CN105779444A - Tandem promoters capable of improving protein expression quantities of bacilli - Google Patents
Tandem promoters capable of improving protein expression quantities of bacilli Download PDFInfo
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Abstract
The invention aims at providing tandem promoters capable of improving protein expression quantities of bacilli. In a QC6 strain which is constructed by a P43-PHpaII-PSPO-I tandem promoter provided by the invention, an alkaline protease expression quantity is significantly higher than that of QC1, QC2 and QC3 strains which are constructed by single promoters P43, PHpaII and PSPO-I, and is even about 70% higher than the sum of the expression quantities of the three strains. In a QC7 strain which is constructed by a P43-PHpaII-PSPO-I-PcryIIIA tandem promoter provided by the invention, an alkaline protease expression quantity is significantly higher than that of a QC6 strain which is constructed by the P43-PHpaII-PSPO-I tandem promoter and that of a QC4 strain which is constructed by a single promoter PcryIIIA, and is even about 50% higher than the sum of the expression quantities of the two strains. The tandem promoters can take an extraordinary technical effect, and the tandem promoters can greatly improve the protein expression quantities of the bacilli.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of Gene expression being remarkably improved bacillus protein expression and application thereof.
Background technology
Bacillus cereus is an excellent protein expression host, it has the high (GenerallyRegardedasSafe of safety, GRAS), growth cycle is short, fermentation costs is low, secretion capacity is strong, without remarkable advantages such as obvious codon preferences, is widely used in the industrialized production of the enzyme preparations such as amylase, protease, pullulanase, pectase.
In order to reduce the production cost of enzyme preparation product, increasing economic efficiency, an important requirement of industrial Bacillusexpression system is that protein expression amount wants height.And the important means improving bacillus cereus expression is to improve the mRNA level in-site of genes of interest, conventional method is the high copy number plasmid expression vector adopting and carrying genes of interest, and the maximum shortcoming of the method is that in commercial process, high copy number plasmid expression vector exists Loss.The problem bad in order to solve plasmid-bearing strains stability, can the expression cassette that contain the single promoter being operatively connected with genes of interest and an amplifiable riddled basins (such as antibiotic-resistance marker's gene) be incorporated on Bacillus chromosome, then pass through expression cassette and riddled basins that screening pressure (such as antibiotic) expands on chromosome, produce the expression cassette of multicopy on chromosome.The method there is also some shortcomings, as passed through to expand the mRNA that can not be obtained saturated level by the gene of single promoters driven.And, the expression cassette potentially unstable of multicopy that chromosome is connected.Additionally, riddled basins is very difficult to remove.And the method that another one obtains saturated mRNA level in-site is to screen specific promoter.But owing to the quantity for bacillus promoter is many, and the effect of different promoters is unpredictable, so screening difficulty is also bigger.
Summary of the invention
It is an object of the invention to provide a kind of Gene expression improving bacillus protein expression, it is use to derive from the Promoter P43 of bacillus cereus, PHpaII and PSPO-I, it is remarkably improved the bacillus cereus expression to foreign protein, thus making 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 described Promoter P43 is classified as SEQIDNO:2；
The nucleotides sequence of described promoter PHpaII is classified as SEQIDNO:3；
The nucleotides sequence of described promoter PSPO-I is classified as SEQIDNO:4；
Above-mentioned promoter, is connected in sequence by Promoter P43, PHpaII and PSPO-I, and its nucleotides sequence is classified as SEQIDNO:7；
In order to obtain better effect, above-mentioned Gene expression, also comprise promoter PcryIIIA.
The nucleotides sequence of described promoter PcryIIIA is classified as SEQIDNO:5.
Described Gene expression, is connected in sequence by Promoter P43, PHpaII, PSPO-I and PcryIIIA, and its nucleotides sequence is classified as SEQIDNO:8.
Present invention also offers a kind of recombiant plasmid, comprise above-mentioned Gene expression.
Present invention also offers a kind of Host Strains, comprise above-mentioned recombiant plasmid.
Described Host Strains is bacillus cereus.
The preferred bacillus subtilis of described Host Strains (Bacillussubtilis).
The application in producing protease of the described Host Strains.
Adopt the QC6 bacterial strain that P43-PHpaII-PSPO-I Gene expression provided by the invention builds, QC1, QC2 and the QC3 bacterial strain that its alkaline protease expression builds significantly greater than with single Promoter P43, PHpaII and PSPO-I, even than the expression sum taller about 70% of this three strains bacterium, create unexpected technique effect, can significantly promote the expressing quantity of bacillus cereus.Adopt the QC7 bacterial strain that P43-PHpaII-PSPO-I-PcryIIIA Gene expression provided by the invention builds, the expression of its alkaline protease is significantly greater than with P43-PHpaII-PSPO-I Gene expression the QC6 bacterial strain built and the QC4 bacterial strain adopting single promoter PcryIIIA structure, even also high by about 50% than the expression sum of this two strains bacterium, thus P43 is described, the tandem compound of these four promoteres of PHpaII, PSPO-I and PcryIIIA creates unexpected technique effect, can significantly promote the expressing quantity of bacillus cereus.
Accompanying drawing explanation
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 that the integrant expression strain fermentation enzyme running water adopting different promoters is put down.
Detailed description of the invention
Applicant by existing promoter is carried out in a large number screening and tandem compound after find that the protein expression of bacillus cereus is had obvious facilitation by some single promoter, some single promoter does not then have remarkable result；The impact of bacillus protein expression be may be unexpected by by the tandem compound of two or more promoteres, after the series connection of some promoter, the protein expression of bacillus cereus there is obvious facilitation, its facilitation effect is even significantly larger than the effect sum of its single promoter comprised, and the facilitation effect after the series connection of some promoter is not as the effect of its single promoter comprised；And) its effect is had a great impact by the series sequence of each single promoter in Gene expression, each promoter only adopts specific order to carry out the effect that series connection competence exertion is best, otherwise, its effect is poor.
Through long-term research, applicant screens two kinds of Gene expression P43-PHpaII-PSPO-I and P43-PHpaII-PSPO-I-PcryIIIA that can be applicable to bacillus cereus, the protein expression of bacillus cereus is had obvious facilitation by both Gene expressions, adopt the bacillus cereus that this Gene expression builds, its alkaline protease expression is far above the expression sum being respectively adopted the bacillus cereus that its single promoter comprised builds, achieve unexpected technique effect, thus facilitating the present invention.
Below in conjunction with example, the method for the present invention is described further, the experimental technique of unreceipted actual conditions in embodiment, generally can condition routinely, such as the condition described in " the Molecular Cloning: A Laboratory guide " write such as J. Pehanorm Brooker (Sambrook), or according to manufacturer it is proposed that condition run.Those skill in the art related can be more fully understood that by embodiment and grasp the present invention.But, it is achieved the method for the present invention should not necessarily be limited by the concrete grammar step described in the embodiment of the present invention.
Embodiment 1 adopts the integrant expression bacterial strain BacillussubtilisQC1 that P43 promoter builds
Primer 1:CGTACGGGATCCGCTGTTTGCGTTTTTGCCGTG(BamHI)
Primer 2: ATTTTCCCCAACGGTTTCTTCATTATCACTTTATATTATAAACA
Primer 3:TGTTTATAATATAAAGTGATAATGAAGAAACCGTTGGGGAAAAT
Primer 4:GTTTTATTTGATGCCTGGCAGTTAGCGTGTTGCCGCTTCTGC
Primer 5:GCAGAAGCGGCAACACGCTAACTGCCAGGCATCAAATAAAAC
Primer 6:CGTACGAAGCTTGAGTTTGTAGAAACGCAAAAAG(HindIII)
With the plasmid containing SEQIDNO:2 sequence of synthesis for template, utilize primer 1, primer 2 amplification P43 promoter.Primer 3, primer 4 is utilized to expand alkaline protease gene aprE (SEQIDNO:1).With bacillus coli DH 5 alpha (EscherichiacoliDH5 α) genome for template, primer 5, primer 6 is utilized to expand rrnBterminators.PCR reaction system is: 5 × PhusionHFBuffer10 μ L, 2.5mMdNTP4 μ L, 10 μMs of ForwardPrimer2.5 μ L, 10 μMs of ReversePrimer2.5 μ L, template DNA 0.5 μ L, NEBPhusionDNAPolymerase0.5 μ L, ddH2O mends to 50 μ L.Pcr amplification 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.
Utilize E.Z.N.A.GelExtractionKit to reclaim P43 promoter, aprE and rrnBterminatorsPCR amplified production, obtained by fusion DNA vaccine and merge fragment P43/aprE/rrnBterminators.Fusion DNA vaccine process is as follows: the first round, and in PCR reaction system, each P43, aprE, rrnBterminators fragment adding 200ng, is not added with primer, pcr amplification 10 circulation, and 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 takes turns, and takes 10 μ L first round PCR primer as template, utilizes primer 1, primer 6PCR to expand 30 circulations, and pcr amplification condition is 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.GelExtractionKit to reclaim the PCR primer of about 1.5kb, obtain P43/aprE/rrnBterminators and merge fragment.
nullP43/aprE/rrnBterminators fragment and pDG1662 plasmid (Fig. 1) are carried out BamHI simultaneously、HindIII double digestion，E.Z.N.A.GelExtractionKit purification respectively is utilized to reclaim the band of 1.5kb and 7.0kb size，Then reclaim product T4DNALigase by two to connect，By connecting the common competence method conversion Bacillussubtilis1A751 Host Strains of product, (alkaline protease gene (aprE) and neutral protease gene (nprE) in this bacterial strain are knocked，Itself does not produce protease substantially，This bacterial strain is to be given by Virginia, US Polytechnics doctor Zhang Xiaozhou)，Conversion detailed process is as follows: by the Bacillussubtilis1A751 of fresh activation by LB (tryptone 1%，Yeast powder 0.5%，NaCl1%) to 5mlGM I, (GMI compound method is plating: 1 × minimum saline solution 95.6ml，20% glucose 2.5ml，5% caseinhydrolysate 0.4ml，10% yeast powder juice 1ml；Wherein the compound method of 1 × minimum saline solution is: K2HPO414g/L, KH2PO46g/L, (NH4)2SO42g/L, trisodium citrate 1g/L, MgSO4·7H2O0.2g/L, in distilled water successively dissolve) solution, 30 DEG C, 125rpm shaken cultivation overnight.Within second day, take 2ml and be transferred in 18mlGM I, 37 DEG C, 250rpm cultivate 3.5h.Take the culture fluid of 10ml previous step again to be transferred to 90mlGM II (GMII compound method is: 1 × minimum saline solution 96.98mL, 20% glucose 2.5mL, 5% caseinhydrolysate 0.08mL, 10% yeast powder juice 0.04mL, 1MMgCl20.25mL, 1MCaCl2In 0.05mL), 37 DEG C, 125rpm cultivate after 90min, 5000g, 10min are centrifugal collects thalline.Suspending gently thalline with 10mL original fluid supernatant, the thalline after suspension is competent cell.Then add in 0.5mL competence appropriate DNA in 37 DEG C, be coated with the LB flat board containing 5 μ g/mL chloromycetin after 200rpm shaken cultivation 30min, then in 37 DEG C of overnight incubation, secondary daily inspection and checking transformant.The positive transformant obtained, after sequence verification is correct, plasmid called after pQSXG-1 (Fig. 2) that will obtain.
Using KpnI enzyme action pQSXG-1 plasmid, carry out linearization process, reclaim the band of 8.5kb size, common competence method converts Bacillussubtilis1A751, the coating LB flat board containing 5 μ g/mL chloromycetin.After pQSXG-1 linearized fragment enters Bacillussubtilis1A751, double crossing over being occurred to integrate in amyE site, P43/aprE/rrnBterminators expression cassette and cat resistant gene are incorporated on genome.The proof procedure of transformant is as follows: the parallel dibbling LB of picking transformant (Cm5 μ g/mL) flat board and LB (Cm5 μ g/mL+Spc100 μ g/mL) flat board, and the positive transformant that double crossing over occurs is CmRSpcS.It is observed that significant transparent circle after 37 DEG C of incubated overnight of positive transformant dibbling 1% defatted milk powder flat board.Extracting the genome of positive transformant, utilize primer 1, primer 6 can amplify the fragment being about 1.5kb containing P43/aprE/rrnBterminators, size, this sequencing fragment is correct.The positive transformant called after BacillussubtilisQC1 obtained.
Embodiment 2 adopts the integrant expression bacterial strain BacillussubtilisQC2 that PHpaII promoter builds
Primer 7:TATAATTTACTTGGAAGTGGTTGCCATGAAGAAACCGTTGGGGAAAATTG
Primer 8:GAAAAAAGAAACAAAAAAACCTGCCGGATCCGATCAGACCAGTTTTTAAT
Primer 9:ATTAAAAACTGGTCTGATCGGATCCGGCAGGTTTTTTTGTTTCTTTTTTC
Primer 10:CAATTTTCCCCAACGGTTTCTTCATGGCAACCACTTCCAAGTAAATTATA
With the plasmid containing SEQIDNO:3 sequence of synthesis for template, primer 9, primer 10 is utilized to expand PHpaII promoter.With pQSXG-1 for template, primer 7, primer 8 is utilized to expand plasmid backbone.E.Z.N.A.GelExtractionKit purification respectively is utilized to reclaim the band of 0.15kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction system is: 5 × PhusionHFBuffer10 μ L, 2.5mMdNTP4 μ L, pQSXG-1 plasmid backbone and each about 200ng of PHpaII promoter, NEBPhusionDNAPolymerase1 μ L, moisturizing is to 50 μ L.Pcr amplification condition is 98 DEG C of 2min；98 DEG C of 10s, 72 DEG C of 3min, 20 circulations；98 DEG C of 10s, 72 DEG C of 6min, 15 circulations；72℃5min.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.5kb size, namely obtains linearisation pQSXG-2 (Fig. 3) fragment.
According to the method for embodiment 1, linearisation pQSXG-2 fragment converting Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of PHpaII/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC2.
Embodiment 3 adopts the integrant expression bacterial strain BacillussubtilisQC3 that PSPO-I promoter builds
Primer 11:CTACAAGGTGTGGTATAATGTGTGGATGAAGAAACCGTTGGGGAAAATTG
Primer 12:TGTAGAACAAAACATCTTTCCGCTCGGATCCGATCAGACCAGTTTTTAAT
Primer 13:ATTAAAAACTGGTCTGATCGGATCCGAGCGGAAAGATGTTTTGTTCTACA
Primer 14:CAATTTTCCCCAACGGTTTCTTCATCCACACATTATACCACACCTTGTAG
With the plasmid containing SEQIDNO:4 sequence of synthesis for template, primer 13, primer 14 is utilized to expand PSPO-I promoter.With pQSXG-1 for template, primer 11, primer 12 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.1kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.5kb size, namely obtains linearisation pQSXG-3 (Fig. 4) fragment.
According to the method for embodiment 1, linearisation pQSXG-3 fragment converting Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of PSPO-I/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC3.
Embodiment 4 adopts the integrant expression bacterial strain BacillussubtilisQC4 that PcryIIIA promoter builds
Primer 15:ACACCAATTAAAGGAGGAATTCAAAATGAAGAAACCGTTGGGGAAAATTG
Primer 16:AGCGATAATTCACATGAACAAGTTCGGATCCGATCAGACCAGTTTTTAAT
Primer 17:ATTAAAAACTGGTCTGATCGGATCCGAACTTGTTCATGTGAATTATCGCT
Primer 18:CAATTTTCCCCAACGGTTTCTTCATTTTGAATTCCTCCTTTAATTGGTGT
With the plasmid containing SEQIDNO:5 sequence of synthesis for template, primer 17, primer 18 is utilized to expand PcryIIIA promoter.With pQSXG-1 for template, primer 15, primer 16 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.37kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.7kb size, namely obtains linearisation pQSXG-4 (Fig. 5) fragment.
Method according to embodiment 1, linearisation pQSXG-4 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of PcryIIIA/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC4.
Embodiment 5 adopts the integrant expression bacterial strain BacillussubtilisQC5 that P43-PHpaII Gene expression builds
Primer 19:AAATCACGGCAAAAACGCAAACAGCGGATCCGATCAGACCAGTTTTTAAT
Primer 2 0:ATTAAAAACTGGTCTGATCGGATCCGCTGTTTGCGTTTTTGCCGTGATTT
With the plasmid containing SEQIDNO:6 sequence of synthesis for template, primer 20, primer 10 is utilized to expand P43-PHpaII Gene expression.With pQSXG-1 for template, primer 7, primer 19 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.3kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.7kb size, namely obtains linearisation pQSXG-5 (Fig. 6) fragment.
Method according to embodiment 1, linearisation pQSXG-5 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of P43-PHpaII/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC5.
Embodiment 6 adopts the integrant expression bacterial strain BacillussubtilisQC6 that P43-PHpaII-PSPO-I Gene expression builds
With the plasmid containing SEQIDNO:7 sequence of synthesis for template, primer 20, primer 14 is utilized to expand P43-PHpaII-PSPO-I Gene expression.With pQSXG-1 for template, primer 11, primer 19 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.4kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.8kb size, namely obtains linearisation pQSXG-6 (Fig. 7) fragment.
Method according to embodiment 1, linearisation pQSXG-6 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of P43-PHpaII-PSPO-I/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC6.
Embodiment 7 adopts the integrant expression bacterial strain BacillussubtilisQC7 that P43-PHpaII-PSPO-I-PcryIIIA Gene expression builds
With the plasmid containing SEQIDNO:8 sequence of synthesis for template, primer 20, primer 18 is utilized to expand P43-PHpaII-PSPO-I-PcryIIIA Gene expression.With pQSXG-1 for template, primer 15, primer 19 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.77kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 9.1kb size, namely obtains linearisation pQSXG-7 (Fig. 8) fragment.
Method according to embodiment 1, linearisation pQSXG-7 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of P43-PHpaII-PSPO-I-PcryIIIA/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC7.
Embodiment 8 adopts the integrant expression bacterial strain BacillussubtilisQC8 that P43-PHpaII-PcryIIIA-PSPO-I Gene expression builds
With the plasmid containing SEQIDNO:9 sequence of synthesis for template, primer 20, primer 14 is utilized to expand P43-PHpaII-PcryIIIA-PSPO-1 Gene expression.With pQSXG-1 for template, primer 11, primer 19 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.77kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 9.1kb size, namely obtains linearisation pQSXG-8 (Fig. 9) fragment.
Method according to embodiment 1, linearisation pQSXG-8 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of P43-PHpaII-PcryIIIA-PSPO-1/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC8.
Embodiment 9 adopts the integrant expression bacterial strain BacillussubtilisQC9 that P43-PHpaII-PSPO-I-PamyQ Gene expression builds
Primer 2 1:ATAATATAATTTGTATAAGAAAATGATGAAGAAACCGTTGGGGAAAATTG
Primer 2 2:CAATTTTCCCCAACGGTTTCTTCATCATTTTCTTATACAAATTATATTAT
With the plasmid containing SEQIDNO:10 sequence of synthesis for template, primer 20, primer 22 is utilized to expand P43-PHpaII-PSPO-I-PamyQ Gene expression.With pQSXG-1 for template, primer 21, primer 19 is utilized to expand plasmid backbone.Utilizing E.Z.N.A.GelExtractionKit purification respectively to reclaim the band of 0.5kb and 8.4kb size, carry out taking turns fusion DNA vaccine more, PCR reaction condition is with embodiment 2.
PCR primer is carried out KpnI enzyme action, and glue reclaims the band of 8.9kb size, namely obtains linearisation pQSXG-9 (Figure 10) fragment.
Method according to embodiment 1, linearisation pQSXG-9 fragment is converted Bacillussubtilis1A751, screening amyE site double crossing over integrates the integrant expression bacterial strain of P43-PHpaII-PSPO-I-PamyQ/aprE/rrnBterminators expression cassette, called after BacillussubtilisQC9.
Embodiment 10 adopts the integrant expression bacterial strain shake flask fermentation of different promoters
Adopt the information of integrant expression bacterial strain of different promoters in Table 1 described in embodiment 1-9.
Table 1 adopts the integrant expression bacterial strain of different promoters
The above-mentioned integrant expression bacterial strain built is carried out shake flask fermentation under the same conditions, and concrete grammar is as follows:
From the LB flat board containing 5 μ g/ml chloromycetin, single bacterium colony of picking above-mentioned bacterial strains is inoculated in 50mL seed culture medium (yeast leaching powder 0.5%, tryptone 0.5%, glucose 1%, K respectively2HPO41.8%, chloromycetin 5 μ g/mL) in, 34 DEG C, 210rpm shaken cultivation 8h.Then take 2.5mL fermentation liquid respectively and be inoculated into 50mL fermentation medium (yeast powder 1～2%, soybean cake 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；Adopting the alkaline protease enzyme that National Standard of the People's Republic of China's protease preparation assay method (GB/T25327-2009) measures above-mentioned bacterial strains fermented supernatant fluid respectively to live, result is shown in Figure 11.
As shown in figure 11, QC1, QC2 and QC3 bacterial strain is respectively adopted single Promoter P43, PHpaII and PSPO-I, the expression of its alkaline protease is all higher, and QC4 bacterial strain adopts single promoter PcryIIIA, its alkaline protease expression is very low, less than the 40% of QC1, QC2 and QC3 bacterial strain expression.Thus single promoter is described, on the impact of bacillus protein expression, because of the difference of promoter, there were significant differences.
QC5 bacterial strain adopts P43-PHpaII Gene expression, the expression of its alkaline protease is substantially suitable with QC1 and the QC2 bacterial strain adopting single Promoter P43 and PHpaII to build, also lower by 10% than the expression of QC1 bacterial strain even, thus illustrating that the protein expression for bacillus cereus of connecting of Promoter P43 and PHpaII does not have obvious facilitation, the impact that bacillus protein is expressed is unpredictable by the tandem compound also further illustrating promoter.
QC6 bacterial strain adopts P43-PHpaII-PSPO-I Gene expression, QC1, QC2 and the QC3 bacterial strain that its alkaline protease expression builds significantly greater than with single Promoter P43, PHpaII and PSPO-I, even than the expression sum taller about 70% of this three strains bacterium.The tandem compound of P43, PHpaII and PSPO-I these three promoter creates unexpected technique effect, can significantly promote the expressing quantity of bacillus cereus.
QC7 bacterial strain adopts P43-PHpaII-PSPO-I-PcryIIIA Gene expression, the expression of its alkaline protease is significantly greater than with P43-PHpaII-PSPO-I Gene expression the QC6 bacterial strain built and the QC4 bacterial strain adopting single promoter PcryIIIA structure, even also high by about 50% than the expression sum of this two strains bacterium.P43, these four promoteres of PHpaII, PSPO-I and PcryIIIA tandem compound create unexpected technique effect, can significantly promote the expressing quantity of bacillus cereus.
Although QC8 bacterial strain is also adopted by P43, these four promoteres of PHpaII, PSPO-I and PcryIIIA are connected, but single promoter put in order and QC7 bacterial strain is slightly different, its series connection after promoter be P43-PHpaII-PcryIIIA-PSPO-I.But the alkaline protease expression of QC8 bacterial strain is only the 20% of QC7 bacterial strain, thus illustrating that in Gene expression, its effect is had a great impact by the series sequence of each single promoter, each promoter only adopts specific order to carry out the effect that series connection competence exertion is best, otherwise, its effect is poor.
QC9 bacterial strain adopts P43-PHpaII-PSPO-I-PamyQ Gene expression, compared with QC7 bacterial strain, it is distinctive in that the selection of the 4th promoter, but the expression of its alkaline protease is not only well below QC7 bacterial strain, also lower by 30% than the QC6 bacterial strain adopting the tandem compound of tri-promoteres of P43-PHpaII-PSPO-I to build even.Thus illustrating, the effect of Gene expression is not directly proportional to the quantity of its promoter, it is critical only that the selection of promoter, and in Gene expression, the change of any one single promoter is all likely to result in significantly alterring of its whole structure, and its impact is unpredictable.
To sum up, Gene expression P43-PHpaII-PSPO-I and the P43-PHpaII-PSPO-I-PcryIIIA of the present invention, the protein expression of bacillus cereus is had obvious facilitation by both Gene expressions.
Claims (10)
1. a Gene expression, it is characterised in that described Gene expression includes Promoter P43, PHpaII and PSPO-I.
2. Gene expression as claimed in claim 1, it is characterised in that the nucleotides sequence of described Promoter P43 is classified as SEQIDNO:2.
3. Gene expression as claimed in claim 1, it is characterised in that the nucleotides sequence of described promoter PHpaII is classified as SEQIDNO:3.
4. Gene expression as claimed in claim 1, it is characterised in that the nucleotides sequence of described promoter PSPO-I is classified as SEQIDNO:4.
5. the Gene expression described in claim 1, it is characterised in that described Gene expression also comprises promoter PcryIIIA；The nucleotides sequence of described promoter PcryIIIA is classified as SEQIDNO:5.
6. Gene expression as claimed in claim 5, it is characterised in that described Gene expression is connected in sequence by Promoter P43, PHpaII, PSPO-I and PcryIIIA, and its nucleotides sequence is classified as SEQIDNO:8.
7. the Gene expression described in claim 1 or 5 is used for the application in the plasmid of transforming bacillus in preparation.
8. a recombiant plasmid, it is characterised in that the promoter of described recombiant plasmid is the Gene expression described in claim 1 or 5.
9. a recombinant bacterial strain, it is characterised in that described recombinant bacterial strain is the Host Strains converting/transfect the recombiant plasmid having the right described in requirement 8.
10. recombinant bacterial strain as claimed in claim 9, it is characterised in that described Host Strains is bacillus subtilis (Bacillussubtilis).
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| CN109486847A (en) * | 2018-12-17 | 2019-03-19 | 江南大学 | The efficient inducible expression of bacillus subtilis based on artificial Gene expression |
| CN109652417A (en) * | 2018-12-17 | 2019-04-19 | 江南大学 | A method of constructing efficient bacillus subtilis promoter |
| CN111926013A (en) * | 2020-08-18 | 2020-11-13 | 湖北大学 | Promoter suitable for bacillus licheniformis and application thereof in high-efficiency expression of target product |
| WO2021020996A1 (en) | 2019-07-26 | 2021-02-04 | Общество с ограниченной ответственностью "ЦЕНТР ТРАНСФЕРА БИОТЕХНОЛОГИЙ ОКА-Биотех" | Tandem promoter, bacterium of the genus bacillus producing a target product containing said promoter, and method of producing the target product |
| CN114277046A (en) * | 2021-12-14 | 2022-04-05 | 河北师范大学 | Tri-gene tandem expression vector for synthesizing tetrahydropyrimidine and application thereof |
| CN114958897A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Bacillus subtilis construction method capable of efficiently expressing low-temperature keratinase for feed |
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| CN107988245A (en) * | 2017-10-25 | 2018-05-04 | 南京福斯弗瑞生物科技有限公司 | The Pullulanase and its expression vector and construction method of a kind of codon optimization |
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| CN109652417A (en) * | 2018-12-17 | 2019-04-19 | 江南大学 | A method of constructing efficient bacillus subtilis promoter |
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| WO2020124831A1 (en) * | 2018-12-17 | 2020-06-25 | 江南大学 | Method for constructing high-efficiency bacillus subtilis promoters |
| US12123007B2 (en) | 2018-12-17 | 2024-10-22 | Jiangnan University | Method for constructing efficient Bacillus subtilis promoter |
| CN109652417B (en) * | 2018-12-17 | 2021-07-27 | 江南大学 | Method for constructing efficient bacillus subtilis promoter |
| WO2021020996A1 (en) | 2019-07-26 | 2021-02-04 | Общество с ограниченной ответственностью "ЦЕНТР ТРАНСФЕРА БИОТЕХНОЛОГИЙ ОКА-Биотех" | Tandem promoter, bacterium of the genus bacillus producing a target product containing said promoter, and method of producing the target product |
| CN111926013A (en) * | 2020-08-18 | 2020-11-13 | 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 |
| CN114277046A (en) * | 2021-12-14 | 2022-04-05 | 河北师范大学 | Tri-gene tandem expression vector for synthesizing tetrahydropyrimidine and application thereof |
| CN114958897A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Bacillus subtilis construction method capable of efficiently expressing low-temperature keratinase for feed |
| 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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