Escherichia coli expression vector capable of controlling self-cracking of host bacterium

An expression vector and Escherichia coli technology, applied in the fields of microorganisms and genetic engineering, can solve the problems of high energy consumption, inactivation of target products, expensive mechanical equipment, etc., and achieve the effect of facilitating product recovery

Inactive Publication Date: 2011-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods often have some inherent disadvantages: the mechanical equipment is relatively expensive, the energy consumption in the process of use is large, and the mechanical shear force generated by it may also deactivate the target product

Method used

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  • Escherichia coli expression vector capable of controlling self-cracking of host bacterium
  • Escherichia coli expression vector capable of controlling self-cracking of host bacterium
  • Escherichia coli expression vector capable of controlling self-cracking of host bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Codon and endonuclease recognition site optimization of T4 lysozyme gene

[0058] 1) Site-directed mutation of the codon AGA located at both ends of the gene

[0059] The full sequence of the original T4 lysozyme structural gene is SEQ ID NO:3. Analysis of the structural gene of T4 lysozyme showed that three of the codons encoding arginine were AGA, which were codons that greatly affected gene expression in Escherichia coli and would seriously reduce the expression level of the gene. One of these three codons is near the 5' end of the gene, and the other is near the 3' end of the gene. These two codons can be directly mutated when amplifying the gene using the PCR method. It contains a Mlu I recognition site downstream of the AGA codon near the 3' end. Mlu I is a commonly used enzyme cutting site in genetic engineering. If it is retained in the gene, it may cause inconvenience to the construction of expression vectors and the application of vectors. The...

Embodiment 2

[0074] Embodiment 2: construction of plasmid pEtac2

[0075] Plasmid pEtac was double-digested with BglII and BamH I, and a fragment of about 5.4 kb was recovered by electrophoresis separation of the digested product. After purification, it was self-ligated, and the ligated product was transformed into E.coli JM109, and the transformants were screened on a kanamycin plate. Four transformants were randomly selected to extract plasmids and digested with Bgl II. The results showed that none of the four plasmids could be cut by Bgl II, which conformed to the characteristics of pEtac2. BglII and BamH I are homologous enzymes, which produce the same sticky ends after digestion, and can be annealed to each other, but the original restriction site disappears after connection, and neither enzyme can cut a new site. There is an Nco I restriction site between the Bgl II and BamH I sites in the original pEtac vector, and Nco I will not be able to cut the new plasmid after the fragment bet...

Embodiment 3

[0077] The construction of embodiment 3pEly expression vector

[0078] The lyMu gene fragment of about 580 bp was recovered by electrophoresis after the plasmid pMD-lyMu was digested with Sal I. Plasmid pEtac2 was digested with Xho I and ligated with the above-mentioned recovered fragment, the ligated product was transformed into Escherichia coli JM109, and transformants were screened on a kanamycin plate. A total of 20 transformants were selected to extract plasmids, and these plasmids were used as templates to carry out PCR with the following primers.

[0079] Ply 03: 5'-TTACGTATAGATGAACGTC-3' (SEQ ID NO: 8)

[0080] Pkan: 5'-GTTTCATTTGATGCTCGATG-3' (SEQ ID NO: 9)

[0081] The PCR amplification conditions were denaturation at 95°C for 5 minutes, 25 cycles of amplification reaction (94°C for 50 s, 56°C for 60 s, 72°C for 1 min), and extension at 72°C for 5 min. The plasmid extracted from one of the transformants was a template PCR to obtain an amplified fragment of about 1...

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Abstract

The invention discloses an Escherichia coli expression vector capable of controlling the self-cracking of a host bacterium, which belongs to the technical field of microbes and gene engineering. In the invention, the composition of the codon of a lysozyme gene in a T4 phage and a restriction endonuclease identification site are optimized, and the optimized T4 lysozyme gene mutation lyMu and a modified Escherichia coli expression vector are recombined to form a recombinant expression vector pEly. The expression vector pEly can be used for inducing expression of an exogenous gene, and when the exogenous gene is induced to express, T4 lysozyme is expressed. The cell undergoing induction expression is treated by ethylene diamine tetraacetic acid (EDTA) solution and then self-cracks, so that the product of expression in the cell is released. The expression vector obtained by implementing the invention can realize the express of various exogenous proteins in Escherichia coli hosts, the host bacteria release the products of expression after undergoing EDTA treatment, and thus, the recovery of the products of the expression can be promoted. The expression vector has an application prospect in fields of enzymic preparation production, polypeptide medicine production and the like.

Description

technical field [0001] The invention relates to the construction of an Escherichia coli expression vector by means of genetic engineering, and belongs to the technical fields of microorganisms and genetic engineering. Background technique [0002] Escherichia coli is the most commonly used host in modern genetic engineering. It has the advantages of convenient operation and high yield, and is the most commonly used host for expressing foreign proteins. However, Escherichia coli also has many disadvantages as a protein expression host, one of which is that when Escherichia coli expresses foreign proteins, the target product generally exists in the cell or in the periplasm, and few products can be secreted outside the cell. The first problem facing the recovery of intracellular products is the problem of cell wall breaking. There is an outer membrane outside the cell wall of Escherichia coli, so when using lysozyme to break the wall, it is necessary to add a surfactant to des...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/56C12N1/21C12R1/19
Inventor 沈微范如意王正祥
Owner JIANGNAN UNIV
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