Unlock instant, AI-driven research and patent intelligence for your innovation.

Monoclonal antibody of cyprinid herpesvirus 2 (CyHV-2) ORF121 protein and application of monoclonal antibody

A technology of ORF121 and GST-ORF121, which is applied in the field of monoclonal antibodies, can solve the problems of relatively little research on detection methods

Active Publication Date: 2020-08-11
SHANGHAI OCEAN UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, relatively mature detection methods for CyHV-2 include electron microscope diagnosis technology and PCR amplification technology [1] , cell culture isolation and identification and loop-mediated isothermal amplification technology [2] etc., but there are relatively few studies on immunological detection methods, among which, some studies use the CyHV-2 shell protein ORF72 [3] , ORF92 [4] Prepared a monoclonal antibody for the antigen, which can effectively detect CyHV-2-infected tissues and cells, and established an immunological detection method for CyHV-2 for the first time

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Monoclonal antibody of cyprinid herpesvirus 2 (CyHV-2) ORF121 protein and application of monoclonal antibody
  • Monoclonal antibody of cyprinid herpesvirus 2 (CyHV-2) ORF121 protein and application of monoclonal antibody
  • Monoclonal antibody of cyprinid herpesvirus 2 (CyHV-2) ORF121 protein and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of polyclonal antibody: The recombinant plasmids used in the present invention were stored in the laboratory of Shanghai Ocean University Pathogen Bank. Competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Restriction enzymes and T4 ligase were purchased from From TaKaRa Company (ie Treasure Bioengineering (Dalian) Co., Ltd.).

[0031] ORF121 prokaryotic expression

[0032] 1.1 ORF121 gene fragment amplification

[0033] Design specific primers according to the gene sequence of CyHV-2ORF121

[0034] (F:5′-CCGGAATTCCATGTCTACAGAAATGGTTTTCATCG-3′(SEQ ID NO.1); R:5′-CCGCTCGAGTCCTACATATCGTCTTCATCCTCGGGT-3′(SEQ ID NO.2)), EcoRI and XhoI restriction sites were inserted into the upstream and downstream primer sequences respectively point. Collect the GiCF cell infection solution infected with CyHV-2 and freeze-thaw repeatedly 2-3 times, centrifuge at 4000r / min at 4°C for 20min, collect the supernatant and centrifuge at 32000r / min...

Embodiment 2

[0042] Monoclonal antibody preparation:

[0043] 2.1 SP2 / 0 mouse myeloma cells and experimental mice

[0044] The SP2 / 0 mouse myeloma cells were preserved in our laboratory, and cultured before immunization, so that they were in the logarithmic growth phase. The SPF-grade BABL / C mice used in the experiment were purchased from Shanghai Legen Biotechnology Co., Ltd., and kept in the laboratory for 7 days before immunization.

[0045] 2.2 Immunization procedure

[0046] The mice were immunized with the purified target protein in four times, 200 μl / mouse each time. For the first immunization, Freund's complete adjuvant and ORF121 recombinant protein were mixed and completely emulsified at 1:1 and injected intraperitoneally; the second immunization was carried out 14 days after the first immunization, and ORF121 recombinant protein was mixed with Freund's incomplete adjuvant The dose was mixed 1:1 and then completely emulsified and injected intraperitoneally; the third and fourt...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a monoclonal antibody of cyprinid herpesvirus 2 (CyHV-2) ORF121 protein and application of the monoclonal antibody. The monoclonal antibody is secreted by hybridoma cells with acollection number of CCTCC NO: C2019330, and the hybridoma cells are formed by fusing spleen cells and myeloma cells of mice immunized with GST-ORF121 recombinant protein as an antigen. A PGEX-4T-3-ORF121 recombinant plasmid is successfully cloned, a cell strain CyHV-2-ORF121-3D9 resisting the CyHV-2 ORF121 protein is screened out, the monoclonal antibody resisting the CyHV-2-ORF121 is obtained for the first time, can specifically purify CyHV-2 virus particles, ORF121 recombinant protein and Cyhv-2-infected carassius auratus gibelio tail fin cell lines, and can be used for preparing a CyHV-2detection test strip or kit.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody to a type II carp herpesvirus ORF121 protein and an application thereof. Background technique [0002] The scale of crucian carp (Crucian carp) farming in my country's freshwater aquaculture industry is increasing. According to the 2018 China Fishery Statistical Yearbook, the annual output of crucian carp in my country is 2.772 million tons, of which the main cultured species is heterogeneous gibel carp (Carassius auratus gibelio ). In recent years, the outbreak of Herpesviralhaematopoietic necrosis (HVHN) has brought huge economic losses to the heterogeneous gibel carp breeding industry at home and abroad. The main pathogen of the disease is carp herpesvirus type Ⅱ ( Cyprinid herpesvirus 2, CyHV-2). At present, relatively mature detection methods for CyHV-2 include electron microscope diagnosis technology and PCR amplification technology [1] , cel...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/70C12N5/20G01N33/577G01N33/569C12R1/91
CPCC07K16/085C12N15/70G01N33/577G01N33/56994
Inventor 姜有声高娃王浩吕利群郑义华
Owner SHANGHAI OCEAN UNIV