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Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation

A phage, pore vector technology, applied in the field of genetic engineering vaccines, can solve problems such as no research reports

Inactive Publication Date: 2009-08-12
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

The present invention includes the use of Salmonella transformed with pBV-mE carrier pBV-mE with high pore efficiency to prepare ghost vaccines and evaluate their immune efficiency, but there is no relevant research report on this aspect

Method used

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  • Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation
  • Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation
  • Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Cloning of rearranged bacteriophage lytic genes

[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.2)

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

[0027] Restriction sites EcoR I and Bam H 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, 10x Taq buffer, Taq TMDNA 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. The E gene amplified by PCR is recovered by gel electrophoresis, and t...

Embodiment 2

[0029] Example 2 Construction of perforated carrier and test of perforated effect

[0030] After purifying the mE lytic gene cloned in Example 1, carry out double digestion with EcoR I / BamH I, and use T 4 DNA ligase (TaKaRa) was ligated with the pBV220 vector digested by EcoR I and BamH I 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 to obtain the perforated vector pBV-mE.

[0031] Select 20 (1-20) colonies of Escherichia coli TG1 containing the punched plasmid vector pBV-mE, and inoculate them in 5 mL of LB containing 50 μg / mL ampicillin, culture overnight at 28°C with shaking (220 r / min), and then Transfer 1-2mL to 50mL LB containing 50μg / mL ampicillin, culture with shaking at 28°C until OD 600nm Up to about 0.4. Take out 100 μl of the culture for later use, quickly raise the...

Embodiment 3

[0033] Example 3 The relationship between the effect of punching plasmid vector pBV-mE and the OD value of Salmonella avian

[0034] The punched plasmid pBV-mE was transferred into Salmonella avian, and the colonies of Salmonella avian containing the punched plasmid vector pBV-E were selected, inoculated in 5 mL of LB containing 50 μg / mL ampicillin, and cultured 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 about 0.4, 0.6, 0.8 and 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 of mE gene, continue to culture for 3-5 hours, and observe the punching efficiency with the naked eye. After observation, it was found that under different OD values, the mE protein has a high hole-punching effect, that is, it presents clear and transparent bacterial fluid and cell debris. It shows that...

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Abstract

The invention discloses a rearranged phage bacteriolytic gene, as well as a perforation vector and application thereof in vaccine preparation. The rearranged phage bacteriolytic gene has a nucleotide sequence shown in SEQ ID NO:1. An efficient perforation vector can be obtained by operationally connecting the rearranged phage bacteriolytic gene with a prokaryotic expression vector. The perforation vector is transformed into avian salmonella and then added with value; the expression of the rearranged phage bacteriolytic gene is induced; and formed bacterial ghosts are collected so as to obtain avian salmonella bacterial-ghost vaccine. The bacterial-ghost vaccine prepared in the invention can be applied without concentration. The avian salmonella bacterial-ghost vaccine is good in safety. After chicks are inoculated with the avian salmonella bacterial-ghost vaccine, the content of antibodies in every immune chick can be remarkably increased, so that every immune chick can be effectively protected against attacking toxins of avian salmonella, which shows that the avian salmonella bacterial-ghost vaccine has good immune protection effect.

Description

technical field [0001] The present invention relates to a bacteriolytic gene and its application in preparing vaccines, in particular to a rearranged bacteriophage lytic gene, and the present invention further relates to a punched carrier containing the gene and the use of the punched carrier in the preparation of avian Salmonella ghost vaccines The invention belongs 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 be ruptured under the action of osmotic pressure. The cytoplasm and nucleic acid components in the bacteria pass through this tunnel. Expelled, an empty bacterial shell is formed, which is the "Bacterial Ghost". Bacterial ghost is composed of inner membrane (cytoplasmic...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/70C12N15/74A61K39/112A61P31/04C12R1/42
CPCY02A50/30
Inventor 刘思国常月红彭伟刘慧芳王春来司微
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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