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Ubiquitin-like substrate fusion protein as well as preparation method and medical application

A fusion protein and fusion gene technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problem that the reactogenicity and immunogenicity of the expression products are not as good as that of soluble proteins, and the purification is inconvenient. Problems such as low protein expression

Inactive Publication Date: 2018-05-08
JILIN UNIV
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Problems solved by technology

[0005] The present invention further provides a method for preparing a fusion protein modified by NEDD8. The pColdTF cold-induced expression vector is used to efficiently prepare the fusion protein Strep tag II-NEDD8-PLA2, which overcomes the low expression of PLA2 protein in the existing method and the expression product exists in the form of inclusion bodies for subsequent use. Purification brings a lot of inconvenience, and the reactogenicity and immunogenicity of expression products are not as good as soluble proteins and other defects

Method used

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  • Ubiquitin-like substrate fusion protein as well as preparation method and medical application
  • Ubiquitin-like substrate fusion protein as well as preparation method and medical application
  • Ubiquitin-like substrate fusion protein as well as preparation method and medical application

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Embodiment 1

[0030] 1. Acquisition of chicken NEDD8 gene

[0031] According to Genbank #XP_015129231.1, the NEDD8 gene was optimally designed and synthesized, and the NdeⅠ restriction site CATATG, enterokinase recognition site: 5'-GATGACGACGACAAG-3', Strep tagⅡ tag: TGGAGCCACCCGCAGTTCGAAAAA, Linker: GGCGGTGGCGGAAGC, KpnI restriction site: GGTACC; Add AgeI restriction site ACCGGT to the C-terminus to form the gene sequence shown in SEQ no.1, artificially synthesized and connected to pUC57 plasmid to form recombinant plasmid pUC57-NEDD8.

[0032] 2. Acquisition of full-length genes

[0033] 1) Acquisition of PLA2-gene

[0034]According to the protein sequence of Genbank#NP_036117.1, a gene sequence encoding the 29th amino acid to the 151st amino acid of the protein was synthesized, and a glycine was added to the N-terminal, and the PLA2 gene fragment was recovered by double digestion with AgeI and XhoI.

[0035] 2) Ligate the two fragments obtained above under the action of T4 DNA Ligase...

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Abstract

The invention discloses ubiquitin-like substrate fusion protein as well as a preparation method and medical application, and provides a novel NEDD8 (neural precursor cell-expressed developmentally downregulated 8) modified fusion protein, which solves the technological problem of dissolvable expression of PLA2 on escherichia coli. A pColdTF cold-induced expression carrier is utilized by the preparation method to effectively prepare the fusion protein Strep tagII-NEDD8-PLA2, so the defects that the PLA2 protein expression amount is low, the expression product existing in a inclusion body form brings the inconvenience for subsequent purification in the prior art, the reactogenicity and immunogenicity of the expression product is poorer than the soluble protein and the like are overcome.

Description

technical field [0001] The invention belongs to the field of biotechnology and provides a ubiquitin-like substrate fusion protein and a preparation method, especially a fusion protein Strep tagII-NEDD8-PLA2 and a preparation method. The invention also provides a medical application of the NEDD8 modified fusion protein. Background technique [0002] NEDD8 (neural precursor cell-expressed developmentally downregulated 8) involved in the present invention is a protein encoded by 81 amino acids, which has a high similarity to ubiquitin. The initial report of NEDD8 is that it is highly enriched in the brain tissue of mouse embryos Northern blot experiments showed that NEDD8 was downregulated during development. In adult tissues NEDD8 is specifically expressed in heart and skeletal muscle. Moreover, the structure of NEDD8 has been analyzed. The overall structure is similar to that of ubiquitin. The main difference is that the surface regions of the two have different electrostati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/62C12N15/70C12Q1/37
CPCC07K14/46C07K2319/22C12N9/18C12Q1/37C12Y301/01004
Inventor 艾永兴杜辉许家翠吕岩周宏达周小露吴山力王铁东于浩王梦云王梦涵周文杰陈浩天王文学
Owner JILIN UNIV
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