阅读说明：本技术 一株ⅱ型鲤疱疹病毒orf121蛋白的单克隆抗体及其应用 (Monoclonal antibody of II-type carp herpesvirus ORF121 protein and application thereof ) 是由 姜有声 高娃 王浩 吕利群 郑义华 于 2020-04-09 设计创作，主要内容包括：本发明提供了一株Ⅱ型鲤疱疹病毒ORF121蛋白的单克隆抗体及其应用,该单克隆抗体由保藏号为CCTCC NO:C2019330杂交瘤细胞分泌,杂交瘤细胞以GST-ORF121重组蛋白为抗原免疫的小鼠脾脏细胞与小鼠骨髓瘤细胞融合而成；本发明成功克隆了PGEX-4T-3-ORF121重组质粒,筛选出一株抗Ⅱ型鲤疱疹病毒ORF121蛋白的细胞株CyHV-2-ORF121-3D9,首次获得了抗CyHV-2-ORF121的单克隆抗体,可特异性识纯化的CyHV-2病毒颗粒、ORF121重组蛋白以及感染CyHV-2的异育银鲫尾鳍细胞系,可用于制备关于CyHV-2检测试纸条或试剂盒的应用。(The invention provides a monoclonal antibody of II type carp herpes virus ORF121 protein and application thereof, wherein the monoclonal antibody is secreted by a hybridoma cell with the preservation number of CCTCC NO: C2019330, and the hybridoma cell is formed by fusing a mouse spleen cell immunized by taking GST-ORF121 recombinant protein as an antigen and a mouse myeloma cell; the invention successfully clones PGEX-4T-3-ORF121 recombinant plasmid, screens out a cell strain CyHV-2-ORF121-3D9 of II-type carp herpes virus ORF121 protein, obtains a monoclonal antibody of anti-CyHV-2-ORF 121 for the first time, can specifically identify purified CyHV-2 virus particles, ORF121 recombinant protein and a crucian carp tail fin cell line infected with CyHV-2, and can be used for preparing application of a CyHV-2 detection test paper strip or a kit.)

1. A monoclonal antibody of II type carp herpesvirus ORF121 protein is characterized in that: the monoclonal antibody is secreted by a hybridoma cell with the preservation number of CCTCC NO: C2019330.

2. The monoclonal antibody of type II herpesvirus of carp ORF121 protein according to claim 1, which is characterized in that: the preparation method of the hybridoma cell comprises the following steps: and the hybridoma cell is formed by fusing a mouse spleen cell immunized by taking the GST-ORF121 recombinant protein as an antigen and a mouse myeloma cell.

3. The monoclonal antibody of the herpes carp virus type ii ORF121 protein according to claim 2, wherein: the method for obtaining the spleen cells of the mice immunized by the GST-ORF121 recombinant protein as the antigen comprises the following steps: the first immunization is that the antigen and Freund complete adjuvant are fully emulsified in equal volume and then injected into the abdominal cavity of the mouse; carrying out second immunization 14 days later, fully emulsifying the antigen and Freund incomplete adjuvant in the same volume, and carrying out intraperitoneal injection on the mice for immunization; the third immunization is carried out 7 days later by adopting antigen intraperitoneal injection without adjuvant; the fourth immunization was carried out 7 days later, and antigen was intraperitoneally injected without adjuvant.

4. The monoclonal antibody of the herpes carp virus type ii ORF121 protein according to claim 2, wherein: the preparation method of the GST-ORF121 recombinant protein comprises the following steps: amplifying ORF121 gene from II type cyprinid herpesvirus PCR, cloning ORF121 gene to PGEX-4T-3 plasmid to obtain recombinant plasmid PGEX-4T-3-ORF 121; the recombinant plasmid PGEX-4T-3-ORF121 is transfected into Escherichia coli to induce and express GST-ORF121 recombinant protein.

5. Use of the monoclonal antibody of any one of claims 1-4 in the preparation of a test strip or kit for detecting herpes cyprinid virus type ii.

6. A hybridoma cell secreting monoclonal antibody of II type carp herpesvirus ORF121 protein is characterized in that: the preservation number of the hybridoma cell is CCTCC NO: C2019330.

7. The hybridoma cell according to claim 6, wherein: the preparation method of the hybridoma cell comprises the following steps: and the hybridoma cell is formed by fusing a mouse spleen cell immunized by taking the GST-ORF121 recombinant protein as an antigen and a mouse myeloma cell.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody of II-type carp herpesvirus ORF121 protein and application thereof.

Background

The breeding scale of Crucian carp (Crucian carp) in freshwater aquaculture industry in China is getting bigger and bigger, according to the identification of the statistical years of fishery in China in 2018, the annual yield of Crucian carp in China is 277.2 million tons, wherein the main breeding variety is Carassius auratus gibelio. In recent years, due to outbreak of Herpes Viral Hematopoietic Necrosis (HVHN), the carp breeding industry at home and abroad is subjected to huge economic loss, and the main pathogen of the disease is carp herpes virus type II (Cyprinid herpesvirus 2, CyHV-2). The existing detection method aiming at CyHV-2 relatively mature comprises electron microscope diagnosis technology and PCR amplification technology^[1]Cell culture separation and identification and loop-mediated isothermal amplification technology^[2]However, the detection method based on immunology is relatively less studied, and among them, there is a study using CyHV-2 coat protein ORF72^[3]，ORF92^[4]Monoclonal antibodies are prepared for antigens, tissues and cells infected with CyHV-2 can be effectively detected, and an immunological detection method for CyHV-2 is established for the first time.

Reference documents:

[1]Goodwin A E,Merry G E,Sadler J.Detection of the herpesviralhematopoietic necrosis disease agent(Cyprinid herpesvirus 2)in moribund andhealthy goldfish:validation of a quantitative PCR diagnostic method[J].Diseases of Aquatic Organisms,2006,69(2-3):137-143.

[2]He J Q,Shi X J,Yu L,et al.Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus2[J].Journal of virological methods,2013, 194(1-2):206-210.

[3]Kong S Y,Jisng Y S,Wang Q,et al.Detection methods of Cyprinidherpesvirus 2infection in silver crucian carp(Carassius auratus gibelio)via apORF72 monoclonal antibody[J].Journal of Fish Diseases,2017,40(12):1791-1798.

[4]Shen Z Y,Jiang Y S,Lu J F,et al.Application of a monoclonalantibody specific for the ORF92 capsid protein of Cyprinid herpesvirus 2[J].Journal of Virological Methods,2018,261:22-27。

disclosure of Invention

Aiming at the defects in the prior art, the invention mainly aims to provide a monoclonal antibody of II type cyprinid herpesvirus ORF121 protein. Therefore, the CyHV-2-ORF121 monoclonal antibody 3D9 cell strain prepared by the invention can provide more theoretical basis for developing a CyHV-2 immunological detection method.

The second purpose of the invention is to provide the application of the monoclonal antibody.

The third purpose of the invention is to provide hybridoma cells secreting the monoclonal antibody.

In order to achieve the above primary object, the solution of the present invention is:

a monoclonal antibody of II type cyprinid herpesvirus ORF121 protein is secreted by hybridoma with the preservation number of CCTCCNO: C2019330, and specifically recognizes purified CyHV-2 virus particles, ORF121 recombinant protein and GiCF cells infected with CyHV-2.

Preferably, the hybridoma cells are prepared by: and the hybridoma cell is formed by fusing a mouse spleen cell immunized by taking the GST-ORF121 recombinant protein as an antigen and a mouse myeloma cell.

Preferably, the method for obtaining spleen cells of mice immunized by GST-ORF121 recombinant protein as antigen comprises the following steps: the first immunization is that after the antigen and Freund's complete adjuvant are fully emulsified in equal volume, the mouse is injected with the immunity in abdominal cavity; carrying out second immunization 14 days later, fully emulsifying the antigen and Freund incomplete adjuvant in the same volume, and carrying out intraperitoneal injection on the mice for immunization; the third immunization is carried out 7 days later by adopting antigen intraperitoneal injection without adjuvant; the fourth immunization was carried out 7 days later, and antigen was intraperitoneally injected without adjuvant.

Preferably, the GST-ORF121 recombinant protein is prepared by the following method: amplifying ORF121 gene from II type cyprinid herpesvirus PCR, cloning ORF121 gene to PGEX-4T-3 plasmid to obtain recombinant plasmid PGEX-4T-3-ORF 121; the recombinant plasmid PGEX-4T-3-ORF121 is transfected into Escherichia coli to induce and express GST-ORF121 recombinant protein.

In order to achieve the second objective, the solution of the invention is:

the monoclonal antibody is used for specifically recognizing purified CyHV-2 virus particles and ORF121 recombinant protein, and is applied to preparation of II type carp herpesvirus detection test paper strips or kits.

In order to achieve the third object, the solution of the invention is:

a hybridoma secreting monoclonal antibody of II type carp herpesvirus ORF121 protein is disclosed, and the preservation number of the hybridoma is CCTCC NO: C2019330.

Preferably, the hybridoma cells are prepared by: and the hybridoma cell is formed by fusing a mouse spleen cell immunized by taking the GST-ORF121 recombinant protein as an antigen and a mouse myeloma cell.

Due to the adoption of the scheme, the invention has the beneficial effects that:

the invention successfully clones PGEX-4T-3-ORF121 recombinant plasmid, screens out a cell strain CyHV-2-ORF121-3D9 of II-type carp herpes virus ORF121 protein, obtains a monoclonal antibody of anti-CyHV-2-ORF 121 for the first time, can specifically identify purified CyHV-2 virus particles, ORF121 recombinant protein and GiCF cells infected with CyHV-2, and can be used for preparing application related to a CyHV-2 detection test strip or kit.

Drawings

FIG. 1 is a graph showing the results of the Dot-Blot screening of the most positive cell lines.

FIG. 2 is a result chart of Western-blot positive cell lines.

FIG. 3 is a graph of GiCF cells infected with CyHV-2 detected by immunofluorescence.

The II type cyprinid herpesvirus (CyHV-2) ORF 1213D 9 hybridoma cell strain is preserved in the China center for type culture Collection of Wuhan university at 12 months and 12 days in 2019 with the preservation number: CCTCC NO: C2019330.

Detailed Description

The invention provides a monoclonal antibody of II type carp herpesvirus ORF121 protein and application thereof.

The present invention will be further described with reference to the following examples.