Wide host perforating plasmid carrier, construction method thereof and applications in bacterial ghost preparation

A plasmid vector and host technology, applied in the field of genetic engineering, can solve problems such as narrow application scope

Inactive Publication Date: 2008-05-21
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

[0005] At present, most of the punching plasmid vectors used in the preparation of ghost sloughs of Gram-negative bacteria abroad are derived from the shuttle plasmid vectors between the target strain and Escherichia coli, and the punching plasmid vectors constructed on this basis can generally only be used to prepare The target strain or the ghost slough of a closely related strain has a relatively narrow application range

Method used

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  • Wide host perforating plasmid carrier, construction method thereof and applications in bacterial ghost preparation

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Embodiment 1

[0024] Example 1 Construction of the broad host punching plasmid vector pBBR-E

[0025] 1.1 Cloning of bacteriophage PhiX174 lytic gene E

[0026] Primers were designed according to the coding sequence of bacteriophage PhiX174 lytic gene E in GenBank:

[0027] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO.1)

[0028] Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO.1)

[0029] Restriction sites EcoR I and BamHI were introduced into the 5' ends of the upstream and downstream primers, 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, 10x Taq buffer, Taq TM DNA polymerase 2 U (TaKaRa), template DNA 10ng. The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 30s, 59°C for 30s, 72°C for 30s, and 72°C for 5 minutes. After ...

Embodiment 2

[0037] Example 2 Preparation of Bordetella bronchiseptica ghost

[0038] The broad-host punching plasmid vector pBBR-E was transformed into the competent cells of Bordetella bronchiseptica phase I "A50-4" strain by electric shock, and the electric shock competent was prepared according to the method of Sambrook et al. (Molecular Cloning Experiment Guide), The electric shock parameters of the electroporation instrument (Micro-pulser, Bio-Rad, USA) are: 12.5 kV / cm, 200Q, 25μf, 5.0ms, and Amp is coated after transformation + The modified Bauer's agar plate (10% defibrated sheep blood), and the transformants were identified by colony PCR. Inoculate the Bordetella bronchiseptica containing the broad-host punching plasmid vector pBBR E into the modified Bordetella (50 μg / mL ampicillin) liquid medium, shake culture overnight at 28°C (220r / min), and then transfer 1 -2mL in 50mL modified Bauer Jiang's liquid medium containing 50μg / mL ampicillin, shake culture at 28℃ until OD 600nm up...

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Abstract

The invention discloses a broad-host perforated plasmid vector p BBR-E and a construction method thereof; the invention also discloses the purpose of the broad-host perforated plasmid vector p BBR-E in the process of preparing the ghost of Bordetella bronchiseptica, which belongs to the gene engineering field. The broad-host perforated plasmid vector p BBR-E of the invention consists of a temperature sensitive controlling gene cI857, a phage bacteriolysis gene E and a broad-host plasmid p BBR4-MCS. The perforated plasmid vector p BBR-E of the invention is constructed based on the plasmid vector p BBR4-MCS which is provided with a broad host range and the vector can be duplicated in mostgram negativebacteria and express the bacteriolysis gene E with a high efficiency; the decomposition rate is high in the process of preparing the ghost of Bordetella bronchiseptica which can reach 99.9997 percent and is about 4 magnitudes high than the prior art.

Description

technical field [0001] The present invention relates to a plasmid vector, in particular to a broad-host perforated plasmid vector and a construction method thereof. The present invention also relates to the use of the broad-host perforated plasmid vector in preparing Bordetella bronchiseptica slough, which belongs to genetic engineering field. Background technique [0002] A "Bacterial Ghost" is an empty bacterial body devoid of 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 membra...

Claims

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Application Information

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