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Recombinant soluble streptococcus hemolyticus haemolysin O gene, recombinant protein and preparation thereof

A hemolytic streptococcus, soluble technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as difficult industrial production, low yield, and large batch-to-batch variation

Inactive Publication Date: 2008-11-12
SICHUAN MACCURA BIOTECH CO LTD
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

The purification of hemolysin O from natural hemolytic streptococci has many insurmountable shortcomings, such as: very low yield; large batch-to-batch variation; difficult industrial production; biological safety hazards, etc.

Method used

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Embodiment Construction

[0011] In order to achieve the purpose of the present invention, the preparation of r-sSLO protein in the present invention comprises the following steps:

[0012] (1) Clone the r-sSLO gene and construct the expression plasmid r-sSLO-PET32a(+)

[0013] Primers were designed according to the amino acid sequence of the SLO gene, the gene was amplified by PCR and connected to the pMD18-T vector, transformed into Escherichia coli DH5a, the single clone was identified by PCR to determine the possible positive plasmid, and then the plasmid was extracted and identified by double enzyme digestion. Prokaryotic expression plasmid r-sSLO-PET32a(+) was constructed and transformed into the expression host strain Rosseta. gamiII. Positive host bacteria were identified by double enzyme digestion. The plasmid vectors used in the above experiments were all kept in our laboratory, the expression strain Rosseta gami II was purchased from Merck Company, and the restriction endonuclease was purc...

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Abstract

The invention relates to a recombinant protein for providing a soluble hemolysis streptolysin O gene and the gene expression, and a method for making the same. The method comprises the steps of: cloning from the streptococcus hemolyticus gene group by using the gene engineering technology to obtain a truncated SLO gene; connecting to an expression vector and transferring into host cell expression to purify a large amount of stable r-s SLO proteins, thereby overcoming various shortcomings of the protein from natural extraction and being favorable for standardization of making various reagents by using the protein as the raw material. The whole-length SLO protein is the protein of about 60 KD containing 571 AA, and the end N is provided with a signal peptide which is used for guiding the mature protein out of the cell. A research shows that the end N containing 77 AA provided with the signal peptide has a hazard function to the host cell, wherein the hazard function is more than a lyticase function which is generated by the latter AA of between 78 and 571.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant soluble hemolytic streptolysin O gene, a recombinant protein expressed by the gene and a preparation method thereof. Background technique [0002] Streptococcus hemolyticus lysin O is a cytolytic protein secreted by various types A, G and C hemolytic streptococci. After human infection with hemolytic streptococcus, the body will produce specific antibodies against hemolytic streptolysin O, and detecting the concentration of the specific antibodies is of great significance for clinical diagnosis; The biological activity of up-punching has been widely used in many frontier fields including cancer treatment in recent years. The purification of hemolysin O from natural hemolytic streptococcus has many insurmountable shortcomings, such as: very low yield; large batch-to-batch variation; difficult industrial production; biological safety hazards and so on. Cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/315C12N15/63C12N15/70
Inventor 喻坤王保宁陈丹杨卫平
Owner SICHUAN MACCURA BIOTECH CO LTD
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