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Bacillus coli periplasm cavity secretion type expression vector

An expression vector, Escherichia coli technology, applied in the use of vectors to introduce foreign genetic material, biochemical equipment and methods, applications, etc., can solve the problem of affecting correct conformation formation, increasing operating steps and production costs, and unfavorable foreign protein formation. Sulfur bridges, etc.

Inactive Publication Date: 2004-11-17
弘业新创抗体技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prokaryotic expression system also has some disadvantages: for example, foreign proteins cannot be glycosylated and easy to form inclusion bodies, etc.
However, the intracellular soluble expression method also has obvious disadvantages: the intracellular environment is a reducing environment, which is not conducive to the formation of correct disulfide bond bridges for foreign proteins, thereby affecting the formation of the correct conformation (Ritz D. et al., 2001); and it needs to break the cell wall by ultrasonic or pressure to release the soluble expression components in the cell, which is not conducive to large-scale culture and purification
However, the fusion expressed foreign protein needs to be digested by protease to remove the fused non-target protein part, which increases the operation steps and production cost, reduces the production efficiency, and is not conducive to large-scale preparation of the target protein.

Method used

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  • Bacillus coli periplasm cavity secretion type expression vector
  • Bacillus coli periplasm cavity secretion type expression vector
  • Bacillus coli periplasm cavity secretion type expression vector

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specific Embodiment approach

[0036] The present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that the embodiments in this specification are only for illustrating the present invention, but not limiting the scope of the present invention in any way.

Embodiment 1

[0039] Example 1. Construction of the periplasmic cavity secreted expression vector pFUW802s (see the construction route figure 1)

[0040] 1. Change the multiple cloning site of the bifunctional expression vector pFUW802.

[0041] according to Figure 1 Construction process, first design a pair of primers (primer 1: 5'-GGCCGCGAATTCAAATTCTATTTCGGATCCGGC-3' (SEQ ID NO: 2); primer 2: 5'-GGCCGCCGGATCCGAAATAGAATTTGAATTCGC-3' (SEQ ID NO: 3)), after annealing A double-stranded DNA fragment is formed, and the restriction site NotI is automatically formed at both ends of the fragment. The specific method is as follows: 1 μl each of primer 1 and primer 2, add 8 μl of water, and repeat the following reaction 10 times on a common PCR instrument: 94°C for 30 sec, 72°C for 30 sec. The reaction product was directly used as an exogenous fragment for the following ligation reaction.

[0042] About 1 μg of the vector pFUW802 was extracted using the plasmid mini-extraction kit from Shangha...

Embodiment 2

[0055] Example 2. Construction of pFUW802s / CEAscFv and expression identification (SDS-PAGE, Western Blot) and activity analysis (ELISA) of anti-CEA single-chain antibody

[0056] 1. Construction of pFUW802s / CEAscFv

[0057] The gene sequence of the anti-CEA single-chain antibody was excised from the intracellular soluble expression vector (pTCEF / CEAscFv) of the anti-CEA single-chain antibody constructed in our laboratory by XhoI / EcoRI double digestion reaction, and used as the foreign source of the following ligation reaction Fragment; pFUW802s and pFUW802 were also subjected to the same enzyme digestion reaction, and after recovering the large fragment, it was used as the carrier fragment for the following ligation reaction. The gene sequence of the anti-CEA single-chain antibody was obtained from reference (Koga H. et al., 1990). Digestion reaction mixture: about 1 μg carrier (10 μl), 2 μl 10× buffer, 1 μl XhoI and 1 μl EcoRI, 6 μl double distilled water. Reaction conditio...

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Abstract

The invention relates to a Bacillus coli periplasm cavity secretion type expression vector pFUW302s, wherein the vector is prepared through adding pectic enzyme (PelB) signal peptide (SEG ID NO:1) to the N end of exogenesis protein, and co-expression is achieved to exogenesis protein with FK06 conjugated protein quasi-peptidul-proryl-cis / trans isomerase (FkpA) to increase the expression level of exogenesis protein in bacillus coli periplasm cavity, a vector pFUW802s / CEA-scFv of the periplasm cavity secretion type expression antitumour-associated antigen single-chain antibody (scFv), a vector pFUW802s / hSLC of the secondary lymphoid-tissue chemokine, and a host cell containing the periplasm cavity secretion type expression vector.

Description

technical field [0001] The present invention relates to an E. coli periplasmic cavity secretion type expression vector, more specifically, relates to a kind of expression vector by adding PelB signal peptide (MKYLLPTAAAGLLLLAAQPAM, (SEQ ID NO: 1)) at the N-terminus of foreign protein, and make the foreign protein Co-expression with FK506-binding protein-like peptidylproline cis-trans isomerase to improve the expression level of foreign proteins in the periplasmic cavity of Escherichia coli; a method for constructing the expression vector; and containing the vector host cells. technical background [0002] Compared with eukaryotic expression systems (such as yeast, insect cells, mammalian cells, etc.), prokaryotic expression systems (mainly E. coli expression systems) have many unique advantages. For example, clear genetic background, rapid cell growth, short fermentation cycle, easy screening and gene recombination operations, rich expression systems, etc. However, the pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/56C12N15/61C12N15/63C12N15/67C12N15/70
Inventor 王祥斌黄华樑周炳赵琦赵宝锋张众
Owner 弘业新创抗体技术股份有限公司
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