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Anti-ihnv monoclonal antibody 6g7 and its preparation and application

An IHNV-6G7, monoclonal antibody technology, applied in antiviral immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of low antigen active protein content, poor antibody specificity, high cost, etc., and achieve affinity The effect of good performance, strong antibody specificity and high sensitivity

Active Publication Date: 2017-01-18
深圳出入境检验检疫局动植物检验检疫技术中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above inspection methods all require certain conditions and techniques, or because of the need for special instruments, or because of the high cost, the promotion of on-site inspection and repeated follow-up inspections are limited.
In addition, using immunological technology detection, such as patent US20050163795A1 and "Zhu Xu et al., Preparation and Application of Infectious Hematopoietic Necrosis Virus Glycoprotein Antiserum, "Advances in Fishery Science", 201206 (33)" using IHNV-G protein (expression Protein) as an antigen to prepare antibodies and detect IHNV, but there are still shortcomings that cannot ensure that the IHNV-G protein has good antigenicity, or the content of antigenic active protein is low, and the specificity of the antibody is poor

Method used

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  • Anti-ihnv monoclonal antibody 6g7 and its preparation and application
  • Anti-ihnv monoclonal antibody 6g7 and its preparation and application
  • Anti-ihnv monoclonal antibody 6g7 and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Expression of Infectious Hematopoietic Necrosis Virus Membrane Protein

[0037] According to the sequence published by NCBI with accession number AAT99394, the membrane protein glycoprotein of infectious tissue necrosis virus was selected as the antigen detection site of infectious tissue necrosis virus, and the whole sequence was analyzed by antigenic determinant analysis software, and finally the immunogenicity and specificity were selected. A high protein is used as an immunogen, the amino acid sequence of the protein is SEQ ID NO.2, and the DNA sequence corresponding to the synthesized protein is SEQ ID NO.1. This DNA (sequence shown in SEQ ID NO.1) was inserted into the prokaryotic expression vector PET28e and identified by sequencing. IPTG is used to induce expression, and the protein IHNV glycoprotein is isolated and purified, which can be used as an immunogen to immunize mice to prepare antibodies. The specific expression purification process is as f...

Embodiment 2

[0059] Example 2: Preparation of monoclonal antibody against infectious tissue necrosis virus:

[0060] (1) A total of 5 8-week-old BALB / C mice were selected, and the immunization procedure was as follows:

[0061] First immunization: IHNV glycoprotein obtained in Example 1 was used as the immunogen, Quick Antibody-Mouse5W was used as the immune adjuvant, the immunization dose was 5 μg / monkey (5 μg immunogen + 5 μL Quick Antibody-Mouse5W + normal saline 90ul), thigh intramuscular injection .

[0062] Booster immunization: carried out on the 21st day after the first immunization, the immunization dose was 5 μg / monkey (5 μg immunogen + 5 μL QuickAntibody-Mouse5W + physiological saline 90ul), a total of 2 immunizations, on the 35th day, the blood was collected by docking the tail to separate the serum, and the indirect ELISA method was used The serum antibody titer of the anti-IHMV suspension was detected. If the titer reaches 1:5000, it can be used for cell fusion.

[0063] T...

Embodiment 3

[0078] Example 3: Preparation of fluorescent nanospheres:

[0079] (1) Preparation of yellow fluorescent nanocrystals - CdTe / CdS core-shell semiconductor nanocrystals

[0080] Add sodium borohydride into the centrifuge tube, inject high-purity water; after the sodium borohydride dissolves, quickly add tellurium powder, and the molar ratio is 2:1. The reaction system was cooled with ice, and an exhaust port was reserved to discharge the hydrogen generated in the reaction. After reacting for 2-8 hours, white sodium tetraborate precipitates are formed at the bottom of the bottle, and the clear night in the upper layer is the required NaHTe solution.

[0081] The reaction equation is:

[0082] 4NaBH 4 +2Te+7H 2 0→2NaHTe+Na 2 B 4 o 7 +14H 2 ;

[0083] CdCl 2 Adjust the pH value of the mixed solution with mercaptopropionic acid to 7-14, and add the newly prepared NaHTe solution with a micro-injector, the molar ratio of which is Cd:Te:mercaptopropionic acid=2:1:2-8. The ab...

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Abstract

The invention discloses an anti-infectious hematopoietic necrosis virus (IHNV) monoclonal antibody 6G7, and preparation and application thereof. The monoclonal antibody 6G7 is secreted by a hybridoma cell strain IHNV-6G7; the preservation number of the cell strain is CCTCC C201360. The hybridoma cell strain IHNV-6G7 can stably generate the monoclonal antibody 6G7. The antibody has the advantages of being high in valence, high in sensitivity, strong in specificity, strong in affinity with natural antigen and the like. The invention also provides a quick test strip containing the monoclonal antibody, and a kit thereof. By adopting a fluorescent nanometer microspheres immune chromatography detection technology, the monoclonal antibody 6G7 has the characteristics of being good in sensitivity, specificity, stability, repeatability, reproducibility and the like. Meanwhile, the test strip and the kit are simple and convenient to use, high in test speeds, and applicable to a field test; the testing requirements of food safety, slaughter, testing mechanisms and the like can be met.

Description

technical field [0001] This application relates to the field of infectious hematopoietic necrosis virus detection, in particular to an anti-infectious hematopoietic necrosis virus (IHNV) monoclonal antibody 6G7 and its preparation and application. Background technique [0002] Infectious hematopoietic necrosis of fish (or infectious hematopoietic necrosis of fish, IHN) is caused by a highly virulent rhabdovirus, Infectious hematopoietic viremia virus (Infectious hematopoietic necrosis virus) Acute, systemic severe infectious disease caused by necrosis virus (IHNV for short), the pathogenic factor IHNV virion is elastic and has a capsule, its inner surface is matrix protein (M), and there is glycoprotein (G) on the capsule ) There are matrix proteins, nucleoproteins, polymerases, etc. in the protruding membrane, and the main target organ of infection is the renal hematopoietic tissue. This outbreak was first seen in juvenile salmonids from Pacific Northwest farms in North Am...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569C07K14/145
Inventor 贾鹏钟松清何俊强刘荭郑晓聪谭攀王津津史秀杰兰文升于力谢冬霞
Owner 深圳出入境检验检疫局动植物检验检疫技术中心