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Gene and method for preparing recombinant fugu rubripes interleukin 2 protein by eukaryotic expression

A technology of red-finned pufferfish and interleukin, which is applied in the biological field to achieve the effect of high repeatability and good stability

Inactive Publication Date: 2017-07-07
DALIAN OCEAN UNIV
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Problems solved by technology

By 2014, the interleukin-2 gene sequences of grass carp, trout, green puffer, red-finned puffer, stickleback, rainbow trout, perch and other interleukin-2 genes had been registered in Genebank, and some of them had been successfully cloned, but the red-finned puffer interleukin-2 gene Expression studies in eukaryotic cells have not been reported

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  • Gene and method for preparing recombinant fugu rubripes interleukin 2 protein by eukaryotic expression
  • Gene and method for preparing recombinant fugu rubripes interleukin 2 protein by eukaryotic expression
  • Gene and method for preparing recombinant fugu rubripes interleukin 2 protein by eukaryotic expression

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Embodiment Construction

[0016] The present invention is based on the natural redfin puffer interleukin 2 gene and optimizes the codon for the codon preference of Pichia pastoris, and adds xho Ⅰ and kex 2 protease cleavage site, the C-terminal retains the stop codon and adds Xba Ⅰ Restriction site. The optimized gene DNA sequence is shown as SEQ ID NO: 1, which is used for eukaryotic expression to prepare recombinant redfin puffer interleukin-2 protein.

[0017] The eukaryotic expression of the present invention prepares the method for recombination red fin puffer interleukin 2 protein, carries out according to the following steps successively:

[0018] 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-IL-2, and carry out the eukaryotic expression recombinant plasmid pPICZαA-IL-2 obtained Double enzyme digestion identification, the results are as follows figure 1 shown.

[001...

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Abstract

The invention discloses a gene for preparing a recombinant fugu rubripes interleukin 2 protein by eukaryotic expression and a preparation method, wherein the protein can be efficiently expressed and an expression product has obvious biological activity. The preparation method comprises the following steps of: cloning a gene as shown in SEQ ID NO: 1 into a prokaryotic expression vector pPICZalphaA to construct an eukaryotic expression plasmid pPICZalphaA-IL-2; electrically converting the eukaryotic expression plasmid into a pichia pastoris GS115 competent cell and screening a positive clone; inoculating the screened positive clone to a BMGY liquid culture medium to be cultured in an amplified manner till OD600nm is equal to 2-6, and centrifugally collecting thalli; and extracting the protein after methanol inducible expression to obtain the recombinant fugu rubripes interleukin 2 protein. Verified by MTT, the recombinant protein has activity of promoting CTLL-2 cell proliferation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene and method for preparing recombinant redfin puffer interleukin 2 protein through eukaryotic expression capable of high-efficiency expression and whose expression product has obvious biological activity. Background technique [0002] Redfin puffer is the main species of fish cultured and exported in China. However, viral diseases such as white-mouth disease and red sea bream iridescent virus disease and bacterial diseases such as vibriosis and streptococcal disease often occur during breeding, which reduces the yield of adult fish and increases the cost of breeding. Therefore, for example, improving the autoimmune function of the fish body during the breeding process of the red-finned puffer can not only ensure the food safety of the fish body, but also avoid the pollution of the environment caused by chemical drugs. [0003] Cytokines are important members of the ...

Claims

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Application Information

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IPC IPC(8): C12N15/26C07K14/55C12N15/81C12R1/84
Inventor 王秀利孙鹤李慧孔德荣刘海映仇雪梅
Owner DALIAN OCEAN UNIV
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