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Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli

A technology of PMAL-E-SN and Escherichia coli, which is applied in the field of genetic engineering, can solve problems such as difficulty in improving lysis efficiency, and achieve the effect of improving lysis efficiency

Inactive Publication Date: 2009-06-17
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many successful precedents in the preparation of ghosts at home and abroad, but the general ghost carrier only uses the E gene, and the lysis efficiency is difficult to improve

Method used

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  • Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli
  • Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli
  • Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The construction of the ghost carrier PMAL-E-SN (according to figure 1 )

[0025] 1. Primer design and synthesis:

[0026] According to the PhiX174 phage cleavage gene E (serial number: 9626372) and Staphylococcus aureus nuclease A (serial number: 21281729) published on Genebank, primers were designed: E1, E2, SN1 and SN2, and 5 and 15 flexible amino acids were designed respectively The primers of Linker (E2', SN1', E2" and SN2") connect the cleavage gene E and the SN gene in series. Italic is the Linker part, and all primers were synthesized by Shanghai Bioengineering Company.

[0027] Primers for PCR reactions

[0028]

[0029]

[0030] 2. PCR amplification of E gene:

[0031] PCR amplification was performed using the PhiX174 phage RFI plasmid as a template: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 45 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min. After the end, take 5 μL of the P...

Embodiment 2

[0045] E. coli O 138 Ghost preparation

[0046] 1. Electrotransformation of E. coli O 138 Preparation of Competent Cells:

[0047] Take the E.Coli O stored at -80℃ 138 The strains were spread on LB plates and cultured at 37°C for 24 hours, picked a single colony, and cultured with shaking for 12-14 hours (approximately OD 600 When the value reaches 0.7, re-inoculate in 200mL fresh LB medium, shake culture at 37°C until OD 600 between 0.1-0.2; take it out, ice-bath for 30min, electroporation transformation medium CYT is placed on ice at the same time; 4°C, 5 000rpm centrifuge for 10min, fully resuspend the pellet with 30mL ice-bathed CYT; 4°C, 5 000rpm centrifuge After 10 minutes, the supernatant was discarded, and the pellet was fully resuspended with 500 μL ice-bathed CYT.

[0048] 2. Transformation of E. coli O by Electroporation 138 :

[0049] Take 20 μL of the ghost carrier PMAL-E-SN, mix with 80 μL of the above-mentioned E. coli O 138 Competent cells were mixed ev...

Embodiment 3

[0057] Escherichia coli O138 ghost vaccine and formaldehyde inactivated vaccine immune effect evaluation

[0058] 1. Escherichia coli O 138 Ghost preparation

[0059] Freeze-dried E. coli O 138 Bacteria, resuspended in an appropriate volume of normal saline, so that the concentration of bacteria shadows is 5.0×10 10 / mL, mix thoroughly.

[0060] 2. Preparation of formaldehyde-inactivated aluminum-adjuvanted vaccines

[0061] coli O 138 Pick purely on ordinary LB plates, inoculate in LB liquid medium and culture for 18-24 hours at 37°C to prepare seed bacterial liquid, inoculate in LB liquid medium, culture for 18-24 hours at 37°C, take out a small amount to count viable bacteria, and the rest The bacterial solution was added with 0.1% formaldehyde, inactivated at 37°C for 24 hours, and stored at 4°C for later use. When in use, centrifuge at 5000rpm, wash twice with normal saline, add aluminum hydroxide gel at a ratio of 1:5, and shake fully to obtain formaldehyde-inactiv...

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Abstract

The invention discloses a bacterial ghost vector PMAL-E-SN construction and application thereof in an escherichia coli comprising an E gene obtained by PCR amplification using a PhiX174 phage RFI plasmid as a template; an SN gene obtained by PCR amplification using a staphylococcus aureus standard strain ATCC25923 genome DNA as a template; an E-SN gene obtained by serial connecting the E gene and the SN gene by the Linker of the fifteen flexible amino acids; a positive plasmid pT-E-SN sequencing screened by coupling an E-SN gene PCR amplification outcome and a pMD18-T vector and the bacterial ghost vector PMAL-E-SN constructed by the recombinant plasmid pT-E-SN with right sequencing and the plasmid vector PMAL-p2X. The bacterial ghost vector PMAL-E-SN of the invention enables the E gene and the SN gene to obtain the high efficiency expression through a ''tac'' strong promoter and a malE start signal, successfully prepares the escherichia coli bacterial ghost and the degradation efficiency can reach 99.99 The invention induces a secondary killing gene SN, obtains good effect by combining the E gene and the SN gene to a high efficiency expression vector PMAL-p2X and is widely used in preparation of the escherichia coli bacterial ghost.

Description

technical field [0001] The invention relates to a method for constructing a bacterial ghost carrier PMAL-E-SN by using E gene and SN gene, and also relates to the application of the bacterial ghost carrier PMAL-E-SN in preparing Escherichia coli ghost, which belongs to the field of genetic engineering. Background technique [0002] Bacterial ghost (BG) is a new type of vaccine and adjuvant form. Its essence is an empty cell body without cytoplasm and nucleic acid. Its biggest feature is that it retains the same The complete inner and outer membrane structure can effectively stimulate the body to produce specific and non-specific immune responses to resist the invasion of pathogenic microorganisms. Compared with traditional inactivated vaccines, its surface antigen structure is preserved to the greatest extent, and its immunogenicity is high. The original antigen becomes an excellent antigen delivery carrier, and at the same time provides a research platform for the research...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/55C12N15/33C12N1/21
Inventor 雷连成杜崇涛韩文瑜孙长江冯新
Owner JILIN UNIV
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