CN101503694B - Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation - Google Patents
Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation Download PDFInfo
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Abstract
The invention discloses a rearranged phage bacteriolytic gene, as well as a perforation vector and application thereof in vaccine preparation. The rearranged phage bacteriolytic gene has a nucleotide sequence shown in SEQ ID NO:1. An efficient perforation vector can be obtained by operationally connecting the rearranged phage bacteriolytic gene with a prokaryotic expression vector. The perforation vector is transformed into avian salmonella and then added with value; the expression of the rearranged phage bacteriolytic gene is induced; and formed bacterial ghosts are collected so as to obtain avian salmonella bacterial-ghost vaccine. The bacterial-ghost vaccine prepared in the invention can be applied without concentration. The avian salmonella bacterial-ghost vaccine is good in safety. After chicks are inoculated with the avian salmonella bacterial-ghost vaccine, the content of antibodies in every immune chick can be remarkably increased, so that every immune chick can be effectively protected against attacking toxins of avian salmonella, which shows that the avian salmonella bacterial-ghost vaccine has good immune protection effect.
Description
Technical field
The present invention relates to bacteriolyze gene and the application in the preparation vaccine thereof, relate in particular to a kind of rearranged bacterial virus bacteriolysis gene, the present invention also further relates to perforating vector and the purposes of this perforating vector in preparation fowl Salmonellas bacterium shadow vaccine that contains this gene, belongs to the recombinant vaccine 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 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 it has kept bacterial membrane structure and the associated antigen protein the same with viable bacteria, and adventitia contains the high conservative structure PAMP (pathogen-associated molecular patterns) of natural immunocyte by the identification of pattern recognition acceptor, lipopolysaccharides for example, peptidoglycan, CPG, OmpA and 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, other compositions such as exotic antigen, nucleic acid or medicine are anchored to the medial and lateral of after birth, or fill 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 bacteria such as various coli strains, Salmonella typhimurium and Salmonella enteritidis, 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.
Salmonellas causes that not only bird infects, and also can cause people's bacterial infection, and then causes diseases such as food poisoning and acute gastroenteritis.At present, countries such as European Union, American and Britain, method and day must not be to the regulation of Salmonellas in meat, egg, the milk animal products and detect.China's stem egg GB-non-polluted egg national standard was put into effect in 2006, and new non-polluted egg standard is very strict, requires must not detect things such as Salmonellas and paraxin in the egg.With attenuation salmonella prevention fowl salmonellosis, exist problems such as food safety and vaccine safety in view of both at home and abroad.Therefore, safe, nontoxic, the effective vaccine of development is very important and timely.Salmonellas bacterium shadow vaccine has the characteristics of safe, nontoxic and validity, is that live attenuated i (salmonella) vaccines is incomparable, and the Salmonellas bacterium shadow vaccine of development is the development trend that meets food safety.The present invention includes and utilize the Salmonellas that transforms the carrier pBV-mE that efficient perforating efficiency is arranged, preparation bacterium shadow vaccine and immune efficiency rating thereof, but this respect is not seen relevant research report.
Summary of the invention
One of the object of the invention provides a kind of rearranged bacterial virus bacteriolysis gene;
Two of the object of the invention provides a kind of perforating vector that contains above-mentioned rearranged bacterial virus bacteriolysis gene;
Three of the object of the invention is that above-mentioned perforating vector is applied to prepare fowl Salmonellas bacterium shadow vaccine;
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of rearranged bacterial virus bacteriolysis gene (mE), its nucleotides sequence are classified as shown in the SEQ ID NO:1;
The present invention utilizes the gene rearrangement technology, and PhiX174 phage E gene is reset, and making reading frame is that the E of 276 Nucleotide is mutated into the mE that reading frame is 201 Nucleotide (promptly since the 76th ATG).Bacterial virus bacteriolysis gene after the rearrangement has remarkable increase on perforating efficiency.
Rearranged bacterial virus bacteriolysis gene of the present invention is exercisable with after prokaryotic expression carrier is connected, promptly obtain perforating vector; As a kind of embodiment preferred, for example, can be connected with the pBV220 carrier the nucleotide sequence shown in the SEQ ID NO:1 is exercisable, can obtain a kind of efficient perforating vector pBV-mE; After testing, the perforating efficiency of this perforating vector pBV-mE can be up to 99.9999%.
The perforating vector that will contain rearranged bacterial virus bacteriolysis gene of the present invention is transformed in the fowl Salmonellas, obtains containing the fowl Salmonellas transformant of perforating vector; After this fowl Salmonellas transformant increment, elevated temperature is induced the expression of rearranged bacterial virus bacteriolysis gene, collects the ghost bacterium shadow that forms, and obtains fowl Salmonellas bacterium shadow vaccine.
Preferably, a kind of method for preparing fowl Salmonellas bacterium shadow vaccine, comprising: (1) is transformed into perforating vector pBV-mE in the fowl Salmonellas, obtains containing the fowl Salmonellas transformant of perforating vector; (2) this fowl Salmonellas transformant is carried out the bacterium increment at 28 ℃; (3) in the expression of 42 ℃ of temperature-induced rearranged bacterial virus bacteriolysis genes, collect the ghost bacterium shadow that forms, obtain fowl Salmonellas bacterium shadow vaccine.
The present invention finds by test, under the situation of different OD values, mE albumen all has very high punching effect (promptly presenting as clear as crystal bacterium liquid and cell debris), and the proteic perforating efficiency of this explanation mE of the present invention is not subjected to the influence of bacterium OD value, even in the OD value is 1.0 (to contain 10,000,000,000 bacterium, 10
10) time still can effectively punch, the bacterium OD value that is higher than external report far away must maintain 0.4 level.
In addition, the prepared bacterium shadow vaccine of the present invention does not need to concentrate and can use.And the present invention prepares the bacterium shadow can use fermentor tank, and the setting through program can reach automatization and large-scale production fully.
Safety experiment is the result show, the prepared fowl Salmonellas bacterium shadow vaccine safety of the present invention is good, and postvaccinal each chicken has no adverse reaction.
The immunoprotection test-results shows; after inoculating fowl Salmonellas bacterium shadow vaccine of the present invention; can significantly improve the intravital anti-body contg of each immunized chicks; can effectively protect each immunized chicks not to be subjected to the attack of fowl Salmonellas, illustrate that fowl Salmonellas bacterium shadow vaccine of the present invention has the good immune protection effect.
Description of drawings
The transmission electron microscope observing result of Fig. 1 fowl Salmonellas bacterium shadow.
Fig. 2 fowl Salmonellas forms the visual inspection result of bacterium shadow in different OD values.
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, fowl Salmonellas are 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 relevant document (Hou Yunde contains the establishment and the application thereof of the protokaryon efficient expression vector of PrPl promotor for Zhang Zhiqing, Yao Lihong, viral journal, 1990,6 (2), 111-116).
The clone of embodiment 1 rearranged bacterial virus bacteriolysis gene
Encoding sequence design primer according to GenBank pnagus medius PhiX174 bacteriolyze gene E:
Lysis?E-U：5′-AGGGAATTCATGGTACGCTGGACTTTGTGG-3′(SEQ?ID?NO.2)
Lysis?E-L：5′-AGGGGATCCGAGCTCTCACTCCTTCCG-3′(SEQ?ID?NO.3)
Upstream and downstream primer 5 ' end has been introduced restriction enzyme site EcoR I and Bam H 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.E gene behind the pcr amplification reclaims test kit with glue and reclaims after gel electrophoresis, with the DNA enzyme E gene is carried out enzyme then and cut, and reclaims the dna fragmentation of 10-50bp.The DNA that gets after 1 μ L reclaims carries out pcr amplification, and the pcr amplification reaction system is 50 μ L: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, 42 ℃ of 30s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 5min.Electrophoresis result is the Smear less than 276bp.
With Auele Specific Primer Lysis E-U and Lysis E-L to carrying out specific amplification with above-mentioned PCR product.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, Taq
TMArchaeal dna polymerase 2U (TaKaRa), template 1 μ L.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.Obtain the E gene after the gene rearrangement, i.e. mE gene, after the order-checking, its nucleotides sequence is classified as shown in the SEQ ID NO:1, reclaims test kit with glue and reclaims.
The test of the structure of embodiment 2 perforating vectors and punching effect
Carry out double digestion with EcoR I/Bam H I after the mE bacteriolyze isogeneity that embodiment 1 is cloned, use T
4Dna ligase (TaKaRa) is connected with pBV220 carrier through the digestion of EcoR I and Bam H I double digestion, 16 ℃ are spent the night and connect and heat shock is transformed into the e. coli tg1 competent cell, be accredited as the male clone through bacterium colony PCR and increase bacterium and utilize alkaline lysis to extract plasmid in a small amount, obtain perforating vector pBV-mE.
Choose the individual bacterium colony that contains the e. coli tg1 of perforating plasmid carrier pBV-mE of 20 (1-20 numbers), be seeded in respectively among the LB that 5mL contains 50 μ g/mL penbritins, (220r/min) cultivated in 28 ℃ of concussions of spending the night, 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, to remain culture and heighten 42 ℃ of cultivations rapidly to induce mE genetic expression, continue to cultivate 3-5 hour, the visual inspection perforating efficiency, can observe different punching results, the high as clear as crystal bacterium liquid of perforating efficiency is promptly arranged, the muddy bacterium liquid that perforating efficiency is low or do not punch is also arranged.Reorganization bacterium perforating efficiency is high and low or that do not punch carries out the nucleotide sequencing of plasmid extraction and mE gene respectively, analyze the variation situation of the mE gene of relatively more different punching effect recombinant plasmids, and obtained the plasmid vector pBV-mE of high perforating efficiency.
The plasmid pBV-mE that will punch is transferred in the fowl Salmonellas (penbritin sensitive strain), choose the bacterium colony of the fowl Salmonellas that contains perforating plasmid carrier pBV-E, be seeded among the LB that 5mL contains 50 μ g/mL penbritins, (220r/min) cultivated in 28 ℃ of concussions of spending the night, 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 mE genetic expression, continues to cultivate the visual inspection perforating efficiency 3-5 hour.Finding by observing the back, is that mE albumen has very high punching effect, promptly presents as clear as crystal bacterium liquid and cell debris under 0.4 the situation in OD value, and the effect of punching reaches 99.9999%.
The relation of embodiment 3 perforating plasmid carrier pBV-mE punching effect and fowl Salmonellas OD value
The plasmid pBV-mE that will punch is transferred in the fowl Salmonellas, choose the bacterium colony of the fowl Salmonellas that contains perforating plasmid carrier pBV-E, be seeded among the LB that 5mL contains 50 μ g/mL penbritins, (220r/min) cultivated in 28 ℃ of concussions of spending the night, 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 0.4,0.6,0.8 and 1.0.It is standby to take out 100 μ l cultures, will remain culture and heighten 42 ℃ of cultivations rapidly to induce mE genetic expression, continues to cultivate the visual inspection perforating efficiency 3-5 hour.Find that by observing the back under the situation of different OD values, mE albumen has very high punching effect, promptly presents as clear as crystal bacterium liquid and cell debris.Show that the proteic perforating efficiency of mE is not subjected to the influence of bacterium OD value, even be 1.0 (to contain 10,000,000,000 bacterium, 10 in the OD value
10) time still can effectively punch, the bacterium OD value that is higher than external report far away must maintain 0.4 level.Bacterium shadow vaccine through the present invention's preparation does not need to concentrate and can use.And the present invention prepares the bacterium shadow can use fermentor tank, and the setting through program can reach automatization and large-scale production fully.
Be coated with the LB flat board after each 100 μ l of culture before and after other gets and induces suitably dilute and carry out viable bacteria CFU detection.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.
With the centrifugal 10min of bacterium liquid 4000g that in 42 ℃, cultivates 3-5 hour in the present embodiment, 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.
The preparation of embodiment 4 fowl Salmonellas bacterium shadow vaccines
Contain the fowl Salmonellas that inoculation among the LB of 50 μ g/mL penbritins contains perforating plasmid carrier pBV-mE 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 0.8 (10
9) about.It is standby to take out 100 μ l cultures, will remain culture and heighten 42 ℃ of cultivations rapidly to induce mE 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.Test-results: the culture bacteriolyze deactivation efficient before and after the bacteriolyze reaches 99.9999%.
Bacteriolyze finishes the bacterium shadow of back formation with PBS washing 3 times, eliminates residual viable bacteria through ultraviolet, and freeze-drying is preserved, and obtains fowl Salmonellas bacterium shadow vaccine, and it is carried out security and the immune efficiency test of following chick.
The safety testing of test example 1 fowl Salmonellas bacterium shadow vaccine
Select 50 1 Japanese instar chicklings, be divided into 5 groups at random, every group of 10 chickens, 4 experimental group, 1 group is control group; 4 experimental group difference subcutaneous injection 0.1ml (10
10), 0.1ml (10
9), 0.1ml (10
8), 0.1ml (10
7) the prepared bacterium shadow vaccine of embodiment 4, control group injection 0.1ml physiological saline cutd open at 10 days respectively respectively to organize 5 chick with extremelying and cutd open in 20 days and respectively organizes remaining 5 with extremelying and organize chick, got internal organs such as liver, lungs and small intestine and carried out the histopathology observation.Test-results sees Table 1.
The safety testing of table 1 fowl Salmonellas bacterium shadow vaccine
Test-results shows that 4 each internal organs of experimental group chick are identical with control group, all do not have pathology, shows that the prepared fowl Salmonellas bacterium shadow vaccine of the present invention is security to the chick of 1 age in days.
The immune efficiency test of test example 2 fowl Salmonellas bacterium shadow vaccines
1, test materials:
(1) positive control: fowl Salmonellas deactivation vaccine;
(2) for test agent: the fowl Salmonellas bacterium shadow vaccine that fowl Salmonellas bacterium shadow vaccine of the present invention: embodiment 4 is prepared;
2, test method:
Select 40 1 Japanese instar chicklings, be divided into 4 groups at random, every group of 10 chickens, 3 experimental group (experimental group 1: fowl Salmonellas deactivation vaccine group; Experimental group 2: fowl Salmonellas bacterium shadow vaccine group of the present invention; Experimental group 3: fowl Salmonellas bacterium shadow vaccine group of the present invention+freund adjuvant group), 1 group is control group (physiological saline).3 experimental group difference subcutaneous injection 0.1ml (10
9) vaccine, control group injection 0.1ml physiological saline.Carry out 2 immunity after 3 weeks, dosage is identical with the first time with approach.Exempt to exempt from 2 weeks of back and 22 weeks of back to gather serum 1 respectively, measure the antibody situation.2 exempted from for 2 weeks after, every group of chicken inoculates 10 respectively
9The fowl Salmonellas, carry out the evaluation of immune efficient.Morbidity and the death condition of respectively organizing chicken behind the poison attacked in observation, and the survival chicken was cutd open and kills attacking poison in back 10 days, gets internal organs such as liver, lungs and small intestine and carries out histopathology and observe.
3, test-results:
Test-results sees Table 2 respectively, table 3 and table 4.
The antibody of table 2 fowl Salmonellas bacterium shadow vaccine immunity chick changes (OD490)
See from table 2, inoculate fowl Salmonellas bacterium shadow vaccine of the present invention after, can significantly improve the intravital anti-body contg of each immunized chicks, compare significant difference with control group;
The immune efficient simultaneous test of table 3 fowl Salmonellas bacterium shadow vaccine
See that from table 3 fowl Salmonellas bacterium shadow vaccine of the present invention can effectively protect each immunized chicks not to be subjected to the attack of fowl Salmonellas, illustrate that fowl Salmonellas bacterium shadow vaccine of the present invention has the good immune protection effect.
The pathological observation result of table 4 fowl Salmonellas bacterium shadow vaccine
See that from table 4 each internal organs of each immunized chicks of inoculating fowl Salmonellas bacterium shadow vaccine of the present invention are normal substantially, illustrate that fowl Salmonellas bacterium shadow vaccine safety of the present invention is good.
Sequence table .txt
Sequence table
＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture
＜120〉rearranged bacterial virus bacteriolysis gene and perforating vector thereof and the application in the preparation vaccine
<130>KLPI089
<160>3
<170>PatentIn?version?3.1
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<213>phage
<400>1
tggtacgct?ggactttgtg?ggataccctc?gctttcctgc?ccctgttgag?tttattgctg 60
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 275
<210>2
<211>30
<212>DNA
<213>artifical
<400>2
agggaattca?tggtacgctg?gactttgtgg 30
<210>3
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<212>DNA
<213>artifical
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aggggatccg?agctctcact?ccttccg 27
Claims (9)
1. rearranged bacterial virus bacteriolysis gene, its nucleotides sequence is classified as shown in the SEQ ID NO:1.
2. the perforating vector that contains the described rearranged bacterial virus bacteriolysis gene of claim 1.
3. according to the described perforating vector of claim 2, it is characterized in that: this perforating vector is pBV-mE, and wherein, the nucleotides sequence of described mE is classified as shown in the SEQ ID NO:1.
4. the application of the described rearranged bacterial virus bacteriolysis gene of claim 1 in preparation fowl Salmonellas bacterium shadow vaccine.
5. method for preparing fowl Salmonellas bacterium shadow vaccine comprises:
(1) makes up the perforating vector that contains the described rearranged bacterial virus bacteriolysis gene of claim 1; (2) this perforating vector is transformed in the fowl Salmonellas, obtains containing the fowl Salmonellas transformant of perforating vector; (3) with this fowl Salmonellas transformant increment; (4) induce the expression of rearranged bacterial virus bacteriolysis gene, collect the ghost bacterium shadow that forms, obtain fowl Salmonellas bacterium shadow vaccine.
6. in accordance with the method for claim 5, it is characterized in that: described perforating vector is pBV-mE, and wherein, the nucleotides sequence of described mE is classified as shown in the SEQ ID NO:1.
7. it is characterized in that in accordance with the method for claim 5: this fowl Salmonellas transformant rises in value at 28 ℃ in the step (3).
8. it is characterized in that in accordance with the method for claim 5: the expression of under 42 ℃ of temperature condition, inducing rearranged bacterial virus bacteriolysis gene in the step (4).
9. the fowl Salmonellas bacterium shadow vaccine for preparing by any one method of claim 5-8.
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| CN102335422B (en) * | 2011-10-11 | 2013-04-24 | 江苏省农业科学院 | Recombinant phage peculiar smell removal vaccine for boars |
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| CN101503686A (en) * | 2009-03-02 | 2009-08-12 | 中国农业科学院哈尔滨兽医研究所 | DNA sequence for reinforcing bacterial virus bacteriolysis gene perforating efficiency and use thereof |
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| CN101302527A (en) * | 2008-06-13 | 2008-11-12 | 中国农业科学院哈尔滨兽医研究所 | Rearranged bacterial virus E gene, perforating plasmid vector containing the same and use thereof |
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