2018 -04-10), there is a large variation between this strain and foreign popular strains. As far as the gB gene is concerned, the foreign popular strain encodes a total of 919 amino acids, while the domestic popular strain JSHA1405gB gene encodes 920 amino acids, and the mutations at 11 amino acid sites. The virus strain was inoculated into 70% confluent MDBK cells (purchased from China Veterinary Drug Administration) at 0.01-0.1 MOI, and placed at 37°C, 5% CO 2 Cell cultures were cultured in a dark place in a cell incubator. Cell cultures were collected 48 hours after inoculation, and the supernatant was obtaine...

Embodiment 2

[0081] The preparation of embodiment 2 ascites

[0082] Inject sterilized liquid paraffin intraperitoneally into 10-12-week-old BALB / c mice (purchased from the Comparative Medicine Center of Yangzhou University), 0.5 mL / mouse, and 7 days later, inject the hybridoma cell line into the peritoneal cavity of mice, each 0.2 mL (including 2×10 6 -3×10 6 hybridoma cells), 7-10 days later, the ascites of the mouse whose abdomen was obviously bulging was collected and centrifuged at 5000rpm for 10min. The supernatant was collected, aliquoted and stored at -20°C for later use.

Embodiment 3

[0083] Example 3 Virus neutralization test

[0084] Ascites prepared from 89 established hybridoma cell lines stably secreting CpHV-1 monoclonal antibody were used for CpHV-1 neutralization test, and hybridoma cell lines secreting high neutralizing titer of monoclonal antibody were further screened. First, measure the TCID of CpHV-1JSHA1405 strain 50 . Using the method of immobilizing virus and diluting antibody, MDBK cells were digested and inoculated in 96-well cell plates. Mix the mouse ascites fluid of each strain of monoclonal antibody diluted 10 times serially with an equal volume containing 200TCID 50 The CpHV-1 suspension was mixed evenly, and reacted at 37°C for 1 hour, and 0.1 mL of the virus-antibody suspension was inoculated in the above-mentioned 96-well cell plate, and CpHV-1 and normal MDBK cell controls were set up, and placed at 37°C , 5%CO 2 Cultivate in the cell incubator, and observe the results after 2 days. Among the 89 CpHV-1-specific monoclonal ant...

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Abstract

The invention relates to a hybridoma cell strain secreting CpHV-1 monoclonal antibodies and application thereof, and belongs to the technical field of immunization. The hybridoma cell strain 4A4 is screened out of a built hybridoma cell bank for secreting the CpHV-1 monoclonal antibodies. The neutralizing titer of the mouse ascitic monoclonal antibodies prepared through the hybridoma cell strain 4A4 to CpHV-1 reaches 28. The monoclonal antibodies secreted by the hybridoma cell strain 4A4 can be used for clinic therapy of CpHV-1 pathogenetic sheep, the curative effect is remarkable, the safe effect is achieved, and adverse side reactions are avoided. After a monoclonal antibody therapeutic agent prepared through the hybridoma cell strain 4A4 is preserved for two years, the neutralizing titer is not lowered, and stability is good. Additionally, the monoclonal antibodies secreted by the hybridoma cell strain 4A4 serve as an indirect immunofluorescence (IFA) detecting method for antibody building detecting, good sensitivity and specificity are achieved, and the hybridoma cell strain can be used for differential diagnosis of CpHV-1 infection.

Description

technical field [0001] The invention belongs to the technical field of immunization, and in particular relates to a hybridoma cell strain secreting monoclonal antibody against goat herpes virus type I (CpHV-1). Background technique [0002] Caprine herpesvirus type 1 (Caprine herpesvirus 1, CpHV-1) is a double-stranded DNA virus belonging to the varicellavirus genus of the herpesviridae subfamily of the family Herpesviridae. Neonatal lambs infected with the virus manifested as severe infection with main symptoms of fever, conjunctivitis, increased ocular discharge, dyspnea, intestinal ulceration and necrotic lesions, accompanied by high morbidity and mortality. Infection of adult goats presents as subclinical infection, which can lead to different symptoms including respiratory disease, fever and leukopenia, vulvovaginitis and balanitis of the foreskin. Infection can induce abortion in goats that are 3 to 4 months pregnant. In 1974, CpHV-1 was first isolated from the sick ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/569A61K39/42A61P31/22C12R1/91
CPCA61K2039/505A61P31/22C07K16/085C07K2317/33C07K2317/35C07K2317/76G01N33/56994
Inventor 郝飞张纹纹李文良刘茂军毛立李基棕杨蕾蕾孙敏袁朗肖蓉
Owner JIANGSU ACAD OF AGRI SCI
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