Rearranged bacterial virus E gene, perforating plasmid vector containing the same and use thereof

A plasmid carrier and phage technology, applied in the field of DNA, can solve the problems of reduced drilling efficiency, no relevant research reports, reduction, etc., and achieve the effect of improving drilling efficiency

Inactive Publication Date: 2008-11-12
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to data, the original high-copy perforation plasmid, when connected to the E gene, the copy number of the perforation plasmid is significantly reduced, and becomes a low copy number plasmid, which also reduces the punching efficiency.
Therefore, apart from factors such as the promoter, the E gene involved in hole punching will be the focus of research, but there are no relevant research reports on this aspect

Method used

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  • Rearranged bacterial virus E gene, perforating plasmid vector containing the same and use thereof
  • Rearranged bacterial virus E gene, perforating plasmid vector containing the same and use thereof
  • Rearranged bacterial virus E gene, perforating plasmid vector containing the same and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Isolation and cloning of embodiment 1PhiX174 bacteriolytic gene E (mE)

[0031] Primers were designed according to the coding sequence of bacteriophage PhiX174 lytic gene E in GenBank:

[0032] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO.2)

[0033] Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO.3)

[0034] Restriction sites EcoR I and Bam HI 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, 10x Taq buffer, Taq TM DNA polymerase 2U (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. The E gene amplified by PCR is recovered by gel el...

Embodiment 2

[0036] The construction of embodiment 2 punching plasmid vector pBV-mE

[0037] After purifying the bacteriolytic gene mE cloned in Example 1, carry out double digestion with EcoRI / Bam HI, and use T 4 DNA ligase (TaKaRa) was ligated with the pBV220 vector digested by EcoR I and Bam HI double enzymes, ligated overnight at 16°C and transformed into Escherichia coli TG1 competent cells by heat shock, and the clones identified as positive by colony PCR were enriched A small amount of plasmid was extracted by alkaline lysis method and named pBV-mE.

experiment example 1

[0038] Experimental example 1 Comparison experiment of the punching efficiency of the punching plasmid vector constructed by the unrearranged phage lytic gene E and the rearranged phage lytic gene mE

[0039] 1. Experimental materials

[0040] (1), the punched plasmid vector pBV-mE constructed in Example 2;

[0041] (2), the unrearranged phage lysoE gene was constructed according to the method of Example 2 to obtain the punched plasmid vector pBV-E;

[0042] 2. Experimental method

[0043] Select 10 colonies (No. 1-10) of Escherichia coli TG1 containing punched plasmid vectors pBV-E and pBV-mE, inoculate them in 5 mL of LB containing 50 μg / mL ampicillin, and culture overnight at 28°C with shaking (220r / mL min), then transfer 1-2mL to 50mL LB containing 50μg / mL ampicillin, culture with shaking at 28℃ until OD 600nm Up to 0.4, 0.6, 0.8, 1.0. Take out 100 μl of the culture for later use, quickly raise the temperature of the remaining culture to 42°C to induce the expression o...

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Abstract

The invention discloses a PhiX174 bacterial virus mE gene obtained by gene rearrangement and a perforating plasmid vector containing the gene as well as a method for building the same. The invention further relates to an application of the perforating plasmid vector for making bacterial ghost, belonging to the gene engineering field. The PhiX174 bacterial virus E gene is rearranged by using the gene rearrangement technology so that a reading frame with E containing 276 nucleotides is mutated as a reading frame with mE (from ATG at the 76th site) containing 201 nucleotides by deleting A of an initial codon ATG at the first site. The made mE gene is maneuverably connected with pBV 220 vector to perforate the vector pBV-mE, and the vector pBV-mE can effectively improve perforation efficiency of the cell and lysis efficiency is up to 99.9613 percent. Compared with the prior perforating plasmid vector, the lysis efficiency of homophytic escherichia coli is more than three orders of magnitude.

Description

technical field [0001] The invention discloses a DNA obtained after gene rearrangement, in particular to a DNA of PhiX174 phage mE gene obtained after gene rearrangement, and the invention also relates to a punched plasmid vector containing the DNA and a construction method thereof, The present invention further relates to the application of the pore plasmid carrier in the preparation of 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 compose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/10C12N15/63C12N1/21C12R1/19
Inventor 刘思国常月红刘慧芳王春来彭伟司薇
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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