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Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines

A technology for lysing genes and bacteriophages, which can be used in applications, genetic engineering, plant genetic improvement, etc. to achieve good safety, high initial induction concentration, and high lysis efficiency.

Inactive Publication Date: 2013-12-18
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

More critically, the cleavage vector pBV-Eprom constructed based on the mutated E gene Eprom can use a fermenter to prepare a large number of bacteria shadows of Actinobacillus pleuropneumoniae, making large-scale production possible, but there is no correlation in this aspect research report

Method used

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  • Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines
  • Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines
  • Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning of mutated phage lytic genes

[0039] Primers were designed according to the coding sequence of phage PhiX174 cleavage gene E in GenBank:

[0040] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO.2)

[0041] Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO.3)

[0042] Restriction sites EcoR I and BamH I were introduced into the 5′ ends of the upstream and downstream primers, which were synthesized by Harbin Boshi. Use bacteriophage PhiX174 double-stranded DNA as a template to amplify cleavage 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 of 94°C for 30s, 59°C for 30s, 72°C for 30s, 72°C for 5min. The E gene amplified by PCR is recovered by gel electrophoresis, and then the E gene is digested with DNase...

Embodiment 2

[0044] Example 2 Construction of cleavage plasmid vector pBV-Eprom and screening of culture conditions

[0045] After purification of the cleavage gene E cloned in Example 1, it was digested with EcoR I / BamH I, and then ligated with the pBV220 vector digested by EcoR I and BamH I with T4 DNA ligase (TaKaRa), overnight at 16°C Ligate and transform into Escherichia coli TG1 competent cells by heat shock. The clones identified as positive by colony PCR were enriched and a small amount of plasmid was extracted by alkaline lysis method to obtain the cleavage vector.

[0046] Select 20 colonies of Escherichia coli TG1 containing the lysis vector, inoculate them in 5 mL of LB containing 50 μg / mL ampicillin, culture overnight at 37°C with shaking (220 r / min), then transfer 1-2 mL to 50 mL containing 50 μg / mL Ampicillin in LB, shake culture at 37°C to OD 600 Up to about 0.4. At this time, the temperature was raised rapidly to 42° C. for culture to induce the expression of the E gene,...

Embodiment 3

[0047] Example 3 Construction of recombinant Actinobacillus pleuropneumoniae containing lysis vector pBV-Eprom

[0048] 1. Construct a lysis vector containing the bacteriophage lysis gene E of the mutation shown in SEQ ID NO: 1;

[0049] (1) Synthesizing the sequence of the mutated phage lytic gene E shown in SEQ ID NO: 1;

[0050] (2) Design primers:

[0051] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO.2)

[0052] Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO.3)

[0053] Using specific primers Lysis E-U and Lysis E-L to specifically amplify the sequence of the mutated phage lytic gene E synthesized above. The 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 1 μL. The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles of 94°C for 30s, 59°C for 30s, 72°C for 30s, 72°C for 5min. Obtain the amp...

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Abstract

The invention discloses a mutant phage lysis gene E, a lysis plasmid vector containing the lysis gene and an application in preparation of bacterial ghost vaccines. The mutant phage lysis gene E (Eprom) disclosed by the invention is obtained after mutation of a promoter region of the lysis gene E of a phage phiX174, the E gene after mutation can turn the temperature of culture bacteria from the existing 28 DEG C to 37 DEG C, the mutant phage lysis gene E further has higher lysis efficiency, higher starting induction concentration and large-scale production capacity, and the culture lysis efficiency of a fermentation tank is as high as 99.99997%. After the Eprom is connected with pBV220, the high-efficient lysis plasmid vector pBV-Eprom can be obtained. The pBV-Eprom is transformed into actinobacillus pleuropneumoniae to induce the gene expression of the Eprom so as to get a bacterial ghost of the actinobacillus pleuropneumoniae. Porcine transmissible pleuropneumonia bacterial ghost vaccines disclosed by the invention has good safety and immune protection efficacy and can stimulate organisms to produce high-titer antibodies and further provide good cross immunoprotection against attacks of different serotypes of actinobacillus pleuropneumoniae virulent strains.

Description

technical field [0001] The present invention relates to the application of bacteriophage lytic gene E in the preparation of vaccines, in particular to a mutated phage lytic gene E, and the present invention also relates to a lytic vector containing the gene and the use of the lytic vector in the preparation of bacteriophage vaccines, especially porcine infectivity The invention relates to a use in pleuropneumonia ghost vaccines, belonging to the field of genetic engineering vaccines. 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 bacterial shell, that is, "bacteria...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/63C12N1/21A61K39/02A61P31/04
Inventor 王春来李刚谢芳张艳禾
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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