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Bacillus anthracis gamma bacterial virus catenase expression method

A phage lytic enzyme, Bacillus anthracis technology, applied in the direction of lytic enzymes, microorganism-based methods, biochemical equipment and methods, etc.

Inactive Publication Date: 2008-12-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the amount of plyG expressed and purified by this method is relatively large, the endotoxin and heat source problems of this system still have certain limitations in its application.

Method used

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  • Bacillus anthracis gamma bacterial virus catenase expression method
  • Bacillus anthracis gamma bacterial virus catenase expression method
  • Bacillus anthracis gamma bacterial virus catenase expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0053] Embodiment 2, construction of recombinant expression plasmid

[0054] The PCR product was cut with SacI and EcoR I, and the secreted yeast expression vector pMEX9K ( figure 2 ) connection, transform Escherichia coli DH5α by TSS method, and screen the positive clones to extract the plasmid. Sent to Takara Company for sequencing, the sequencing results showed that the 702bp fragment was PlyG gene, two nucleotide positions were mutated, but they were all synonymous mutations, the amino acid sequences were completely consistent after comparison, the expression vector was successfully constructed, and the recombinant plasmid was named is pMEXplyG.

Embodiment 3

[0055] Embodiment 3, the efficient expression of PlyG gene in yeast

[0056] The recombinant plasmid pMEXplyG DNA was extracted, then digested with Sac I to linearize it, separated and purified, and then transformed into Pichia pastoris GS115 by electroporation. The transformed GS115 was screened by MD plate to obtain HIS + For recombinant clones, the clones grown on the MD plate were inoculated on the YPD--G418 plate and cultured in a 30°C incubator. Recombinant clones screened from MD plates and YPD plates with different G418 concentrations were inoculated in 5ml BMGY respectively, and cultured at 30°C and 300r / min until OD 600 2.0~6.0; centrifuge at 4000r / min for 4min at room temperature. The collected bacteria were suspended with BMMY and diluted to OD 600 After reaching 1.0, induce at 30°C 300r / min. Add methanol to 0.5% every 12h. After 48 h of induction, the culture supernatant was collected, and the protein expression was detected by SDS-PAGE ( image 3 ) and meas...

Embodiment 4

[0057] Embodiment 4, three-step chromatographic purification of PlyG protein

[0058] Chromatographic purification methods mainly include ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and gel filtration chromatography. In the present invention, PlyG is secreted and expressed in the fermentation supernatant, and bacteria and large particle impurities are removed by centrifugation, filtration, etc.; then small molecule pigments and salts are removed by ultrafiltration, and the volume is concentrated, and the fermentation broth is adjusted A suitable buffer is beneficial for the next step of chromatographic purification.

[0059] The first step of chromatographic purification uses cation exchange medium SP Sepharose XL. SP Sepharose XL medium has the characteristics of high loading, fast flow rate and easy handling, and can quickly and effectively capture plyG from the ultrafiltrate. After loading the sample, in consideration of th...

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Abstract

The invention discloses a method for expressing anthrax bacteria gamma bacteriophage lysins. The invention connects an anthrax bacteria bacteriophage gene and pMEX9K, by adopting a pichia pastoris expression system and an expression vector pMEX9K (ZL02117906.9) with intellectual property right, to construct a new expression plasmid pMEXplyG; reservoir host Pichia pastoris GS115 undergoes electrotransformation after linearization; a producing strain G6 is obtained after random selection and pressure filtering, so that the gamma bacteriophage lysins are expressed in pichia pastoris G6 supernatant, and a expression product does not contain superfluous amino acid; and the gamma bacteriophage lysins with the purity of over 95 percent are obtained after centrifuging, ultrafiltering, ion-exchange chromatography, dewatering chromatography and gel chromatography. The anthrax bacteria gamma bacteriophage lysins have cleavage activity to anthrax bacteria and fungus gemma in sprouting, and can be used to develop anthrax decontaminating agents and anthrax medicines.

Description

technical field [0001] The invention relates to a method for expressing Bacillus anthracis gamma phage lyase Background technique [0002] Bacillus anthracis is a Gram-positive large bacillus that can form spores. Anthrax caused by its infection is a fatal and major infectious disease that endangers humans. Bacillus anthracis forms a large number of spores under aerobic conditions. After the spores are inhaled by the human body, they germinate in the lymphocytes around the alveoli, and their propagules and toxins enter the blood, causing more than 99% of unprotected people to die. [0003] The spores have strong viability and are still infectious after surviving in dry soil for more than 40 years. At present, new outbreaks of bacterial pathogens continue to appear, and antibiotic resistance is getting higher and higher, and they are widespread in the world. Lyase can kill bacteria The mechanism of action is different from that of antibiotics. It cracks the bacterial cell wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/88C12R1/84
Inventor 陈薇曹晓梅李曼付玲候利华李建民张晓鹏董大勇宋小红
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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