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A kind of hybridoma cell line secreting anti-cphv-1 monoclonal antibody and its application

A monoclonal antibody and hybridoma cell technology, applied in the field of immunity, can solve the problems of easy false positives and long time consumption, and achieve no adverse reactions, good sensitivity and specificity, and no reduction in antibody neutralization titer Effect

Active Publication Date: 2021-06-22
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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  • A kind of hybridoma cell line secreting anti-cphv-1 monoclonal antibody and its application
  • A kind of hybridoma cell line secreting anti-cphv-1 monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1 Establishment of monoclonal antibody hybridoma cell line

[0071] 1, Preparation of CPHV-1 antigen

[0072] The inventors have been separated from a new type of CPHV-1, a new type of CPHV-1, a cultivation of a new type of CPHV-1, is separated from a cultivated respiratory tract infection. -04-10) The poisonous strain has a large variation of foreign floating drug strains. Only 919 amino acids are encoded in foreign epidemic strains, while domestic epidemic strains JSHA1405GB gene encodes 920 amino acids, and occurs 11 mutations of 11 amino acid sites. The strain was inoculated by a 0.01-0.1MoI in vaccination of MDBK cells (purchased from China Veterinary Drug Supervision), placed in 37 ° C, 5% CO 2 The cell culture is collected in the cell culture box, and the cell culture was collected at 48 hours after the drug, repeatedly frozen three times, and centrifugally, 40 000 g speed centrifugation 2h, discarded the supernatant, precipitated with an appropriate amount o...

Embodiment 2

[0081] Example 2 Preparation of Ascites

[0082] The sterilized liquid paraffin abdominal cavity injected 10-12 week old BALB / C mice (purchased from Yangzhou University Comparative Medicine Center), 0.5ml / only, 7d, after 7 days, the hybridoma cell line was injected into mice, each 0.2 ML (including 2 × 10 6 -3 × 10 6 Hybridoma cells), after 7-10 days, the abdomen of the mouse in the abdomen was taken after 7-10 days, and 5000 rpm was centrifuged for 10 min. Collect the supernatant, place the spare in -20 ° C after dispensing.

Embodiment 3

[0083] Example 3 Virus neutralization test

[0084] Ascites prepared by stably secreting CPHV-1 monoclonal antibodies were used to carry out CPHV-1 neutralized and experiments, respectively, and a monoclonal antibody hybridoma cell line with high secretion and high titers were further screened. First, determine the TCID of CPHV-1JSHA1405 strains 50 . The MDBK cells were digeulated using a fixed viral dilution antibody, and the MDBK cell was digested in a 96-well cell plate. The mice of the 10x series diluted monoclonal antibodies are 200tcid respectively. 50 The CPHV-1 suspension was mixed uniform, 37 ° C for 1 h, and 0.1 ml of the viral-antibody suspension was taken in the 96-well cell plate, and CphV-1 and normal MDBK cell control were set, placed in 37 ° C. 5% CO 2 Culture in the cell incubator and observed after 2D. In the hybridoma cell line of 89 CPHV-1 specific monoclonal antibodies, mice prepared by 4A4 strains of hybridoma cells were the highest, neutralized and high of 2...

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Abstract

The invention relates to a hybridoma cell line secreting an anti-CpHV-1 monoclonal antibody and an application thereof, belonging to the technical field of immunization. The invention screens a hybridoma cell 4A4 strain from the established hybridoma cell library secreting anti-CpHV-1 monoclonal antibody. The neutralizing titer of mouse ascitic fluid monoclonal antibody to CpHV‑1 reached 2 8 . The monoclonal antibody secreted by the hybridoma cell 4A4 strain of the present invention can be used for the clinical treatment of CpHV-1-affected sheep, has remarkable curative effect, is safe and has no adverse side effects. The monoclonal antibody therapeutic agent prepared by the hybridoma cell 4A4 strain of the present invention has good stability and does not decrease in neutralization titer after two years of storage. In addition, the indirect immunofluorescence (IFA) detection method established by using the monoclonal antibody secreted by the hybridoma cell 4A4 strain of the present invention as the detection antibody has good sensitivity and specificity, and can be used for differential diagnosis of CpHV‑1 infection .

Description

Technical field [0001] The present invention belongs to the field of immunization, and more particularly to hybridoma cell lines secreting anti-goat herpes-type I type (CPHV-1) monoclonal antibody. Background technique [0002] Goat herpes virus type I (Caprine Herpesvirus 1, CPHV-1) belongs to the double-stranded DNA virus of herpes virus pessine virus. The virus is infected with new lambs, as severe infections, main symptoms are fever, conjunctivitis, increased eye secretion, difficulty breathing, intestinal ulcerative and necrotic lesions, with higher incidence and mortality. Adult goats are infected with subclinical infections, which can result in different symptoms, including respiratory diseases, heat and leukocyte reduction, outer virgitis and foreskin balanitis. Walkmen can be induced after 3 to 4 months of goats infection. In 1974, CPHV-1 was first isolated in the United States, California, is isolated from newborn lamb diseases in Enteritis, and then there will be a pre...

Claims

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Application Information

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Patent Type & Authority Patents(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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