CN109402155A - A kind of dual control delay cracking performance plasmid and its construction method and application - Google Patents
A kind of dual control delay cracking performance plasmid and its construction method and application Download PDFInfo
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- CN109402155A CN109402155A CN201811340755.0A CN201811340755A CN109402155A CN 109402155 A CN109402155 A CN 109402155A CN 201811340755 A CN201811340755 A CN 201811340755A CN 109402155 A CN109402155 A CN 109402155A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Abstract
The present invention provides construction method and the application of a kind of pUC pUC and its plasmid with dual control delay cracking.Dual control delay cracking performance plasmid of the invention contains the gene order as shown in SEQ ID NO.1.The present invention also provides the construction methods of above-mentioned plasmid, the plasmid is used for the application of the vaccine submission by bacteria mediated or the molecular drug treatment aspect by bacteria mediated, the dual control delay cracking for tentatively realizing bacterium, may be implemented normal growth when containing inducer (arabinose)；When not containing inducer, bacterium is in low concentration normal growth, and bacterium starts to crack at high concentrations.On the one hand it is allowed to safer for host, on the other hand can effectively discharge vaccine or molecular drug of bacteria mediated etc., and obtain better immune effect and therapeutic effect, there is great application prospect.
Description
Technical field
The invention belongs to gene therapy vector technical fields, are related to plasmid and its building and application, and in particular to Yi Zhongshuan
Control delay cracking performance plasmid and its construction method and application.
Background technique
Plasmid is a kind of gene, is a kind of cell dyeing ring-shaped DNA molecule of very little in vitro, is able to carry out autonomous
Duplication, does not have the extracellular phase, is not that the vital movements such as the growth, metabolism, breeding of host cell institute is required, but can assign place
Chief cell certain special attributes and function, such as the tolerance to antibiotic, the degradation to toxic compounds or utilize.It is existing
Studies have shown that many phenomenons in nature, such as degradation of the microorganism to heteroplasia body or the resistance to heavy metal and micro- life
The biological phenomenas such as function, characteristic variations and the gene evolution of object are all often related with plasmid.
The research carried out at present for plasmid is fairly common and deeply, also has much for the construction method of plasmid
Kind, method is increasingly mature, and presently found have famous plasmid ecology such as TOL, NAH etc., r plasmid such as ColE1, F because
Son etc., research contents are related to the duplication mechanism of plasmid, the expression of the functional gene carried on plasmid, regulation etc..New matter
The discovery and building of grain are still the advanced subject and research hotspot of current life science.
Another important function of plasmid is the carrier that can be used as genetic engineering, since plasmid can be in host appropriate
Self-replacation is carried out in cell, and in most cases host will not be caused to damage, and can easily would be integrated into certainly
The foreign gene of body is introduced into current recipient cell, therefore plasmid usually can be used as current gene cloning and expression carrier.
It is directed to the application of this aspect, some grind has been carried out in terms of realizing cracking for the expression of foreign gene
Study carefully, but the means regulated and controled at present are all more single, there is no the method that can realize multiple regulation cracking very well.
Summary of the invention
The object of the invention is in order to solve the above-mentioned technical problem, and provide a kind of plasmid with dual control delay cracking
The construction method and application of system and its plasmid.The dual control postpones cracking performance plasmid by two kinds of regulating and controlling effects, one is group
Incude promoter Pluxi regulation, when bacterial concentration reaches threshold value, bacterium is cracked；The second is being adjusted by arabinose (ara)
Control, when containing ara environment, lacI is expressed and in conjunction with lacO, thus bacteria lysis Protein S RRZ expression by inhibitation system；It is being free of
When ara, LacI is not expressed, and bacterioprotein expression is only controlled by Pluxi.The plasmid is applied to the vaccine submission by bacteria mediated
Or aspect is treated by the molecular drug of bacteria mediated, can be realized bacterium and cultivate in vitro is, ara is added, can be with normal growth；
In vivo without under ara environment, normal growth when low concentration makes bacterium start to crack in high concentration.Tentatively realize bacterium
Dual control delay cracking, has broad prospects in terms of gene therapy.
An object of the present invention is to provide a kind of dual control delay cracking performance plasmid, and the plasmid contains such as SEQ ID
Gene order shown in NO.1.
Further, the plasmid contains araC-Para regulatory protein gene, SRRZ lysis genes, three lacO behaviour
Vertical sequence, the encoding gene of intervention school-based promoter Pluxi and GAP-associated protein GAP LuxI/LuxR
Further, above-mentioned plasmid also contains resistant gene and replication origin, such as ampicillin resistance gene
Ampr and ori initiation site.
The second object of the present invention is to provide the construction method of above-mentioned dual control delay cracking performance plasmid, specific construction step
It is as follows:
(1) it is expanded using 12pHB plasmid as template, a lactose operon is added behind its Pluxi promoter
LacO manipulates sequence, obtains 5pHB plasmid；The sequence table of the 12pHB plasmid is in pUC57 as shown in SEQ ID NO.2
One section of core sequence is introduced in plasmid；
(2) it is expanded using 5pHB plasmid as template, PlacI-LacI sequence is introduced behind GFP green fluorescent protein,
Obtain 6pHB plasmid；
(3) it is expanded using 6pHB plasmid as template, is re-introduced into a lacO manipulation sequence in lacO manipulation sequence downstream,
Obtain 6-2pHB plasmid；Again using 6-2pHB plasmid as template, last time operation is repeated, 6-3pHB plasmid is obtained；
(4) PlacI is substituted with araC-Para, obtains 7pHB plasmid；
(5) GFP is substituted with SRRZ, obtains 8pHB plasmid, as target product dual control postpones cracking performance plasmid.
Further, being to use PCR using 5FG3P1/5FG3P2 and 5FG1P1/5FG1P2 as primer in above-mentioned steps (1)
Kit amplified fragments 5FG3 and 5FG1；Using pRE112 as template, using 5FG2P1/5FG2P2 as primer, expanded with PCR kit
Segment 5FG2；3 segments use DNA Purification Kit to recycle respectively, with seamless connection kit to 3 pieces of above-mentioned purifying
Section is attached, and connection product is transformed into bacillus coli DH 5 alpha, is screened in LB+Amp culture medium, the positive identified through PCR
Clone extracts plasmid, is sent to raw work sequencing.
Further, being with 5FG1P1/5FG1P2,5FG2P1/5FG2P2 and 5FG3P1/ in above-mentioned steps (2)
6FG3P2 is primer, with PCR kit amplified fragments 5FG1,5FG2 and 6FG3；Using pET32a as template, with 6FG4P1/
6FG4P2 is primer, with PCR kit amplified fragments 6FG4；4 segments use DNA Purification Kit to recycle respectively, with nothing
Seam connection kit is attached 4 segments of above-mentioned purifying, and connection product is transformed into bacillus coli DH 5 alpha, trains in LB+Amp
Feeding base is screened, and the positive colony through PCR identification extracts plasmid, is sent to raw work sequencing.
Further, the 6-2pHB plasmid is with 62FG1P1/62FG1P2 and 62FG2P1/ in step (3)
62FG2P2 is primer, and with PCR kit amplified fragments 62FG1 and 62FG2,2 segments use DNA Purification Kit respectively
Recycling, 62FG1 and 62FG2 use restriction enzyme NotI and BglII to digest, digestion product DNA Purification Kit
Recycling, then be attached with T4DNA ligase, connection product is transformed into bacillus coli DH 5 alpha, is sieved in LB+Amp culture medium
Choosing, the positive colony through PCR identification extract plasmid, are sent to raw work sequencing.
Further, the 6-3pHB plasmid be using 62FG1P1/63FG1P2 and 63FG2P1/62FG2P2 as primer,
With PCR kit amplified fragments 63FG1 and 63FG2,2 segments use DNA Purification Kit to recycle respectively, 63FG1 and
63FG2 uses restriction enzyme NotI and EcoRI to digest, and digestion product is recycled with DNA Purification Kit, then is used
T4DNA ligase is attached, and connection product is transformed into bacillus coli DH 5 alpha, in LB+Amp culture because being screened, is reflected through PCR
Fixed positive colony extracts plasmid, is sent to raw work sequencing.
Further, being using 6-3pHB plasmid as template, with 5FG1P1/5FG2P2 and 5FG3P1/ in step (4)
7FG2P2 is primer, with PCR kit amplified fragments 7FG1 and 7FG2；It is to draw with 7FG3P1/7FG3P2 using pBAD24 as template
Object, with PCR kit amplified fragments 7FG3；3 segments use DNA Purification Kit to recycle respectively, with seamless connection reagent
Box is attached 3 segments of above-mentioned purifying, and connection product is transformed into bacillus coli DH 5 alpha, cultivates in LB+Amp because being sieved
Choosing, the positive colony through PCR identification extract plasmid, are sent to raw work sequencing.
Further, being using 7pHB plasmid as template, with 7FG3P1/8FG1P2 and 8FG3P1/ in step (5)
7FG2P2 is primer, with super fidelity PCR kit amplified fragments 8FG1 and 8FG3；With pSRRZ (gene chemical synthesis, sequence such as SEQ
Shown in ID NO.3, map such as Figure 15) it is template, using 8FG2P1/8FG2P2 as primer, with PCR kit amplified fragments 8FG2；3
A segment uses DNA Purification Kit to recycle respectively, is attached with seamless connection kit to 3 segments of above-mentioned purifying,
Connection product is transformed into bacillus coli DH 5 alpha, extracts matter in the positive colony that LB+Amp is cultivated because being screened, through PCR identification
Grain is sent to raw work sequencing.
The present invention is also claimed above-mentioned dual control delay cracking performance plasmid or is postponed by the dual control that the above method constructs
The application of cracking performance plasmid can be and be applied to preparation by the vaccine submission of bacteria mediated or by the molecular drug of bacteria mediated
The application of aspect, or it is used for other fields.
Beneficial effects of the present invention are as follows: the present invention has constructed a kind of matter that can be carried out dual control and reconcile delay bacteria lysis
Grain system, the plasmid are mainly used for the vaccine submission by bacteria mediated or the molecular drug treatment by bacteria mediated etc..When containing
When the bacterium (bacterium does not crack when containing ara in vitro) of this plasmid is entered in host, do not contain ara (arabinose),
Bacterium normal growth in low concentration makes bacterium start to crack, is on the one hand allowed to safer for host in high concentration, another
Aspect can effectively discharge vaccine or molecular drug of bacteria mediated etc., and obtain better immune effect and therapeutic effect,
With great application prospect.
Detailed description of the invention
Fig. 1 and 2 is the OD600 value of each bacterial strain with the measurement result of incubation time；
Fig. 3 and 4 is measurement result of each bacterium viable bacteria concentration with incubation time；
Fig. 5 is measurement result of the viable bacteria ratio with incubation time；
Fig. 6 and 7 is the measurement result of viable bacteria ratio and viable bacteria concentration with incubation time；
Fig. 8-14 is respectively the map of plasmid 12-pHB, 5-pHB, 6-pHB, 6-2-pHB, 6-3-pHB, 7-pHB, 8-pHB；
Figure 15 is the map of plasmid pSSRZ.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments to the present invention
It is specifically described, it is necessary to, it is noted that following embodiment is used only for that the present invention is explained and illustrated, be not used to
Limit the present invention.Some nonessential modifications and adaptations that those skilled in the art are made according to foregoing invention content, still belong to
In protection scope of the present invention.
Such as the following table 1 of primer sequence involved in this specification and following embodiment:
Table 1
Embodiment 1
The building of 5-pHB plasmid:
The sequence table of 12-pHB plasmid is one section of core sequence of introducing in pUC57 plasmid as shown in SEQ ID NO.2
Column, plasmid map are as shown in Figure 8.
Using 12-pHB plasmid as template, using 5FG3P1/5FG3P2 and 5FG1P1/5FG1P2 as primer, tried with super fidelity PCR
Agent box (Quan Shijin, AP231, similarly hereinafter) amplified fragments 5FG3 and 5FG1.It is to draw with 5FG2P1/5FG2P2 using pRE112 as template
Object, with PCR kit amplified fragments 5FG2.3 segments use DNA purification kit (day with, DP214-03, similarly hereinafter) purifying respectively
Recycling.3 segments of above-mentioned purifying are attached with seamless connection kit (Quan Shijin, CU101, similarly hereinafter).Connection product turns
Change to bacillus coli DH 5 alpha, screened in LB+Amp, the positive colony through PCR identification extracts plasmid, is sent to raw work sequencing (matter
Grain map such as Fig. 9).
5-pHB plasmid is on the basis of 12-pHB, plus the lacO manipulation of a lactose operon behind Pluxi promoter
Sequence, the sequence can be in conjunction with aporepressor LacI, thus the initiation of transcription for preventing promoter from mediating.
Embodiment 2
The building of 6-pHB plasmid:
Using 5-pHB plasmid as template, with 5FG1P1/5FG1P2,5FG2P1/5FG2P2 and 5FG3P1/6FG3P2 for primer,
With super fidelity PCR kit amplified fragments 5FG1,5FG2 and 6FG3.It is to draw with 6FG4P1/6FG4P2 using pET32a as template
Object, with PCR kit amplified fragments 6FG4.4 segments use DNA Purification Kit to recycle respectively.With seamless connection reagent
Box is attached 3 segments of above-mentioned purifying.Connection product is transformed into bacillus coli DH 5 alpha, is screened in LB+Amp, through PCR
The positive colony of identification extracts plasmid, is sent to raw work sequencing (plasmid map such as Figure 10).
6-pHB plasmid introduces PlacI-LacI sequence on the basis of 5-pHB, makes the structural expression of aporepressor lacI.
When exogenous inducer IPTG is not added, LacI expresses that GFP not in conjunction with LacO manipulation sequence.When IPTG is added, this suppression
System is released, GFP expression.
Embodiment 3
The building of 6-2-pHB plasmid:
Using 6-pHB plasmid as template, using 62FG1P1/62FG1P2 and 62FG2P1/62FG2P2 as primer, with super fidelity
PCR kit amplified fragments 62FG1 and 62FG2.2 segments use DNA Purification Kit to recycle respectively.62FG1 and 62FG2
It is digested with restriction enzyme NotI and BglII (Quan Shijin, JN401, JB201), digestion product is pure with DNA purification kit
Change recycling, then is attached with T4DNA ligase (Quan Shijin, FL101, similarly hereinafter).Connection product is transformed into bacillus coli DH 5 alpha,
It is screened in LB+Amp, the positive colony through PCR identification extracts plasmid, is sent to raw work sequencing (plasmid map such as Figure 11).
Embodiment 4
The building of 6-3-pHB plasmid:
Using 6-2-pHB plasmid as template, using 62FG1P1/63FG1P2 and 63FG2P1/62FG2P2 as primer, with super fidelity
PCR kit amplified fragments 63FG1 and 63FG2.2 segments use DNA Purification Kit to recycle respectively.63FG1 and 63FG2
It is digested with restriction enzyme NotI and EcoRI (Quan Shijin, JE201), digestion product is returned with DNA Purification Kit
It receives, then is attached with T4DNA ligase.Connection product is transformed into bacillus coli DH 5 alpha, is screened in LB+Amp, through PCR
The positive colony of identification extracts plasmid, is sent to raw work sequencing (plasmid map such as Figure 12).
6-2-pHB plasmid is re-introduced into a lacO manipulation sequence on the basis of 5-pHB, in lacO manipulation sequence downstream；6-
3-pHB plasmid is re-introduced into a lacO manipulation sequence on 6-2-pHB plasmid basic, in lacO manipulation sequence downstream.Have 3 altogether
A lacO manipulation sequence reduces the background expression level of GFP to reinforce the inhibiting effect of aporepressor LacI.
Embodiment 5
The building of 7-pHB plasmid:
Using 6-3-pHB plasmid as template, using 5FG1P1/5FG2P2 and 5FG3P1/7FG2P2 as primer, with super fidelity PCR
Kit amplified fragments 7FG1 and 7FG2.Using pBAD24 as template, using 7FG3P1/7FG3P2 as primer, expanded with PCR kit
Segment 7FG3.3 segments use DNA Purification Kit to recycle respectively.With seamless connection kit to 3 segments of above-mentioned purifying
It is attached.Connection product is transformed into bacillus coli DH 5 alpha, is screened in LB+Amp, and the positive colony through PCR identification extracts
Plasmid is sent to raw work sequencing (plasmid map such as Figure 13).
7-pHB plasmid, with the PlacI on araC-Para substitution 6-3-pHB, makes aporepressor on the basis of 6-3-pHB
LacI becomes inducible expression (arabinose regulation) from structural expression.Aporepressor araC is combined and can be tied with promoter Para
It closes, inhibits it to transcribe downstream gene, in the presence of having exogenous arabinose, arabinose can be in conjunction with araC, and releases this
Transcripting suppressioning action.
So the GFP in 7-pHB is regulated and controled by two kinds.One is quorum sensing promoter Pluxi regulates and controls, when bacterium is dense
When degree reaches threshold value, GFP could be expressed.The second is being regulated and controled by arabinose (ara), when containing ara environment, lacI is expressed simultaneously
In conjunction with lacO, thus GFP expression by inhibitation system；When being free of ara, LacI is not expressed, and GFP expression is only controlled by Pluxi.
Embodiment 6
The building of 8-pHB plasmid:
Using 7-pHB as template, using 7FG3P1/8FG1P2 and 8FG3P1/7FG2P2 as primer, with super fidelity PCR kit
Amplified fragments 8FG1 and 8FG3.Using pSRRZ as template (pSRRZ come from gene chemical synthesis, sequence table as shown in SEQ ID NO.3,
It is that introducing SRRZ sequence, the map of the plasmid are as shown in figure 15 in plasmid pUC57), using 8FG2P1/8FG2P2 as primer,
With PCR kit amplified fragments 8FG2.3 segments use DNA Purification Kit to recycle respectively.With seamless connection kit pair
3 segments of above-mentioned purifying are attached.Connection product is transformed into bacillus coli DH 5 alpha, is screened in LB+Amp, identifies through PCR
Positive colony extract plasmid, be sent to raw work sequencing (plasmid map such as Figure 14).
8-pHB plasmid substitutes GFP on the basis of 7-pHB, with SRRZ.SRRZ is the key gene of λ bacteriophage cracking bacterium.
In 8-pHB plasmid, SRRZ is controlled by above two regulator control system, i.e., when containing ara environment, SRRZ expression by inhibitation system, carefully
Bacterium normal growth；When being free of ara, when bacterial concentration reaches threshold value, SRRZ expression, bacteria lysis discharges content.
This dual control delay cracking system of 8-pHB plasmid is mainly used for by the vaccine submission of bacteria mediated or by bacteria mediated
Molecular drug treatment etc..In vitro when mass propgation bacterium, be added ara enable bacterium normal growth, when contain this control
When the bacterium of system is entered in host, ara is not contained, bacterium normal growth in low concentration starts bacterium in high concentration
Cracking, is on the one hand allowed to safer for host, on the other hand can effectively discharge the vaccine or molecule medicine of bacteria mediated
Object etc., and obtain better immune effect and therapeutic effect.
Preliminary dual control lytic effect verifying such as experimental example 1-4 to plasmid
Experimental example 1
OD600The measurement of value and incubation time:
Method: each bacterium, which is inoculated in LB+Amp+Ara, to be incubated overnight.Next day, with brine 3 times.105It dilutes again
It is inoculated in fresh culture culture.OD is measured per hour600Value, measurement result are as depicted in figs. 1 and 2.
Note: ST is salmonella typhimurium 14028s plants；Ara is 0.2% arabinose；Amp is 100 μ g/ml；Cultivate item
Part is 37 DEG C, 180r/min.Similarly hereinafter.
As a result: DH5 α pHB8 (and STpHB8) is under the conditions of no Ara, under at 12 hours (10 hours), OD value started afterwards
Drop；Under the conditions of having Ara, culture for 24 hours can reach similar OD value with wild strain.
Experimental example 2
The measurement of viable bacteria concentration and incubation time:
Method: while above-mentioned OD detection test, each bacterium viable bacteria concentration is detected, as a result such as Fig. 3 and Fig. 4.
As a result: under the conditions of no Ara, at 11 hours (7 hours), viable bacteria concentration started DH5 α pHB8 (and STpHB8) afterwards
Decline；Under the conditions of having Ara, culture for 24 hours can reach similar viable bacteria concentration with wild strain.
Conclusion: Ara can release the cracking of bacterium；When not having Ara, bacterium starts to crack when viable count reaches threshold value.
Experimental example 3
The measurement of viable bacteria ratio and incubation time:
Method brief introduction: DH5 α pHB8 (or ST pHB8) is cultivated in LB+Amp and LB+Amp+Ara respectively, each small time-division
It Jian Ce not viable bacteria concentration.It is illustrated in figure 5 the viable count ratio of two kinds of bacterium respectively in different time points.
As a result with conclusion: DH5 α pHB8 (or ST pHB8) (9 hours) 11 hours start, viable count ratio be lower than 1, and
It is gradually reduced with incubation time, illustrates that bacterium starts largely to crack.When being less than lower than above-mentioned concentration the time, bacterium viable bacteria ratio
1 or so, illustrate not crack.
Experimental example 4
The measurement of viable bacteria ratio and viable bacteria concentration:
Method brief introduction: DH5 α pHB8 (or ST pHB8) is cultivated in LB+Amp and LB+Amp+Ara respectively, each small time-division
Not Jian Ce viable bacteria concentration, as a result as shown in Figure 6 and Figure 7.
Fig. 6 and Fig. 7 is that in various concentration, (viable bacteria concentration cultivated in LB+Amp+Ara per hour is horizontal to two kinds of bacterium respectively
Coordinate) under viable count ratio.Viable count algorithm is the same.
As a result with conclusion: DH5 α pHB8 (or ST pHB8) concentration reaches 9.03 × 107CFU/ml(2.22×109CFU/ml)
When, viable count ratio is lower than 1, and is gradually reduced with concentration increase, illustrates that bacterium starts largely to crack.When concentration is lower than above-mentioned dense
When spending, bacterium viable count illustrates not crack 1 or so.
Sequence table
<110>Chuanbei Medical College
<120>a kind of dual control delay cracking performance plasmid and its construction method and application
<130> 2018
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7756
<212> DNA
<213>(8-pHB plasmid)
<400> 1
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 60
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 120
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 180
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 240
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 300
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 360
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 420
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 480
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 540
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 600
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 660
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 720
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 780
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 840
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 900
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 960
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 1020
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 1080
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 1140
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 1200
cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 1260
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 1320
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 1380
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 1440
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 1500
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 1560
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 1620
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 1680
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 1740
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 1800
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 1860
atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa aattcgcgtt 1920
aaatttttgt taaatcagct cattttccag atatcgacgt cagtcctttg attctaataa 1980
attggatttt tgtcacacta ttgtatcgct gggaatacaa ttacttaaca taagcacctg 2040
taggatcgta caggtttacg caagaaaatg gtttgttata gtcgaataaa cgcaagggag 2100
ggaattgtga gcggataaca atttcgaaga tcttcggaat tgtgagcgga taacaatttc 2160
cggaattccg ggaattgtga gcggataaca atttcgatcc tctagattta agaaggagat 2220
atacatatga agatgccaga aaaacatgac ctgttggccg ccattctcgc ggcaaaggaa 2280
caaggcatcg gggcaatcct tgcgtttgca atggcgtacc ttcgcggcag atataatggc 2340
ggtgcgttta caaaaacagt aatcgacgca acgatgtgcg ccattatcgc ctggttcatt 2400
cgtgaccttc tcgacttcgc cggactaagt agcaatctcg cttatataac gagcgtgttt 2460
atcggctaca tcggtactga ctcgattggt tcgcttatca aacgcttcgc tgctaaaaaa 2520
gccggagtag aagatggtag aaatcaataa tcaacgtaag gcgttcctcg atatgctggc 2580
gtggtcggag ggaactgata acggacgtca gaaaaccaga aatcatggtt atgacgtcat 2640
tgtaggcgga gagctattta ctgattactc cgatcaccct cgcaaacttg tcacgctaaa 2700
cccaaaactc aaatcaacag gcgccggacg ctaccagctt ctttcccgtt ggtgggatgc 2760
ctaccgcaag cagcttggcc tgaaagactt ctctccgaaa agtcaggacg ctgtggcatt 2820
gcagcagatt aaggagcgtg gcgctttacc tatgattgat cgtggtgata tccgtcaggc 2880
aatcgaccgt tgcagcaata tctgggcttc actgccgggc gctggttatg gtcagttcga 2940
gcataaggct gacagcctga ttgcaaaatt caaagaagcg ggcggaacgg tcagagagat 3000
tgatgtatga gcagagtcac cgcgattatc tccgctctgg ttatctgcat catcgtctgc 3060
ctgtcatggg ctgttaatca ttaccgtgat aacgccatta cctacaaagc ccagcgcgac 3120
aaaaatgcca gagaactgaa gctggcgaac gcggcaatta ctgacatgca gatgcgtcag 3180
cgtgatgttg ctgcgctcga tgcaaaatac acgaaggagt tagctgatgc taaagctgaa 3240
aatgatgctc tgcgtgatga tgttgccgct ggtcgtcgtc ggttgcacat caaagcagtc 3300
tgtcagtcag tgcgtgaagc caccaccgcc tccggcgtgg ataatgcagc ctccccccga 3360
ctggcagaca ccgctgaacg ggattatttc accctcagag agaggctgat cactatgcaa 3420
aaacaactgg aaggaaccca gaagtatatt aatgagcagt gcagatagag ttgcccatat 3480
atctcctgca ggcatgcaag cttaatgcgg tagtttatca cagttaaatt gctaacgcag 3540
tcaggcaccg tgtatcgatc gttaaagagg agaaaggtac ccatgaaaaa cataaatgcc 3600
gacgacacat acagaataat taataaaatt aaagcttgta gaagcaataa tgatattaat 3660
caatgcttat ctgatatgac taaaatggta cattgtgaat attatttact cgcgatcatt 3720
tatcctcatt ctatggttaa atctgatatt tcaattctag ataattaccc taaaaaatgg 3780
aggcaatatt atgatgacgc taatttaata aaatatgatc ctatagtaga ttattctaac 3840
tccaatcatt caccaattaa ttggaatata tttgaaaaca atgctgtaaa taaaaaatct 3900
ccaaatgtaa ttaaagaagc gaaaacatca ggtcttatca ctgggtttag tttccctatt 3960
catacggcta acaatggctt cggaatgctt agttttgcac attcagaaaa agacaactat 4020
atagatagtt tatttttaca tgcgtgtatg aacataccat taattgttcc ttctctagtt 4080
gataattatc gaaaaataaa tatagcaaat aataaatcaa acaacgattt aaccaaaaga 4140
gaaaaagaat gtttagcgtg ggcatgcgaa ggaaaaagct cttgggatat ttcaaaaata 4200
ttaggctgca gtgagcgtac tgtcactttc catttaacca atgcgcaaat gaaactcaat 4260
acaacaaacc gctgccaaag tatttctaaa gcaattttaa caggagcaat tgattgccca 4320
tactttaaaa attaagcggc cggaataaac gcaagggagg ttggtatgac tataatgata 4380
aaaaaatcgg attttttggc aattccatcg gaggagtata aaggtattct aagtcttcgt 4440
tatcaagtgt ttaagcaaag acttgagtgg gacttagttg tagaaaataa ccttgaatca 4500
gatgagtatg ataactcaaa tgcagaatat atttatgctt gtgatgatac tgaaaatgta 4560
agtggatgct ggcgtttatt acctacaaca ggtgattata tgctgaaaag tgtttttcct 4620
gaattgcttg gtcaacagag tgctcccaaa gatcctaata tagtcgaatt aagtcgtttt 4680
gctgtaggta aaaatagctc aaagataaat aactctgcta gtgaaattac aatgaaacta 4740
tttgaagcta tatataaaca cgctgttagt caaggtatta cagaatatgt aacagtaaca 4800
tcaacagcaa tagagcgatt tttaaagcgt attaaagttc cttgtcatcg tattggagac 4860
aaagaaattc atgtattagg tgatactaaa tcggttgtat tgtctatgcc tattaatgaa 4920
cagtttaaaa aagcagtctt aaattaatgc tgctaaccag taaggcaacc ccgccagcct 4980
agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg 5040
ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg 5100
agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga 5160
aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg 5220
ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag 5280
ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc 5340
taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 5400
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag 5460
ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc 5520
ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt 5580
gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac 5640
cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc 5700
catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat 5760
ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat 5820
ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact 5880
taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc 5940
cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac 6000
atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc 6060
atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc 6120
cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag 6180
ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact 6240
ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt 6300
gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac 6360
gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc 6420
gacatcgtat aacgttactg gtttcacatg tatatctcct tcttaacggg tatggagaaa 6480
cagtagagag ttgcgataaa aagcgtcagg taggatccgc taatcttatg gataaaaatg 6540
ctatggcata gcaaagtgtg acgccgtgca aataatcaat gtggactttt ctgccgtgat 6600
tatagacact tttgttacgc gtttttgtca tggctttggt cccgctttgt tacagaatgc 6660
ttttaataag cggggttacc ggtttggtta gcgagaagag ccagtaaaag acgcagtgac 6720
ggcaatgtct gatgcaatat ggacaattgg tttcttctct gaatggcggg agtatgaaaa 6780
gtatggctga agcgcaaaat gatcccctgc tgccgggata ctcgtttaat gcccatctgg 6840
tggcgggttt aacgccgatt gaggccaacg gttatctcga tttttttatc gaccgaccgc 6900
tgggaatgaa aggttatatt ctcaatctca ccattcgcgg tcagggggtg gtgaaaaatc 6960
agggacgaga atttgtttgc cgaccgggtg atattttgct gttcccgcca ggagagattc 7020
atcactacgg tcgtcatccg gaggctcgcg aatggtatca ccagtgggtt tactttcgtc 7080
cgcgcgccta ctggcatgaa tggcttaact ggccgtcaat atttgccaat acggggttct 7140
ttcgcccgga tgaagcgcac cagccgcatt tcagcgacct gtttgggcaa atcattaacg 7200
ccgggcaagg ggaagggcgc tattcggagc tgctggcgat aaatctgctt gagcaattgt 7260
tactgcggcg catggaagcg attaacgagt cgctccatcc accgatggat aatcgggtac 7320
gcgaggcttg tcagtacatc agcgatcacc tggcagacag caattttgat atcgccagcg 7380
tcgcacagca tgtttgcttg tcgccgtcgc gtctgtcaca tcttttccgc cagcagttag 7440
ggattagcgt cttaagctgg cgcgaggacc aacgtatcag ccaggcgaag ctgcttttga 7500
gcaccacccg gatgcctatc gccaccgtcg gtcgcaatgt tggttttgac gatcaactct 7560
atttctcgcg ggtatttaaa aaatgcaccg gggccagccc gagcgagttc cgtgccggtt 7620
gtgaagaaaa agtgaatgat gtagccgtca agttgtcata attggtaacg aatcagacaa 7680
ttgacggctt gacggagtag catagggttt gcagaatccc tgcttcgtcc atttgacagg 7740
cacattatgc atcgat 7756
<210> 2
<211> 4347
<212> DNA
<213>(12-pHB plasmid)
<400> 2
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 60
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 120
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 180
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 240
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 300
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 360
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 420
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 480
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 540
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 600
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 660
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 720
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 780
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 840
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 900
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 960
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 1020
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 1080
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 1140
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 1200
cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 1260
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 1320
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 1380
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 1440
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 1500
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 1560
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 1620
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 1680
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 1740
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 1800
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 1860
atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa aattcgcgtt 1920
aaatttttgt taaatcagct cattttccag atatcgacgt cagtcctttg attctaataa 1980
attggatttt tgtcacacta ttgtatcgct gggaatacaa ttacttaaca taagcacctg 2040
taggatcgta caggtttacg caagaaaatg gtttgttata gtcgaataaa cgcaagggag 2100
gttggtgcta gctcacacag gaaacagcta tgacctctag atttaagaag gagatataca 2160
tatgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg 2220
tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg 2280
aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact 2340
tgtcactact ttcgcgtatg gtcttcaatg ctttgcgaga tacccagatc atatgaaaca 2400
gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt 2460
caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt 2520
taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa 2580
attggaatac aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg 2640
aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga 2700
ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta 2760
cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct 2820
tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat acaaataaat 2880
ctcctgcagg catgcaagct taatgcggta gtttatcaca gttaaattgc taacgcagtc 2940
aggcaccgtg tatcgatcgt taaagaggag aaaggtaccc atgaaaaaca taaatgccga 3000
cgacacatac agaataatta ataaaattaa agcttgtaga agcaataatg atattaatca 3060
atgcttatct gatatgacta aaatggtaca ttgtgaatat tatttactcg cgatcattta 3120
tcctcattct atggttaaat ctgatatttc aattctagat aattacccta aaaaatggag 3180
gcaatattat gatgacgcta atttaataaa atatgatcct atagtagatt attctaactc 3240
caatcattca ccaattaatt ggaatatatt tgaaaacaat gctgtaaata aaaaatctcc 3300
aaatgtaatt aaagaagcga aaacatcagg tcttatcact gggtttagtt tccctattca 3360
tacggctaac aatggcttcg gaatgcttag ttttgcacat tcagaaaaag acaactatat 3420
agatagttta tttttacatg cgtgtatgaa cataccatta attgttcctt ctctagttga 3480
taattatcga aaaataaata tagcaaataa taaatcaaac aacgatttaa ccaaaagaga 3540
aaaagaatgt ttagcgtggg catgcgaagg aaaaagctct tgggatattt caaaaatatt 3600
aggctgcagt gagcgtactg tcactttcca tttaaccaat gcgcaaatga aactcaatac 3660
aacaaaccgc tgccaaagta tttctaaagc aattttaaca ggagcaattg attgcccata 3720
ctttaaaaat taagcggccg gaataaacgc aagggaggtt ggtatgacta taatgataaa 3780
aaaatcggat tttttggcaa ttccatcgga ggagtataaa ggtattctaa gtcttcgtta 3840
tcaagtgttt aagcaaagac ttgagtggga cttagttgta gaaaataacc ttgaatcaga 3900
tgagtatgat aactcaaatg cagaatatat ttatgcttgt gatgatactg aaaatgtaag 3960
tggatgctgg cgtttattac ctacaacagg tgattatatg ctgaaaagtg tttttcctga 4020
attgcttggt caacagagtg ctcccaaaga tcctaatata gtcgaattaa gtcgttttgc 4080
tgtaggtaaa aatagctcaa agataaataa ctctgctagt gaaattacaa tgaaactatt 4140
tgaagctata tataaacacg ctgttagtca aggtattaca gaatatgtaa cagtaacatc 4200
aacagcaata gagcgatttt taaagcgtat taaagttcct tgtcatcgta ttggagacaa 4260
agaaattcat gtattaggtg atactaaatc ggttgtattg tctatgccta ttaatgaaca 4320
gtttaaaaaa gcagtcttaa attaatg 4347
<210> 3
<211> 4597
<212> DNA
<213>(pSSRZ plasmid)
<400> 3
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc acggatggca acatattaac ggcatgatat tgacttattg 480
aataaaattg ggtaaatttg actcaacgat gggttaattc gctcgttgtg gtagtgagat 540
gaaaagaggc ggcgcttact accgattccg cctagttggt cacttcgacg tatcgtctgg 600
aactccaacc atcgcaggca gagaggtctg caaaatgcaa tcccgaaaca gttcgcaggt 660
aatagttaga gcctgcataa cggtttcggg attttttata tctgcacaac aggtaagagc 720
attgagtcga taatcgtgaa gagtcggcga gcctggttag ccagtgctct ttccgttgtg 780
ctgaattaag cgaataccgg aagcagaacc ggatcaccaa atgcgtacag gcgtcatcgc 840
cgcccagcaa cagcacaacc caaactgagc cgtagccact gtctgtcctg aattcattag 900
taatagttac gctgcggcct tttacacatg accttcgtga aagcgggtgg caggaggtcg 960
cgctaacaac ctcctgccgt tttgcccgtg catatcggtc acgaacaaat ctgattacta 1020
aacacagtag cctggatttg ttctatcagt aatcgacctt attcctaatt aaatagagca 1080
aatcccctta ttgggggtaa gacatgaaga tgccagaaaa acatgacctg ttggccgcca 1140
ttctcgcggc aaaggaacaa ggcatcgggg caatccttgc gtttgcaatg gcgtaccttc 1200
gcggcagata taatggcggt gcgtttacaa aaacagtaat cgacgcaacg atgtgcgcca 1260
ttatcgcctg gttcattcgt gaccttctcg acttcgccgg actaagtagc aatctcgctt 1320
atataacgag cgtgtttatc ggctacatcg gtactgactc gattggttcg cttatcaaac 1380
gcttcgctgc taaaaaagcc ggagtagaag atggtagaaa tcaataatca acgtaaggcg 1440
ttcctcgata tgctggcgtg gtcggaggga actgataacg gacgtcagaa aaccagaaat 1500
catggttatg acgtcattgt aggcggagag ctatttactg attactccga tcaccctcgc 1560
aaacttgtca cgctaaaccc aaaactcaaa tcaacaggcg ccggacgcta ccagcttctt 1620
tcccgttggt gggatgccta ccgcaagcag cttggcctga aagacttctc tccgaaaagt 1680
caggacgctg tggcattgca gcagattaag gagcgtggcg ctttacctat gattgatcgt 1740
ggtgatatcc gtcaggcaat cgaccgttgc agcaatatct gggcttcact gccgggcgct 1800
ggttatggtc agttcgagca taaggctgac agcctgattg caaaattcaa agaagcgggc 1860
ggaacggtca gagagattga tgtatgagca gagtcaccgc gattatctcc gctctggtta 1920
tctgcatcat cgtctgcctg tcatgggctg ttaatcatta ccgtgataac gccattacct 1980
acaaagccca gcgcgacaaa aatgccagag aactgaagct ggcgaacgcg gcaattactg 2040
acatgcagat gcgtcagcgt gatgttgctg cgctcgatgc aaaatacacg aaggagttag 2100
ctgatgctaa agctgaaaat gatgctctgc gtgatgatgt tgccgctggt cgtcgtcggt 2160
tgcacatcaa agcagtctgt cagtcagtgc gtgaagccac caccgcctcc ggcgtggata 2220
atgcagcctc cccccgactg gcagacaccg ctgaacggga ttatttcacc ctcagagaga 2280
ggctgatcac tatgcaaaaa caactggaag gaacccagaa gtatattaat gagcagtgca 2340
gatagagttg cccatataag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 2400
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 2460
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 2520
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 2580
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 2640
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 2700
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 2760
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 2820
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 2880
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 2940
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 3000
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 3060
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 3120
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 3180
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 3240
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 3300
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 3360
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 3420
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 3480
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 3540
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 3600
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 3660
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 3720
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3780
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3840
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 3900
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 3960
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 4020
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 4080
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 4140
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 4200
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 4260
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 4320
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 4380
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 4440
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 4500
cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 4560
acctataaaa ataggcgtat cacgaggccc tttcgtc 4597
Claims (10)
1. a kind of dual control postpones cracking performance plasmid, which is characterized in that the plasmid contains the gene sequence as shown in SEQID NO.1
Column.
2. dual control according to claim 1 postpones cracking performance plasmid, which is characterized in that the plasmid contains araC-Para
Regulatory protein gene, SRRZ lysis genes, three lacO manipulation sequences and intervention school-based promoter Pluxi and GAP-associated protein GAP
The encoding gene of LuxI/LuxR；The plasmid also contains resistant gene and replication origin.
3. a kind of construction method of dual control delay cracking performance plasmid as claimed in claim 1 or 2, which is characterized in that its specific structure
Build that steps are as follows:
(1) it is expanded using 12pHB plasmid as template, the lacO of a lactose operon is added behind its Pluxi promoter
Sequence is manipulated, 5pHB plasmid is obtained；The sequence table of the 12pHB plasmid is in pUC57 plasmid as shown in SEQ ID NO.2
One section of core sequence of middle introducing；
(2) it is expanded using 5pHB plasmid as template, PlacI-LacI sequence is introduced behind GFP green fluorescent protein, is obtained
6pHB plasmid；
(3) it is expanded using 6pHB plasmid as template, is re-introduced into a lacO manipulation sequence in lacO manipulation sequence downstream, obtains
6-2pHB plasmid；Again using 6-2pHB plasmid as template, last time operation is repeated, 6-3pHB plasmid is obtained；
(4) PlacI is substituted with araC-Para, obtains 7pHB plasmid；
(5) GFP is substituted with SRRZ, obtains 8pHB plasmid, as target product dual control postpones cracking performance plasmid.
4. according to claim 3 dual control delay cracking performance plasmid construction method, which is characterized in that in step (1), be with
5FG3P1/5FG3P2 and 5FG1P1/5FG1P2 is primer, amplifies segment 5FG3 and 5FG1；Using pRE112 as template, with
5FG2P1/5FG2P2 is primer, amplifies segment 5FG2；3 segments are subjected to purification and recovery respectively, with seamless connection kit
3 segments after purification are attached, connection product is transformed into bacillus coli DH 5 alpha, it is screened in LB+Amp culture medium,
Positive colony through PCR identification extracts plasmid, is sent to raw work sequencing.
5. according to claim 3 dual control delay cracking performance plasmid construction method, which is characterized in that in step (2), be with
5FG1P1/5FG1P2,5FG2P1/5FG2P2 and 5FG3P1/6FG3P2 are primer, amplified fragments 5FG1,5FG2 and 6FG3；With
PET32a is template, using 6FG4P1/6FG4P2 as primer, amplified fragments 6FG4；4 segments use DNA purification kit pure respectively
Change recycling, 4 segments of above-mentioned purifying is attached with seamless connection kit, connection product is transformed into bacillus coli DH 5
α is screened in LB+Amp culture medium, and the positive colony through PCR identification extracts plasmid, is sent to raw work sequencing.
6. the construction method of dual control delay cracking performance plasmid according to claim 3, which is characterized in that described in step (3)
6-2pHB plasmid is using 62FG1P1/62FG1P2 and 62FG2P1/62FG2P2 as primer, with PCR kit amplified fragments 62FG1
And 62FG2,2 segments use DNA Purification Kit to recycle respectively, 62FG1 and 62FG2 use restriction enzyme NotI and
BglII digestion, digestion product are recycled with DNA Purification Kit, then are attached with T4DNA ligase, and connection product turns
Change to bacillus coli DH 5 alpha, screened in LB+Amp culture medium, the positive colony through PCR identification extracts plasmid, is sent to raw work
Sequencing.
7. the construction method of dual control delay cracking performance plasmid according to claim 3, which is characterized in that the 6-3pHB plasmid
It is using 62FG1P1/63FG1P2 and 63FG2P1/62FG2P2 as primer, amplified fragments 63FG1 and 63FG2,2 segments carry out pure
Change recycling, then digested with restriction enzyme NotI and EcoRI, digestion product is recycled with DNA Purification Kit, then is used
T4DNA ligase is attached, and connection product is transformed into bacillus coli DH 5 alpha, in LB+Amp culture because being screened, is reflected through PCR
Fixed positive colony extracts plasmid, is sent to raw work sequencing.
8. according to claim 3 dual control delay cracking performance plasmid construction method, which is characterized in that in step (4), be with
6-3pHB plasmid is template, using 5FG1P1/5FG2P2 and 5FG3P1/7FG2P2 as primer, with PCR kit amplified fragments 7FG1
And 7FG2；Using pBAD24 as template, using 7FG3P1/7FG3P2 as primer, with PCR kit amplified fragments 7FG3；3 segments point
Not Yong DNA Purification Kit recycling, with seamless connection kit 3 segments of above-mentioned purifying are attached, connection product
It is transformed into bacillus coli DH 5 alpha, plasmid is extracted in the positive colony that LB+Amp is cultivated because being screened, through PCR identification, is sent to life
Work sequencing.
9. according to claim 3 dual control delay cracking performance plasmid construction method, which is characterized in that in step (5), be with
7pHB plasmid is template, using 7FG3P1/8FG1P2 and 8FG3P1/7FG2P2 as primer, with super fidelity PCR kit amplified fragments
8FG1 and 8FG3；Using pSRRZ plasmid as template, using 8FG2P1/8FG2P2 as primer, with PCR kit amplified fragments 8FG2, institute
The sequence table of pSRRZ plasmid is stated as shown in SEQ IDNO.3；3 segments use DNA Purification Kit to recycle respectively, and use is seamless
Connection kit 3 segments of above-mentioned purifying are attached, connection product is transformed into bacillus coli DH 5 alpha, in LB+Amp culture because
It is screened, the positive colony through PCR identification extracts plasmid, is sent to raw work sequencing.
10. dual control postpones any one of cracking performance plasmid or claim 4-9 the method structure as described in claim any one of 1-2
Build dual control delay fragility plasmid application, which is characterized in that be applied to preparation by bacteria mediated vaccine submission or
By bacteria mediated molecular drug in terms of application.
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