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Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof

A technology of ORF049L and monoclonal antibody, which is applied in the field of monoclonal antibody and preparation of ORF049L recombinant protein of striped sea bream iris virus, and achieves the effects of high sensitivity and strong specificity

Inactive Publication Date: 2013-09-11
DALIAN OCEAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no monoclonal antibody against the transmembrane protein ORF049L has been reported so far.

Method used

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  • Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof
  • Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof
  • Monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins and preparation method thereof

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Embodiment 1

[0031] The preparation method of the present invention is carried out according to the following steps:

[0032] a. The ORF049L recombinant protein of the 16.9kDa stonefish iridescent virus ORF049L was obtained by gene cloning and prokaryotic expression:

[0033] a.1 Take 100 mg of the spleen of the dying rockfish, put it in a 1.5 ml Eppendorf tube, and extract the viral DNA according to the CTAB method at 10°C;

[0034] a.2 Design primers according to the restriction site of plasmid pET-28a (+) as follows:

[0035] Upstream primer P1: 5'-GAATTCATGT ACCCTGACTGTCCCAG-3';

[0036] Downstream primer P2: 5'-GTCGACTTATTTCATAAGCCTTGCAC A-3';

[0037] The total PCR reaction system was 25 μl, and the reaction conditions were: pre-denaturation at 95°C for 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, and a total of 30 cycles, and then extended at 72°C for 8 min;

[0038] PCR products were identified by electrophoresis, recovered and purified, cloned into pMD...

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Abstract

The invention discloses a monoclonal antibody with rock bream iridovirus ORF049L recombinant proteins. The monoclonal antibody is produced by secreting rock bream iridovirus ORF049L recombinant protein hybridoma of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.7304. A preparation method of the monoclonal antibody comprises the following steps of: obtaining the rock bream iridovirus ORF049L recombinant proteins with the molecular weight of 16.9 kDa through gene cloning and prokaryotic expression; taking the obtained recombinant proteins as antigens to immune mice; performing cell fusion on mice spleens and myeloma cells, and screening and secreting the hybridoma of the rock bream iridovirus ORF049L recombinant protein monoclonal antibody by an immunoblotting method, a dot immunobinding assay and an indirect immunofluorescence; and cloning positive hybridoma by a finite dilution method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal antibody to the recombinant protein of the redhead iridescent virus ORF049L and a preparation method thereof. Background technique [0002] Red bream iridovirus (RBIV) is the main pathogen that causes severe economic losses in red bream aquaculture. Its transmembrane protein is an important epitope recognized by cells, so it is also a key target for drug design and an antigen for vaccine preparation . ORF049L is a very important transmembrane protein of the redhead iridescent virus. Kim et al. (2007) found that the polyclonal antibody prepared using the recombinant protein of the redhead iridescent virus ORF049L could block RBIV's effect on the gill cells of blue sunfish (BF- 2) Infection, has a certain neutralizing effect, and relying on the protein antibody can establish a specific immunological detection technology for the iridescent virus of the scorpion. Although ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08
Inventor 李强王友娟李华叶仕根孙志鹏
Owner DALIAN OCEAN UNIV
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