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Gene and method for preparing recombinant fugu rubripes IFN-gamma protein

A technology of redfin puffer and gamma protein, which is applied in the field of biogenetic engineering to achieve high repeatability and good stability

Inactive Publication Date: 2017-05-24
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there is no relevant report on eukaryotic expression of recombinant redfin puffer IFN-γ protein

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  • Gene and method for preparing recombinant fugu rubripes IFN-gamma protein
  • Gene and method for preparing recombinant fugu rubripes IFN-gamma protein
  • Gene and method for preparing recombinant fugu rubripes IFN-gamma protein

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Embodiment Construction

[0017] The present invention uses bioinformatics software to analyze the IFN-γ DNA sequence of pufferfish (GenBank: AJ616216.2) to obtain its mature peptide sequence, which encodes 167 amino acids. Codon-optimized target gene sequence and added at its N-terminus xho Ⅰ and kex 2 protease cleavage site, the C-terminal retains the stop codon and adds Xba Ⅰ Restriction site. The optimized IFN-γ DNA sequence is shown as SEQ ID NO: 1, which is used to prepare recombinant redfin puffer IFN-γ protein.

[0018] The preparation method of recombinant redfin puffer IFN-γ protein of the present invention is carried out according to the following steps successively:

[0019] a. Synthesize the gene shown in SEQ ID NO:1 and clone it into the eukaryotic expression plasmid pPICZαA to obtain the eukaryotic expression recombinant plasmid pPICZαA-IFN-γ.

[0020] The obtained eukaryotic expression recombinant plasmid pPICZαA-IFN-γ was identified by double enzyme digestion, and the results w...

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Abstract

The invention discloses a gene and method for preparing recombinant fugu rubripes IFN-gamma protein. The gene has a DNA sequence as shown in SEQ ID NO:1. The method comprises the following steps: cloning the gene having a DNA sequence as shown in the SEQ ID NO:1 onto eukaryotic expression plasmids pPICZ alpha A to obtain eukaryotic expression recombinant plasmids pPICZ alpha A-IFN-gamma; electronically transforming the eukaryotic expression recombinant plasmids pPICZ alpha A-IFN-gamma into a pichia pastoris GS115 competent cell, and screening positive clone; inoculating the screened positive clone into a BMGY liquid culture medium, enlarging and culturing until OD600nm is 2-6, and centrifuging to collect strains; and extracting protein after methanol induced expression to obtain recombinant fugu rubripes IFN-gamma protein.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to a gene and a method for preparing recombinant redfin puffer IFN-γ protein. Background technique [0002] Redfin puffer is an important cultured species in China, with high economic value and mature breeding and breeding technology. With the continuous expansion of breeding scale, diseases, especially viral, parasitic and bacterial diseases, are very harmful to red fin puffer and cause serious losses to the breeding industry; disease prevention and control has become a more difficult problem in the breeding process. At present, the research on the disease of pufferfish is still immature, and the prevention and control of the disease is still based on prevention. Therefore, it is particularly important to improve the disease resistance of the fish itself in the breeding process. [0003] Interferon gamma (interferon gama, IFN-γ) belongs to type II interferon and i...

Claims

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Application Information

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IPC IPC(8): C12N15/23C12N15/81C07K14/57
Inventor 王秀利孔德荣李慧孙鹤刘海映仇雪梅
Owner DALIAN OCEAN UNIV
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