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Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof

A technology of genetically engineered bacteria and monophosphoric acid lipids, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as numerous steps, unfavorable large-scale production, and difficult operation, so as to reduce production costs, Facilitate the effect of large-scale industrial production

Inactive Publication Date: 2012-04-04
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 to operate; second, Salmonella can simultaneously synthesize several lipid A with different structures, which brings many difficulties to subsequent separation and purification; 3. The method involves chemical treatment, and there are many steps, which affect product quality and yield
This strain created a new method for MPLA production, but it needs to be induced by adding IPTG in the production process, and the production cost is high, which is not conducive to large-scale production

Method used

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  • Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof
  • Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof
  • Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Construction of Mutant E.coli W3110 ΔlacI

[0016] 1. Obtaining the lacI gene knockout fragment

[0017] 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 fragment was cloned into pBlueScript II SK(+) to obtain the recombinant plasmid pBlueScript II SK(+)-lacI(U)-pkan-lacI(D).

[0018] 2. Preparation and electrotransformation of knockout competent cells

[0019] 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) Escherichia coli W3110 (ATCC39936) was cultured overnight in LB liquid medium containing 100 μg / mL ampicillin at ...

Embodiment 2

[0025] Construction of embodiment 2 mutant strain HW001

[0026] 1. Obtaining the knock-in fragment FnlpxE-Fkan

[0027] According to the sequence of pWSK29-FnlpxE-Fkan (Jiuzhou Chen et al.2010), artificially synthesize or PCR amplify the knock-in fragment FnlpxE-Fkan with lacZ-a natural homology arm, its nucleotide sequence is shown in SEQ IN NO.2 shown. Among them, there are FRT sites on both sides of the kan gene.

[0028] 2. Preparation and electrotransformation of knockout competent cells

[0029] The W3110 ΔlacI strain carrying the pKD46 plasmid was used as the starting strain to prepare competent cells, and the method was the same as above. 500-1000ng of the FnlpxE-Fkan knock-in fragment was electrotransformed into competent cells, and the mutant strain W3110 ΔlacI lacZ::FnlpxE-Fkan knocked in and expressing FnlpxE-Fkan inside the chromosome lacZ gene was obtained by kan resistance screening.

[0030] 3. Removal of mutant resistance markers

[0031]By transferring ...

Embodiment 3

[0032] Lipid A structure analysis of embodiment 3 mutant strain HW001

[0033] 1. Lipid A extraction and thin layer chromatography (TLC) analysis of mutant strain HW001

[0034] 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, cultivated to OD at 37°C 600 = 1 hour 8000rpm centrifugal 10min collects bacterial cell, ddH 2 After washing the cells once, use the Bligh-Dyer one-phase system (chloroform / methanol / water, 1:2:0.8, v / v / v) to suspend the cells, magnetically stir for 1 h, and centrifuge at 2000 rpm for 20 min 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, shake for 10min, and cleavage the sugar chains in a water bath at 100°C for 30min. After cooling to room temperature, add 30 mL of chloroform and 30 mL of methanol to form a Bligh-Dyer two-phase ...

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Abstract

The invention discloses a genetic engineering bacterium for producing a monophosphoryl lipid A as well as a construction method and application thereof, belonging to the field of a genetic engineering. The genetic engineering bacterium is E.coli W3110 delta lacI lacZ::FnlpxE, wherein the lacI takes place a deletion mutation to lose activity and an expression FnlpxE gene is knocked into the lacZ gene. According to the bacterial strain constructed in the invention, on the basis of keeping a lipid structure single, the production cost is also reduced; no exogenous resistance genes are introduced into the bacterial strain so that the industrial production of the bacterial strain is easier to realize in a large scale.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing monophosphoric acid lipid A and its construction method and application, in particular to an Escherichia coli directly producing monophosphoric acid lipid A 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. MPL is a liposome adjuvant that has been used in clinical trials. The frequency of adverse reactions in human clinical trials is as low as that of aluminum adjuvants, and its inflammatory toxicity is 0.1% of that of LPS. In addition to MPL, monoph...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/64C12R1/19
Inventor 王小元韩雅宁陈久洲李烨
Owner JIANGNAN UNIV
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