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Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation

A technology of chloramphenicol and lysate, applied in the field of genetic engineering, can solve the problem of inability to use antibiotic screening markers, etc., and achieve the effect of wide application and high lysis efficiency

Inactive Publication Date: 2012-07-18
SHANDONG NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the growing phenomenon of bacterial resistance, many bacteria are resistant to commonly used antibiotics such as ampicillin and kanamycin, and cannot use these antibiotics as selection markers

Method used

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  • Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation
  • Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation
  • Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation

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Embodiment 1

[0018] Embodiment 1, construction of chloramphenicol resistance temperature-controlled lysis plasmid pBV-geneE-cat

[0019] 1.1 Cloning of PhiX174 cleavage gene E

[0020] Primers were designed according to the coding sequence of PhiX174 gene E in GenBank:

[0021] geneE-F: 5'-ATCA GAATTC ATGGTACGCTGGACTTTGTG-3' (SEQ No.1), the introduction of EcoR I restriction enzyme site;

[0022] geneE-R: 5'-GGC CTGCAG CAGAACGTTTTTACCTTTAG-3' (SEQ No. 2), the Pst I restriction enzyme site was introduced.

[0023] Primers were synthesized by BGI. Gene E was amplified by PCR using PhiX174RFIDNA as a template. The PCR reaction system is TaKaRa Ex Taq 1.2U, 10×Ex Taq Buffer (Mg 2+ Plus) 5 μL, dNTP Mixture (2.5 mM each) 4 μL, PhiX174RFI DNA 2ng, geneE-F (20 μM) 1 μL, geneE-R (20 μM) 1 μL, and water up to 50 μL. The PCR reaction program was pre-denaturation at 94°C for 5 min, 28 cycles at 94°C for 45 s, 56°C for 45 s, and 72°C for 40 s, and extension at 72°C for 1 min. The PCR product ...

Embodiment 2

[0033] Embodiment 2, plasmid pBV-geneE-cat transformation Escherichia coli BL21 (DE3) prepares slough

[0034] 2.1 Electrotransformation of chloramphenicol-resistant temperature-controlled lysis plasmid pBV-geneE-cat

[0035] The chloramphenicol-resistant temperature-controlled lysis plasmid pBV-geneE-cat was electroporated to transform Escherichia coli BL21(DE3) competent cells. The electrotransformation competent cells were purchased from TaKaRa, referring to the method of Sambrook et al. (Molecular Cloning Experiment Guide) Preparation, electric shock parameters of electroporation instrument (Bio-Rad, California, USA): 1.25kV / cm, 25μF, 200Ω, 6.0ms, coated with LB agar plate containing 37μg / mL chloramphenicol after electroporation, 30℃ After culturing for 16 hours, transformants were identified by colony PCR.

[0036] Inoculate BL21(DE3) single clone containing chloramphenicol-resistant temperature-controlled cleavage plasmid pBV-geneE-cat into 5 mL LB liquid medium contain...

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Abstract

The invention discloses a chloramphenicol resistant temperature controlled lytic plasmid pBV-geneE-cat and its construction method, and also discloses application of the plasmid pBV-geneE-cat in preparing a bacterial ghost of Ecoli. BL21 (DE3). The chloramphenicol resistant temperature controlled lytic plasmid pBV-geneE-cat of the invention is composed of a lytic gene E of phage PhiX174, a chloramphenicol acetyl transferase gene (cat) and a prokaryotic temperature controlled expression vector pBV220. The plasmid pBV-geneE-cat constructed in the invention can be replicated stablely in Ecoli., and can efficiently express the chloramphenicol acetyl transferase so as to generate resistance to chloramphenicol, which can be taken as a resistance selection marker to achieve efficient expression of the lytic gene E. The lytic plasmid provided in the invention can reach lytic efficiency up to 99.98% during prepaing a chloramphenicol sensitive bacterial ghost of Ecoli.

Description

technical field [0001] The invention relates to a plasmid carrier, in particular to a chloramphenicol-resistant temperature-controlled lysis plasmid and its construction method, as well as its use in preparing Escherichia coli slough, belonging to the field of genetic engineering. Background technique [0002] Bacterial ghost is a complete bacterial empty shell formed after Gram-negative bacteria are cleaved by cleavage protein E of bacteriophage PhiX174. This non-denaturing gene inactivation method makes the bacterial ghost retain the same bacterial cells as live bacteria Membrane structures and related antigenic proteins, thereby inducing the body's humoral and cellular immune responses. The natural highly conserved structure PAMP (pathogen-associated molecular patterns) of the decidual outer membrane, such as lipopolysaccharide, peptidoglycan, CPG, pili, etc., can be recognized by immune cells through pattern recognition receptors, and can be effectively recognized by den...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N1/20
Inventor 祝文兴安利国杨桂文袁金铎
Owner SHANDONG NORMAL UNIV
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