Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof

A technology of genetically engineered bacteria and monophosphoric acid lipids, applied in the field of genetically engineered bacteria, can solve problems affecting product quality and yield, numerous steps, and difficult operations

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the existing production method are: first, Salmonella is a pathogenic bacterium, and it is difficult t

Method used

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  • Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof
  • Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof
  • Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Construction of mutant E.coli W3110 △lacl

[0018] 1. Obtaining the lacI gene knockout fragment

[0019] The lacI gene knockout fragment is obtained by chemical total synthesis or PCR step-by-step amplification, and its two ends are the upstream and downstream homology arms of the lacI gene, and the middle is a kan fragment. The nucleotide sequence of the lacI gene knockout fragment is shown in SEQ ID NO.1. The lacI gene knockout fragments XhoI and PstI were cloned into pBlueScript II SK(+) to obtain the recombinant plasmid pBlueScript II SK(+)-lacI(U)-pkan-lacI(D). Among them, there are loxP sites on both sides of the kan gene.

[0020] 2. Preparation and electrotransformation of knockout competent cells

[0021] Inoculate with Red recombinant helper plasmid pKD46 (Datsenko K A, Wanner B L. One-Step inactivation of chromosome genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): 6640-6645) coli W3110 (ATCC39936) was cult...

Embodiment 2

[0027] Construction of embodiment 2 mutant strain HW002

[0028] 1. Obtaining the knock-in fragment pagl-lpxE-pagP-Fkan

[0029] The PCR amplification method was used to obtain pagL, lpxE, pagP, and Fkan gene fragments, which were cloned into pWSK29 in turn to obtain the recombinant plasmid pWSK29-pagL-lpxE-pagP-Fkan. The knock-in fragment pagL-lpxE-pagP-Fkan with lacZ-α natural homology arm was amplified by PCR, and its nucleotide sequence is shown in SEQ IN NO.2. Among them, there are FRT sites on both sides of the kan gene.

[0030] 2. Preparation of knock-in competent cells and electrotransformation

[0031] The W3110△lacI strain carrying the pKD46 plasmid was used as the starting strain to prepare competent cells, and the method was the same as above. 500-1000ngpagL-lpxE-pagP-Fkan knock-in fragment was electrotransformed into competent cells, and the mutant strain W3110△lacI lacZ:: pagL-lpxE-pagP-Fkan.

[0032] 3. Removal of mutant resistance markers

[0033] By trans...

Embodiment 3

[0034] Lipid A structure analysis of embodiment 3 mutant strain HW002

[0035] 1. Lipid A extraction and thin layer chromatography (TLC) analysis of mutant strain HW002

[0036] Lipid A was extracted by chloroform / methanol / water mixed phase extraction. The overnight cultured bacterial solution was divided into initial OD 600 =0.02 transferred to 200mL LB liquid medium, cultured to OD at 37°C 600 = 1 hour 8000rpm centrifuge 10min to collect bacteria, ddH 2 After washing the cells once, use Bligh-Dyer one-phase system (chloroform / methanol / water, 1:2:0.8, v / v / v) to suspend the cells, magnetically stir for 1 hour, and centrifuge at 2000rpm for 20 minutes to separate the phases. Use a one-phase system Wash cell debris 2-3 times. Add 27mL of 12.5mM sodium acetate (PH4.5) solution, ultrasonically shake for 10min, and cleavage the sugar chains in a 100°C water bath for 30min. After cooling to room temperature, add 30mL chloroform and 30mL methanol to form a Bligh-Dyer two-phase s...

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Abstract

The invention discloses a genetically engineered bacterium of a colon bacillus for producing arabinoside-cytidine monophosphate lipoid A. The genetically engineered bacterium is characterized in that a deletion mutation deactivation is carried out on lacz genes in a genome of the genetically engineered bacterium; and express exogenous pagL and lpxE and pagP genes are implanted into the lacz genes. According to the invention, the formed bacterial strain cannot produce a pathogenic bacterium and is free of the use of an inductive agent during a production process. Therefore, the genetically engineered bacterium is suitable for mass production of MPL adjuvants in earlier stages.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing monophosphate lipid A (MPL), in particular to an Escherichia coli directly producing MPL without induction. technical background [0002] Vaccine adjuvants can non-specifically enhance the body's immune response to antigens and improve vaccine efficiency. Aluminum adjuvant is currently recognized globally as a vaccine adjuvant widely used in humans. It is safe and reliable, and can significantly enhance humoral immune response, but its effect on cellular immunity is not ideal. Therefore, people are devoting themselves to developing more efficient and safer vaccine adjuvants. Lipid A can be recognized by Toll Like Receptor 4 (TLR-4) on the surface of host cells, and then trigger a series of physiological and biochemical reactions in the cells, producing TNF-α, IL-6, IL-8, etc. Various inflammatory cytokines. The types and quantities of cytokines mainly depend on the structure of li...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/87C12P19/26C12R1/19
Inventor 王小元韩雅宁陈久洲李烨
Owner JIANGNAN UNIV
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