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Antigen protein, monoclonal antibody or polyclonal antibody thereof and application

A technology of monoclonal antibody and polyclonal antibody, applied in the field of monoclonal antibody or polyclonal antibody and its application, antigen protein, can solve the problem that serological diagnosis has not been widely used, and achieve high sensitivity, strong specificity, good repeatability

Pending Publication Date: 2022-07-01
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are many detection and diagnosis methods for CyHV-2, but in the actual widespread application, they are mainly based on molecular detection technology, and serological diagnosis has not been widely used.

Method used

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  • Antigen protein, monoclonal antibody or polyclonal antibody thereof and application
  • Antigen protein, monoclonal antibody or polyclonal antibody thereof and application
  • Antigen protein, monoclonal antibody or polyclonal antibody thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Sequence analysis of antigenic protein and expression of antigenic protein

[0041] By comparing the original ORF153 protein (amino acid sequence such as Figure 1A The hydrophobicity, antigenicity and signal peptide analysis of the original ORF153 protein, namely: amino acids 1-44, amino acids 57-75, amino acids 96-123, 146-175 position amino acid, and each remaining amino acid sequence is expressed by connecting 6 histidines (amino acid sequence such as Figure 1B and SEQ ID NO.5), on the one hand, histidine plays the role of connecting the amino acid sequence, and on the other hand, adding histidine is also to use a nickel column when purifying the protein, and finally express the antigen of the sumo-tagged antigen protein ORF153. The results of the characterization analysis showed that it was superior to the original ORF153 protein, and the final expressed recombinant antigen protein with the sumo tag was referred to as ORF153 hereinafter in this applica...

Embodiment 2

[0046] Example 2. Preparation of monoclonal antibodies

[0047] Balb / c mice were immunized with ORF153 protein, the serum titer of the mice was detected by ELSIA, and the serum titer of the mice was detected by extracting the supernatant from the disease material. Antibody pairing experiments.

[0048] 1. Animal immunity

[0049] 5 female BALB / c mice (18±2g) aged 6-8 weeks were selected and immunized with ORF153 protein mixed with Freund's adjuvant, subcutaneously injected with 100ug, and boosted once every 2-3 weeks. After four immunizations, blood was collected for detection, and the titer of mouse antiserum against ORF153 protein was determined by indirect ELISA method, and the titer was normal. The results showed that the immunized mice all produced high-titer antibodies, and the titer of the mouse antiserum was over 1:121,500 whether it was directed against ORF153 or the supernatant of the diseased material.

[0050] 2. Cell fusion

[0051] Mix myeloma cells and immun...

Embodiment 3

[0059] Example 3, polyclonal antibody preparation

[0060] Using ORF153 protein, 2 New Zealand rabbits were immunized, the serum titer of the rabbits was detected by ELSIA, and the rabbit serum titer was detected by extracting the supernatant from the disease material; the rabbit polyclonal antibody was prepared by antigen affinity purification.

[0061] 1. Animal immunity

[0062] After measuring BCA protein concentration with ORF153 protein, 2 New Zealand white rabbits (2-2.5kg) were immunized subcutaneously at 400ug / time, once every 2-3 weeks, and immunized 4 times. The titer of rabbit antiserum against ORF153 protein was determined by indirect ELISA, and the titer was normal. Then, the titer of rabbit antiserum was detected by extracting the supernatant from the disease material, and the titer was normal. The results showed that after immunization, the rabbits all produced high titer antibodies, and the titer of the rabbit antiserum was more than 1:121,500 whether it was ...

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Abstract

The embodiment of the invention relates to an antigen protein as well as a monoclonal antibody or a polyclonal antibody and application thereof. The antigen protein comprises 45th to 56th amino acids, 76th to 95th amino acids, 124 to 145th amino acids and 176th to 281 amino acids of cyprinid herpesvirus type 2 ORF153 protein which are sequentially connected through connecting sequences from an N segment to a C segment. According to the antigen protein with the hydrophobic region expressed in the original ORF153 protein removed, the antigenicity analysis result shows that the antigen protein is superior to the original ORF153 protein, and the monoclonal antibody or the polyclonal antibody obtained by utilizing the antigen protein can be used for detecting the cyprinid herpesvirus type 2.

Description

technical field [0001] The present invention relates to the field of immune detection, in particular to an antigen protein, its monoclonal antibody or polyclonal antibody and application. Background technique [0002] Carp herpesvirus 2 (Cyprinid Herpesvirus 2, CyHV-2), also known as goldfish hematopoietic necrosis virus (goldfish haematopoietic necrosis virus, GFHNV), is a highly pathogenic virus that causes hematopoietic necrosis in goldfish and crucian carp. Serious harm to goldfish, crucian carp aquaculture. Since the virus was first discovered in Japan in 1992, it has rapidly spread to Asia, North America, Oceania, and Europe. It has spread on a large scale in the main breeding areas of crucian carp in my country, causing serious economic losses to the goldfish and crucian carp aquaculture industries. [0003] The virus is the second herpes virus isolated from cyprinids. According to the systematic naming rules of the International Committee for Taxonomy of Viruses, th...

Claims

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Application Information

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IPC IPC(8): C07K14/03C07K16/08C12N15/38C12N5/20G01N33/569G01N33/577C12R1/91
CPCC07K14/005C07K16/085G01N33/56994G01N33/577G01N2333/03G01N2469/10
Inventor 王娜张旻景宏丽吕继洲吴绍强林祥梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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