Anti-SVCV (Spring Viraemia of Carp Virus) monoclonal antibody 3E1 as well as preparation and application thereof

A technology of SVCV-3E1 and monoclonal antibody, which is applied in the field of carp spring viremia virus detection, can solve the problems of on-site detection promotion, repeated tracking and review restrictions, high operating costs, etc., and achieves intuitive detection results, fast speed, The effect of high sensitivity

Inactive Publication Date: 2014-01-15
SHENZHEN AUDAQUE DATA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adopt the method for RT-PCR to detect carp spring viremia virus as CN102876810A; CN102876811A patent has also announced a kind of RT-LAMP detection kit, above detection method all needs certain condition and technology, in addition, also needs special instrument, operating cost Large, its on-site testing promotion and repeated follow-up review are limited

Method used

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  • Anti-SVCV (Spring Viraemia of Carp Virus) monoclonal antibody 3E1 as well as preparation and application thereof
  • Anti-SVCV (Spring Viraemia of Carp Virus) monoclonal antibody 3E1 as well as preparation and application thereof
  • Anti-SVCV (Spring Viraemia of Carp Virus) monoclonal antibody 3E1 as well as preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Expression of membrane protein of carp spring viremia virus

[0035]According to the sequence published by NCBI with the accession number AY527273, the membrane protein glycoprotein [Spring viraemia of carp virus] was selected as the antigen detection site of carp spring viraemia virus, and the whole sequence was analyzed by antigenic determinant analysis software. Finally, a protein with strong immunogenicity and high specificity is selected as the immunogen. The amino acid sequence of the protein is SEQ ID NO.1, and the DNA sequence corresponding to the synthesized protein is SEQ ID NO.2. This DNA (sequence shown in SEQ ID NO.2) was inserted into the prokaryotic expression vector PET28e and identified by sequencing. IPTG is used to induce expression, and the protein SVCV glycoprotein is obtained by separation and purification, which can be used as an immunogen to immunize mice to prepare antibodies. The specific expression purification process is as follow...

Embodiment 2

[0057] Embodiment 2: Preparation of anti-carp spring viremia virus monoclonal antibody:

[0058] (1) A total of 5 8-week-old BALB / C mice were selected, and the immunization procedure was as follows:

[0059] First immunization: SVCV-glycoprotein was used as the immunogen, Quick Antibody-Mouse5W was used as the immune adjuvant, and the immunization dose was 5 μg / monkey (5 μg immunogen + 5 μL Quick Antibody-Mouse5W + saline 90ul), injected into the thigh muscle.

[0060] Booster immunization: carried out on the 21st day after the first immunization, the immunization dose was 5 μg / monkey (5 μg immunogen + 5 μL Quick Antibody-Mouse5W + normal saline 90ul), a total of 2 immunizations, and on the 35th day, the blood was collected by docking the tail to separate the serum, and the serum was separated by indirect ELISA The serum antibody titer of the anti-SVCV suspension was detected by the method. If the titer reaches 1:5000, it can be used for cell fusion.

[0061] The immunized m...

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 0 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 abo...

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Abstract

The invention discloses an anti-SVCV (Spring Viraemia of Carp Virus) monoclonal antibody 3E1 as well as preparation and application thereof. The monoclonal antibody 3E1 is secreted by a hybridoma cell strain SVCV-3E1, and the preservation number of the cell strain is CCTCC C201366. The hybridoma cell strain SVCV-3E1 can stably generate the monoclonal antibody 3E1, and the antibody has the advantages of high antibody titer, high sensitivity, strong specificity, strong affinity with natural antigen and the like. The invention also provides a rapid detection test strip containing the monoclonal antibody and a kit thereof. The rapid detection test strip adopts a fluorescent nanometer microsphere immunochromatography assay technology and has the characteristics of good sensitivity, specificity, stability, repeatability, reproducibility and the like; meanwhile, the test strip and the kit have the advantages of simplicity and convenience in use and rapid detecting speed, can meet the detection requirements for food safety, slaughter, detection mechanisms and the like, and are suitable for field detection.

Description

technical field [0001] This application relates to the field of carp spring viremia virus detection, in particular to an anti-carp spring viremia virus (SVCV) monoclonal antibody 3E1 and its preparation and application. Background technique [0002] Spring viraemia of carp (SVC) belongs to the Rhabdoviridae Vesivirus genus, contains a linear, antisense, single-stranded RNA, and contains five structural proteins, namely nucleoprotein (N) , phosphoprotein (P), membrane protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L-protein). SVCV is a serious contagious disease affecting carps, mainly in some European countries in the past (Austria, Belgium, France, Germany, Great Britain, Hungary, Italy, Spain and parts of former Slovakia, Soviet Union and Yugoslavia regions) and cause severe economic losses to farmed carp in these countries. The diseased fish have black body color, dyspnea, ataxia (side swimming, drifting along the water or abnormal swimming), enlarged a...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569C07K14/145
Inventor 郑晓聪钟松清何俊强谭攀贾鹏王津津史秀杰兰文升于力谢冬霞刘荭
Owner SHENZHEN AUDAQUE DATA TECH
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