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Recombinant human cell restin MHD fusion protein and preparation method thereof

A fusion protein and resting technology, applied in the biological field, can solve problems such as large molecular weight, affecting gene drug efficiency, complex secondary structure of mRNA, etc., and achieve the effect of easy purification and wide target effect

Inactive Publication Date: 2011-02-09
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complex secondary structure of the mRNA of the restin gene, it is not conducive to the exogenous expression of the prokaryotic system; and the molecular weight of the full-length Restin protein is relatively large, which is 24.4kD, which will also affect the efficiency of the gene drug in the later stage; in addition, the Restin-MHD polypeptide can For Π-like MAGE molecules, it has a wider range of target effects than Restin

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  • Recombinant human cell restin MHD fusion protein and preparation method thereof
  • Recombinant human cell restin MHD fusion protein and preparation method thereof
  • Recombinant human cell restin MHD fusion protein and preparation method thereof

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Embodiment Construction

[0038] The present invention realizes the preparation of recombinant human Restin melanoma-associated antigen homology domain through biotechnology, and constructs the prokaryotic expression vector pET44a(+)-Restin- MHD , and transformed into Escherichia coli to obtain transformants, and then the Escherichia coli transformants were induced and expressed, and the Restin melanoma-associated antigen homology domain fusion protein was obtained after separation and purification. SDS-PAGE and Western Blot analysis of the Restin domain fusion protein showed the same molecular weight results; and biological activity verification showed that the Restin domain fusion protein had a cell cycle arrest effect on tumor cells. The present invention will be described in detail below, which is an explanation of the present invention rather than a limitation.

[0039] 1. Restin- MHD gene cloning

[0040] The human Restin gene (NM 014061) was searched in GenBank, and its melanoma-associated ant...

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Abstract

The invention discloses a recombinant human cell restin MHD fusion protein and a preparation method thereof. The fusion protein comprises a human cell restin MHD structure domain, and an N end of the human cell restin MHD structure domain is connected with a purification label sequence, the enzyme digesting sites of thrombase and enterokinase and a Nus sequence. In the invention, the transformant of the restin-MHD fusion protein is obtained by constructing a pronucleus expression vector pET44a(+)-Restin-MHD-MHD and transferring the pronucleus expression vector Escherichia coli, the expression of the transformant is induced in the Escherichia coli, and the restin-MHD fusion protein is obtained by separating and purifying. Bioactivity verification proves that the restin fusion protein has a cell cycle arrest effect on tumuor cells and can be used for preparing antitumor medicaments.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the expression of a recombinant fusion protein, in particular to a recombinant human cell resting factor MHD fusion protein and a preparation method thereof. Background technique [0002] Restin is a new member of the melanoma associated antigen (MAGE) family cloned by Zhu et al. in 1999. The full length of the Restin gene is 1475bp, encoding 219 amino acids. The gene product has different localizations in different cell lines and is expressed in both the cytoplasm and the nucleus. [0003] The 8-25 amino acids at the N-terminal of the Restin protein is a typical binary nuclear localization signal sequence, and the 42-52 amino acids are the casein kinase II phosphorylation site, and the phosphorylation of this site also affects the nuclear localization of the Restin protein;88 The -176th amino acid is the homology domain MHD of the MAGE family, and the theoretical pI value of this doma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/18C12N15/70C12N15/12C07K1/22
Inventor 路凡王卫华吴有盛王莉王孝功沈琦药立波
Owner FOURTH MILITARY MEDICAL UNIVERSITY