CN102994435A - Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof - Google Patents
Genetically engineered bacterium of colon bacillus for producing arabinoside-cytidine monophosphate lipoid A, and application thereof Download PDFInfo
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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
The present invention relates to the genetic engineering bacterium of a kind of product MPLA (MPL), particularly a kind of intestinal bacteria that need not to induce direct production MPL.
Technical background
Vaccine adjuvant can nonspecific enhancing body to the immune response of antigen, improve vaccine efficient.Aluminium adjuvant is the vaccine adjuvant that is widely used in human body that generally acknowledge in the present whole world, and is safe and reliable, can significantly strengthen humoral immune reaction, but not ideal enough to the effect of cellular immunization.Therefore, people are devoted to develop the more vaccine adjuvant of highly effective and safe.Lipoid A can be by the Toll sample acceptor 4(Toll Like Receptor 4 of host cell surface, TLR-4) identification, and a series of biochemical reactions in the trigger cell produces TNF-α, the multiple inflammatory cytokines such as IL-6, IL-8 then.The kind of cytokine and quantity depend primarily on the structure of lipoid A, and therefore, the lipoid A of some special construction can be used as vaccine adjuvant.
It is a kind of MPLA mixture that contains 6 and 7 fatty acid chains; its resulting structure is 3 deacylations, 2 MPLAs that add 16 carbon hydroxy fatty acid chains; it is the Liposome Adjuvant that has been applied to clinical trial; human clinical trial's untoward reaction frequency is the same with aluminium adjuvant low, and its inflammation toxicity is 0.1% of LPS.At present, the Corixa company commercialization of production is only arranged
Its production method is to extract lipopolysaccharides from Salmonellas, carries out the processes such as a series of hydrolysis, chromatography, purifying again.The shortcoming of existing production method is: the first, Salmonellas is pathogenic bacterium, and relatively difficulty operates; The second, the method relates to chemical treatment, and step is various, affects quality product and output.
Summary of the invention
The technical problem to be solved in the present invention provides the genetic engineering bacterium of a kind of novel product MPL, does not relate to pathogenic bacterium in its production process, and need not inductor, can be used for the scale operation in early stage of MPL adjuvant.
For solving the problems of the technologies described above, genetic engineering bacterium provided by the invention is E.coli W3110 △ lacI lacZ::pagL-lpxE-pagP, and the deletion mutantion inactivation occurs lacI in its genome, and lacZ gene inside is knocked in and expressed pagL, lpxE and pagP gene.
Lipoid A begins to synthesize from cell inner membrance inboard, and route of synthesis is more conservative, but in the process that is transported to the adventitia outside multiple modification can occur, to adapt to external environment.At present, modifying relevant gene with quasi-grease A structure has been reported.Utilize these genes, according to the synthetic of lipoid A molecule and modify mechanism, by genetic engineering technique the structure of modification of lipoid A in the intestinal bacteria is become MPL.
Described lpxE derives from Francisella, and the protein of its coding can be hydrolyzed the phosphoric acid on the lipoid A molecule 1 position; PagL derives from Salmonellas, and the protein of its coding can be hydrolyzed the fatty acid chain of removing 3 of lipoid A molecules; PagP derives from Salmonellas, and the protein of its coding can add 16 carbon fatty acid chains 2 of lipoid A molecule β.
For making foreign gene constitutive expression in intestinal bacteria, knock out the lacI gene on the karyomit(e), the adjusting albumen in the lacI genes encoding lac operon knocks out this gene and can remove the effect of checking of regulating albumen.
The present invention also provides a kind of method of utilizing described genetic engineering bacterium fermentative production MPL.Wherein fermentation process is the normal fermentation method, and the extracting method of MPL is: the bacterium liquid of incubated overnight is pressed initial OD
600=0.02 is transferred in the 200mL LB liquid nutrient medium, and 37 ° of C are cultured to OD
600The centrifugal 10min of 8000rpm collected thalline, ddH in=1 o'clock
2O washing thalline is once used Bligh-Dyer one phase system (chloroform/methanol/water, 1:2:0.8, v/v/v) suspension thalline afterwards, magnetic agitation 1h, and the centrifugal 20min phase-splitting of 2000rpm is used a phase system washed cell fragment 2-3 time; Sodium-acetate (PH4.5) solution that adds 27mL 12.5mM, ultrasonic concussion 10min, 100 ° of C water-bath 30min cracking sugar chains.Be chilled to and add the 30mL chloroform after the room temperature and 30mL methyl alcohol is made into Bligh-Dyer two-phase system (chloroform/methanol/water, 2:2:1.8, v/v/v), the centrifugal 10min of 2000rpm takes off to move into mutually to revolve and steams in the bottle rotary evaporation; Adding at last chloroform/methanol solution (4:1, v/v) washes out lipoid A.
The detection of lipoid A, analysis:
Thin-layer chromatography (TLC) detects: on Gel 60 TLC plates, developing agent is chloroform/methanol/water/ammoniacal liquor (40:25:4:2, v/v/v/v) with sample spot.Chromatography finishes residual developing agent on the after-blow dry plate, carries out carbonization with 10% sulfuric acid that is dissolved in ethanol, places on the hot-plate and develops the color.
ESI/MS analyzes: the lipoid sample is dissolved in the chloroform/methanol solution (4:1, v/v), carries out mass spectrometric detection at WATERS SYNAPT Q-TOF Mass Spectrometer mass spectrograph.Adopt the ESI ion source, the negatively charged ion detecting pattern, sensing range is less than m/z2500.
Description of drawings
Fig. 1 wild-type e. coli and genetic engineering bacterium lipoid A TLC analyze
1:W3110 lipoid A sample; 2: mutant strain HW002 lipoid A sample
Fig. 2 genetic engineering bacterium lipoid A ESI/MS analyzes
A:W3110 lipoid A sample; B: mutant strain HW002 lipoid A sample
Fig. 3 wild-type e. coli and genetic engineering bacterium lipopolysaccharides Stimulated Macrophages RAW264.7 produce the analysis of IL-6
Embodiment
The structure of embodiment 1 mutant strain E.coli W3110 △ lacl
1, the acquisition of lacI gene knockout fragment
Adopt the method that chemistry is complete synthesis or PCR increases step by step to obtain lacI gene knockout fragment, its two ends are that lacI gene upstream and downstream homology arm, centre are the kan fragment.The nucleotide sequence of lacI gene knockout fragment is shown in SEQ ID NO.1.LacI gene knockout fragment XhoI, PstI are cloned into pBlueScript II SK (+), obtain recombinant plasmid pBlueScript II SK (+)-lacI (U)-pkan-lacI (D).Wherein, kan gene both sides are with the loxP site.
2, knock out competent preparation and electricity conversion
Inoculation is with Red restructuring helper plasmid pKD46(Datsenko K A, Wanner B L.One-Step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci USA, 2000,97 (12): intestinal bacteria W3110(ATCC39936 6640-6645)) in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, 30 ° of C, the 200rpm incubated overnight.By 2% inoculum size switching 100mL LB liquid nutrient medium, 30 ° of C, 200rpm is cultured to OD
600Added the expression that L-arabinose (final concentration is 30mmol/L) is induced recombinase, and continued to be cultured to OD in=0.2 o'clock
600=0.5, the nutrient solution ice bath is changed in the 50mL centrifuge tube of precooling over to 4 ° of C after half an hour, the centrifugal 10min of 8000rpm collects thalline, precipitation is washed 3 times with 10% glycerine of precooling, suspends with 1mL 10% glycerine at last, and every pipe 80 μ L divide in the aseptic EP pipe that is filled to precooling.
500-1000ng lacI is knocked out fragment to add in the competent cell, mixing, ice bath 15min, 1.5kv electric shock 5ms, 30 ° of C are hatched 2h, are coated with the LB solid plate of 30 μ g/mL kantlex, and 30 ° of C cultivate, the picking transformant is cultivated the PCR the result in the LB liquid nutrient medium that contains 30 μ g/mL kantlex and 100 μ g/mL penbritins.
3, the structure of temperature sensitive type expression vector pCRE
According to (the Guldener U of pSH47 in the ncbi database, Heck S, Fielder T, Beinhauer J, Hegemann JH.A new efficient gene disruption cassette for repeated use in budding yeast.Nucleic Acids Res, 1996,24:25192524) carrier sequence, adopt the complete synthesis or PCR method of chemistry to obtain the cre gene and pass through SacI, the NcoI restriction enzyme site is cloned into pKD46 plasmid multiple clone site, transform escherichia coli jm109 competent cell, be coated with the LB solid plate of 100 μ g/mL penbritins, 30 ° of C incubated overnight, the picking transformant is proposed the plasmid enzyme restriction checking, obtains Cre recombinase temperature sensitive type expression vector, and called after pCRE.
4, the removal of mutant strain resistance marker
The positive inoculation of PCR checking is contained in the LB liquid nutrient medium of 30 μ g/mL kantlex, 42 ° of C incubated overnight, with the rule LB solid plate of 30 μ g/mL kantlex of nutrient solution, 37 ° of C cultivate, picking list bacterium colony is verified it to the susceptibility of penbritin, will contain kalamycin resistance and to amp-S bacterial strain called after W3110 lacI::kan and preservation.Do competence as starting strain, change the temperature sensitive type expression vector pCRE with recombinase Cre over to, positive strain is cultivated in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, add L-arabinose (final concentration is 30mmol/L) and induce the expression of Cre recombinase, the restructuring of mediation loxP locus specificity, transformant is selected in the PCR checking.PCR is verified that line LB is dull and stereotyped after 42 ° of C heat shocks of correct bacterial strain, and picking list bacterium colony is verified it to the susceptibility of penbritin and kantlex, chooses all responsive bacterial strain called after W3110 △ lacI and the preservations of two kinds of microbiotic.
The structure of embodiment 2 mutant strain HW002
1, knocks in the acquisition of fragment pagl-lpxE-pagP-Fkan
Adopt the method for pcr amplification to obtain to obtain respectively pagL, lpxE, pagP, Fkan gene fragment, successively it is cloned into pWSK29, obtain recombinant plasmid pWSK29-pagL-lpxE-pagP-Fkan.Pcr amplification is knocked in fragment pagL-lpxE-pagP-Fkan with the natural homology arm of lacZ-α, and its nucleotide sequence is shown in SEQ IN NO.2.Wherein, kan gene both sides are with the FRT site.
2, knock in competent preparation and electricity conversion
With with the W3110 △ lacI bacterial strain of pKD46 plasmid as starting strain, the preparation competence, method is the same.500-1000ngpagL-lpxE-pagP-Fkan is knocked in the fragment electricity change in the competent cell, obtain karyomit(e) lacZ gene inside by the kan resistance screening and knock in the mutant strain W3110 △ lacI lacZ::pagL-lpxE-pagP-Fkan that expresses pagL-lpxE-pagP-Fkan.
3, the removal of mutant strain resistance marker
By changing pCP20 plasmid (Cherepanov P P over to, Wackernagel W.Gene disruption in Escherichia coli:TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.Gene, 1995,158 (1): 9-14), the FLP enzyme is expressed in 42 ° of C heat shocks, the specificity restructuring in mediation FRT site can be removed the kan resistance marker, screening method is the same, the mutant strain called after HW002 and the preservation that obtain.
The quasi-grease A structure analysis of embodiment 3 mutant strain HW002
1, the lipoid A of mutant strain HW002 extracts and thin-layer chromatography (TLC) analysis
Chloroform/methanol/water mixed phase extraction process is adopted in the extraction of lipoid A.The bacterium liquid of incubated overnight is pressed initial OD
600=0.02 is transferred in the 200mL LB liquid nutrient medium, and 37 ° of C are cultured to OD
600The centrifugal 10min of 8000rpm collected thalline, ddH in=1 o'clock
2O washing thalline is once used Bligh-Dyer one phase system (chloroform/methanol/water, 1:2:0.8, v/v/v) suspension thalline afterwards, magnetic agitation 1h, and the centrifugal 20min phase-splitting of 2000rpm is used a phase system washed cell fragment 2-3 time.Sodium-acetate (PH4.5) solution that adds 27mL 12.5mM, ultrasonic concussion 10min, 100 ° of C water-bath 30min cracking sugar chains.Be chilled to and add the 30mL chloroform after the room temperature and 30mL methyl alcohol is made into Bligh-Dyer two-phase system (chloroform/methanol/water, 2:2:1.8, v/v/v), the centrifugal 10min of 2000rpm takes off to move into mutually to revolve and steams in the bottle rotary evaporation.Adding chloroform/methanol solution (4:1, v/v) washes out lipoid A.Then carry out thin-layer chromatography (TLC) and detect, on Gel 60 TLC plates, developing agent is chloroform/methanol/water/ammoniacal liquor (40:25:4:2, v/v/v/v) with sample spot.Chromatography finishes residual developing agent on the after-blow dry plate, carries out carbonization with 10% sulfuric acid that is dissolved in ethanol, places colour developing (Fig. 1) on the hot-plate.
TLC result shows wild-type e. coli lipoid A(swimming lane 1) structure is more single, being the bis phosphoric acid form, the lipoid A(swimming lane 2 of mutant strain HW002) structure has four kinds, wherein comprises the MPL structure, other structures are because the incomplete expression of pagL, pagP gene causes, this and business-like
Composition is consistent, illustrates that mutant strain can produce the lipoid A mixture that contains MPL.
2, the ESI/MS of quasi-grease A structure analyzes
The lipoid sample is dissolved in the chloroform/methanol solution (4:1, v/v), carries out mass spectrometric detection at WATERS SYNAPT Q-TOF Mass Spectrometer mass spectrograph.Adopt the ESI ion source, the negatively charged ion detecting pattern, sensing range is less than m/z 2500(Fig. 2).The wild-type e. coli quasi-grease A structure is the bis phosphoric acid form, and the m/z value is 1797.2, can cause a small amount of 1 phosphate group disappearance in the leaching process; The MPL structure is the MPLAs that 3 deacylations, 2 add 16 carbon hydroxy fatty acid chains, and m/z value is 1729.3, and m/z value to be 1491.1,1717.3,1955.5 peak be the incomplete product that acts on of PagL, PagP.Therefore, mutant strain HW002 can produce the lipoid A mixture that contains MPL.
Embodiment 4 mutant strain HW002 lipopolysaccharides (LPS) oxicity analysis
1, the extraction of mutant strain HW002 LPS and purifying
Hot phenol method is adopted in the extraction of LPS.The bacterium liquid of incubated overnight is pressed initial OD
600=0.02 is transferred in the 800mL LB liquid nutrient medium, and 37 ° of C cultivate the centrifugal 10min collection of 8000rpm thalline after 12 hours, ddH
2O washing thalline is rear weighing thalline weight in wet base once, and every 3g wet thallus is dissolved in 10mL ddH
2Among the O, 68 ℃ of preheatings.90% phenol that adds the equal-volume preheating, 68 ℃ of thermal agitations 1 hour.After the subcooling, 4 ℃, centrifugal 20 minutes of 4000rpm, supernatant liquor ddH
2Vacuum lyophilization obtained the LPS crude product to remove phenol in three days in the O dialysis.An amount of ddH of crude product
2After O is resuspended, add RNase A(final concentration 50 μ g/ml) and DNase I(final concentration 100 μ g/ml) 37 ℃ of processing 2 hours, add again 37 ℃ of processing of Proteinase K (final concentration 100 μ g/ml) and spend the night.Add 5mL water saturation phenol in the sample after enzyme is processed, mixing, centrifugal 30 minutes of 4000rpm, supernatant liquor ddH
2O dialysis one day, vacuum lyophilization obtains LPS.LPS is redissolved to chloroform/methanol solution (2:1, v/v), vortex vibration 30 seconds, centrifugal 10 minutes of 12000rpm removes supernatant, and precipitation is redissolved in water, and vacuum lyophilization obtains the LPS sterling.
2, the analysis RAW264.7 that produces interleukin-6 (IL-6) behind the LPS Stimulated Macrophages leukemia cell RAW264.7 is inoculated in 96 orifice plates, every hole 10
5Individual cell, 37 ℃, 5%CO
2Cultivate after 12 hours, cell attachment renews bright nutrient solution, add that different concns LPS(final concentration is respectively 10,100,1000ng/ml), stimulate and get supernatant liquor after 24 hours, use ELISA test kit (eBioscience, 88-7064) to detect IL-6 content (Fig. 3).Wild-type e. coli W3110 and mutant strain HW002 utilize minimum showing the method for difference to have carried out variance analysis, * P＜0.05, * * P＜0.01.Because change, the especially appearance of MPL structure of mutant strain quasi-grease A structure, so that the IL-6 that the RAW264.7 cell produces significantly descends, weakened inflammatory reaction, therefore, the MPL mixture that mutant strain HW002 produces has the effect of attenuation, can be applicable to the exploitation of follow-up vaccine adjuvant.
The bacterial strain that the present invention makes up has avoided the use pathogenic bacterium to produce lipoid A, has guaranteed simultaneously the generation of MPL resulting structure, more is conducive to large-scale industrial production.
Although the present invention with preferred embodiment openly as above; but it is not to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (7)
1. a Recombinant organism that produces MPLA is characterized in that the deletion mutantion inactivation occurs lacI in the described genetic engineering bacterium genome, and lacZ gene inside is knocked in and expressed external source pagL, lpxE and pagP gene.
2. the construction process of the described genetic engineering bacterium of claim 1 is characterized in that comprising the steps:
1) artificial constructed lacI is knocked out fragment Transformed E .coli W3110, obtain mutant strain E.coli W3110 lacI::Pkan, remove kalamycin resistance gene by the locus specificity restructuring;
2) pagL-lpxE-pagP-Fkan fragment electricity is changed in the E.coli W3110 △ lacI competent cell of step 1) preparation, obtain E.coli W3110 △ lacI lacZ::pagL-lpxE-pagP-Fkan, again by locus specificity restructuring removal kalamycin resistance gene wherein.
3. method claimed in claim 2 is characterized in that described lacI knocks out the fragment nucleotide sequence shown in SEQ ID NO.1.
4. method claimed in claim 2 is characterized in that described pagL-lpxE-pagP-Fkan fragment fragment nucleotide sequence is shown in SEQID NO.2.
5. the arbitrary described method of claim 2-4 is characterized in that the described locus specificity of step 1) is reassembled as the loxP locus specificity restructuring of Cre enzyme mediation.
6. the arbitrary described method of claim 2-4 is characterized in that step 2) described locus specificity is reassembled as the FRT locus specificity restructuring of FLP enzyme mediation.
7. the described genetic engineering bacterium of claim 1 is applied to MPLA production.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104844665A (en) * | 2015-05-28 | 2015-08-19 | 江南大学 | Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains |
| CN105001276A (en) * | 2015-05-28 | 2015-10-28 | 江南大学 | Preparation and application of novel low-toxic Kdo2-monphosphate lipoid A |
| CN113151354A (en) * | 2021-03-22 | 2021-07-23 | 中国农业科学院兰州兽医研究所 | Vector for conditional knockout of target gene and method for conditional knockout of target gene |
| CN113337529A (en) * | 2020-03-03 | 2021-09-03 | 北京安百胜生物科技有限公司 | Engineering bacterium of hormone and construction expression method |
| CN115181715A (en) * | 2022-08-10 | 2022-10-14 | 江南大学 | Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant |
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| CN102399736A (en) * | 2011-10-18 | 2012-04-04 | 江南大学 | Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844665A (en) * | 2015-05-28 | 2015-08-19 | 江南大学 | Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains |
| CN105001276A (en) * | 2015-05-28 | 2015-10-28 | 江南大学 | Preparation and application of novel low-toxic Kdo2-monphosphate lipoid A |
| CN113337529A (en) * | 2020-03-03 | 2021-09-03 | 北京安百胜生物科技有限公司 | Engineering bacterium of hormone and construction expression method |
| CN113151354A (en) * | 2021-03-22 | 2021-07-23 | 中国农业科学院兰州兽医研究所 | Vector for conditional knockout of target gene and method for conditional knockout of target gene |
| CN113151354B (en) * | 2021-03-22 | 2022-03-08 | 中国农业科学院兰州兽医研究所 | Vector for conditional knockout of target gene and method for conditional knockout of target gene |
| CN115181715A (en) * | 2022-08-10 | 2022-10-14 | 江南大学 | Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant |
| CN115181715B (en) * | 2022-08-10 | 2023-08-08 | 江南大学 | Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant |
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