CN105779444B - A kind of Gene expression improving bacillus protein expression quantity - Google Patents
A kind of Gene expression improving bacillus protein expression quantity Download PDFInfo
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- CN105779444B CN105779444B CN201410777879.0A CN201410777879A CN105779444B CN 105779444 B CN105779444 B CN 105779444B CN 201410777879 A CN201410777879 A CN 201410777879A CN 105779444 B CN105779444 B CN 105779444B
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Abstract
The object of the present invention is to provide a kind of Gene expressions for improving bacillus protein expression quantity.The QC6 bacterial strain constructed using P43-PHpaII-PSPO-I Gene expression provided by the invention, QC1, QC2 and QC3 bacterial strain that its alkali protease expression quantity is constructed significantly greater than with single Promoter P43, PHpaII and PSPO-I, or even more taller than the sum of the expression quantity of three plants of bacterium 70% or so.The QC7 bacterial strain constructed using P43-PHpaII-PSPO-I-PcryIIIA Gene expression provided by the invention, the QC6 bacterial strain that the expression quantity of its alkali protease is constructed significantly greater than with P43-PHpaII-PSPO-I Gene expression and the QC4 bacterial strain using single promoter PcryIIIA building, in addition it is also higher by 50% or so than the sum of expression quantity of two plants of bacterium.Above-mentioned Gene expression produces unexpected technical effect, can significantly promote the expressing quantity of bacillus.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of bacillus protein expression quantity of being remarkably improved
Gene expression and its application.
Background technique
Bacillus is an excellent protein expression host, it has highly-safe (Generally Regarded as
Safe, GRAS), growth cycle is short, and fermentation costs are low, and secretion capacity is strong, without remarkable advantages such as apparent codon preferences, extensively
The general industrialized production applied to enzyme preparations such as amylase, protease, Pullulanase, pectases.
In order to reduce the production cost of enzyme preparation product, increase economic efficiency, the one of industrial Bacillusexpression system
A important requirement is that protein expression quantity wants high.And an important means for improving bacillus expression is to improve purpose base
The mRNA level in-site of cause, common method are that this method is maximum to be lacked using the high copy number plasmid expression vector for carrying target gene
Point is that there are Loss for high copy number plasmid expression vector in commercial process.It is bad in order to solve plasmid-bearing strains stability
The problem of, it can be by expression cassette and an amplifiable screening mark containing the single promoter being operatively connected with target gene
Note gene (such as antibiotic-resistance marker's gene) is integrated on Bacillus chromosome, then passes through screening pressure (such as antibiotic)
The expression cassette and riddled basins on chromosome are expanded, generates the expression cassette of multicopy on chromosome.This method there is also
Some disadvantages can not such as obtain the mRNA of saturated level by the gene that amplification is driven by single promoter.Moreover, chromosome
The expression cassette potentially unstable of upper series connection multicopy.In addition, riddled basins are difficult to remove.And another is saturated
The method of mRNA level in-site is the specific promoter of screening.But since the quantity for bacillus promoter is more, and different promoters
Effect it is unpredictable, so screening difficulty it is also larger.
Summary of the invention
The object of the present invention is to provide a kind of Gene expressions for improving bacillus protein expression quantity, are using deriving from
Promoter P43, PHpaII and the PSPO-I of bacillus are remarkably improved bacillus to the expression quantity of foreign protein, thus
Make up the deficiencies in the prior art.
One aspect of the present invention provides a kind of Gene expression, includes Promoter P43, PHpaII and PSPO-I；
Wherein, the nucleotides sequence of the Promoter P43 is classified as SEQ ID NO：2；
The nucleotides sequence of the promoter PHpaII is classified as SEQ ID NO：3；
The nucleotides sequence of the promoter PSPO-I is classified as SEQ ID NO：4；
Above-mentioned promoter is connected in sequence by Promoter P43, PHpaII and PSPO-I, nucleotide sequence
For SEQ ID NO：7；
In order to obtain better effect, above-mentioned Gene expression also includes promoter PcryIIIA.
The nucleotides sequence of the promoter PcryIIIA is classified as SEQ ID NO：5.
The Gene expression is connected in sequence by Promoter P43, PHpaII, PSPO-I and PcryIIIA, core
Nucleotide sequence is SEQ ID NO：8.
The present invention also provides a kind of recombinant plasmids, include above-mentioned Gene expression.
The present invention also provides a kind of host strains, include above-mentioned recombinant plasmid.
The host strain is bacillus.
The preferred bacillus subtilis of host strain (Bacillus subtilis).
Application of the host strain in production protease.
The QC6 bacterial strain constructed using P43-PHpaII-PSPO-I Gene expression provided by the invention, alkali protease
QC1, QC2 and QC3 bacterial strain that expression quantity is constructed significantly greater than with single Promoter P43, PHpaII and PSPO-I, or even than being somebody's turn to do
The sum of the expression quantity of three plants of bacterium taller 70% or so, produces unexpected technical effect, can significantly promote gemma bar
The expressing quantity of bacterium.The QC7 constructed using P43-PHpaII-PSPO-I-PcryIIIA Gene expression provided by the invention
Bacterial strain, the QC6 bacterial strain that the expression quantity of alkali protease is constructed significantly greater than with P43-PHpaII-PSPO-I Gene expression
The QC4 bacterial strain constructed with the single promoter PcryIIIA of use, in addition it is also higher by 50% or so than the sum of expression quantity of two plants of bacterium,
To illustrate that the tandem compound of this four promoters of P43, PHpaII, PSPO-I and PcryIIIA produces unexpected technology
Effect can significantly promote the expressing quantity of bacillus.
Detailed description of the invention
Fig. 1 is pDG1662 plasmid map；
Fig. 2 is pQSXG-1 plasmid map；
Fig. 3 is pQSXG-2 plasmid map；
Fig. 4 is pQSXG-3 plasmid map；
Fig. 5 is pQSXG-4 plasmid map；
Fig. 6 is pQSXG-5 plasmid map；
Fig. 7 is pQSXG-6 plasmid map；
Fig. 8 is pQSXG-7 plasmid map；
Fig. 9 is pQSXG-8 plasmid map；
Figure 10 is pQSXG-9 plasmid map；
Figure 11 is horizontal using the integrant expression strain fermentation enzyme activity of different promoters.
Specific embodiment
Applicant by existing promoter largely screened and tandem compound after find some single promoters to bud
The protein expression of spore bacillus has apparent facilitation, and some single promoters are then without remarkable result；It is two or more to open
Influence of the tandem compound of mover to bacillus protein expression quantity may be unexpected by, to bacillus after the series connection of some promoters
Protein expression have an apparent facilitation, facilitation effect even significantly larger than the effect of its single promoter for being included it
With, and some promoters series connection after facilitation effect be not so good as it includes single promoter effect；Moreover) tandem promoter
The series sequence of each single promoter has a great impact to its effect in son, and each promoter is only carried out using specific sequence
The best effect of series connection competence exertion, conversely, its effect is poor.
By long-term research, applicant screens two kinds of Gene expression P43- that can be applied to bacillus
PHpaII-PSPO-I and P43-PHpaII-PSPO-I-PcryIIIA, albumen table of both Gene expressions to bacillus
The aobvious facilitation of Da Youming, the bacillus constructed using the Gene expression, alkali protease expression quantity, which is much higher than, to be divided
Not Cai Yong it includes the building of single promoter the sum of the expression quantity of bacillus, unexpected technical results have been achieved,
To facilitate the present invention.
Method of the invention is described further below with reference to example, the experiment side of actual conditions is not specified in embodiment
Method, usually can routinely condition, as J. Pehanorm Brooker (Sambrook) etc. is write《Molecular Cloning:A Laboratory guide》Described in
Condition, or run according to the normal condition proposed by manufacturer.Those skill in the art related can be more preferable by embodiment
Ground understands and grasps the present invention.But realize that method of the invention should not necessarily be limited by specific method documented by the embodiment of the present invention
Step.
The integrant expression bacterial strain Bacillus subtilis QC1 that embodiment 1 is constructed using P43 promoter
Primer 1：CGTACGGGATCCGCTGTTTGCGTTTTTGCCGTG(BamHI)
Primer 2：ATTTTCCCCAACGGTTTCTTCATTATCACTTTATATTATAAACA
Primer 3：TGTTTATAATATAAAGTGATAATGAAGAAACCGTTGGGGAAAAT
Primer 4：GTTTTATTTGATGCCTGGCAGTTAGCGTGTTGCCGCTTCTGC
Primer 5：GCAGAAGCGGCAACACGCTAACTGCCAGGCATCAAATAAAAC
Primer 6：CGTACGAAGCTTGAGTTTGTAGAAACGCAAAAAG(HindIII)
With the NO of ID containing SEQ of synthesis:The plasmid of 2 sequences is template, expands P43 promoter using primer 1, primer 2.Benefit
Alkaline protease gene aprE (SEQ ID NO is expanded with primer 3, primer 4:1).With bacillus coli DH 5 alpha (Escherichia
Coli DH5 α) genome be template, utilize primer 5, primer 6 expand rrnB terminators.PCR reaction system is：5×
Phusion HF Buffer 10 μ L, 2.5mMdNTP4 μ L, 10 μM of Forward Primer2.5 μ L, 10 μM of Reverse
Primer2.5 μ L, template DNA 0.5 μ L, NEB Phusion DNA Polymerase0.5 μ L, ddH2O is mended to 50 μ L.PCR expands
Increasing condition is 98 DEG C of 2min；98 DEG C of 10s, 54 DEG C of 20s, 72 DEG C of 1min, 30 circulations；72℃5min.
P43 promoter, aprE and rrnB terminators are recycled using E.Z.N.A.Gel Extraction Kit
Pcr amplification product obtains fusion segment P43/aprE/rrnB terminators by fusion DNA vaccine.Fusion DNA vaccine process is as follows：
The first round, P43, aprE, rrnB the terminators segment of 200ng were respectively added in PCR reaction system, and primer is not added, and PCR expands
Increase 10 circulations, PCR amplification condition is 98 DEG C of 2min；98 DEG C of 10s, 54 DEG C of 20s, 72 DEG C of 4min, 10 circulations；72℃5min.
Second wheel, takes 10 μ L first round PCR products as template, expands 30 circulations, PCR amplification condition using primer 1, primer 6PCR
For 98 DEG C of 2min；98 DEG C of 10s, 54 DEG C of 20s, 72 DEG C of 2min, 30 circulations；72℃5min.Utilize E.Z.N.A.Gel
Extraction Kit recycles the PCR product of about 1.5kb, obtains P43/aprE/rrnB terminators fusion segment.
P43/aprE/rrnB terminators segment and pDG1662 plasmid (Fig. 1) are carried out simultaneously BamHI,
HindIII double digestion utilizes the item of E.Z.N.A.Gel Extraction Kit difference purification and recovery 1.5kb and 7.0kb size
The common competence method of connection product is converted Bacillus then by the T4DNA Ligase connection of two recovery products by band
Subtilis1A751 host strain (alkaline protease gene (aprE) and neutral protease gene (nprE) in the bacterial strain by
Knock out, itself does not produce protease substantially, which given by Virginia, US Polytechnics doctor Zhang Xiaozhou), conversion tool
Body process is as follows：By the Bacillus subtilis1A751 of fresh activation by LB (tryptone 1%, yeast powder 0.5%,
NaCl1%) to 5ml GM I, (GM I preparation method is plating：1 × minimum salting liquid 95.6ml, 20% glucose 2.5ml,
5% caseinhydrolysate 0.4ml, 10% yeast powder juice 1ml；Wherein 1 × minimum salting liquid preparation method is：K2HPO414g/L,
KH2PO46g/L, (NH4)2SO42g/L, trisodium citrate 1g/L, MgSO4·7H2O 0.2g/L successively dissolves in distilled water)
Solution is stayed overnight in 30 DEG C, 125rpm shaken cultivation.2ml is taken to be transferred in 18ml GM I within second day, 37 DEG C, 250rpm culture
3.5h.Taking the culture solution of 10ml previous step to be transferred to 90ml GM II again, (GM II preparation method is：1 × minimum salting liquid
96.98mL, 20% glucose 2.5mL, 5% caseinhydrolysate 0.08mL, 10% yeast powder juice 0.04mL, 1M
MgCl20.25mL, 1M CaCl2In 0.05mL), 37 DEG C, 125rpm culture 90min after, thalline were collected by centrifugation by 5000g, 10min.
With 10mL original fluid supernatant, gently suspension thalline, the thallus after suspension are competent cell.Then in 0.5mL competence
The middle appropriate DNA of addition is coated with the LB plate containing 5 μ g/mL chloramphenicol after 37 DEG C, 200rpm shaken cultivation 30min, then at 37 DEG C
Overnight incubation, secondary daily inspection and verifying transformant.After sequence verification is correct, the plasmid of acquisition is ordered for obtained positive transformant
Entitled pQSXG-1 (Fig. 2).
Using KpnI digestion pQSXG-1 plasmid, linearization process is carried out, recycles the band of 8.5kb size, common competence
Method converts Bacillus subtilis 1A751, and coating contains the LB plate of 5 μ g/mL chloramphenicol.PQSXG-1 linearized fragment
Into after Bacillus subtilis 1A751, double crossing over integration, P43/aprE/rrnB can occur in the site amyE
Terminators expression cassette and cat resistant gene are integrated on genome.The verification process of transformant is as follows：Picking transformant
Parallel dibbling LB (Cm5 μ g/mL) plate and LB (Cm5 μ g/mL+Spc100 μ g/mL) plate, occur the positive transformant of double crossing over
For CmRSpcS.It can be observed that significant transparent circle after 37 DEG C of 1% skimmed milk power plate of positive transformant dibbling is incubated overnight.It mentions
The genome for taking positive transformant, using primer 1, primer 6 can amplify containing P43/aprE/rrnB terminators,
The segment of size about 1.5kb, the sequencing fragment are correct.The positive transformant of acquisition is named as Bacillus subtilis QC1.
The integrant expression bacterial strain Bacillus subtilis QC2 that embodiment 2 is constructed using PHpaII promoter
Primer 7：TATAATTTACTTGGAAGTGGTTGCCATGAAGAAACCGTTGGGGAAAATTG
Primer 8：GAAAAAAGAAACAAAAAAACCTGCCGGATCCGATCAGACCAGTTTTTAAT
Primer 9：ATTAAAAACTGGTCTGATCGGATCCGGCAGGTTTTTTTGTTTCTTTTTTC
Primer 10：CAATTTTCCCCAACGGTTTCTTCATGGCAACCACTTCCAAGTAAATTATA
With the NO of ID containing SEQ of synthesis:The plasmid of 3 sequences is template, expands PHpaII using primer 9, primer 10 and starts
Son.Using pQSXG-1 as template, plasmid backbone is expanded using primer 7, primer 8.Utilize E.Z.N.A.Gel Extraction Kit
0.15 kb of purification and recovery and the band of 8.4 kb sizes respectively, carry out more wheel fusion DNA vaccines, and PCR reaction system is：5×
The each about 200ng of 10 μ L, 2.5mMdNTP4 μ L, pQSXG-1 plasmid backbone of Phusion HF Buffer and PHpaII promoter,
NEB Phusion DNA Polymerase1 μ L, moisturizing to 50 μ L.PCR amplification condition is 98 DEG C of 2min；98 DEG C of 10s, 72 DEG C
3min, 20 circulations；98 DEG C of 10s, 72 DEG C of 6min, 15 circulations；72℃5min.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.5kb size to get linearisation pQSXG-2 (Fig. 3) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-2 segment is converted into Bacillus subtilis1A751, screening
The integrant expression bacterial strain of PHpaII/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE, is named as
Bacillus subtilis QC2。
The integrant expression bacterial strain Bacillus subtilis QC3 that embodiment 3 is constructed using PSPO-I promoter
Primer 11：CTACAAGGTGTGGTATAATGTGTGGATGAAGAAACCGTTGGGGAAAATTG
Primer 12：TGTAGAACAAAACATCTTTCCGCTCGGATCCGATCAGACCAGTTTTTAAT
Primer 13：ATTAAAAACTGGTCTGATCGGATCCGAGCGGAAAGATGTTTTGTTCTACA
Primer 14：CAATTTTCCCCAACGGTTTCTTCATCCACACATTATACCACACCTTGTAG
With the NO of ID containing SEQ of synthesis:The plasmid of 4 sequences is template, expands PSPO-I using primer 13, primer 14 and starts
Son.Using pQSXG-1 as template, plasmid backbone is expanded using primer 11, primer 12.Utilize E.Z.N.A.Gel Extraction
Kit distinguishes the band of purification and recovery 0.1kb and 8.4kb size, carries out more wheel fusion DNA vaccines, PCR reaction condition is the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.5kb size to get linearisation pQSXG-3 (Fig. 4) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-3 segment is converted into Bacillus subtilis1A751, screening
The integrant expression bacterial strain of PSPO-I/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE, is named as
Bacillus subtilis QC3。
The integrant expression bacterial strain Bacillus subtilis QC4 that embodiment 4 is constructed using PcryIIIA promoter
Primer 15：ACACCAATTAAAGGAGGAATTCAAAATGAAGAAACCGTTGGGGAAAATTG
Primer 16：AGCGATAATTCACATGAACAAGTTCGGATCCGATCAGACCAGTTTTTAAT
Primer 17：ATTAAAAACTGGTCTGATCGGATCCGAACTTGTTCATGTGAATTATCGCT
Primer 18：CAATTTTCCCCAACGGTTTCTTCATTTTGAATTCCTCCTTTAATTGGTGT
With the NO of ID containing SEQ of synthesis:The plasmid of 5 sequences is template, expands PcryIIIA using primer 17, primer 18 and opens
Mover.Using pQSXG-1 as template, plasmid backbone is expanded using primer 15, primer 16.Utilize E.Z.N.A.Gel Extraction
Kit distinguishes the band of purification and recovery 0.37kb and 8.4kb size, carries out more wheel fusion DNA vaccines, PCR reaction condition is the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.7kb size to get linearisation pQSXG-4 (Fig. 5) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-4 segment is converted into Bacillus subtilis1A751, screening
The integrant expression bacterial strain of PcryIIIA/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE, is named as
Bacillus subtilis QC4。
The integrant expression bacterial strain Bacillus subtilis that embodiment 5 is constructed using P43-PHpaII Gene expression
QC5
Primer 19：AAATCACGGCAAAAACGCAAACAGCGGATCCGATCAGACCAGTTTTTAAT
Primer 20：ATTAAAAACTGGTCTGATCGGATCCGCTGTTTGCGTTTTTGCCGTGATTT
With the NO of ID containing SEQ of synthesis:The plasmid of 6 sequences is template, expands P43-PHpaII using primer 20, primer 10
Gene expression.Using pQSXG-1 as template, plasmid backbone is expanded using primer 7, primer 19.Utilize E.Z.N.A.Gel
Extraction Kit distinguishes the band of purification and recovery 0.3kb and 8.4kb size, carries out more wheel fusion DNA vaccines, PCR reaction condition
With embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.7kb size to get linearisation pQSXG-5 (Fig. 6) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-5 segment is converted into Bacillus subtilis1A751, screening
The integrant expression bacterial strain of P43-PHpaII/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE, is named as
Bacillus subtilis QC5。
The integrant expression bacterial strain Bacillus that embodiment 6 is constructed using P43-PHpaII-PSPO-I Gene expression
subtilis QC6
With the NO of ID containing SEQ of synthesis:The plasmid of 7 sequences is template, expands P43- using primer 20, primer 14
PHpaII-PSPO-I Gene expression.Using pQSXG-1 as template, plasmid backbone is expanded using primer 11, primer 19.It utilizes
E.Z.N.A.Gel Extraction Kit distinguishes the band of purification and recovery 0.4kb and 8.4kb size, carries out more wheel fusion DNA vaccines,
PCR reaction condition is the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.8kb size to get linearisation pQSXG-6 (Fig. 7) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-6 segment is converted into Bacillus subtilis1A751, screening
The integrant expression bacterium of P43-PHpaII-PSPO-I/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE
Strain, is named as Bacillus subtilis QC6.
The integrant expression bacterial strain that embodiment 7 is constructed using P43-PHpaII-PSPO-I-PcryIIIA Gene expression
Bacillus subtilis QC7
With the NO of ID containing SEQ of synthesis:The plasmid of 8 sequences is template, expands P43- using primer 20, primer 18
PHpaII-PSPO-I-PcryIIIA Gene expression.Using pQSXG-1 as template, plasmid bone is expanded using primer 15, primer 19
Frame.Using the band of E.Z.N.A.Gel Extraction Kit difference purification and recovery 0.77kb and 8.4kb size, more wheels are carried out
Fusion DNA vaccine, PCR reaction condition is the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 9.1kb size to get linearisation pQSXG-7 (Fig. 8) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-7 segment is converted into Bacillus subtilis1A751, screening
The whole of P43-PHpaII-PSPO-I-PcryIIIA/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE
Expression bacterial strain is closed, Bacillus subtilis QC7 is named as.
The integrant expression bacterial strain that embodiment 8 is constructed using P43-PHpaII-PcryIIIA-PSPO-I Gene expression
Bacillus subtilis QC8
With the NO of ID containing SEQ of synthesis:The plasmid of 9 sequences is template, expands P43- using primer 20, primer 14
PHpaII-PcryIIIA-PSPO-1 Gene expression.Using pQSXG-1 as template, plasmid bone is expanded using primer 11, primer 19
Frame.Using the band of E.Z.N.A.Gel Extraction Kit difference purification and recovery 0.77kb and 8.4kb size, more wheels are carried out
Fusion DNA vaccine, PCR reaction condition is the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 9.1kb size to get linearisation pQSXG-8 (Fig. 9) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-8 segment is converted into Bacillus subtilis1A751, screening
The whole of P43-PHpaII-PcryIIIA-PSPO-1/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE
Expression bacterial strain is closed, Bacillus subtilis QC8 is named as.
The integrant expression bacterial strain that embodiment 9 is constructed using P43-PHpaII-PSPO-I-PamyQ Gene expression
Bacillus subtilis QC9
Primer 21：ATAATATAATTTGTATAAGAAAATGATGAAGAAACCGTTGGGGAAAATTG
Primer 22：CAATTTTCCCCAACGGTTTCTTCATCATTTTCTTATACAAATTATATTAT
With the NO of ID containing SEQ of synthesis:The plasmid of 10 sequences is template, expands P43- using primer 20, primer 22
PHpaII-PSPO-I-PamyQ Gene expression.Using pQSXG-1 as template, plasmid backbone is expanded using primer 21, primer 19.
Using the band of E.Z.N.A.Gel Extraction Kit difference purification and recovery 0.5kb and 8.4kb size, more wheel fusions are carried out
PCR, PCR reaction condition are the same as embodiment 2.
PCR product is subjected to KpnI digestion, glue recycles the band of 8.9kb size to get linearisation pQSXG-9 (Figure 10) is arrived
Segment.
According to the method for embodiment 1, linearisation pQSXG-9 segment is converted into Bacillus subtilis1A751, screening
The integration table of P43-PHpaII-PSPO-I-PamyQ/aprE/rrnB terminators expression cassette is integrated in the double crossing over of the site amyE
Up to bacterial strain, it is named as Bacillus subtilis QC9.
Embodiment 10 uses the integrant expression bacterial strain shake flask fermentation of different promoters
The information of integrant expression bacterial strain described in embodiment 1-9 using different promoters is shown in Table 1.
Table 1 uses the integrant expression bacterial strain of different promoters
The above-mentioned integrant expression bacterial strain of building is subjected to shake flask fermentation under the same conditions, the specific method is as follows：
It is inoculated in 50mL seed culture respectively from the single colonie of picking above-mentioned bacterial strains on the LB plate containing 5 μ g/ml chloramphenicol
Base (yeast extract 0.5%, tryptone 0.5%, glucose 1%, K2HPO41.8%, 5 μ g/mL of chloramphenicol) in, 34 DEG C,
210rpm shaken cultivation 8h.Then 2.5mL fermentation liquid is taken to be inoculated into 50mL fermentation medium (yeast powder 1~2%, soya-bean cake respectively
Powder 2~5%, maltodextrin 5~10%, sodium citrate 0.1~0.5%, CaCl20.1~0.5%, MgSO40.1~0.5%,
K2HPO40.5~2%) in, 34 DEG C, 250rpm shaken cultivation 72h；Centrifuging and taking supernatant；It is marked using country of the People's Republic of China (PRC)
Quasi- protease preparation measuring method (GB/T25327-2009) measures the alkali protease enzyme of above-mentioned bacterial strains fermented supernatant fluid respectively
It is living, the result is shown in Figure 11.
As shown in figure 11, single Promoter P43, PHpaII and PSPO-I, alkali is respectively adopted in QC1, QC2 and QC3 bacterial strain
Property protease expression quantity it is higher, and QC4 bacterial strain use single promoter PcryIIIA, alkali protease expression quantity is very
It is low, less than the 40% of QC1, QC2 and QC3 bacterial strain expression quantity.To which one promoter of instruction sheet is to bacillus protein expression quantity
There were significant differences due to the difference of promoter for influence.
QC5 bacterial strain is using P43-PHpaII Gene expression, the expression quantity of alkali protease and using single promoter
QC1 the and QC2 bacterial strain of P43 and PHpaII building is substantially suitable, even also lower by 10% than the expression quantity of QC1 bacterial strain, to say
The series connection of bright Promoter P43 and PHpaII do not have apparent facilitation for the protein expression of bacillus, furtherly yet
The influence that the tandem compound of clear promoter expresses bacillus protein is unpredictable.
QC6 bacterial strain use P43-PHpaII-PSPO-I Gene expression, alkali protease expression quantity significantly greater than with
QC1, QC2 and QC3 bacterial strain of single Promoter P43, PHpaII and PSPO-I building, or even the sum of the expression quantity than three plants of bacterium
Taller 70% or so.The tandem compound of these three promoters of P43, PHpaII and PSPO-I produces unexpected technology effect
Fruit can significantly promote the expressing quantity of bacillus.
QC7 bacterial strain uses P43-PHpaII-PSPO-I-PcryIIIA Gene expression, the expression quantity of alkali protease
QC6 bacterial strain and the single promoter PcryIIIA of use significantly greater than with the building of P43-PHpaII-PSPO-I Gene expression
The QC4 bacterial strain of building, in addition it is also higher by 50% or so than the sum of expression quantity of two plants of bacterium.P43, PHpaII, PSPO-I and
The tandem compound of this four promoters of PcryIIIA produces unexpected technical effect, can significantly promote bacillus
Expressing quantity.
It is single although QC8 bacterial strain is also connected using this four promoters of P43, PHpaII, PSPO-I and PcryIIIA
Putting in order for a promoter is slightly different with QC7 bacterial strain, and the promoter after series connection is P43-PHpaII-PcryIIIA-
PSPO-I.But the alkali protease expression quantity of QC8 bacterial strain is only the 20% of QC7 bacterial strain, to illustrate each list in Gene expression
The series sequence of one promoter has a great impact to its effect, and each promoter only sequentially carries out series connection ability using specific
Best effect is played, conversely, its effect is poor.
QC9 bacterial strain uses P43-PHpaII-PSPO-I-PamyQ Gene expression, and compared with QC7 bacterial strain, difference is
The selection of 4th promoter, but the expression quantity of its alkali protease is not only well below QC7 bacterial strain, even than using P43-
The QC6 bacterial strain of the tandem compound building of tri- promoters of PHpaII-PSPO-I also wants low 30%.To illustrate, Gene expression
Effect and the quantity of its promoter it is not directly proportional, key is the selection of promoter, any one in Gene expression is single
The variation of promoter is likely to result in significantly alterring for its overall effect, influences unpredictable.
To sum up, Gene expression P43-PHpaII-PSPO-I and P43-PHpaII-PSPO-I-PcryIIIA of the invention,
Both Gene expressions have apparent facilitation to the protein expression of bacillus.
Claims (10)
1. a kind of Gene expression, which is characterized in that the Gene expression is by Promoter P43, PHpaII and PSPO-I
It is connected in sequence.
2. Gene expression as described in claim 1, which is characterized in that the nucleotides sequence of the Promoter P43 is classified as SEQ
ID NO：2.
3. Gene expression as described in claim 1, which is characterized in that the nucleotides sequence of the promoter PHpaII is classified as
SEQ ID NO：3.
4. Gene expression as described in claim 1, which is characterized in that the nucleotides sequence of the promoter PSPO-I is classified as
SEQ ID NO：4.
5. a kind of Gene expression, which is characterized in that the Gene expression be by Promoter P43, PHpaII, PSPO-I and
PcryIIIA is connected in sequence.
6. Gene expression as claimed in claim 5, which is characterized in that the nucleotide sequence of the promoter PcryIIIA
For SEQ ID NO：5.
7. application of the Gene expression described in claim 1 or 5 in the plasmid that preparation is used for transforming bacillus.
8. a kind of recombinant plasmid, which is characterized in that the promoter of the recombinant plasmid is series connection described in claim 1 or 5
Promoter.
9. a kind of recombinant bacterial strain, which is characterized in that the recombinant bacterial strain is recombination described in conversion/transfection has the right to require 8
The host strain of plasmid.
10. recombinant bacterial strain as claimed in claim 9, which is characterized in that the host strain is bacillus subtilis
(Bacillus subtilis)。
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| CN107988245B (en) * | 2017-10-25 | 2021-11-19 | 南京福斯弗瑞生物科技有限公司 | Codon-optimized pullulanase, expression vector and construction method thereof |
| CN109652417B (en) * | 2018-12-17 | 2021-07-27 | 江南大学 | Method for constructing efficient bacillus subtilis promoter |
| CN109486847B (en) * | 2018-12-17 | 2021-03-02 | 江南大学 | Bacillus subtilis efficient induction expression system based on artificial tandem promoter |
| RU2723722C1 (en) | 2019-07-26 | 2020-06-17 | Общество с ограниченной ответственностью "ЦЕНТР ТРАНСФЕРА БИОТЕХНОЛОГИЙ ОКА-Биотех" | Tandem promoter functioning in bacteria of bacillus genus, transformed bacillus bacterium - target product producer containing said promoter, and method for producing desired product using said bacterium |
| CN111926013B (en) * | 2020-08-18 | 2021-11-02 | 湖北大学 | Promoter suitable for bacillus licheniformis and application thereof in high-efficiency expression of target product |
| CN114277046B (en) * | 2021-12-14 | 2024-05-28 | 河北师范大学 | Three-gene tandem expression vector for synthesizing tetrahydropyrimidine and application thereof |
| CN114958897B (en) * | 2022-06-14 | 2023-12-22 | 中农华威生物制药(湖北)有限公司 | Construction method of bacillus subtilis capable of efficiently expressing feed low-temperature keratinase |
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