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Tobacco etch virus protease active inclusion body as well as preparation method and application thereof

A technology of tobacco etch virus and protease activity, applied in the biological field

Inactive Publication Date: 2016-08-24
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this strategy is easy to think of, it faces the following technical problems in the specific design: how to construct an N-terminal self-polypeptide fusion expression vector, whether the natural TEVP coding sequence is suitable for expression in Escherichia coli, and whether fusion with self-polypeptide can form What are the best conditions for active inclusion bodies, separation and purification, and enzyme digestion reactions?

Method used

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  • Tobacco etch virus protease active inclusion body as well as preparation method and application thereof
  • Tobacco etch virus protease active inclusion body as well as preparation method and application thereof
  • Tobacco etch virus protease active inclusion body as well as preparation method and application thereof

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Experimental program
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Embodiment Construction

[0033] Source of biological material:

[0034] 1. pET-30a vector: imported from Novagen, USA, and preserved in our laboratory.

[0035] 2. Escherichia coli DH5a: imported from BD Biosciences Clontech, USA, and preserved in our laboratory.

[0036] 3. BL21(DE3) Escherichia coli: imported from Novagen, USA, and preserved in our laboratory.

[0037] 4. MDBK cells: imported from ATCC in the United States and preserved in our laboratory.

[0038] 5. Vesicular stomatitis virus (VSV): preserved by our laboratory (document: Xu Yaoxian, Zhou Xiaofeng, Liu Lide. Molecular Virology. Wuhan: Hubei Science and Technology Press, 2000.300-302).

[0039] 6. pGEX-BoIFNg vector: Constructed and preserved by our laboratory (document: Xu Jinjun, Qin Aijian, Jin Wenjie, etc. Cloning of cow gamma interferon gene and its expression in Escherichia coli. Journal of Yangzhou University, 2003, 24(1): 5-10).

[0040] The specific operation steps are as follows:

[0041] 1. Construction of pP16P-TEVP ...

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Abstract

The invention belongs to the field of biotechnology research, and particularly relates to a TEVP (Tobacco Etch Virus Protease) active inclusion body as well as a preparation method thereof and application thereof in the preparation of a recombinant protein. The TEVP active inclusion body consists of an ELK16 self-polymeric peptide and TEVP that are separated by two PT connectors. The preparation method of the TEVP active inclusion body comprises the steps of an expression vector construction, an active inclusion body expression and purification, recombinant protein cleavage and target protein recovery. Compared with existing TEVP, the TEVP active inclusion body proviced by the invention has the advantages of simple preparation, easiness for being removed from an end product, low cost and the like, and can be applied to prepare various recombinant proteins.

Description

technical field [0001] The invention relates to the field of biotechnology research, in particular to a preparation method of tobacco etch virus protease activity inclusion body and its application in the preparation of recombinant protein. technical background [0002] Purification of recombinant proteins has always been one of the bottlenecks restricting molecular biology research and production of biological products. Gene fusion technology can simplify recombinant protein purification, and the most commonly used fusion tag is an affinity tag. Although affinity tag fusion proteins can be purified by chromatography, there are disadvantages such as time-consuming, expensive and difficult to scale up. In most cases, these fusion proteins require removal of the affinity tag in order for the protein of interest to fold properly or become active. A common practice is to introduce a protease recognition site between the fusion tag and the target protein, and then use protease ...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/70C12N15/57C12P21/06
CPCC12N9/506C07K14/57C12N15/70C12N2800/101C12N2800/22C12P21/06
Inventor 孙怀昌李光亚卢会鹏肖真真李洋洋周晓慧谭笑张鑫宇夏晓莉
Owner YANGZHOU UNIV
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