High-expression streptolysin O (SLO) gene as well as secreting expression vector and application thereof

A technology of hemolysin gene and expression vector, applied in the field of streptococcus pyogenes hemolysin gene nslo, can solve the problems of difficult application and low expression yield, and achieve the effect of significant antigenicity and high-efficiency secretory expression

Inactive Publication Date: 2013-06-12
YANCHENG TEACHERS UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, in the current recombinant expression method of SLO, there is a significant disadvantage that the expression yield is always relatively low, below 0.3mg / mL, which makes it difficult to purify and apply in the later stage

Method used

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  • High-expression streptolysin O (SLO) gene as well as secreting expression vector and application thereof
  • High-expression streptolysin O (SLO) gene as well as secreting expression vector and application thereof
  • High-expression streptolysin O (SLO) gene as well as secreting expression vector and application thereof

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Experimental program
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Embodiment 2

[0022] Embodiment 2 nslo gene synthesis and cloning

[0023] Compared with the original slo gene, the nslo gene has a large change compared with the original slo gene, and it is not convenient to use mutation technology. Therefore, the nslo gene was synthesized in one step by chemical synthesis, and the synthesized nslo gene was connected to the pMD18T (Takara) vector, transformed into JM109, and the transformed product was coated. The LB plate containing 100g / mL ampicillin was cultured overnight at 37°C to obtain a single clone transformant nslo / pMD18T / JM109, which was identified by colony PCR, plasmid digestion and sequencing, and the newly synthesized nslo gene was 1485bp in length , encoding 495 amino acids, the determined sequence is exactly the same as the designed sequence.

Embodiment 3

[0024] Embodiment 3 The vector construction of secreting and expressing SLO protein

[0025] The vector used to realize the secretory expression of nslo gene in Escherichia coli is pET-20b (+), which has a pelB signal peptide and a His-tag tag. The pET-20b (+) plasmid and nslo / pMD18T are used with NocI and NotI cut, digested products were gel-recovered, then ligated with T4 ligase, transformed E.coli JM109 competent cells with ligated products, cultured overnight at 37°C, selected transformants, identified by colony PCR, and identified by plasmid digestion (see figure 1 ) and sequencing identification, save the identified nslo / pET-20b(+) / E.coli JM109 bacteria, and extract nslo / pET-20b(+) for future use.

Embodiment 4

[0026] Example 4 The construction of engineering bacteria that secretes and expresses the SLO protein

[0027] The plasmid nslo / pET-20b(+) was transformed into Escherichia coli host BL21(DE3), then cultured on 100g / mL ampicillin LB plate overnight at 37°C, and colony PCR identification was performed to obtain a single clone transformant nslo / pET- 20b(+) / BL21(DE3).

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Abstract

The invention discloses a high-expression streptolysin O (SLO) gene as well as a secreting expression vector and application thereof, belonging to the field of genetic engineering. In the invention, a slo gene sequence derived from streptococcus pyogenes NZ131 strain is used as a template (streptococcus pyogenes NZ131, complete genome: NCBI accession: NC_011375.1); escherichia coli optimized codons replace 56 escherichia coli rare codons; a new gene nslo of codified streptococcus pyrogenes SLO is designed and synthesized; and a secreting expression vector containing the nslo gene and an engineering bacterium containing the secreting expression plasmid are constructed. Indicated by an expression result, the protein expression yield can reach more than 3mg / mL, and enzyme activity can reach 2.4*106HU / mL. The invention realizes the efficient expression of the nslo, can be widely used for medical research and disease diagnosis and is suitable for industrialized production.

Description

technical field [0001] The invention relates to an artificially modified and synthesized highly expressed streptolysin gene nslo, which belongs to the field of medical microbial genetic engineering. The invention also relates to a secreted expression vector constructed according to the above gene and its application, belonging to the field of genetic engineering. technical background [0002] Hemolysin mainly comes from Streptococcus, Staphylococcus aureus and Bacillus. The hemolysin (Streptolysin O, SLO) from Streptococcus pyogenes (Streptococcuspyogenes) is cholesterol-dependent, and can bind to cholesterol on the cell membrane of humans and other animals, and the SLO protein molecule bound to the cell membrane will form a circular oligomerization body, forming large pores, leading to the dissolution of the cell membrane, and mediating some macromolecules (such as NADase) through the cell membrane, so SLO is an important toxin protein in the process of streptococcal infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/63C12N5/10C12N1/21C07K16/12C07K16/06G01N33/569
Inventor 赵庆新王欢莉康贻军
Owner YANCHENG TEACHERS UNIV
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