CN101503686B

CN101503686B - DNA sequence for reinforcing bacterial virus bacteriolysis gene perforating efficiency and use thereof

Info

Publication number: CN101503686B
Application number: CN2009101183808A
Authority: CN
Inventors: 刘思国; 彭伟; 常月红; 刘慧芳; 王春来; 司微
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2009-03-02
Filing date: 2009-03-02
Publication date: 2011-03-30
Anticipated expiration: 2029-03-02
Also published as: CN101503686A

Abstract

The invention discloses a DNA sequence for enhancing the perforation efficiency of phage bacteriolytic genes, and application thereof. The DNA sequence utilizes a gene rearrangement technique to rearrange phage E genes so as to change E processus, of which a reading frame is 276 nucleotides intodeltaE(SEQ ID NO:3), of which the reading frame is 201 nucleotides and change anterior 746bp genes into a non-coding region sequence (SEQ ID NO:1), thereby having the effects of enhancers. The non-coding region DNA sequence can improve the perforation efficiency of the phage bacteriolytic genes for escherichia coli and avian salmonella. Perforation plasmid constructed by mE genes containing the non-coding region DNA sequence has high lysis efficiency in the escherichia coli, while the perforation plasmid pBV-deltaE constructed by deltaE genes not containing the non-coding region DNA sequence only has weak perforation efficiency, and the perforation efficiency of the two differs by over 1,000 times.

Description

Strengthen the dna sequence dna and the application thereof of bacterial virus bacteriolysis gene perforating efficiency

Technical field

The present invention relates to a kind of enhancing increment dna sequence dna, relate in particular to a kind of dna sequence dna of the non-coding region that after gene rearrangement, obtains, this dna sequence dna can effectively improve the perforating efficiency of bacterial virus bacteriolysis gene (mE) proteins encoded to bacterium, the invention still further relates to the purposes of this dna sequence dna in preparation bacterium ghost, belong to the genetically engineered field.

Background technology

Enhanser (enhancer) is meant the dna sequence dna of increase with its chain genetic transcription frequency, and enhanser increases by promotor transcribes.Effectively enhanser can be positioned at 5 ' end of gene, also can be positioned at 3 ' end of gene, the intron that also can be arranged in gene that has.The effect of enhanser clearly generally can make the genetic transcription frequency increase by 10～200 times, have in addition can be up to thousands of times.

Enhanser is the cis-acting elements that strengthens promoter function in the eukaryotic cell.Along with the further investigation of people, it is found that in procaryotic regulator control system and some virus genom DNA fragment, also to have the increment of enhancing function to the prokaryote gene expression regulation and control.These researchs have important significance for theories and practical value to gene expression regulation and the expression level of enhancing foreign gene in prokaryotic system that discloses prokaryotic system.

1981, Benerji found the sequence of a 140bp in SV40DNA, and it can improve the expression level of SV40DNA/ rabbit β-oxyphorase fusion gene greatly, and this is first enhanser of finding.It is positioned at the upstream of SV40 early gene, is made of each long 72bp two forward tumor-necrosis factor glycoproteinss.At present the enhanser of finding is tumor-necrosis factor glycoproteins mostly, generally long 50bp, and common " core " sequence of being made up of a 8-12bp is 5 '-GGTGTGGAAAG-3 ' as the core sequence of SV40 enhanser.Enhanser is different from the promotor place 2 points: 1) enhanser is unfixing for the position of promotor, and very big change can be arranged; 2) it can produce effect mutually at both direction.An enhanser is not limited to promote transcribing of a certain special promoter, and it can stimulate near the arbitrary promotor it.

Enhanser can be divided into cell specificity enhanser and inducibility enhanser two classes: 1) tissue and cell specificity enhanser.The reinforcing effect of many enhansers has very high histocyte specificity, only under specific transcription factor (protein) participates in, and its function of competence exertion.For example only in B lymph born of the same parents, activity is just the highest for the enhanser of immunoglobulin gene.In addition, found all that in the enhanser of insulin gene and Quimotrase gene very strong tissue specificity is arranged.In addition, in all enhansers all by one section by the DNA that the alternative pyrimidine-the purine residue is formed, this DNA very easily forms the Z-DNA type; So there is the people to think that enhanser just has function after forming a bit of Z-DNA.The order that similar enhanser is arranged in yeast is called upstream activation sequence (UAS).UAS can work to both direction.And be positioned at any distance of promotor upstream, but then there is not effect (being different from general enhanser) in the promotor downstream.Mouse mammary tumor virus (MMTV) DNA transcribes the stimulation that can be subjected to the carbohydrate steroid hormone.This can be subjected to the order of hormonal effects to be positioned at about 100bp place, transcriptional start point upstream.The mixture that this order energy and hormone and protein receptor thereof are formed combines.When the either side that this is placed in proper order the promotor of certain gene (being upstream or downstream) and various apart from the time, it still can stimulate this gene transcription.So the enhanser activation may be the mechanism that the carbohydrate sterol is regulated and control one group of target gene: the carbohydrate steroid hormone enters behind the cell promptly and its receptors bind.The keying action activated receptor makes it can discern the common sequence that is present in the enhanser, and then has activated near enhanser the gene that can react to the carbohydrate sterol.Promptly when carbohydrate sterol-receptor complex and enhanser in conjunction with the time, near the promotor it is initial transcribing.2) inducibility enhanser.The activity of this enhanser will have specific promotor to participate in usually.For example, metallothionein gene can be transcribed in multiple histocyte, can be subjected to inducing of steroid hormone, zinc, cadmium and somatomedin etc. again and improves transcriptional level.

1, strengthens the research of increment sequence in the prokaryotic gene group

Lack in the substratum at nitrogen, the transcriptional activation of intestinal bacteria gln ALG operon needs the ntrc gene product NR1 of lower concentration.Reifzer and Magasam etc. discover that the glnAP promotor upstream of this operon has two strong binding sites of NR1.Deletion mutantion studies show that it is closely related to be positioned 2100bp and 2150bp neighbouring these two strong binding sites of NR1 and transcriptional activity.Their disappearance can make the transcriptional activation forfeiture of lower concentration NR1.With move up 1400bp or move down 2000bp of this segment DNA sequence, do not influence their replying to NR1.Drummond etc. find also that to the research of K.pneumoniae operon the disappearance at this 159bp place, promotor upstream can make its transcription initiation that relies on high density NR1 weaken 4 times.The nucleotides sequence of the 2170bp to 2156bp of disappearance is classified GCACN as ₅CGGCCC, only the binding sequence GCACN of 3 bases and glnAP2 ₅The TGGTGC difference, this may be its same high density rather than lower concentration NR1 activated reason of needing with other nitrogen regulatory gene.Ames and Nikaido have also found a strong binding site of NR1 when research S.typhmurium system, be positioned ArgT gene downstream 60bp and hisJ upstream region of gene 120bp place, and NR1 can activate the promotor of these two genes in conjunction with this site.The binding site of these prokaryotic gene control regions all has the feature that does not rely on position and direction activation promoter transcription, and is very similar to the enhanser in the eukaryotic cell.Discoveries such as Buckd, the disappearance between the nif H promotor upstream 272bp to 2184bp of K.pneumoniae can greatly reduce the transcription activating of the special activator nif of nif A to it.Nif H, nif U and researching and analysing of nif B promoter upstream sequence are shown this upstream sequence only is present in the nif A activated gene.They have determined a common conserved sequence that exists in the nif of K.pneumoniae, Rhizobium and Azotobacter promotor, and it is a protein binding site, and nif A interacts at this and nif promotor.This sequence is positioned at 100bpA place, transcripting start point upstream, is one and is rich in the TGTNACA sequence that sequence separates by AT.Itself is not had a promoter activity, must could start efficiently with 224 districts of transcripting start point upstream and 212 district's conserved sequences to transcribe.Studies show that further this sequence is to transcription activating effect and the position and the orientation independent of downstream promotor,, also have a transcription activating effect even be displaced to beyond the 2000bp.But its optimum position is near 2136bp.Better etc. find that also the gene nif A product of K.Pneumoniae needs the existence of upstream sequence to the maximum activation effect of the nifHDK promotor of Rhizobium meliloti, similar upstream sequence requires also to be present among the nif H and the activation of nif D promoter transcription of Rhizobium juponium, and visible nif A binding sequence also is a kind of enhanser similar sequences.Goreiarrubio and Grarrubias have carried out comparative study to the transcriptional control of the 82kD protein gene of intestinal bacteria gln ALG and vicinity.The transcription initiation site of 82kD protein gene is positioned at 2272bp place, upstream, and transcriptional orientation is opposite with glnALG, and two of the glnAP upstream strong binding sites of NR1 are in similar position, 82kD upstream too like this.Only gln AP2 is had effect but result of study shows in conjunction with the NR1 site, whether it exists does not have obviously influence to transcribing of 82kD, the pronucleus enhancer sample sequence of this explanation gln AP2 upstream make apparatus promotor specificity.Further research and analyse and show that this specific specificity is by RNA polymerase Sigma Factors kind and the common decision of promoter structure.Gln AP2 promotor conserved regions is positioned at the TTGGCACANTCGCT between 227bp to 2410bp, and is similar with 212bp conserved regions preface CTGGYAYRNTTGCA to the 224bp of Ntr-type promotor, can be by 60 identifications.And 82P promotor conserved regions is the TTGTAGNTACAAT in 235 districts and 210 intervals, belongs to Pribnow2 type promotor, and the Sigma Factors of discerning it is σ 70.Herendeew etc. are transcribing after controlling mechanism furthers investigate the T4 phage, further illustrate the different promotor specificitys that are that its tool is stronger of pronucleus enhancer and eucaryon enhanser, can be divided into two big classes: the conserved regions of Pribnow-type promotor is 210 districts and 235 districts, be σ 70 identifications, the enhancing increment sequence that is positioned at their upstreams is the abundant district of AT; The conserved regions of Ntr-type promotor is 212 districts and 224 districts, can be σ 60 identifications, and the enhancer sequence that is positioned at its upstream is TGTNACA.

Discoveries such as nineteen ninety Chinese scholar Pan Wei, the long fragment (M) of 1000bp can promote the expression to CAT gene and β 2 galactosidase genes of vaccinia virus P715 and N promotor with the ind characteristic of direction in the intestinal bacteria JM103 chromosomal DNA, but the position dependence that tool is certain, 3 ends or the upstream 2000bp that are positioned at gene then do not have enhancement outward.It also has certain host specificity, and it is roughly close to strengthen the effect of expressing in JM103, MC1061 and C600, but does not then have tangible enhancement in HB101.Simultaneously, itself also has promoter function this segment, but is positioned at the CAT expression of gene in its downstream in the start detection carrier.This segmental enhancement of transcribing can not explain with the tandem promoter subfunction simply, because when arranging in the M2P7.52CAT mode, M can strengthen the transcriptional activity of P7.5; And when arranging in the P7.52M2CAT mode, CAT expresses not obvious increase.Above-mentioned result of study shows that this section has the pronucleus enhancer sample function.Gong Qinghong etc. make further research this M fragment, they are digested to LM (2535bp) and two small segments of SM (2355bp) with BstI with the M fragment, find after being cloned into the detection carrier that the segmental pronucleus enhancer sample function of M mainly concentrates on the SM small segment of 5 ' end 355bp.This small segment can make the CAT genetic expression under the control of vaccine virus N promotor improve 3-7 doubly in intestinal bacteria, and its enhanced activity is similar to the M fragment, also directionless dependency.Its host's dependency that also tool is relative, the enhancement in different intestinal bacteria is JM103＞DH5 α＞LE392＞HB101 successively.Segmental research further proves to M in employing deletion mutantions such as Xie Ming, and the segmental reactive site of M is positioned at 5 ' end of this sequence, is multidigit point synergy.Holding upstream 440bp-2500bp, 580bp-2640bp and 740bp-2800bp place respectively to have a length in M sequence 3 ' is the avtive spot of 60bp, gets intermediate value and demarcates 410bp, 610bp and the left and right sides, 770bp place scope 30bp.Three sites exist enhanced activity the highest simultaneously, and enhanced activity is lost when lacking two sites simultaneously.

The research of pronucleus enhancer sample sequence in the 2 eukaryotic viral gene groups

Pronucleus enhancer sample sequence removes and is present in prokaryotic gene group China and foreign countries, has found also that in some eukaryotic viral gene group some sequences can strengthen the increment function and promote the expression of foreign gene in intestinal bacteria.Chinese scholar Hou Yun moral in 1985 finds that the expression in intestinal bacteria has tangible enhancement to SV40Hind IIIB fragment to people α D type IFN gene when inquiring into the SV40 enhanser to the influencing of prokaryotic expression system.The SV40 genomic dna can produce A, B, C, D, six fragments of E, F after Hind III enzyme is cut, wherein B fragment (4002bp-2517bp) contains complete t antigen gene, and C fragment (517bp-21046bp) contains ori and the 721bp tumor-necrosis factor glycoproteins of SV40DNA.They are inserted the IFN upstream region of gene 1200bp place of PL promotor control respectively, and the segmental insertion of Hind IIIB can make the expression of IFN improve 711 times, and the segmental insertion of C does not have obvious enhancement.The research of Wu Jihua etc. shows that also the HindIIIB fragment can make the expression level of IFN-β under the PL control improve 515 times in intestinal bacteria.Wu Ji China in 1996 etc. find not only SV40Hind IIIB1 100bp fragment (SV40-HB) tool pronucleus enhancer sample function again, and the 0.7KB fragment (RSV-G) of the 211000bpBgl fragment (vv-k) of the Hind IIIK of vaccinia virus and respiratory syncytial virus (RSV) BamH I-G also has the pronucleus enhancer sample function.These fragment forwards insert and can make exogenous gene expression strengthen 3.7-7.3 doubly, and oppositely insertion can strengthen 1.5-7.6 doubly, and strengthening the intensity order is RSV2G＞vv-k＞SV40-HB.These fragments also all have the prokaryotic promoter function, can make the CAT genetic expression that is cloned into the downstream.Segmental 212kb complete sequence of vv-k and JM103-M fragment 3 end 308bp sequences are carried out sequential analysis, and compare with SV40Hind IIIB fragment sequence, adopt the DNA binding site of microcomputer and known protokaryon and eukaryotic transcription activator to compare the retrieval discovery, the common sequences of Ap-l binding site among the tool SV40 in the SV40-HB segment, the bacterium F13 factor and eucaryon NF-III binding site are arranged among the vv-k, two SPI of place binding sites (GCbox) and the Ap-l of a place binding site are arranged among the JM103-M, point out these sequences to interact, come the expression of enhancing gene in prokaryotic cell prokaryocyte with cis-acting elements mode and trans-acting factor.Liu Zhewei etc. 1992 upstream regulatory region of finder's papilloma virus 6b type (HPV-6b) again also have the increment of enhancing function in intestinal bacteria.Be positioned at the 540bp fragment between the ATG upstream from start codon 310bp to 852bp of E6 in the HPV-6b genome, be the upstream extremity of its non-coding region (uuR), in intestinal bacteria JM103 and MC1061, can make CAT and beta-galactosidase gene under the control of vaccinia virus P715 promotor and P11 promotor express significantly enhancing.Forward inserts and beta-galactosidase gene to be expressed in improve 10 times and 6 times among JM103 and the MC1061 respectively, and reverse insertion can improve about 3 times.Sequential analysis shows that putting in order to purine pyrimidine is spaced of 74bp arranged in this segment DNA sequence, can be divided into three groups, and preceding two groups are 24bp, is the repeated arrangement order, and the 3rd group is 26bp, but array mode is slightly different with the above two.Once had report to point out that this purine pyrimidine arrangement architecture tends to form Z-DNA, in the eucaryon enhanser, the SV40 enhanser also contains this form to be arranged, and this may be exactly the architecture basics of this 540bp fragment enhanser function.

Enhanser is as a kind of cis-regulating element, the enforcement of its function must be unable to do without the trans factor, as the correct selection of enhanser to corresponding promotor, just need with the synergy of some tissue specificities and public protein factor, these protein factors may be responsible for discerning the core sequence of promotor and the conserved sequence of enhanser.

From above-mentioned research as can be seen, be present in and strengthen the increment sequence in pronucleus enhancer sample sequence and the prokaryotic gene group in the eukaryotic virus genome and have different characteristics.They do not have significant host specificity, have certain direction specificity, though the equal tool of the insertion of two kinds of directions strengthens the increment function, generally more oppositely to insert enhancement stronger but forward inserts.Be more significantly, the pronucleus enhancer sample sequence in the eukaryotic virus genome also has the prokaryotic promoter function simultaneously, points out two class pronucleus enhancer sample sequences that certain difference is arranged on the structure and the mechanism of action.Further illustrating of these mechanism is all will be very important, valuable to the theoretical investigation of prokaryotic expression regulation and control or to the practical application of prokaryotic gene engineering.

Summary of the invention

One of the object of the invention provides a kind of dna sequence dna that can strengthen the bacterial virus bacteriolysis gene proteins encoded to the perforating efficiency of bacterium;

Two of the object of the invention is that above-mentioned dna sequence dna is applied to prepare bacterium ghost (comprising intestinal bacteria ghost and fowl Salmonellas ghost etc.).

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

Through obtaining PhiX174 phage E gene (mE) non-coding sequence dna sequence dna after the gene rearrangement, this dna sequence dna length is 74bp (SEQ ID NO:1), has the enhanser effect with PhiX174 phage E gene in the present invention.

The phage mE gene (SEQ IDNO:2) that the present invention will include enhancer sequence of the present invention (SEQ ID NO:1) is connected with plasmid pBV220 and obtains perforating vector pBV-mE; The phage Δ E gene (SEQ ID NO:3) that will not comprise enhancer sequence of the present invention is connected with plasmid pBV220 and obtains perforating vector pBV-Δ E; With perforating vector pBV-mE be transformed into intestinal bacteria by the bacterium colony of choosing is carried out 28 ℃ of concussions of spending the night and cultivates and 42 ℃ of cultivations inducing mE genetic expression, the visual inspection perforating efficiency can be observed the punching result of highly significant.Promptly present as clear as crystal bacterium liquid, perforating efficiency reaches 99.9999%.Show that dna sequence dna of the present invention (SEQID NO:1) has the effect that strengthens the mE gene transcription, give expression to more mE albumen, thereby have more mE albumen to participate in punching, perforating efficiency is significantly increased bacterium.

Same, E is transformed into intestinal bacteria with perforating vector pBV-Δ, by to choose that bacterium colony carries out that 28 ℃ of concussions of spending the night are cultivated and 42 ℃ of cultivations to induce Δ E genetic expression, visual inspection perforating efficiency.The result shows that under the situation of disappearance SEQ ID NO:1 sequence, perforating plasmid carrier pBV-Δ E presents low-down perforating efficiency, the bacterium liquid muddiness of cultivation; The perforating efficiency of perforating vector pBV-mE will be higher than the perforating efficiency of perforating vector pBV-Δ E more than 1000 times, illustrates that SEQ ID NO:1 sequence has the effect of the increment of enhancing really.

Description of drawings

The visual inspection result of Fig. 1 intestinal bacteria bacterium shadow

The visual inspection result of Fig. 2 fowl Salmonellas bacterium shadow

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-mE, screening and punching effect observation

1.1 the gene rearrangement technology prepares PhiX174 bacteriolyze gene E (mE)

Encoding sequence design primer according to GenBank pnagus medius PhiX174 bacteriolyze gene E:

Lysis?mE-U：5′-AGGGAATTCTGGTACGCTGGACTTTGTGG-3′(SEQ?IDNO.4)

Lysis?mE-L：5′-AGGGGATCCGAGCTCTCACTCCTTCCG-3′(SEQ?ID?NO.5)

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.ME gene behind the pcr amplification reclaims test kit with glue and reclaims after gel electrophoresis, size is 275bp (SEQ ID NO:2), and wherein, enhancer sequence is shown in the SEQ ID NO:1.

1.2 the structure of perforating plasmid carrier pBV-mE

With carrying out double digestion with EcoR I/Bam H I behind the bacteriolyze gene mE purifying, 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, 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-mE.

Observe the dependency that reaches with the non-encoding gene of 74bp 1.3 contain the colibacillary perforating efficiency of perforating plasmid carrier pBV-mE

Choose the bacterium colony of the e. coli tg1 that contains perforating plasmid carrier pBV-mE, be seeded among the LB that 5mL contains 50 μ g/mL penbritins, (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; to remain culture and heighten 42 ℃ of cultivations rapidly to induce mE genetic expression; continue to cultivate 3-5 hour; naked eyes can be observed the perforating efficiency of highly significant; promptly present as clear as crystal bacterium liquid; perforating efficiency reaches 99.9999%, and control group then is very muddy bacterium liquid.Show that SEQ ID NO:1 can strengthen the punching effect of perforating plasmid carrier pBV-mE, have the effect of the increment of enhancing.

The correlation test of the punching effect of embodiment 2 perforating plasmid carrier pBV-Δ E and the non-encoding gene of 74bp (SEQ ID NO:1)

1.1PCR the E gene (Δ E) of amplification PhiX174 disappearance 74bp

LysisΔE-U：5′-AATGGATCCTCACTCCTTCCGCAC-3′(SEQ?ID?NO.6)

LysisΔE-L：5′-GTGGAATTCATGTTCATCCCGTCAAC-3′(SEQ?ID?NO.7)

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 Δ 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.ME Δ gene behind the pcr amplification reclaims test kit with glue and reclaims after gel electrophoresis, size is 201bp (SEQ ID NO.3).

1.2 the structure of perforating plasmid carrier pBV-Δ E

E carries out double digestion with EcoR I/Bam H I with purifying bacteriolyze gene Δ, uses 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, 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-Δ E.

The colibacillary perforating efficiency of pBV-Δ E reaches and the dependency of the non-encoding gene of 74bp 1.3 contain

Choose the bacterium colony of the e. coli tg1 that contains perforating plasmid carrier pBV-Δ 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, 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 Δ E genetic expression, continues to cultivate 3-5 hour; it is faint that naked eyes can be observed perforating efficiency; DeGrain promptly presents muddy bacterium liquid, and is not remarkable with the difference of the very muddy bacterium liquid of control group.Δ E gene under the situation of disappearance 74bp non-encoding gene, not only do not improve perforating efficiency, reduced perforating efficiency on the contrary.Show the mE gene and the proteins encoded perforating efficiency highly significant that contain the non-encoding gene of 74bp, and the Δ E gene of disappearance 74bp non-encoding gene and the albumen effect of not punching.The result has proved that the non-encoding gene of 74bp has the effect of the increment of enhancing.

The preparation of embodiment 3 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 ⁹Cfu) 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 99.99%.Bacteriolyze finishes the bacterium shadow of back formation with PBS washing 3 times, eliminates residual viable bacteria through ultraviolet, and freeze-drying is preserved, promptly.

The preparation of embodiment 4 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-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 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 99.99%.

KLPI090141_2.txt

Sequence table

＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture

＜120〉dna sequence dna and the application thereof of enhancing bacterial virus bacteriolysis gene perforating efficiency

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KLPI090141_2.txt

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Claims

1. strengthen the DNA of bacterial virus bacteriolysis gene proteins encoded perforating efficiency, it is characterized in that: its Nucleotide is shown in the SEQ ID NO:1.

2. the purposes of the described DNA of claim 1 in preparation bacterium ghost.

3. according to the described purposes of claim 2, it is characterized in that: described bacterium ghost comprises intestinal bacteria ghost and fowl Salmonellas ghost.