CN101182538A - Bacillus coli perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation - Google Patents
Bacillus coli perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation Download PDFInfo
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- CN101182538A CN101182538A CN 200710193754 CN200710193754A CN101182538A CN 101182538 A CN101182538 A CN 101182538A CN 200710193754 CN200710193754 CN 200710193754 CN 200710193754 A CN200710193754 A CN 200710193754A CN 101182538 A CN101182538 A CN 101182538A
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Abstract
The invention discloses a colon bacillus perforated plasmid vector p BV-E and a construction method thereof; the invention also discloses the purpose of the colon bacillus perforated plasmid vector in the process of preparing the ghost of the colon bacillus, which belongs to the gene engineering field. The colon bacillus perforated plasmid vector of the invention adopts two promoters of pR and pL serial double promoters to express E gene and successfully prepares the ghost of the colon bacillus; the decomposition rate can reach 99.9613 percent which is 3 magnitudes higher than of the present perforated plasmid vector with pR or pL single promoter.
Description
Technical field
The present invention relates to a kind of plasmid vector, relate in particular to a kind of bacillus coli perforating plasmid vector and construction process thereof, the invention still further relates to the purposes of bacillus coli perforating plasmid vector in preparation intestinal bacteria ghost, belong to the genetically engineered field.
Background technology
Bacterium ghost (Bacterial Ghost) is a kind of empty bacterial body that does not have cytoplasm and nucleic acid.PhiX174 phage E lysis genes is expressed in bacterium, this gene coded protein can form on bacterial cell membrane and cell wall wears the film tunnel, under the effect of osmotic pressure, thalline is broken, bacterium born of the same parents inner cell slurry and nucleic acid component are discharged from by this tunnel, form a kind of bacterium shell of sky, be " Bacterial Ghost ".Bacterial ghost is by inner membrance (cytoplasmic membrane), and endochylema gap (periplasmic space) and adventitia (outer membrane) are formed, so cell walls is got off by complete preservation to a great extent.On the adventitia of some bacterial strain, also have one deck S-layer, also become one of composition of bacterial ghost.Bacterial Ghost itself can be used as a kind of good vaccine, because this form has kept bacterial membrane structure and the associated antigen protein the same with viable bacteria, and adventitia contains the high conservative structure PAMP (pathogen-associatedmolecular patterns) of natural immunocyte by the identification of pattern recognition acceptor, lipopolysaccharides for example, peptidoglycan, CPG, OmpA, pili etc. can be engulfed by DC and scavenger cell effectively.At present, ghost has obtained the good immune protection effect by approach immunity such as vein, subcutaneous, gases in animal models such as mouse, rabbit, pig.With A.pleuropneumoniae ghosts gas immunization pig, induced the protection fully of the pleuropneumonia that causes at A.pleuropneumoniae.Cholera bacteria ghost (VCG) is through the high-caliber anti-cholera specific IgG antibodies of the blood serum induced generation of subcutaneous injection mouse.Show simultaneously, be enough to protect newborn mice to exempt from the infection of vibrio cholerae at the antibody of VCG.In addition, Bacterial Ghost can also be used as a kind of fabulous delivery system, by before the bacterium cracking bacterium being carried out the artificial reconstructed of adventitia, with exotic antigen, other composition such as nucleic acid or medicine is anchored to the medial and lateral of after birth, or fills in the endochylema pericentral siphon.Zhi Bei reorganization ghost has intact, natural bacterial outer membrane structure like this, can simultaneous excitation body fluid and cellullar immunologic response.Surface adhesion structures such as its pili, make it target again and be attached on specific tissue, mucous membrane surface as gi tract and respiratory tract, and then more easily by the body phagocytic cell, discern as the M cell of PP knot (Peyer ' s Patches) and to catch, so effectively delivery of vaccines antigen to mucous membrane surface with bring out relevant mucosal immune response.People such as Eko are with the interior film expression of chlamydia trachomatis (C.trachomatis) antigen cholera bacteria, and the reorganization ghost (VCG) of cracking preparation has excited the Th1 type immunne response of reproductive tract mucous membrane effectively then, is setting about entering clinical experiment now.Compare with traditional prokaryotic system expressing protein, reorganization bacterial ghost has several big advantages: (1) recombinant protein is incorporated in the environment with hyperimmunization originality; (2) to proteic size requirements wide scope, but proteic molecular weight is preferably in 2000 to 200, between the 000Da; (3) recombinant protein is incorporated on the film of bacterium after directly expressing, and just can be directly used in immune animal after the cracking, and unnecessary the elephant wanted first separation and purification recombinant protein when preparing immunogen in the past; (4) recombinant protein that is incorporated into cell walls is with natural conformation, so kept its original activity form.And the albumen of general gene recombination by prokaryotic system mostly with the formal representation of inclusion body, method that can only be by sex change, renaturation is the recoverin activity to a certain extent.(5) preparation of ghost is simple relatively, and available fermentation technique obtains, and need not carry out complicated purifying work; Can freeze dried form be stored in room temperature.
The expression of dissolving gene E can or start transcribing under the control of repressible system at lacPO-lacIq at λ pL/pR-cI857 and finish.Some SL plasmids that contain different resistance markers, ori and gene E expression control are fabricated out.λ pR promotor and temperature sensitive repressor cI857 can suppressor gene below 30 ℃ the expression of E, be higher than 30 ℃ and can cause the hot deactivation of repressor cI857 and induce E genetic expression.In order to prepare ghost, bacterium must grow into logarithmic phase under 28 ℃ of conditions, be warmed up to 42 ℃ then and induce its dissolving.
The protein mediated dissolving of E successfully has been applied to various coli strains, Salmonella typhimurium, Salmonella enteritidis, klepsiella pneumoniae, segmental bronchus sepsis bordetella, Hp, vibrio cholerae, actinobacillus pleuropneumoniae, hemophilus influenzae, haemolysis pasteurellosis bacillus, pasteurella multocida, Pseudomonas aeruginosa, pseudomonas putida etc.Scope is like this bigly to have illustrated that the protein mediated dissolving of E may take place as long as E dissolving box is introduced in the appropriate carriers in each Gram-negative bacteria.
At present, abroad the ghost of actinobacillus pleuropneumoniae prepare aspect existing a lot of successful precedents.In these researchs, the perforating plasmid carrier of use has only a promotor mostly in the process that makes up, and promptly pR or pL promotor start the E expression of gene, cause E expression of gene amount not high enough, during with its preparation ghost, there are defectives such as lysis efficiency is lower, have to be overcome.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, a kind of new bacillus coli perforating plasmid vector is provided, this perforating plasmid carrier adopts two series connection double-promoters to express the E gene, effectively raises the lysis efficiency in intestinal bacteria.
The present invention's technical problem at first to be solved is achieved through the following technical solutions:
A kind of bacillus coli perforating plasmid vector pBV-E is made up of bacterial virus bacteriolysis gene E (SEQ ID NO.5) and pBV220 carrier.
Another technical problem to be solved by this invention provides a kind of method that makes up above-mentioned bacillus coli perforating plasmid vector pBV-E.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method that makes up bacillus coli perforating plasmid vector pBV-E comprises:
With phage PhiX174 double-stranded DNA is template, is that primer carries out pcr amplification with SEQID NO.1 and SEQ ID NO.2, obtains bacterial virus bacteriolysis gene E; To be connected with pBV220 carrier with the T4 dna ligase behind the bacteriolyze gene E purifying, promptly through the digestion of EcoRI and BamH I double digestion.
Another technical problem to be solved by this invention provides a kind of preparation method of intestinal bacteria ghost.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of preparation method of intestinal bacteria ghost comprises: inoculation contains the e. coli tg1 of perforating plasmid carrier pBV-E in containing the LB of penbritin, and 28 ℃ of concussions of spending the night are cultivated, and transfer then in the LB that contains penbritin, and 28 ℃ of concussions are cultured to OD
600nmReach about 0.4; It is standby to take out 100 μ l cultures, will remain culture and heighten 42 ℃ of cultivations rapidly to induce E genetic expression, continues to cultivate 3-5 hour; Getting each 100 μ l of culture before and after inducing suitably is coated with the LB flat board after the dilution and carries out viable bacteria CFU and detect; The ghost ghost that bacteriolyze finishes back formation washs 3 times with PBS, and freeze-drying is preserved.
Perforating plasmid carrier pBV-E of the present invention has adopted two promotors, be that pR, pL series connection double-promoter is expressed the E gene, successfully prepared intestinal bacteria Ghost (ghost), lysis efficiency can exceed 3 orders of magnitude than external perforating plasmid carrier with pR or the single promotor of pL at the colibacillary lysis efficiency of homophyletic up to 99.9613%.
Description of drawings
The structure synoptic diagram of Fig. 1 bacillus coli perforating plasmid vector pBV-E of the present invention;
The transmission electron microscope observing result of Fig. 2 intestinal bacteria Ghost (ghost).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Test materials
Bacterial strain and plasmid: e. coli tg1 and be the Zi Zang of this research department; Phage PhiX174 purchases the Bioisystech Co., Ltd in Promega (Beijing).The building process of plasmid pBV220 can make up according to following document: Zhang Zhiqing, and Yao Lihong, Hou Yunde contains the establishment and the application thereof of the protokaryon efficient expression vector of PrPl promotor, viral journal, 1990,6 (2), 111-116.
The structure of embodiment 1 perforating plasmid carrier pBV-E
1.1 the clone of phage PhiX174 bacteriolyze gene E
Encoding sequence design primer according to GenBank pnagus medius PhiX174 bacteriolyze gene E:
Lysis?E-U：5’-AGGGAATTCATGGTACGCTGGACTTTGTGG-3’(SEQ?ID?NO.1)
Lysis?E-L：5’-AGGGGATCCGAGCTCTCACTCCTTCCG-3’(SEQ?ID?NO.1)
Primer 5 ' end in upstream and downstream has been introduced restriction enzyme site EcoR I and BamH I respectively, and it is synthetic to give birth to the worker by Shanghai.With phage PhiX174 double-stranded DNA is that template amplification bacteriolyze gene E:PCR amplification reaction system is 50 μ L, wherein MgSO
42mM, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10xTaq buffer, Taq
TMArchaeal dna polymerase 2U (TaKaRa), template DNA 10ng.The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 5min.The PCR product reclaims test kit with glue and reclaims after gel electrophoresis, carry out enzyme with EcoR I and BamH I then and cut, and reclaims test kit with glue and reclaims.
1.2 the structure of perforating plasmid carrier pBV-E
With the bacteriolyze gene E of purifying with T4 dna ligase (TaKaRa) be connected with the pBV220 carrier of BamH I double digestion digestion through EcoR I, 16 ℃ are spent the night and connect and heat shock is transformed into the e. coli tg1 competent cell, being accredited as the male clone through bacterium colony PCR increases bacterium and utilizes alkaline lysis to extract plasmid in a small amount, called after pBV-E is to clone's the bacteriolyze gene E evaluation of checking order.
The preparation of embodiment 2 intestinal bacteria Ghost (ghost)
Contain the e. coli tg1 that inoculation among the LB of 50 μ g/mL penbritins contains perforating plasmid carrier pBV-E at 5mL, (220r/min) cultivated in 28 ℃ of concussions of spending the night, and the 1-2mL that transfers then contains among the LB of 50 μ g/mL penbritins in 50mL, and 28 ℃ of concussions are cultured to OD
600nmReach about 0.4.It is standby to take out 100 μ l cultures, will remain culture and heighten 42 ℃ of cultivations rapidly to induce E genetic expression, continues to cultivate 3-5 hour; Getting each 100 μ l of culture before and after inducing suitably is coated with the LB flat board after the dilution and carries out viable bacteria CFU and detect.The ghost ghost that bacteriolyze finishes back formation washs 3 times with PBS, and freeze-drying is preserved, and the ghost after the freeze-drying is carried out viable bacteria CFU detect.
Test-results: the culture before and after the bacteriolyze is from 3.1 * 10
8Drop to 1.2 * 10
4(CFU/ml), bacteriolyze deactivation efficient reaches 99.9613%.
The transmission electron microscope observing of test example 1 intestinal bacteria Ghost (ghost)
With the centrifugal 10min of bacterium liquid 4000g that in 42 ℃, cultivates 3-5 hour among the embodiment 2, with 2.5% the eleventh of the twelve Earthly Branches dialdehyde fixation of bacteria, put 4 ℃ of following 2h, 0.01mol/L PBS washing 3 times, centrifugal after perosmic anhydride is fixing again, carry out electron microscopic observation after the step process such as ethanol dewaters step by step, embedding medium embedding.Electron microscopic observation the results are shown in Figure 2.
Sequence table
SEQUENCE?LISTING
＜110〉Scientia Agricultura Sinica research institute Harbin veterinary institute
＜120〉bacillus coli perforating plasmid vector, its construction process and the application in the preparation ghost
<130>22
<160>2
<170>PatentIn?version?3.4
<210>1
<211>30
<212>DNA
<213>phage?E
<400>1
agggaattca?tggtacgctg?gactttgtgg 30
<210>2
<211>27
<212>DNA
<213>phage?E
<400>2
aggggatccg?agctctcact ccttccg 27
Claims (3)
1. a bacillus coli perforating plasmid vector pBV-E is characterized in that: be made up of bacterial virus bacteriolysis gene E and pBV220 carrier.
2. method that makes up the bacillus coli perforating plasmid vector pBV-E of claim 1, comprising: with phage PhiX174 double-stranded DNA is template, is that primer carries out pcr amplification with SEQ ID NO.1 and SEQ ID NO.2, obtains bacteriolyze gene E; To use T behind the bacteriolyze gene E purifying
4Dna ligase is connected with pBV220 carrier through the digestion of EcoR I and BamH I double digestion, promptly.
3. the application of the bacillus coli perforating plasmid vector pBV-E of claim 1 in preparation intestinal bacteria ghost, comprise: inoculation contains the intestinal bacteria of perforating plasmid carrier pBV-E in containing the LB of penbritin, 28 ℃ of concussions of spending the night are cultivated, transfer then in the LB that contains penbritin, 28 ℃ of concussions are cultured to OD
600nmReach about 0.4; It is standby to take out 100 μ l cultures, will remain culture and heighten 42 ℃ of cultivations rapidly to induce E genetic expression, continues to cultivate 3-5 hour; Getting each 100 μ l of culture before and after inducing suitably is coated with the LB flat board after the dilution and carries out viable bacteria CFU and detect; The ghost ghost that bacteriolyze finishes back formation washs 3 times with PBS, and freeze-drying is preserved.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286106A (en) * | 2011-07-11 | 2011-12-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Sandwich outer membrane protein display vector and escherichia coli vaccine prepared by applying same |
| CN103045614A (en) * | 2012-12-12 | 2013-04-17 | 肇庆大华农生物药品有限公司 | Mutant phage E gene, punching plasmid vector containing the gene and application thereof in preparation of vaccine |
| CN103146732A (en) * | 2013-01-28 | 2013-06-12 | 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 |
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
| 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 |
-
2007
- 2007-11-26 CN CN 200710193754 patent/CN101182538A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286106A (en) * | 2011-07-11 | 2011-12-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Sandwich outer membrane protein display vector and escherichia coli vaccine prepared by applying same |
| CN103045614A (en) * | 2012-12-12 | 2013-04-17 | 肇庆大华农生物药品有限公司 | Mutant phage E gene, punching plasmid vector containing the gene and application thereof in preparation of vaccine |
| CN103045614B (en) * | 2012-12-12 | 2014-04-09 | 肇庆大华农生物药品有限公司 | Mutant phage E gene, punching plasmid vector containing gene and application thereof in preparation of vaccine |
| CN103146732A (en) * | 2013-01-28 | 2013-06-12 | 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 |
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
| 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 |
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Open date: 20080521 |