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Bacillus coli perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation

A technology of Escherichia coli and plasmid vectors, applied in the field of genetic engineering, can solve the problems of insufficient expression of E gene and low lysis efficiency

Inactive Publication Date: 2008-05-21
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

In these studies, most of the punched plasmid vectors used had only one promoter during the construction process, that is, the pR or pL promoter to promote the expression of the E gene, resulting in insufficient expression of the E gene. When using it to prepare ghost, There are defects such as low cracking efficiency, which need to be overcome

Method used

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  • Bacillus coli perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation

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Effect test

Embodiment 1

[0022] Example 1 Construction of the punched plasmid vector pBV-E

[0023] 1.1 Cloning of bacteriophage PhiX174 lytic gene E

[0024] Primers were designed according to the coding sequence of bacteriophage PhiX174 lytic gene E in GenBank:

[0025] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO.1)

[0026] Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO.1)

[0027] Restriction sites EcoR I and BamH I were introduced into the 5' ends of the upstream and downstream primers respectively, which were synthesized by Shanghai Sangong. Use bacteriophage PhiX174 double-stranded DNA as a template to amplify the lytic gene E: PCR amplification reaction system is 50 μL, in which MgSO 4 2mM, 1μM each for upstream and downstream primers, dNTP 200μM, 10xTaq buffer, Taq TM DNA polymerase 2U (TaKaRa), template DNA 10ng. The PCR reaction program was: 95°C pre-denaturation for 5 min, 30 cycles at 94°C for 30 s, 59°C for 30 s, 72°C for 30 s, and 72°C for 5 min. After gel ...

Embodiment 2

[0030]The preparation of embodiment 2 Escherichia coli Ghost (sludge)

[0031] Inoculate Escherichia coli TG1 containing punched plasmid vector pBV-E in 5 mL of LB containing 50 μg / mL ampicillin, culture overnight at 28°C with shaking (220 r / min), then transfer 1-2 mL to 50 mL containing 50 μg / mL ampicillin Incubate LB with shaking at 28°C to OD 600nm Up to about 0.4. Take out 100 μl of the culture for later use, quickly increase the remaining culture to 42°C to induce the expression of the E gene, and continue to cultivate for 3-5 hours; take 100 μl of the culture before and after the induction and properly dilute it, and spread it on an LB plate to detect the CFU of live bacteria. The ghost slough formed after lysis was washed 3 times with PBS, stored in a freeze-dried manner, and the CFU of live bacteria was detected on the freeze-dried slough.

[0032] Test results: the culture before and after lysis from 3.1×10 8 down to 1.2×10 4 (CFU / ml), the bacteriolysis and inacti...

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Abstract

The invention discloses a colon bacillus perforated plasmid vector p BV-E and a construction method thereof; the invention also discloses the purpose of the colon bacillus perforated plasmid vector in the process of preparing the ghost of the colon bacillus, which belongs to the gene engineering field. The colon bacillus perforated plasmid vector of the invention adopts two promoters of pR and pL serial double promoters to express E gene and successfully prepares the ghost of the colon bacillus; the decomposition rate can reach 99.9613 percent which is 3 magnitudes higher than of the present perforated plasmid vector with pR or pL single promoter.

Description

technical field [0001] The invention relates to a plasmid vector, in particular to an Escherichia coli perforated plasmid vector and a construction method thereof, and also relates to the use of the Escherichia coli perforated plasmid vector in preparing Escherichia coli slough, which belongs to the field of genetic engineering. Background technique [0002] Bacterial Ghost is an empty bacterial body without cytoplasm and nucleic acid. The PhiX174 phage E cleavage gene is expressed in bacteria. The protein encoded by the gene can form a transmembrane tunnel on the bacterial cell membrane and cell wall, and the bacteria will rupture under the action of osmotic pressure. The cytoplasm and nucleic acid components in the bacterial cell pass through this tunnel. Expelled, forming an empty shell of bacteria, that is, "Bacterial Ghost". Bacterial ghost is composed of inner membrane (cytoplasmic membrane), cytoplasmic space (periplasmic space) and outer membrane (outer membrane), s...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N1/21
Inventor 刘思国王春来常月红刘惠芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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