CN101182537A - Pctinobacillus pleuropneumoniae perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation - Google Patents
Pctinobacillus pleuropneumoniae perforating plasmid vector, construction method thereof and applications in bacterial ghost preparation Download PDFInfo
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- CN101182537A CN101182537A CN 200710193753 CN200710193753A CN101182537A CN 101182537 A CN101182537 A CN 101182537A CN 200710193753 CN200710193753 CN 200710193753 CN 200710193753 A CN200710193753 A CN 200710193753A CN 101182537 A CN101182537 A CN 101182537A
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Abstract
The invention discloses a pleuropneumonia actinobacillus perforated plasmid vector p GZRS-E and a construction method thereof; the invention also discloses the purpose of the pleuropneumonia actinobacillus perforated plasmid vector p GZRS-E in the process of preparing the ghost of the pleuropneumonia actinobacillus, which belongs to the gene engineering field. The perforated plasmid vector p GZRS-E of the invention is constructed based on the pleuropneumonia actinobacillus and the plasmid vector p GZRS-18 which can be duplicated in a shuttled way in colon bacillus; the genetic stability of the plasmid vector in the pleuropneumonia actinobacillus is much higher than that of the plasmid vector pA12 which is used at present. The perforated plasmid vector of the invention p GZRS-E can duplicate a large amount of bacteriolysis genes E stably in the pleuropneumonia actinobacillus and successfully prepare the ghost of the pleuropneumonia actinobacillus; the decomposition rate can reach 99.9997 percent which is about 4 magnitudes higher than the prior art.
Description
Technical field
The present invention relates to a kind of plasmid vector, relate in particular to a kind of actinobacillus pleuropneumoniae perforating plasmid carrier and construction process thereof, the invention still further relates to the purposes of actinobacillus pleuropneumoniae perforating plasmid carrier in preparation actinobacillus pleuropneumoniae 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 successful precedent.In the research, the perforating plasmid carrier that preparation actinobacillus pleuropneumoniae ghost uses is based on hemophilus influenzae-bacillus coli shuttle plasmid vector pAL2, and pAL2 derives from hemophilus influenzae.Though the sibship of hemophilus influenzae and actinobacillus pleuropneumoniae is closer, but the genetic stability of pAL2 in actinobacillus pleuropneumoniae will be weaker than the genetic stability in hemophilus influenzae widely, cause the expression amount of bacteriolyze gene E in actinobacillus pleuropneumoniae lower, finally cause this class perforating plasmid carrier when preparation actinobacillus pleuropneumoniae ghost, to have the not high defective of lysis efficiency.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, and a kind of new actinobacillus pleuropneumoniae perforating plasmid carrier is provided.
The present invention's technical problem at first to be solved is achieved through the following technical solutions:
A kind of actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E is made up of temperature sensitive regulatory gene cI857 (SEQIDNO.6), bacterial virus bacteriolysis gene E (SEQ ID NO.5) and actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS-18.
Another technical problem to be solved by this invention provides a kind of method that makes up above-mentioned actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method that makes up actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E comprises:
With phage PhiX174 double-stranded DNA is template, is that primer carries out pcr amplification with SEQ ID NO.1 and SEQID NO.2, obtains bacteriolyze gene E; With being connected with pBV220 carrier with the T4 dna ligase behind the bacteriolyze gene E purifying, obtain perforating plasmid carrier pBV-E through the digestion of EcoR I and BamH I double digestion; With perforating plasmid carrier pBV-E is template, with SEQ ID NO.3 and SEQ ID NO.4 is that primer carries out pcr amplification, BamH I and SacI double digestion will be used behind the pcr amplification product purifying, be connected with the actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS-18 of same double digestion then, promptly.
Another technical problem to be solved by this invention is to utilize constructed actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E to be applied to prepare the actinobacillus pleuropneumoniae ghost.
Another technical problem to be solved by this invention is achieved through the following technical solutions: actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E electric shock is transformed in actinobacillus pleuropneumoniae serum 1 type reference strain shope 4074 competent cells, the method (molecular cloning experiment guide) of competent preparation with reference to Sambrook etc. shocks by electricity, electroporation apparatus (Micro-pulser, Bio-Rad, USA) shock parameters is: 12.5 kV/cm, 200 Ω, 25 μ f, 5.2ms transform back coating Amp
+TSB agar plate (10%NAD), transformant is identified by bacterium colony PCR.The actinobacillus pleuropneumoniae that will contain perforating plasmid carrier pGZRS-E is inoculated into TSB meat soup (10%NAD, 50 μ g/mL penbritins) in the liquid nutrient medium, (220r/min) cultivated in 28 ℃ of concussions of spending the night, 1 ~ the 2mL that transfers then contains in the TSB meat soup liquid nutrient medium of 50 μ g/mL penbritins and 10%NAD in 50mL, and 28 ℃ of concussions are cultured to OD
600nmReach about 0.5.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 5 ~ 7 hours.Getting each 100 μ l of culture before and after inducing suitably is coated with the TSB agar plate 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.
Perforating plasmid carrier pGZRS-E of the present invention is based on actinobacillus pleuropneumoniae plasmid self and also can shuttles back and forth to make up on the basis of the plasmid vector pGZRS-18 that duplicates in intestinal bacteria and form, and the genetic stability of this plasmid vector in actinobacillus pleuropneumoniae will be higher than the existing plasmid vector pAL2 that uses widely.The perforating plasmid carrier pGZRS-E that the present invention utilizes pGZRS-18 to make up to obtain can stablize in actinobacillus pleuropneumoniae and duplicates and great expression bacteriolyze gene E, the ghost ghost that has successfully prepared actinobacillus pleuropneumoniae, lysis efficiency can be up to 99.9997%, than having exceeded about 4 orders of magnitude abroad.
Description of drawings
The structure synoptic diagram of Fig. 1 actinobacillus pleuropneumoniae perforating plasmid carrier of the present invention pGZRS-E;
The transmission electron microscope observing result of Fig. 2 actinobacillus pleuropneumoniae 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.
Bacterial strain and plasmid
Actinobacillus pleuropneumoniae serum 1 type reference strain shope 4074 is the Zi Zang of this research department, and 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.
(University of Wisconsin, Madison U.S.A) is so kind as to give actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS 18 by Dr.Susan E.H.West.
PGZRS-18 also can make up according to following document and obtain: West SE et al.Construction ofActinobacillus pleuropneumoniae-Escherichia coli shuttle vectors:expression of antibiotic-resistance genes, Gene, 1995,160 (1): 81-6.
The structure of embodiment 1 actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-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?N0.2)
Primer 5 ' end in upstream and downstream has been introduced restriction enzyme site EcoRI 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, 10x Taq 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 EcoRI 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.
1.3 the structure of perforating plasmid carrier pGZRS-E
According to the sequences Design primer cI-U of temperature sensitive regulatory gene cI857 on the pBV220 plasmid vector, according to the sequences Design primer E L of bacteriolyze gene E, sequence is as follows:
cI-U：5’-GGGGGGATCCTCAGCCAAACGTCTC-3’(SEQ?ID?NO.3)
E-L：5’-ACTGTTGAGCTCTCACTCCTTCCGCA-3’(SEQ?ID?NO.4)
Primer 5 ' end in upstream and downstream has been introduced restriction enzyme site BamH I and Sac I respectively, and it is synthetic to give birth to the worker by Shanghai.With perforating plasmid carrier pBV-E is template, and cI-U and E-L are that primer carries out pcr amplification, and gained PCR product comprises temperature sensitive regulatory gene cI857 and bacteriolyze gene E, called after cI+E.The pcr amplification reaction system is 50 μ L, wherein MgSO
42mM, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10x Taq buffer, LA 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 1min, 72 ℃ of 1.5min, 30 circulations, 72 ℃ of 10min.Use BamH I and Sac I double digestion behind the PCR product cI+E purifying, be connected with the actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS-18 of same double digestion then, 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 pGZRS-E is to clone's temperature sensitive regulatory gene cI857 and the bacteriolyze gene E evaluation of checking order.
The preparation of embodiment 2 actinobacillus pleuropneumoniae ghost
Actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E electric shock is transformed in actinobacillus pleuropneumoniae serum 1 type reference strain shope 4074 competent cells, the method (molecular cloning experiment guide) of competent preparation with reference to Sambrook etc. shocks by electricity, electroporation apparatus (Micro-pulser, Bio-Rad, USA) shock parameters is: 12.5kV/cm, 200 Ω, 25 μ f, 5.2ms transform back coating Amp
+TSB agar plate (10%NAD), transformant is identified by bacterium colony PCR.The actinobacillus pleuropneumoniae that will contain perforating plasmid carrier pGZRS-E is inoculated into TSB meat soup (10%NAD, 50 μ g/mL penbritins) in the liquid nutrient medium, (220r/min) cultivated in 28 ℃ of concussions of spending the night, 1 ~ the 2mL that transfers then contains in the TSB meat soup liquid nutrient medium of 50 μ g/mL penbritins and 10%NAD in 50mL, and 28 ℃ of concussions are cultured to OD
600nmReach about 0.5.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 5 ~ 7 hours.Getting each 100 μ l of culture before and after inducing suitably is coated with the TSB agar plate 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.
Detected result: the culture before and after the bacteriolyze is from 1.2 * 10
9Drop to 2.6 * 10
3(CFU/ml), bacteriolyze deactivation efficient reaches 99.9997%.
The scanning electron microscopic observation of test example 1 actinobacillus pleuropneumoniae ghost
Get and induce preceding normal actinobacillus pleuropneumoniae cell of bacteriolyze and ghost cell suitably to concentrate through inducing the bacteriolyze preparation, PBS washing 5 times, glutaraldehyde is 60min fixedly, the multistep ethanol dehydration, acetone dehydration, after critical point drying, metal spraying, use emission scan electron microscopic observation, photograph.The results are shown in Figure 2.
SEQUENCE?LISTING
＜110〉solid research of agricultural science institute Harbin veterinary institute in
＜120〉actinobacillus pleuropneumoniae perforating plasmid carrier, its construction process reach in the preparation ghost and use
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<170>PatentIn?version?3.4
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ccgtcattgc?ttattatgtt?catcccgtca?acattcaaac?ggcctgtctc?atcatggaag 120
gcgctgaatt?tacggaaaac?attattaatg?gcgtcgagcg?tccggttaaa?gccgctgaat 180
tgttcgcgtt?taccttgcgt?gtacgcgcag?gaaacactga?cgttcttact?gacgcagaag 240
aaaacgtgcg?tcaaaaatta?cgtgcggaag?gagtga 276
<210>6
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<213>
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tcagccaaac?gtctcttcag?gccactgact?agcgataact?ttccccacaa?cggaacaact 60
ctcattgcat?gggatcattg?ggtactgtgg?gtttagtggt?tgtaaaaaca?cctgaccgct 120
atccctgatc?agtttcttga?aggtaaactc?atcaccccca?agtctggcta?tgcagaaatc 180
acctggctca?acagcctgct?cagggtcaac?gagaattaac?attccgtcag?gaaagcttgg 240
cttggagcct?gttggtgcgg?tcatggaatt?accttcaacc?tcaagccaga?atgcagaatc 300
actggctttt?ttggttgtgc?ttacccatct?ctccgcatca?cctttggtaa?aggttctaag 360
cttaggtgag?aacatccctg?cctgaacatg?agaaaaaaca?gggtactcat?actcacttct 420
aagtgacggc?tgcatactaa?ccgcttcata?catctcgtag?atttctctgg?cgattgaagg 480
gctaaattct?tcaacgctaa?ctttgagaat?ttttgtaagc?aatgcggcgt?tataagcatt 540
taatgcattg?atgccattaa?ataaagcacc?aacgcctgac?tgccccatcc?ccatcttgtc 600
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gcgacgtgcg?tcctcaagct?gctcttgtgt?taatggtttc?ttttttgtgc?tcatacgtta 720
aatctatcac?cgcaagggat?aaatatctaa?caccgtgcgt?gttgactatt?ttacctctgg 780
cggtgataat?ggttgcatgt?actaaggagg?ttgtatggaa?caacgcataa?ccctgaaaga 840
ttatgcaatg?cgctttgggc?aaaccaagac?agctaaagat?ctctcaccta?ccaaacaatg 900
cccccctgca?aaaaataaat?tcatataaaa?aacatacaga?taaccatctg?cggtgataaa 960
ttatctctgg?cggtgttgac?ataaatacca?ctggcggtga?tactgagcac?atcagcagga 1020
cacactgacc?accatgaagg?tgacgctctt?aaaaattaag?ccctgaagaa?gggcagcatt 1080
caaagcagaa?ggctttgggg?tgtgtgatac?gaaacgaagc?attggttaaa?aattaaggag 1140
gaattc 1146
Claims (3)
1. an actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E is characterized in that: be made up of temperature sensitive regulatory gene cI857, bacterial virus bacteriolysis gene E and actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS-18.
2. method that makes up the actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E of claim 1, comprising: with phage PhiX174 double-stranded DNA is template, is that primer carries out pcr amplification with SEQID NO.1 and SEQID NO.2, obtains bacteriolyze gene E; With being connected with pBV220 carrier with the T4 dna ligase behind the bacteriolyze gene E purifying, obtain perforating plasmid carrier pBV E through the digestion of EcoRI and BamHI double digestion; With perforating plasmid carrier pBV-E is template, with SEQ ID NO.3 and SEQ ID NO.4 is that primer carries out pcr amplification, BamH I and SacI double digestion will be used behind the pcr amplification product purifying, be connected with the actinobacillus pleuropneumoniae-bacillus coli shuttle plasmid vector pGZRS-18 of same double digestion then, promptly.
3. the application of the actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E of claim 1 in preparation actinobacillus pleuropneumoniae ghost, comprise: actinobacillus pleuropneumoniae perforating plasmid carrier pGZRS-E electric shock is transformed in actinobacillus pleuropneumoniae serum 1 type reference strain shope 4074 competent cells, transforms the back coating and contain Amp
+The TSB agar plate, transformant is identified by bacterium colony PCR; The actinobacillus pleuropneumoniae that will contain perforating plasmid carrier pGZRS-E is inoculated in the TSB meat soup liquid nutrient medium, 28 ℃ of concussions of spending the night are cultivated, transfer then in the TSB meat soup liquid nutrient medium that contains 50 μ g/mL penbritins and 10%NAD, 28 ℃ of concussions are cultured to OD
600nmReach about 0.5; 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 5-7 hour; Getting each 100 μ l of culture before and after inducing suitably is coated with the TSB agar plate 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451195A (en) * | 2013-09-10 | 2013-12-18 | 中国农业科学院哈尔滨兽医研究所 | Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines |
| CN110904021A (en) * | 2019-12-20 | 2020-03-24 | 北京市农林科学院 | Preparation and transformation method of porcine pleuropneumonia actinobacillus competent cells |
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2007
- 2007-11-26 CN CN 200710193753 patent/CN101182537A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451195A (en) * | 2013-09-10 | 2013-12-18 | 中国农业科学院哈尔滨兽医研究所 | Mutant phage lysis gene E, lysis plasmid vector containing lysis gene and application in preparation of bacterial ghost vaccines |
| CN110904021A (en) * | 2019-12-20 | 2020-03-24 | 北京市农林科学院 | Preparation and transformation method of porcine pleuropneumonia actinobacillus competent cells |
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