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Determination method of effective antigen contents of infectious spleen and kidney necrosis virus inactivated vaccine and kit

A technology for spleen-kidney necrosis virus and inactivated vaccines, which is applied in the field of determination method and kit for the effective antigen content of inactivated vaccines for infectious spleen-kidney necrosis virus, and can solve problems such as practicability and biosafety drawbacks, antigen quantification, and virus diffusion , to achieve the effect of improving vaccine production efficiency, shortening the required time, and ensuring the quality of vaccines

Active Publication Date: 2018-05-01
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the production process of the inactivated vaccine of mandarin fish infectious spleen and kidney necrosis virus, the quality evaluation of the vaccine mainly uses the determination of the virus titer before antigen inactivation and the immune challenge test of fish after inactivation, but the determination of the virus titer depends on the live virus. , semi-finished products and finished products after virus inactivation cannot be used to quantify antigens by this method, and the fish body immune challenge test cycle is long, and strong virus attacks are required to pose biosafety risks such as virus spread, which has disadvantages in terms of practicality and biosafety

Method used

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  • Determination method of effective antigen contents of infectious spleen and kidney necrosis virus inactivated vaccine and kit
  • Determination method of effective antigen contents of infectious spleen and kidney necrosis virus inactivated vaccine and kit
  • Determination method of effective antigen contents of infectious spleen and kidney necrosis virus inactivated vaccine and kit

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Experimental program
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Effect test

Embodiment 1

[0031] Proliferation and purification of embodiment one ISKNV

[0032] CPB cells grew to the mid-logarithmic growth phase, inoculated with ISKNV at an MOI of 0.2, the final serum concentration was 6%, and cultured at a constant temperature at 28°C. When the cells produced 90% of the lesions, the virus was collected. Virus purification by density gradient centrifugation.

[0033] (1) Virus precipitation: collect the virus-infected cell suspension, centrifuge at 7500rpm 4°C for 30min; take the supernatant and centrifuge at 150,000×g (about 28000rpm) at 4°C for 1h (BECKMAN 70Ti), discard the supernatant; add appropriate amount of TN buffer, pipette, resuspend the pellet, (at this time, the pellet is often agglomerated, which is not conducive to the gradient centrifugation below), and ultrasonically break several times until no obvious granular precipitate appears.

[0034] (2) Over a sucrose cushion: Add the precipitated virus suspension on top of 35% sucrose solution, centrifug...

Embodiment 2

[0038] Embodiment 2 Recombinant protein expression and purification

[0039] The ISKNV mcp gene fragment was connected to the pET-SUMO vector, and the correct recombinant plasmid was identified by sequencing to transform the expression host. Pick a single colony containing the recombinant plasmid and culture it overnight at 37°C in 3ml LB (containing antibiotics). Take 30 μL of the overnight culture solution and add it to 20 mL of LB medium, shake and culture at 37°C until OD 600 About 0.6, part of the liquid was taken as the uninduced control group, and the rest was added with IPTG inducer to a final concentration of 0.5mM as the experimental group, and the two groups continued to culture with shaking at 37°C for 3h. The fermentation broth was collected, and the cells were collected by centrifugation at 6000g for 10min. Suspend the cells in 40 mL of pre-cooled NTA-0 buffer. Bacteria were crushed by ultrasonic waves in an ice bath, the control power was 300W, ultrasonic wav...

Embodiment 3

[0041] The screening of embodiment tri-hybridoma cell line

[0042] Use the mixture of ISKNV-QY strain purified virus and recombinant ISKNV MCP protein as antigen, immunize BalB / c mice by intraperitoneal cavity, 50-100 μg / mouse for the first immunization, the adjuvant is complete Freund's adjuvant, boost immunization mice 50- 80 μg / monkey, the adjuvant is incomplete Freund's adjuvant, a total of four immunizations, the first immunization interval is 2-3 weeks, and then the booster immunization is 2 times with an interval of 1 week, and then the titer is tested, and it is higher than 1:10000 after 1 week Intraperitoneal percussion. Three days after the last impact, the eyeballs were removed and sacrificed, the positive control blood was collected, the spleen was taken out, and a single cell suspension was prepared, and the splenocytes were fused with SP2 / 0 cells with PEG1450 (1:5-1:10); detected by indirect ELISA The antibody titer in the supernatant of hybridoma cells, select...

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Abstract

The invention discloses a determination method of effective antigen contents of an infectious spleen and kidney necrosis virus inactivated vaccine. According to the method, an active antigen in a to-be-tested sample is captured by taking a monoclonal antibody which is capable of recognizing ISKNV virus major capsid protein (MCP) as a capture antibody; and the captured effective antibody is quantified by taking another monoclonal antibody which is capable of recognizing different antigen epitopes of the infectious spleen and kidney necrosis virus MCP as a detection antibody. The method and a kit adopting the method provided by the invention have the advantages of being high in sensitivity, broad in linear range, convenient to use, short in required time and the like; the method is applicable to determination of effective antigen contents in a semi-finished product and a finished product in an infectious spleen and kidney necrosis virus inactivated vaccine production process; therefore,a novel method is provided for the quality control of the infectious spleen and kidney necrosis virus inactivated vaccine; and the quality of the vaccine can be guaranteed, and the production efficiency of the vaccine can be improved.

Description

technical field [0001] The invention relates to a method for measuring the effective antigen content of a vaccine, in particular to a method for measuring the effective antigen content of an infectious spleen and kidney necrosis virus inactivated vaccine and a kit. Background technique [0002] Mandarin fish (Siniperca chuatsi) is an important species of high-quality freshwater fish consumption and export earnings in my country. It is deeply loved by consumers because of its delicious taste and high protein content. However, the serious disease problem has become the main bottleneck restricting the development of mandarin fish aquaculture. Since 1997, the outbreak of infectious diseases of mandarin mandarin fish has brought huge economic losses to the mandarin fish aquaculture. Wu Shuqin and others confirmed for the first time that a large spherical virus particle with a hexagonal cross section and a diameter of about 150nm is the main pathogen of the fulminant infectious di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20G01N33/577G01N33/569
CPCC07K16/081G01N33/56983G01N2333/01G01N2469/10
Inventor 李宁求付小哲张醴溪林强刘礼辉梁红茹黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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