Fusion protein of MCP of nervous necrosis virus and ompN1 of edwarsiella ictaluri as well as preparation method of fusion protein

A technology of nerve necrosis and fusion protein, applied in the field of protein separation and purification

Pending Publication Date: 2020-10-16
王大勇
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the VNN virus vaccine must be injected to play an effective preventive role. At present, there is no genetically engineered vaccine and antigen that can achieve effective vaccination through mucosal immunity, such as soaking or feeding.

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  • Fusion protein of MCP of nervous necrosis virus and ompN1 of edwarsiella ictaluri as well as preparation method of fusion protein
  • Fusion protein of MCP of nervous necrosis virus and ompN1 of edwarsiella ictaluri as well as preparation method of fusion protein
  • Fusion protein of MCP of nervous necrosis virus and ompN1 of edwarsiella ictaluri as well as preparation method of fusion protein

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specific Embodiment approach

[0027] Specific embodiments: In this embodiment, the expression and separation and purification of gene recombinant MCP-ompN1 fusion protein are carried out according to the following steps:

[0028] 1. The MCP-ompN1 CDS sequence was optimized and designed according to the codon preference in the prokaryotic expression system, so as to be suitable for high-efficiency expression in the E. coli BL21-DE3 strain. For the CDS sequence, see Figure 3-8 . BamHI and SalI are used as restriction sites at the upstream and downstream ends of the CDS sequence of the fusion expression protein, the pET-28a vector has a His tag, and the thrombin substrate sequence "LVPRGS" between the His tag and the MCP-ompN1 fusion protein " Nucleotide sequence.

[0029] 2. Digest the CDS fragment BamHI-MCP-link-ompN1-SalI and pET-28a blank vector with BamHI and SalI respectively.

[0030] 3. Ligate the BamHI-MCP-link-ompN1-SalI fragment and the pET-28a vector with T4 ligase to construct the gene recombi...

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Abstract

Existing commercial fish nervous necrosis virus vaccine must be vaccinated by injection. The invention relates to a gene recombinant fusion protein MCP-ompN1 of a major coat protein (MCP) of nervous necrosis virus and ompN1, having a transmembrane effect, of edwarsiella ictaluri as well as expression and a preparation method of the fusion protein. The MCP and ompN1 protein are flexibly linked by asequence composed of four amino acids ''GGGS''". A nucleotide sequence encoding MCP-ompN1 is first optimized according to the codon preference of a BL21-DE3 engineering strain, then an expression plasmid is constructed and expressed in the BL21-DE3 strain, and then the MCP-ompN1 fusion protein is separated and purified through bacterial disruption, inclusion body treatment, protein renaturation,nickel column purification, removal of histone tags with biotin-labeled thrombin, removal of thrombin with avidin resin and other steps. The gene recombinant MCP-ompN1 fusion protein can be used as avaccine antigen of nervous necrosis virus, or a vaccine antigen of nervous necrosis virus and edwarsiella ictalurid, and the ompN1 protein in the MCP-ompN1 fusion protein mediates mucosal immunity.

Description

technical field [0001] The invention belongs to the field of genetic engineering. It specifically involves the primary structure of DNA and protein, the construction and expression of expression vector, and the separation and purification of protein. Background technique [0002] Viral neuronecrosis is the most common and most harmful infectious disease in the southeast coastal areas of my country, especially in Hainan. It often leads to the death of a large number of grouper fry and larvae. The infectious disease has typical neurological symptoms, causing vacuolar necrotic lesions in the central nervous and retinal tissues of fish, and abnormal motility. In addition to neurological abnormalities, the diseased fish had no other clinical symptoms, and no obvious pathological changes or parasites on the surface and inside of the body. The disease can be transmitted vertically through fish eggs, and can also be transmitted horizontally through aquaculture water. Vertical tra...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C07K1/14
CPCC07K14/005C07K14/24C12N2770/30022C07K2319/00
Inventor 王大勇张转丹裴业春
Owner 王大勇
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