CN102586307B - Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation - Google Patents
Chlorampenicol resistant temperature controlled lytic plasmid, its construction and application in bacterial ghost preparation Download PDFInfo
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Abstract
The invention discloses a chloramphenicol resistant temperature controlled lytic plasmid pBV-geneE-cat and its construction method, and also discloses application of the plasmid pBV-geneE-cat in preparing a bacterial ghost of Ecoli. BL21 (DE3). The chloramphenicol resistant temperature controlled lytic plasmid pBV-geneE-cat of the invention is composed of a lytic gene E of phage PhiX174, a chloramphenicol acetyl transferase gene (cat) and a prokaryotic temperature controlled expression vector pBV220. The plasmid pBV-geneE-cat constructed in the invention can be replicated stablely in Ecoli., and can efficiently express the chloramphenicol acetyl transferase so as to generate resistance to chloramphenicol, which can be taken as a resistance selection marker to achieve efficient expression of the lytic gene E. The lytic plasmid provided in the invention can reach lytic efficiency up to 99.98% during prepaing a chloramphenicol sensitive bacterial ghost of Ecoli.
Description
Technical field
The present invention relates to a kind of plasmid vector, particularly a kind of chlorampenicol resistant temperature control cracking plasmid and construction process thereof, with and purposes in preparation intestinal bacteria ghost, belong to genetically engineered field.
Background technology
Bacterium ghost (Bacterial ghost) is the intact bacterial ghost that Gram-negative bacteria forms after by the crack protein E cracking of Phage PhiX174, the inactivation of gene mode of this non-sex change makes ghost retain bacterial membrane structure and the associated antigen protein the same with viable bacteria, thus humoral immunization and the cellullar immunologic response of induction body.The natural high conservative structure PAMP (pathogen-associated molecular patterns) of ghost adventitia, for example: lipopolysaccharides, peptidoglycan, CPG, pili etc., can be identified by pattern recognition receptors by immunocyte, effectively by dendritic cell and macrophage phagocytic, and effectively promote the ripe and activation of dendritic cell.These Biological characteristics of ghost can be directly use it as vaccine, also can be used as the recombiant vaccine of submission heterologous antigen and as the even delivery vector of medicine of DNA vaccination.In addition, ghost can be produced by fermentation mode, and cryodesiccated ghost at room temperature can keep for a long time, and can not cause the loss of efficiency.
The formation of bacterium ghost is to realize by the strict expression regulation of the Lysis gene E to Phage PhiX174.Lysis gene E can be controlled by multiple expression systems, as λ pL/pR-cI857, LacPO-LacI
q(starting and barrier), P
tol-xylS and P
bAD-araC etc., wherein most widely used is to transcribe at λ pL/pR-c1857 the controlled expression of realizing gene under control.The bacteria lysis of the controlled expression mediation of gene E has successfully been applied to various coli strains, Salmonella typhimurium, Salmonella enteritidis, vibrio cholerae, Klebsiella pneumonia, Hp, pleuropneumonia radiation bacillus, hemophilus influenzae, haemolysis pasteurellosis bacillus, pasteurella multocida, edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila etc.The wide explanation of range of application is as long as be incorporated into gene E cracking frame in suitable carrier, and the protein mediated cracking of E may occur in every kind of Gram-negative bacteria.
The cracking plasmid majority of the gene E mediation having built at present utilizes penbritin as resistance screening mark, also has some to have kantlex, tsiklomitsin or gentamicin resistance screening mark.Because bacterial drug resistance phenomenon is more and more common, many bacteriums produce resistance to the common antibiotics such as penbritin, kantlex, cannot utilize these microbiotic as selection markers.But many pathogenic bacterias, if Vibrio parahemolyticus, vibrio cholerae, Yersinia enterocolitica, Escherichia coli O 157, Bacillus proteus, salmonella, shigella etc. are all to paraxin sensitivity, also have most of hydrobiont pathogenic bacterias as all responsive or extremely sensitive to paraxin in Cytophaga Columnaris, the sticky fiber bacterium of column, Pseudomonas fluorescens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas punctata, aeromonas salmonicida and Yersinia ruckeri etc.Therefore, utilize paraxin can make the protein mediated cracking of E be applied to widely in bacterial isolates as resistance screening mark.The temperature control cracking plasmid that builds chlorampenicol resistant is the prerequisite that many pathogenic bacterias can successfully form ghost.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of new chlorampenicol resistant temperature control cracking plasmid, and its construction process be provided, with and application in ghost preparation.
The present invention is achieved by the following technical solutions:
A kind of chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat, by Phage PhiX174 Lysis gene E (as shown in SEQ No.3), chloramphenicol acetyl transferasegene (cat, as shown in SEQ No.4) and protokaryon Human liver glutathione carrier pBV220 composition.
The construction process of described chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat is: taking Phage PhiX174 RFI DNA as template, shown in SEQ No.1 and SEQ No.2, DNA carries out pcr amplification as primer, obtain Phage PhiX174 Lysis gene E, then by its be connected through the pBV220 carrier of EcoR I and Pst I double digestion, obtain temperature control cracking plasmid pBV-geneE; Plasmid pLysS is cut through Acc II enzyme, obtain the cat gene that comprises flank regulating and controlling sequence of 1787bp, then with T4 DNA ligase by cat gene be connected through the pBV-geneE of Sca I single endonuclease digestion, obtain chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat.
Described chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat can be used in and prepares ghost, and concrete application mode is exemplified below: chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat electric shock is transformed in competent escherichia coli cell; After electricity transforms, coating, containing the LB agar plate of paraxin, is cultivated, by bacterium colony PCR qualification transformant; Then, be inoculated in the LB liquid nutrient medium containing paraxin by transforming the successfully intestinal bacteria mono-clonal containing pBV-geneE-cat, shaking culture, and then transfer in containing the LB liquid nutrient medium of paraxin, shaking culture is to OD
600nmreach 0.4-0.6, culture is adjusted to rapidly to the expression of 42 DEG C of cultivations with induction E gene, continue to cultivate, obtain intestinal bacteria ghost.
The above-mentioned method of preparing ghost is specific as follows: chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat electric shock is transformed in e. coli bl21 (DE3) competent cell, described Electroporation-competent cells is purchased from TaKaRa, or with reference to method (molecular cloning experiment guide) preparation of Sambrook etc., electricity conversion instrument (Bio-Rad, California, USA) shock parameters is: 1.5kV/cm, 25 μ F, 200 Ω, 4.0ms, after electricity transforms, coating is containing the LB agar plate of 37 μ g/mL paraxin, cultivate 16 hours for 30 DEG C, by bacterium colony PCR qualification transformant.The LB liquid nutrient medium of 5mL containing 37 μ g/mL paraxin will be inoculated in containing BL21 (DE3) mono-clonal of pBV-geneE-cat, 30 DEG C of vibrations (200r/min) overnight incubation, then transfer and contain in the LB liquid nutrient medium of 37 μ g/mL paraxin in 50mL by 1-2% (v/v), 30 DEG C of shaking culture are to OD
600nmreach 0.4-0.6.Culture is adjusted to rapidly to the expression of 42 DEG C of cultivations with induction E gene, continues to cultivate 4h, obtain intestinal bacteria ghost.
The chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat that the present invention builds can be in to the intestinal bacteria of paraxin sensitivity stable existence, and can induce it that cracking of greater efficiency occurs, intestinal bacteria ghost is prepared in success, and lysis efficiency can be up to 99.998%.
The chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat that the present invention builds, can stable copying in intestinal bacteria, and can high efficient expression E.C. 2.3.1.28, thereby generation chlorampenicol resistant, be used as resistance screening mark with this, be applicable to most pathogenic bacterias, as Vibrio parahemolyticus, vibrio cholerae, Yersinia enterocolitica, Escherichia coli O 157, Bacillus proteus, salmonella, shigella, Cytophaga Columnaris, column is glued fiber bacterium, Pseudomonas fluorescens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas punctata, aeromonas salmonicida and Yersinia ruckeri etc., be widely used, and can high efficient expression Lysis gene E, the ghost lysis efficiency of preparation is high, can reach 99.998%.
Brief description of the drawings
Fig. 1: the present invention builds the schematic flow sheet of chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat;
Fig. 2: the cracking kinetic curve that contains the e. coli bl21 (DE3) of chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat;
Fig. 3: the transmission electron microscope photo of e. coli bl21 (DE3) ghost.
Embodiment
Further explain the present invention below in conjunction with specific embodiment, constitutional features and the advantage of the chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat that the present invention builds will be more clear along with description.In following embodiment, method therefor if no special instructions, is ordinary method.
Test materials: plasmid pBV220, plasmid pLysS, bacterial strain BL21 (DE3) are for preserving in this laboratory; Enterobacteria phage phiX174 RFI DNA, restriction enzyme, T4DNA ligase, PCR related reagent, Agarose Gel DNA Fragment Recovery Kit, TaKaRa MiniBEST Plasmid Purification Kit is all purchased from the precious biology of TaKaRa (engineering) Dalian company limited; Primer is synthetic to be completed by Hua Da gene (BGI) with order-checking.
The structure of embodiment 1, chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat
The clone of 1.1PhiX174 Lysis gene E
Encoding sequence design primer according to PhiX174 gene E in GenBank:
GeneE-F:5 '-ATCA
gAATTCaTGGTACGCTGGACTTTGTG-3 ' (SEQ No.1), introduces EcoR I restriction enzyme site;
GeneE-R:5 '-GGC
cTGCAGcAGAACGTTTTTTACCTTTAG-3 ' (SEQ No.2), introduces Pst I restriction enzyme site.
Primer is synthesized by Hua Da gene.Pass through pcr amplification gene E taking PhiX174RFIDNA as template.PCR reaction system is TaKaRa Ex Taq 1.2U, 10 × Ex Taq Buffer (Mg
2+plus) 5 μ L, dNTP Mixture (each 2.5mM) 4 μ L, PhiX174RFI DNA2ng, geneE-F (20 μ M) 1 μ L, geneE-R (20 μ M) 1 μ L, moisturizing is to 50 μ L.PCR response procedures is 94 DEG C of denaturation 5min, 94 DEG C of 45s, and 56 DEG C of 45s, 72 DEG C of 40s, 28 circulations, 72 DEG C are extended 1min.PCR product is through agarose gel electrophoresis, cuts glue and reclaim the fragment of about 300bp, carries out double digestion with EcoR I and Pst I, reclaims enzyme and cuts product, obtains geneE.
The structure of 12 temperature control cracking plasmid pBV-geneE
Plasmid pBV220 is through EcoR I and Pst I double digestion, and endonuclease reaction system is EcoR I 10U, Pst I 10U, 10 × H Buffer, 2 μ L, pBV2200.8 μ g, moisturizing to 20 μ L, and 37 DEG C of enzymes are cut 1h, reclaims enzyme and cuts product, obtains linearizing pBV220.Linearizing pBV220 is connected with geneE, ligation system is T4DNA ligase 350U, geneE 0.4pmol, pBV 0.03pmol, 10 × T4DNALigase Buffer, 2.5 μ L, moisturizing to 25 μ L, 16 DEG C connect 12h, and thermal shock is transformed into bacillus coli DH 5 alpha.The DH5 α transforming is shaking culture 1h under 30 DEG C, 180r/min condition in the LB liquid nutrient medium without penbritin first, is then coated on the LB flat board that contains penbritin 50 μ g/ml, cultivates 16h for 30 DEG C.By bacterium liquid PCR screening positive clone, and order-checking qualification.The transformant that gene E sequence is correct increases bacterium to be cultivated, and extracts plasmid, called after pBV-geneE.
The acquisition of the flat terminal fragment of 1.3 chloramphenicol acetyl transferasegene cat
Plasmid pLysS is through Acc II single endonuclease digestion, and the enzyme system of cutting is Acc II 10U, 10 × M Buffer, 2 μ L, pLysS 0.6 μ g, moisturizing to 20 μ L, and 37 DEG C of enzymes are cut 1h.Agarose gel electrophoresis enzyme is cut product, cuts glue and reclaims about 1800bp band, obtains the chloramphenicol acetyl transferasegene cat fragment that comprises promoter region.
The acquisition of the flat terminal fragment of 14pBV-geneE linearizing
Plasmid pBV220-geneE cuts through Sca I enzyme, and the enzyme system of cutting is Sca I 10U, 10 × H Buffer, 2 μ L, pBV2200.8 μ g, moisturizing to 20 μ L, and 37 DEG C of enzymes are cut 1h.Enzyme is cut product through agarose gel electrophoresis, reclaims about 3.6kb band, obtains the flat terminal fragment of pBV-geneE linearizing.
The structure of 1.5pBV-geneE-cat plasmid
By cat gene fragment phosphatizing treatment, reaction system is T4polynucleotide Kinase 12U, cat 10pmol, 5 μ L10 × T4 Polynucleotide Kinase Buffer, moisturizing to 50 μ L, processes 1h for 37 DEG C.By linearization plasmid pBV-geneE dephosphorylation, reaction system is Alkaline Phosphatase 4U, linearization plasmid pBV-geneE 15pmol, 10 × SAPBuffer, 5 μ L, and moisturizing to 50 μ L processes 1h for 37 DEG C.
Connect the cat fragment of dephosphorylized pBV-geneE fragment and phosphorylation with T4 DNA ligase, ligation system is T4DNAligase 350U, phosphorylation cat fragment 04pmol, dephosphorylation pBV-geneE fragment 0.03pmol, 10 × T4DNA Ligase Buffer, 2.5 μ L, moisturizing to 25 μ L, 16 DEG C of reaction 12h.Connection product is transformed into bacillus coli DH 5 alpha by thermal shock.By paraxin and bacterium liquid PCR screening positive clone, and order-checking qualification.The sequence of gene E and gene cat all correct transformant increases bacterium and cultivates, and extracts plasmid, called after pBV-geneE-cat.The schematic flow sheet of above-mentioned structure chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat as shown in Figure 1.
Embodiment 2, plasmid pBV-geneE-cat transform e. coli bl21 (DE3) and prepare ghost
The electricity of 2.1 chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat transforms
Chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat electric shock is transformed to e. coli bl21 (DE3) competent cell, described Electroporation-competent cells is purchased from TaKaRa, with reference to method (molecular cloning experiment guide) preparation of Sambrook etc., electricity conversion instrument (Bio-Rad, California, USA) shock parameters is: 1.25kV/cm, 25 μ F, 200 Ω, 6.0ms, after electricity transforms, coating is containing the LB agar plate of 37 μ g/mL paraxin, cultivate 16 hours for 30 DEG C, by bacterium colony PCR qualification transformant.
The LB liquid nutrient medium of 5mL containing 37 μ g/mL paraxin will be inoculated in containing BL21 (DE3) mono-clonal of chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat, 30 DEG C of vibrations (200r/min) overnight incubation, then transfer and contain in the LB liquid nutrient medium of 37 μ g/mL paraxin in 50mL by 1% (v/v), 30 DEG C of shaking culture are to OD
600nmreach 0.4-0.6.Culture is adjusted to rapidly to the expression of 42 DEG C of cultivations with induction E gene, continues to cultivate 4h.Different time points sampling before and after induction, the OD of monitoring culture
600nmchange with viable bacteria CFU, viable bacteria CFU counts by coating LB flat board after the each 100 μ L of culture before and after induction are suitably diluted.The ghost ghost PBS that induction finishes rear formation washs 3 times, and freeze-drying is preserved, and the ghost after freeze-drying is carried out to viable bacteria CFU detection.
The kinetic curve of plasmid pBV-geneE-cat induction BL21 (DE3) cracking is shown in Fig. 2.Heat up after induction 30min, the OD of BL21 (DE3) culture
600with the equal fast reducing of viable bacteria CFU, be slowly reduced to subsequently minimum level.Before induction, the viable count of culture is 3.45 × 10
7cFU/mL, when induction finishes, the viable count of culture is 5.26 × 10
2cFU/mL, the lysis efficiency of this plasmid reaches 99.998%.Ghost after freeze-drying does not detect viable bacteria.
The transmission electron microscope observing of test example 1, e. coli bl21 (DE3) ghost
By the centrifugal 10min of bacterium liquid 4000g of 42 DEG C of induction 4h in embodiment 2, with fixation of bacteria 3h at 4 DEG C of 2.5% glutaraldehyde, 0.01mol/LPBS washs 3 times, centrifugal rear direct electron microscopic observation.The results are shown in Figure 3.
Claims (4)
1. the construction process of a chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat, it is characterized in that: taking Phage PhiX174 RFI DNA as template, shown in SEQ No.1 and SEQ No.2, DNA carries out pcr amplification as primer, obtain Phage PhiX174 Lysis gene E, then by its be connected through the pBV220 carrier of EcoR I and Pst I double digestion, obtain temperature control cracking plasmid pBV-geneE; Plasmid pLysS is cut through Acc II enzyme, obtain the cat gene that comprises flank regulating and controlling sequence of 1787bp, then with T4DNA ligase by cat gene be connected through the pBV-geneE of Sca I single endonuclease digestion, obtain chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat;
The concrete mode of described acquisition plasmid pBV-geneE-cat is: plasmid pLysS is through Acc II single endonuclease digestion, and the enzyme system of cutting is Acc II10U, 10 × M Buffer2 μ L, pLysS0.6 μ g, moisturizing to 20 μ L, and 37 DEG C of enzymes are cut 1h; Agarose gel electrophoresis enzyme is cut product, cuts glue and reclaims band, obtains the chloramphenicol acetyl transferasegene cat fragment that comprises promoter region;
Plasmid pBV-geneE cuts through Sca I enzyme, and the enzyme system of cutting is Sca I10U, 10 × H Buffer2 μ L, pBV-geneE0.8 μ g, moisturizing to 20 μ L, and 37 DEG C of enzymes are cut 1h; Enzyme is cut product through agarose gel electrophoresis, reclaims band, obtains the flat terminal fragment of pBV-geneE linearizing;
By cat gene fragment phosphatizing treatment, reaction system is T4polynucleotide Kinase12U, cat10pmol, 5 μ L10 × T4Polynucleotide Kinase Buffer, moisturizing to 50 μ L, processes 1h for 37 DEG C; By linearization plasmid pBV-geneE dephosphorylation, reaction system is Alkaline Phosphatase4U, linearization plasmid pBV-geneE15pmol, 10 × SAP Buffer5 μ L, and moisturizing to 50 μ L processes 1h for 37 DEG C;
Connect the cat fragment of dephosphorylized pBV-geneE fragment and phosphorylation with T4DNA ligase, ligation system is T4DNA ligase350U, phosphorylation cat fragment 0.4pmol, dephosphorylation pBV-geneE fragment 0.03pmol, 10 × T4DNA Ligase Buffer2.5 μ L, moisturizing to 25 μ L, 16 DEG C of reaction 12h; Connection product is transformed into bacillus coli DH 5 alpha by thermal shock; By paraxin and bacterium liquid PCR screening positive clone, and order-checking qualification; The sequence of gene E and gene cat all correct transformant increases bacterium and cultivates, and extracts plasmid, called after pBV-geneE-cat.
2. utilize the construction process described in claim 1 to build the chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat obtaining.
3. chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat claimed in claim 2 is in the application of preparing in ghost.
4. application according to claim 3, is characterized in that: the method for preparing ghost is as follows: chlorampenicol resistant temperature control cracking plasmid pBV-geneE-cat electric shock is transformed in competent escherichia coli cell; After electricity transforms, coating, containing the LB agar plate of paraxin, is cultivated, by bacterium colony PCR qualification transformant; Then, be inoculated in the LB liquid nutrient medium containing paraxin by transforming the successfully intestinal bacteria mono-clonal containing pBV-geneE-cat, shaking culture, and then transfer in containing the LB liquid nutrient medium of paraxin, shaking culture is to OD
600nmreach 0.4-0.6, culture is adjusted to rapidly to the expression of 42 DEG C of cultivations with induction E gene, continue to cultivate, obtain intestinal bacteria ghost.
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| CN103146732B (en) * | 2013-01-28 | 2014-11-12 | 山东省农业科学院畜牧兽医研究所 | Efficient splitting tandem gene, efficient splitting plasmid and construction method and appliance |
| CN106635930A (en) * | 2017-02-15 | 2017-05-10 | 北京市水产科学研究所 | Culture medium for fermenting bacterial ghosts of aeromonas veronii as well as preparation method and application of culture medium |
| CN109402156A (en) * | 2018-11-30 | 2019-03-01 | 浙江省海洋水产研究所 | A kind of temperature control cracking plasmid and its building and the application in ghost preparation |
| CN110408581A (en) * | 2019-07-11 | 2019-11-05 | 江苏农牧科技职业学院 | A kind of preparation method of O24 type duck enteropathogenic E. Coli ghost |
| CN112608934A (en) * | 2020-12-16 | 2021-04-06 | 华南理工大学 | Efficient preparation method of escherichia coli bacterial ghost |
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