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Hybridoma cell line and anti-canine distemper virus N protein monoclonal antibody produced through hybridoma cell line

A hybridoma cell line, monoclonal antibody technology, applied in the field of microorganism-related genetic engineering, can solve the problems of seldom use, unsafe allergy, danger of puppies and wild carnivores

Inactive Publication Date: 2014-08-20
JL TEYAN BIOLOGICAL TECH LIMITED LIABILITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Canine distemper is distributed worldwide, and there is cross-infection among different species. Canine distemper cannot be completely eliminated. At present, there is no specific treatment drug and method for canine distemper. Vaccination is an effective preventive measure , but conventional vaccines have disadvantages to varying degrees: inactivated canine distemper virus vaccines have poor antigenicity and are rarely used now; measles virus heterologous vaccines have short immunity duration; attenuated vaccines are unstable to heat and immune to certain Defective puppies and wild predators are unsafe and dangerous for allergic reactions

Method used

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  • Hybridoma cell line and anti-canine distemper virus N protein monoclonal antibody produced through hybridoma cell line
  • Hybridoma cell line and anti-canine distemper virus N protein monoclonal antibody produced through hybridoma cell line
  • Hybridoma cell line and anti-canine distemper virus N protein monoclonal antibody produced through hybridoma cell line

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0022] Main experimental materials and sources

[0023] 1. Proteins, cells, viruses

[0024] The prokaryotic expressed and purified CDV-N protein, SP2 / 0 cells, Vero cells and CDV strains are all preserved by our laboratory.

[0025] 2. Main reagents and medicines

[0026] Fetal bovine serum and DMEM medium were purchased from GIBCO; diaminobenzidine (DAB) chromogenic kit, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, FITC-labeled goat anti-mouse IgG antibody, glue The recovery kit was purchased from Kangwei Century Biotechnology Co., Ltd.; 50% PEG, 50×HAT, and 50×HT were purchased from Sigma; o-phenylenediamine and prestained protein marker were purchased from Fermentas; SBAClonotypingTM System / HRP antibody subclass Identification kits were purchased from Southern Biotechnology Company; plasmid extraction kits were purchased from AXYGEN Company, T-E1, reverse transcriptase, Ex Taq DNA polymerase, T4 DNA ligase, and HIS tag purification reagents were purc...

Embodiment 1

[0029] Prokaryotic expression and purification of embodiment 1 CDV-N protein

[0030] 1. Primer Design

[0031] According to the CDV-N gene sequence registered in Genbank (accession number: EF375619), design PCR amplification primers, the sequence is as follows: upstream primer 5-GAAACTATGTATCCGGCT-3, downstream primer 5-TGACTCACTCCATTCGGA-3

[0032] 2. Extraction and reverse transcription of CDV viral RNA

[0033]Viral genomic RNA was extracted from CDV-infected Vero cells by Trizol method as a template, and viral cDNA was synthesized by reverse transcription with random primers.

[0034] RNA extraction step by Trizol method: harvest Vero cells infected with CDV 1-5×10 7 Add 1mL Trizol, mix well, let stand at room temperature for 5min, add 0.2mL chloroform, shake vigorously for 15s, incubate at room temperature for 2-3min, centrifuge at 12000g, 4°C for 15min, carefully remove the upper layer of colorless liquid, add an equal volume of pre-cooled iso Propanol, after mixing,...

Embodiment 2

[0063] The preparation of embodiment 2 monoclonal antibody

[0064] 1. Mice Immunization

[0065] Five 6-week-old female BALB / c mice were immunized with affinity-purified prokaryotic recombinant CDV-N protein for 3 times with a two-week interval between each immunization. The immunization dose was 50 μg / mouse, and the immunization route was intraperitoneal immunization .

[0066] One week after the second and third immunizations, blood was collected from the tail of the mice, the serum was separated (4°C, 10,000 rpm, 20 min), and the antibody level was detected by indirect ELISA. Three days before cell fusion, BALB / c mice with good immune effect were boosted again, and each mouse was intraperitoneally injected with 50 μg of immune antigen.

[0067] 2. Cell Fusion

[0068] The feeder layer cells were prepared 1 day before fusion, and BALB / c mouse peritoneal macrophages were plated in 96-well cell culture plates according to conventional methods for use. Kill the mice to be ...

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Abstract

The present invention discloses a hybridoma cell line and an anti-canine distemper virus N protein monoclonal antibody CDV-1N8 produced through the hybridoma cell line, and belongs to the field of CDV prevention and treatment. According to the present invention, the hybridoma cell line stably secreting the anti-CDV N protein monoclonal antibody is screened, wherein the microorganism preservation number is CGMCC No.8793, and experiment results prove that the monoclonal antibody CDV-1N8 secreted by the hybridoma cell line can specifically react with the CDV N protein, and does not react with the Vero cell protein; the monoclonal antibody can specifically recognize the CDV-N protein, and accurate positioning of the CDV-N protein B-cell epitope recognized by the monoclonal antibody is QITFLHSERS; and the monoclonal antibody CDV-1N8 and the CDV N protein virus-specific conservative B cell epitope polypeptide recognized by the monoclonal antibody can be made into the CDV infection diagnosis reagent so as to establish the foundation for establishment of the CDV serological differential diagnosis method.

Description

technical field [0001] The invention belongs to the field of genetic engineering related to microorganisms, and in particular relates to a hybridoma cell line and a monoclonal antibody secreted by it against the N protein of canine distemper virus. Background technique [0002] Canine distemper (Canine distemper, CD) CD is an acute, febrile, highly contagious infectious disease caused by Canine Distemper Virus (CDV), which belongs to the genus Morbillivirus in the Paramyxoviridae family. The natural infection hosts of CDV include the traditional Canidae, Mustelidae and Procyonidae, but now it has expanded to all 8 families of Carnivora, Suidae of Artiodactyla, Rhesus of Primates and Sealidae of Pinnipeds and other animals. The high incidence rate can reach almost 100%. Due to the variety of clinical symptoms of the disease, it is easy to cause mixed infection and secondary infection of other bacteria and viruses, and the mortality rate can even be as high as 80%. It is known...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/68G01N33/577G01N33/569C12R1/91
Inventor 程世鹏易立陈立志程悦宁王建科仝明薇
Owner JL TEYAN BIOLOGICAL TECH LIMITED LIABILITY
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