pORF65 recombinant protein and preparation method and application thereof

A recombinant protein, porf65 technology, applied in the field of pORF65 recombinant protein and its preparation, can solve the problem that carp serum IgM has not yet been applied

Active Publication Date: 2018-05-11
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although carp is an important economic fish, the purification of its serum IgM has only been reported by gel filtration chromatography, and affinity chromatography, as a method for rapid preparation of high-purity antibodies, has not been applied to the purification of carp serum IgM

Method used

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  • pORF65 recombinant protein and preparation method and application thereof
  • pORF65 recombinant protein and preparation method and application thereof
  • pORF65 recombinant protein and preparation method and application thereof

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Experimental program
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Embodiment

[0030] 1. Method

[0031] 1.1 Design and synthesis of primers

[0032] Primers were designed according to the sequence of CyHV419 strain ORF65 (GenBank No. KP004895) (see Table 1). Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0033] Table 1 ORF65 amplification primer sequence

[0034] Table 1 Sequence of ORF65 gene amplification

[0035]

[0036]

[0037] 1.2 ORF65 gene and protein sequence analysis and codon optimization

[0038] Send the gene coding sequence truncating the signal peptide and the C-terminal transmembrane hydrophobic segment to Jinweizhi Company for codon optimization and sequence synthesis.

[0039] The full length of ORF65 of CyHV-3419 strain is 1785bp, encoding 594 amino acids. Finally, five B cell epitope dominant segments were determined, namely Phe97-Gly172 (A), Ala199-Val284 (B), Lys307-Ala361 (C), Tyr426-Thr445 (D), Thr473-Asn546 (E) (see figure 1 ).

[0040] figure 1 Middle ↓ indicates the signal peptide cleavage...

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Abstract

The invention discloses a pORF65 recombinant protein and a preparation method and application thereof. The pORF65 recombinant protein comprises amino acid sequences shown in the formulas of SEQ ID NO.17 to SEQ ID NO.21 orderly connected by connexins, and the connexin is composed of 4-7 hydrophilic amino acids with small molecular weight and polarity. The ELISA result shows that the pORF65 recombinant protein can be used as a coating antigen and can well detect a serum-specific antibody produced by a cryprinus carpiod immunized by a DNA vaccine constructed from a complete coding sequence of CyHV-3 pORF65 and diagnose whether the cryprinus carpiod infects cyprinid herpesvirus III.

Description

technical field [0001] The invention relates to the field of antigens and antibodies, in particular to pORF65 recombinant protein and its preparation method and application. Background technique [0002] Carp herpesvirus type 3 (CyHV-3), also known as koi herpesvirus (KHV), is a double-stranded linear DNA virus with an envelope structure. It has been prevalent worldwide since it was reported in the 1990s. The koi herpesvirus disease (KHVD) caused by the virus has caused huge economic losses and seriously threatened the safety of carp and koi industries. In the research of CyHV-3 prevention and treatment, the DNA vaccine based on the viral envelope protein has become an important research direction. The viral envelope protein participates in the process of recognition, interaction and infection between the virus and host cells, and can induce a strong humoral immune response , is an important candidate molecule for the development of DNA vaccines. In order to accurately eva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03C12N15/38C12N15/70G01N33/569
Inventor 马艳平刘振兴马江耀梁志凌曹俊明郝乐柯浩
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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