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Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof

A technology of Nodavirus, capsid protein, applied in the field of applied biology

Inactive Publication Date: 2014-08-27
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the protein in the natural conformation has more antigenic epitopes than the inclusion body form, and the immunogenicity is also higher. Therefore, it is expected to obtain a better immune effect by using the refolded capsid protein. There are few reports on the analysis of protein inclusion body renaturation conditions

Method used

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  • Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof
  • Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof
  • Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Injection of immunized cultured flounder to improve immune protection rate

[0041] 1. Construction of expression vector RNA2-Pet21a of recombinant flounder β nodavirus capsid protein:

[0042] (1) Design PCR primers according to the sequence of flounder β nodavirus (Genbank number: KF841612), forward primer NYQf0001e:

[0043] The sequence is 5'- CATATG GTACGCAAAGGTGAG-3', the underline is the Nde I restriction site; reverse primer NYQr1014He:

[0044] The sequence is 5'- AAGCTT AGTGGTGGTGGTGGTGGTGGTTTTCCGAGTCAACCCT-3', the underline is the Hind III restriction site;

[0045] (2) Using the plasmid vector RNA2-PMD18T preserved by the Tianjin Aquatic Animal Disease Prevention and Control Center and the complete cDNA sequence of the flounder beta nodavirus genome RNA2 segment as a template (available externally), use the method described in (1) The primers amplify the coding region of the viral capsid protein, and the amplified product is loaded into the ...

Embodiment 2

[0054] Preparation of polyclonal antibody by recombinant flounder β nodavirus capsid protein for Western-Blot analysis

[0055] 1. Polyclonal Antibody Preparation

[0056] The renatured protein is sent to Kangwei Century Company for polyclonal antibody preparation. The specific steps are:

[0057] (1) Injection immunization

[0058] Two adult New Zealand white rabbits were purchased from a local experimental animal center. The flounder beta nodavirus capsid protein prepared by the method of the present invention is dissolved in 1 ml of phosphate buffer solution according to the dose of 100 μg antigen per rabbit. Add mycobacteria to 1ml of Freund's incomplete adjuvant to make a complete adjuvant (source: all purchased from Huashengyuan Bioreagent Company), and add 1ml of antigen solution, shake vigorously to fully emulsify it, and use a 3ml syringe to draw the emulsified Liquid, connected to a 25G needle, to get rid of the air bubbles in the syringe. Rabbits were removed f...

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Abstract

The invention takes beta nodaviruses infected with paralichthys olivaceus and with the fatality rate close to 100% as objects. By means of primer design, a His tag needed by affinity purification is connected with the C end of reconstructed nodavirus capsid protein to construct an expression vector, and it is guaranteed that expression products obtained due to purification are the capsid protein with complete translation of an amino acid sequence presented in a sequence table SEQIDNo.2; efficient expression of objective protein can be obtained by means of BL21(DE3)ply S host bacteria, and separated, authentication and quality control are carried out on products by means of SDS-PAGE and mass spectrums; under the condition of 4 DEG C, urea, a buffer solution with the PBS concentration of 0.8 M and a buffer solution with the PBS concentration of 0.05 M are used, the pH value of each buffer solution is 7.0, and efficient renaturation of inclusion bodies is achieved. In the prior art, effective method against the nodaviruses infected with paralichthys olivaceus does not exist. After Routine immunization is carried out by means of the reconstructed capsid protein prepared in the method, nodavirus resistance of cultured paralichthys olivaceus can be remarkably improved. Compared with the method of purification achieved step by step in infected hosts, the method has the advantages that the yield is large, batches are stable and the method is not limited by raw materials, and the method has good application prospects.

Description

[0001] This patent application was completed by the Tianjin Key Laboratory of Animal and Plant Resistance, School of Life Sciences, Tianjin Normal University, and was supported by the National Science and Technology Support Program (2011BAD13B04, 2011BAD13B07), the National 973 Project (2012CB114405), the National 863 Project (2012AA092205, 2012AA10A401) and the National Natural Science Foundation of China (30901094) funding. technical field [0002] The invention belongs to the field of applied biotechnology, and relates to a high-efficiency preparation method of a recombinant protein that can be used to improve the immunity of cultured flounder to beta-noda virus. More specifically: the construction of the capsid protein expression vector of flounder-infected beta noda virus, the product purification, the optimization of renaturation conditions and the evaluation of immune protection effect. Background technique [0003] Nodaviradae, also known as Nervous Necrosis Virus (N...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/40C12N15/70A61K39/12A61P31/14C12R1/93
CPCC07K14/005A61K39/00C12N15/70C12N2770/00022C12N2770/00034
Inventor 张亦陈刘逸尘耿绪云蒋嫣冉孙金生
Owner TIANJIN NORMAL UNIVERSITY