CN104232675A - Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof - Google Patents
Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof. The bacillus subtilis shuttle expression vector provided by the invention comprises the following elements: a bacillus subtilis formed strong promoter P43, a sequence which encodes signal peptide, multiple cloning sites, an antibiotics resistance gene (kalamycin or chloramphenicol), a bifunctional promoter sequence which can start resistance gene expression of kalamycin or chloramphenicol in escherichia coli and bacillus subtilis, and copy homing sequences of bacillus subtilis and escherichia coli. The expression vector disclosed by the invention not only can be stably copied in escherichia coli, but also can be stably copied in bacillus subtilis, and expresses homologous and heterologous proteins. All vectors just contain one resistance gene, the plasmid of which is less than 400bp, so that gene operation and plasmid conversion are easy to carry out, and the conversion ratio is high. Besides the expression vector of an exogenous gene, the vector can be further used for screening promoters and constructing a gene knock-out vector.
Description
Technical field
The invention belongs to microorganism and bioengineering field.Be specifically related to a kind of Shuttling expression vector of bacillus coli-bacillus subtilis and application thereof.
Background technology
In the expression study and industrial fermentation production of industrial enzyme, with the recombinant expressed enzyme of Microbial Expression Systems, activated protein and single-chain antibody, be with a wide range of applications.Escherichia expression system and yeast expression system apply recombinant expression system very widely at present, the commercialization company of lot of domestic and international and scientific research institution develop expression vector and the expressive host bacterium of multiple series, and deeply systematized transformation is carried out to escherichia expression system and yeast expression system, these two kinds of expression systems are made great progress in the expression of recombinant protein.But still there is many problems in these two kinds of expression systems, as E. coli secretion ability, exogenous protein expression is easy to form inclusion body, the renaturation process of inclusion body easily makes zymoprotein inactivation, intestinal bacteria are that Gram-negative bacteria exists intracellular toxin, be not suitable for Expression and Application in the enzyme of foodstuffs industry and activated protein, endotoxic removal adds the cost of protein purification.Although it is strong that yeast expression system has secretion capacity, the advantage of expression of recombinant proteins amount height and posttranslational modification, yeast growth is comparatively slow, and yeast expression system expression efficiency is general lower, and fermentation period is long, and recombinant protein productivity is low.
Subtilis expression system mainly contains following advantage [Biochim Biophys Acta, 2004,1694 (1-3): 299-310]: (1) is GRAS (the Generally Recognized as Safe) bacterium that U.S. FDA is assert, the composition of cell walls is simple, only containing peptidoglycan and phosphorus ancient piece of jade, round, flat and with a hole in its centre matter, therefore can not mix in the proteinaceous product of secretion and have intracellular toxin (pyrogenicity lipopolysaccharides), there is biological safety; (2) plasmid and phage can as the carriers of clone; (3) it has a lot of secreting signal peptide, the secretion that can be recombinant protein provides sufficient selectable secreting signal peptide, some extracellular protein can be secreted in a large number, after albumen crosses over cytolemma, processed and be directly released in substratum and do not assemble, reclaim and purifying protein comparatively simple; (4) subtilis has good fermentation basis and production technology, can grow into very high density, utilize fermentation of bacillus manufacture enzyme to have the history of decades in relatively simple substratum; (5) genus bacillus does not have obvious codon-bias, and expression product is not easy to form inclusion body.In E.coli expression system, if containing a large amount of continuous print rare codon in recombinant expressed exogenous protein, then usually cause expression amount low, or translation premature termination, target protein product forms inclusion body and causes protein soluble, causes trouble to separation and purification and recovery target protein.(6) had at present many subtilis full-length genome gene orders to announce, this be that the genetic modification of subtilis provides convenience.
But subtilis expression system remains in some problems at present:
(1) there is restriction and modification system in subtilis expression system, and recombinant plasmid is unstable, easily loses in copying.
(2) sequence optimisation of promotor, the optimization of ribosome bind site (ribosome-binding site, RBS) sequence, the distance between RBS sequence and ATG are treated in further improvement.
(3) signal peptide is for effective secretion of recombinant protein, plays very important effect, needs find more effective signal peptide and find that new promotor and signal peptide combine, and makes secreting function stronger, reduces the formation of inclusion body.
(4) subtilis expression system transformation efficiency is low, is generally less than 1 × 103CFU/ μ g DNA, far below colibacillary transformation efficiency (generally at 1 × 106-1 × 109CFU/ μ g DNA).Although constructed the plasmid vector that some can express foreign protein in subtilis at present, but it is alternative and the carrier of high-efficient expression and host cell species can remain considerably less at present, the property transformed difference, its alternative is far below conventional coli expression carrier and Yeast expression carrier.
The expression plasmid that current subtilis is conventional is pUB110, pWB980, pHT01 and pHT43 etc.Wherein pUB110 and pWB980 is non-shuttle plasmid, can only copy in subtilis, extract plasmid difficulty, enzyme cut after carrier segments be connected with foreign gene after, because plasmid concentration is low, conversion can be caused very difficult; The Shuttling expression vector of bacillus coli-bacillus subtilis that current business-like carrier and scientific research personnel build voluntarily generally all more than 5kb, if pHT01 and pHT43 is at about 8000bp.For the ease of of screening positive clone in intestinal bacteria and subtilis, these Shuttling expression vector of bacillus coli-bacillus subtilis (use ammonia benzyl resistance containing two kinds of resistance gene fragment usually in intestinal bacteria, with kalamycin resistance or chlorampenicol resistant in withered grass) and two kinds of replicon fragments, therefore plasmid is caused larger, copy number is low, extract and genetic transformation all more difficult.Generally plasmid size and transformation frequency are inversely proportional to, and the plasmid that molecular weight is little is easy to transform, and transformation efficiency is high; And plasmid transformation efficiency low [biotechnology circular, 2011 (5): 227-230 comparatively speaking that molecular weight is large; Appl Microbiol Biotechnol, 2008,78 (1): 181-188.], the operational restriction enzyme site of multiple clone site MCS is fewer, and the foreign gene size that can insert also can be limited.Therefore object of the present invention is exactly to solve the excessive problem of current Shuttling expression vector of bacillus coli-bacillus subtilis, structure molecular weight, size at the Shuttling expression vector of bacillus coli-bacillus subtilis of below 4kb, to improving the efficiency of genetic transformation efficiency and exogenous gene expression.These shuttle expression carriers are also applicable to and build stable integrating expression vector and gene knockout carrier in addition.
Summary of the invention
Technical problem to be solved by this invention is to provide the Shuttling expression vector of bacillus coli-bacillus subtilis of the single resistance of a kind of molecular weight little (being less than 4kb).
The invention provides a kind of expression vector, comprise following element: the composing type strong promoter of subtilis, the encoding sequence of signal peptide, multiple clone site and replication initiation sequence, colibacillary replication initiation sequence, the terminator sequence of antibiotics resistance gene, it is characterized in that: be also included in intestinal bacteria and subtilis difunctional promotor, a kind of antibiotics resistance gene that all can start antibiotics resistance gene and express, the size of carrier is less than 4kb.
The composing type strong promoter of described subtilis, the signal peptide of subtilis, multiple clone site is all cloned from pWB980 carrier together with the replication initiation sequence of subtilis, its strong promoter is P43, signal peptide is SacB, colibacillary replication initiation sequence is from pUC57 carrier, namely the difunctional promotor that all can start antibiotics resistance gene expression in intestinal bacteria and subtilis can start kalamycin tolerant gene expression, also can start chloramphenicol resistance gene to express, antibiotics resistance gene is the kalamycin resistance gene (kan gene) in pET-28a (+) carrier or the chloramphenicol resistance gene (Cat gene) in Bacillus licheniformis ATCC14580 genome, Cat gene has following 1) or 2) any one in DNA sequence dna:
1) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.10;
2) with 1) DNA sequence dna that limits has more than 90% homology.
Further, described in intestinal bacteria and subtilis, all can start the genome that difunctional promotor that antibiotics resistance gene expresses derives from subtilis (Bacillus subtilis) BS168 or Bacillus licheniformis (Bacillus licheniformis) ATCC14580, have following 1)-21) any one in DNA sequence dna:
1) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.1;
2) under strict conditions with 1) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
3) with 1) or 2) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
4) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.2;
5) under strict conditions with 4) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
6) with 4) or 5) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
7) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.3;
8) under strict conditions with 7) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
9) with 7) or 8) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
10) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.4;
11) under strict conditions with 10) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
12) with 10) or 11) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
13) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.5;
14) under strict conditions with 13) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
15) with 13) or 14) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
16) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.6;
17) under strict conditions with 16) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
18) with 16) or 17) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
19) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.7;
20) under strict conditions with 19) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
21) with 19) or 20) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
Further, described expression vector has following 22)-27) DNA sequence dna shown in any one:
22) DNA sequence dna is nucleotide sequence shown in SEQ ID NO:8;
23) with 22) DNA sequence dna that limits has more than 90% homology;
24) with 22) sequence in the DNA sequence dna that limits outside antibiotics resistance gene promoter sequence (6-415bp) has more than 90% homology;
25) DNA sequence dna is nucleotide sequence shown in SEQ ID NO:9;
26) with 25) DNA sequence dna that limits has more than 90% homology;
27) with 25) sequence in the DNA sequence dna that limits outside antibiotic resistance gene promoter sequence (6-415bp) has more than 90% homology;
Another technical problem to be solved by this invention is to provide the application of the carrier of described expression.
The above expression vector is building the application in transgenic cell line.
The above expression vector is building the application in engineering strain.
The application of the above expression vector in promoting goal gene to transcribe and translate.
Carrier provided by the invention can copy in intestinal bacteria, can copy in subtilis and express again, and only have a kind of resistant gene in carrier, same antibiotic-screening can be used when expressing in clone and subtilis in intestinal bacteria, the size of plasmid is than bacillus coli-bacillus subtilis shuttle plasmid little (being less than 4kb) common at present, be easy to genetic manipulation and Plastid transformation, enrich the carrier that can be used for subtilis expression system; Serial E.coli-B.subtilis shuttle vector provided by the invention, except for except exogenous gene expression carrier, also can be used for the carrier of promotor screening and builds gene knockout carrier; This vector expression reporter gene is bacillus subtilis alkali proteinase gene, its extracellular expression amount is higher, the enzyme live birth rate of shake flask fermentation is 1600-1850U/mL, higher than the expression level (enzyme activity is respectively 140U/mL and 195U/mL) using other commercialization carrier pHT01 and pHT43.
Accompanying drawing explanation
Fig. 1 Kan gene fragment and the pUC57 carrier electrophorogram of EcoR V and Pci I double digestion.
In figure, symbolic significance is as follows: M, DNA molecular amount Marker; 1, the pUC57 plasmid of double digestion; 2, the Kan gene fragment of double digestion.
Fig. 2 increases the electrophorogram of 7 kinds of promoter fragments.
In figure, symbolic significance is as follows: M, DNA molecular amount Marker; 1-7 is promotor pEB1, pEB2, pEB3, pEB4, pEB5, pEB6 and pEB7 gene fragment respectively.
The intestinal bacteria of the single resistance of Fig. 3 kalamycin and bacillus subtilis shuttle expression vector physical map.
In figure, symbolic significance is as follows: pEB1-PEB7, seven kinds of difunctional promoter sequences coming from genus bacillus; Kan, kalamycin resistant gene; Ori, the replication sequence in intestinal bacteria; RepB, the replicon sequence in withered grass gemma; P43, the constitutive promoter sequence in subtilis, SacB SP is the signal coding sequence of withered grass gemma levansucrase.
Fig. 4 Cat gene fragment and the pWEBK15 carrier electrophorogram of EcoR V and Pci I double digestion.
In figure, symbolic significance is as follows: M, DNA molecular amount Marker; 1, the Cat gene fragment of double digestion; 2, the pWEBK15 plasmid of double digestion.
The intestinal bacteria of the single resistance of Fig. 5 paraxin and bacillus subtilis shuttle expression vector physical map.
In figure, symbolic significance is as follows: pEB1-PEB7, seven kinds of difunctional promoter sequences coming from genus bacillus; Cat, chloramphenicol resistance gene; Ori, the replication sequence in intestinal bacteria; RepB, the replicon sequence in withered grass gemma; P43, the constitutive promoter sequence in subtilis, SacB SP is the signal coding sequence of withered grass gemma levansucrase
The intestinal bacteria of the single resistance of Fig. 6 and bacillus subtilis shuttle expression vector pWEBK15 and pWEBC27 are for expressing Sumizyme MP.
In figure, symbolic significance is as follows: swimming lane M, molecular weight of albumen Marker; Swimming lane 1, empty map WB800N (pWEBK15); Swimming lane 3, the fermented liquid supernatant of empty map WB800N (pWEBC27); The fermented liquid supernatant of swimming lane 2,4-6:WB800N (pWEBK15-aprE); 7-10, WB800N (pWEBC27-aprE) fermented liquid supernatant.Arrow instruction target protein position.
Embodiment
material
Strains B. subtilis (Bacillus subtilis) BS168 and WB800N and plasmid pHT01 and pHT43 used in the present invention all purchased from MoBiTec company, Bacillus licheniformis (Bacillus licheniformis) ATCC14580 purchased from American Type Culture Collection.
PHY300PLK plasmid is purchased from Takara company.Other plasmid pET-28a (+), pUC57 and pWB980 is all purchased from Bao Sai bio tech ltd, Hangzhou.
Competent escherichia coli cell DH5 α is purchased from Hubei Jing Mao Bioisystech Co., Ltd.
Other conventional biochemical reagents are all commercially available analytical pure.The method of PCR primer recovery and glue recovery DNA all adopts the method for the test kit of omega company.Gene fragment amplification all uses Thermo Scientific Phusion high-fidelity DNA polymerase kit method, and the connection of gene fragment all adopts the method for Thermo Scientific T4DNA ligase enzyme test kit.
Table 1 amplimer and sequencing primer
| Title | Sequence (5 '-3 ') | Restriction enzyme site |
| P1 | CGA GATATCATGAGCCATATTCAACGGGA | EcoR?V |
| P2 | CCC ACATGTCAGGTGGCACTTTTCGGGGA | Pci?I |
| P3 | GAGAAAGGCGGACAGGTATC | ? |
| P4 | CGC GGATCCATGAGCCATATTCAACGGGA | BamH?I |
| P5 | TAG GTTAACCAGGTGGCACTTTTCGGGGA | Hpa?I |
| P6 | GAGCGTCAGATTTCGTGATG | ? |
| P7 | AACTACGTCTGCCCTCATTA | ? |
| P8 | CCG GAATTCACACAGGGATAAAATCGGCG | EcoR?I |
| P9 | CGA GATATCTATGCGCTGCATCTCCTCAC | EcoR?V |
| P10 | CCG GAATTCGTAAGGTTCCAAGGCTACAC | EcoR?I |
| P11 | CGA GATATCAAAGCCGCAGCAGATTAAAT | EcoR?V |
| P12 | CCG GAATTCAGACAGCACAGCCTCCAG | EcoR?I |
| P13 | CGA GATATCTCAACCACTCCCCACGTT | EcoR?V |
| P14 | CCG GAATTCATGCTGTCTATGGTCTGC | EcoR?I |
| P15 | CGA GATATCTGTAAAACCCTTCTTCCT | EcoR?V |
| P16 | CCG GAATTCCGCGTCCAGTTAAGAGCA | EcoR?I |
| P17 | CGA GATATCGAAATGATCCTCCACAAA | EcoR?V |
| P18 | CCG GAATTCTGACTGTCTTTCTTTTTCCCG | EcoR?I |
| P19 | CGA GATATCGATATGATAGAAGGAGCGGA | EcoR?V |
| P20 | CCG GAATTCAGTGAAGAAGCAGAGAGGCT | EcoR?I |
| P21 | CGA GATATCGATTCTCCTCCCCTTTCAAT | EcoR?V |
| P22 | ACCTGACGTCTAAGAAACCA | ? |
| P23 | GTGAGTTTTCGTTCCACTGA | ? |
| P24 | CAGAGTTCTTGAAGTGGTGG | ? |
| P25 | CCG GAATTCGAGCTCAGCATTAT | EcoR?I |
| P26 | CTG GACGTCAGCATCTAATCTTCAACAAAC | Aat?II |
| P27 | CGA GATATCATGAATTTTCAAACAATCGAGC | EcoR?V |
| P28 | CCC ACATGTACAGAAAGTTTGTTGAGAGC | Pci?I |
| P29 | CCC AAGCTTTTGCCGCCGGAAAAAGCAGTACAGA | Hind?III |
| P30 | CGC GGATCCTTATTGTGCAGCTGCTTGTA | BamH?I |
| P31 | CGC GGATCCGTGAGAAGCAAAAAATTGTGG | BamH?I |
| P32 | CGC GGATCCGCCGGAAAAAGCAGTACAGA | BamH?I |
| P33 | TCC CCCGGGTTATTGTGCAGCTGCTTGTA | Sma?I |
[embodiment 1] clone of kalamycin resistance gene and the structure of promoter probe vector
Extract pET-28a (+) vector plasmid and pUC57 plasmid.With pET-28a (+) plasmid DNA for masterplate, with the P1/P2 (table 1) synthesized for the Kan gene fragment on primer amplification pET-28a (+) carrier, this Kan gene fragment is not containing promotor.EcoR V and Pci I double digestion Kan gene fragment 1 (Fig. 1), PCR primer reclaims product; EcoR V and Pci I double digestion pUC57 plasmid reclaim 2200bp large fragment (Fig. 1); be connected with Kan gene endonuclease bamhi; transformation of E. coli DH5 α; by Inoue legal system for the super competence of DH5 α; method is with reference to " Molecular Cloning: A Laboratory guide (the 3rd edition) "; be applied on the LB solid medium flat board containing 100 μ g/mL penbritins and screen; by PCR checking and sequence verification (pUC57 carrier universal sequencing primer thing M13F and P3), the successful carrier called after pUCKan of final order-checking.
Same working method, with pET-28a (+) plasmid DNA for masterplate, with the P4/P5 (table 1) synthesized for the Kan gene fragment 2 on primer amplification pET-28a (+) carrier, this Kan gene fragment is not containing promotor.BamH I and Hpa I double digestion Kan gene fragment 2, PCR primer reclaims product; BamH I and Hpa I double digestion pHY300PLK plasmid reclaim 3600bp large fragment; be connected with Kan gene endonuclease bamhi 2; transformation of E. coli DH5 α; be applied on the LB solid medium flat board containing 100 μ g/mL penbritins and screen; by PCR checking and sequence verification (sequencing primer P6/P7), the successful carrier called after pHYKan of final order-checking.The intermediate carrier that two carrier pUCKan and pHYKan more than built can build as downstream, also can be used as the sonde-type carrier in the various bacillus gene group of screening with the difunctional strong promoter all worked in intestinal bacteria and genus bacillus.
The clone of [embodiment 2] promoter active fragment
CTAB method (cetyl trimethylammonium bromide) is adopted to extract template [the microorganism journal of genomic dna as gene amplification of subtilis (Bacillus subtilis) BS168 and Bacillus licheniformis (Bacillus licheniformis) ATCC14580,2006,46 (1): 7-12.]; By partially digested for the genomic dna Sau3A I of subtilis BS168 and Bacillus licheniformis ATCC14580, endonuclease bamhi is concentrated between 0.5-5kb.With pHYKan carrier for promoter probe vector, from the genome of subtilis, screen promotor [microorganism journal, 2004,44 (4): 457-460; University of Fuzhou's journal (natural science edition), 2013,41 (3): 391-396.].Concrete grammar is: with BamH I complete degestion pHYKan carrier, dephosphorylation process, be connected with suitable proportion with the partially digested fragment of said gene group DNA again, transformation of E. coli DH5 α, be applied to screening positive clone on the LB solid medium flat board containing 50 μ g/mL kantlex, obtain the recon (wherein 41 fragments from BS168,16 fragments from ATCC14580) that 57 have kalamycin resistance altogether.All intestinal bacteria positive colony are inoculated in the LB substratum of 50 μ g/mL kantlex and cultivate 10h, extract Plastid transformation subtilis BS168 bacterial strain, be applied to screening positive clone on the LB solid medium flat board containing 50 μ g/mL kantlex, screen 7 strain positive colony (wherein 5 fragments from BS168,2 fragments from ATCC14580) altogether.Finally bacterium colony PCR checking is carried out to 7 bacillus subtilis positive colony and sample presentation order-checking after extracting plasmid, the genome database of sequencing result and subtilis BS168 and Bacillus licheniformis ATCC14580 is carried out Blast comparison and promoter function analysis, finally determine to measure the initial sum termination area that in sequence, promotor is possible, as the sequence that SEQ ID NO.1-7 limits.
Extract pUCKan plasmid, after EcoR I and EcoR V double digestion pUCKan plasmid, reclaim large fragment.
Respectively for SEQ ID NO.1-5 limit primers to P8/P9, P10/P11, P12/P13, P14/P15 and P16/P17 (table 1), with subtilis BS168 genomic dna for masterplate, amplification pEB1, pEB2, pEB3, pEB4 and pEB5 promoter fragment (Fig. 2), with the genomic dna of Bacillus licheniformis ATCC14580 for masterplate, respectively for SEQ ID NO.6,7 limit primers to P18/P19 and P20/P21 (table 1), amplification pEB6 and pEB7 promoter fragment (Fig. 2), EcoR I and EcoR V double digestion pEB1, pEB2, pEB3, pEB4, pEB5, after pEB6 and pEB7 promoter fragment, DNA fragmentation after cutting with PCR primer recovery test kit recovery enzyme, be connected with the pUCKan plasmid fragments after double digestion more respectively, transformation of E. coli DH5 α, be applied on the LB solid medium flat board containing 50 μ g/mL kalamycins and 100 μ g/mL penbritins and screen, by PCR checking and sequence verification (pUC57 carrier universal sequencing primer thing M13F), final order-checking successful carrier called after pUCKanEB1, pUCKanEB2, pUCKanEB3, pUCKanEB4, pUCKanEB5, pUCKanEB6 and pUCKanEB7.
[embodiment 3] full plasmid PCR removes penbritin Amp resistance gene fragment in carrier
The T4Polynucleotide Kinase of Takara company is adopted to carry out phosphatizing treatment to primer P22/P23 (table 1).20 μ L reaction systems are: P22/P23 primer mixture (each primer final concentration 12.5 μMs) 4 μ L; ATP (10mM); 10 × Reaction buffer 2 μ L; 10 × T4Polynucleotide Kinase (10U/ μ L) 2 μ L; DH
2o 9.6 μ L.
Extract pUCKanEB1, pUCKanEB2, pUCKanEB3, pUCKanEB4, pUCKanEB5, pUCKanEB6 and pUCKanEB7 plasmid DNA, respectively with it for masterplate, increase with the primer P22/P23 of phosphatizing treatment, amplified production reclaims after kits through glue, adopt the condition of blunt end cloning, 2h is connected at 22 DEG C, transformation of E. coli DH5 α, be applied on the LB solid medium flat board containing 50 μ g/mL kalamycins and screen, by PCR checking and sequence verification (sequencing primer P24, table 1), final order-checking successful carrier called after pKanEB8, pKanEB9, pKanEB10, pKanEB11, pKanEB12, pKanEB13 and pKanEB14.
The intestinal bacteria of the single resistance of [embodiment 4] kalamycin and bacillus subtilis shuttle expression vector build
With pWB980 plasmid DNA for masterplate, with the P25/P26 of synthesis for primer (table 1) amplification vector including the gene fragment psr of the replicon repB in P43 promotor, SacB signal peptide, multiple clone site and genus bacillus, primer two ends are with the restriction enzyme site of EcoR I and Aat II, and primer is in table 1.Psr gene fragment connects carrier T after adding A, the accuracy of its sequence of universal primer sequence verification.EcoR I and Aat II double digestion psr fragment, PCR primer reclaim test kit reclaim enzyme cut after DNA fragmentation (about 2200bp).
Extract pKanEB8, pKanEB9, pKanEB10, pKanEB11, pKanEB12, pKanEB13 and pKanEB14 plasmid DNA, use EcoR I and Aat II double digestion respectively, glue reclaims kits carrier DNA large fragment.
EcoR I is connected with above-mentioned carrier segments with the psr fragment of Aat II double digestion, transformation of E. coli DH5 α, be applied on the LB solid medium flat board containing 50 μ g/mL kalamycins and screen, positive colony extracts and to carry out EcoR I and the checking of Aat II double digestion and sequence verification (sequencing primer P23, table 1) after plasmid, final order-checking successful carrier called after pWEBK15, pWEBK16, pWEBK17, pWEBK18, pWEBK19, pWEBK20 and pWEBK21 (Fig. 3).
Extract the plasmid that above order-checking is correct, adopt method [the J Microbiol Meth.1999 that electricity transforms, 34 (3): 183-191.] its electricity is transformed in subtilis BS168 bacterial strain, be applied on the LB solid medium flat board containing 25 μ g/mL kalamycins and screen, detect its resistance and transformation efficiency.Select business-like pHT01 and pHT43 carrier as control vector, extract plasmid, plasmid concentration is measured with nucleic acid quantification instrument, its electricity is transformed in subtilis BS168 bacterial strain, be applied on the LB solid medium flat board containing 10 μ g/mL paraxin and screen, each plasmid is coated with three flat boards.The clone that flat board grows counted and calculates mean number, calculating transformation efficiency.The amount (μ g) of Plastid transformation rate (CFU/ μ g DNA)=positive colony quantum count (CFU)/plasmid; The transformation efficiency of pWEBK15, pWEBK16, pWEBK17, pWEBK18, pWEBK19, pWEBK20 and pWEBK21 is 5.8 × 10
3-8.3 × 10
3cFU/ μ g DNA, the transformation efficiency of control plasmid is only 0.7 × 10
3-0.9 × 10
3cFU/ μ g DNA (see table 2).
The intestinal bacteria of the single resistance of table 2. kalamycin and bacillus subtilis shuttle expression vector and control plasmid transformation efficiency compare
| Plasmid | The amount (ng) of plasmid | Clone quantity (CFU) | Transformation efficiency (CFU/ μ g DNA) |
| pHT01 | 100 | 90 | 0.9×10 3 |
| pHT43 | 100 | 70 | 0.7×10 3 |
| pWEBK15 | 50 | 298 | 5.9×10 3 |
| pWEBK16 | 50 | 290 | 5.8×10 3 |
| pWEBK17 | 50 | 329 | 6.6×10 3 |
| pWEBK18 | 50 | 365 | 7.3×10 3 |
| pWEBK19 | 50 | 389 | 7.8×10 3 |
| pWEBK20 | 50 | 312 | 6.2×10 3 |
| pWEBK21 | 50 | 415 | 8.3×10 3 |
The intestinal bacteria of the single resistance of [embodiment 5] paraxin and bacillus subtilis shuttle expression vector build
Extract pWEBK15, pWEBK16, pWEBK17, pWEBK18, pWEBK19, pWEBK20 and pWEBK21 plasmid, EcoR V and Pci I double digestion, DNA glue reclaims the large fragment (Fig. 4) that test kit reclaims about 1500bp.
With the genomic dna of Bacillus licheniformis ATCC14580 for masterplate, with the P27/P28 synthesized for primer (table 1) amplification chloramphenicol resistance gene Cat, EcoR V and Pci I double digestion, PCR primer Purification Kit endonuclease bamhi.
The carrier segments of EcoR V and Pci I double digestion and Cat gene fragment (Fig. 4), connect, transformation of E. coli DH5 α, be applied on the LB solid medium flat board containing 10 μ g/mL paraxin and screen, carry out bacterium liquid PCR after positive colony extracts plasmid to verify and sequence verification (pUC57 carrier universal sequencing primer thing M13F and P3), final order-checking successful carrier called after pWEBC22, pWEBC23, pWEBC24, pWEBC25, pWEBC26, pWEBC27 and pWEBC28 (Fig. 5).
Extract the plasmid that above order-checking is correct, adopt method [the J Microbiol Meth.1999 that electricity transforms, 34 (3): 183-191.] its electricity is transformed in subtilis BS168 bacterial strain, is applied on the LB solid medium flat board containing 10 μ g/mL paraxin and screens.Select business-like pHT01 and pHT43 carrier carrier in contrast, extract plasmid, measure plasmid concentration with nucleic acid quantification instrument, its electricity is transformed in subtilis BS168 bacterial strain, is applied to and screens containing on the LB solid medium flat board be applied to containing 10 μ g/mL paraxin.The clone that flat board grows is counted, calculates transformation efficiency.The amount (μ g) of Plastid transformation rate (CFU/ μ gDNA)=positive colony quantum count (CFU)/plasmid; The transformation efficiency of pWEBC22, pWEBC23, pWEBC24, pWEBC25, pWEBC26, pWEBC27 and pWEBC28 is 6.5 × 10
3-9.7 × 10
3cFU/ μ g DNA, the transformation efficiency of control plasmid is only 0.7 × 10
3-0.9 × 10
3cFU/ μ g DNA (see table 3).
The intestinal bacteria of the single resistance of table 3. paraxin and bacillus subtilis shuttle expression vector and control plasmid transformation efficiency compare
| Plasmid | The amount (ng) of plasmid | Clone quantity (CFU) | Transformation efficiency (CFU/ μ g DNA) |
| pHT01 | 100 | 90 | 0.9×10 3 |
| pHT43 | 100 | 70 | 0.7×10 3 |
| pWEBC22 | 50 | 325 | 6.5×10 3 |
| pWEBC23 | 50 | 335 | 6.7×10 3 |
| pWEBC24 | 50 | 489 | 9.7×10 3 |
| pWEBC25 | 50 | 367 | 7.3×10 3 |
| pWEBC26 | 50 | 352 | 7.0×10 3 |
| pWEBC27 | 50 | 384 | 7.8×10 3 |
| pWEBC28 | 50 | 407 | 8.1×10 3 |
The intestinal bacteria of [embodiment 6] single resistance and bacillus subtilis shuttle expression vector are for expressing Sumizyme MP
With subtilis BS168 genomic dna for masterplate, be that the encoding gene aprE of primer (see table 1) amplification bacillus subtilis alkali proteinase is as test cdna with P29/P30, Hind III and BamH I double digestion, PCR primer reclaim kits enzyme cut after gene fragment.
Extract pWEBK15 and the pWEBC27 plasmid expression vector as test, Hind III and BamH I double digestion, PCR primer reclaim kits enzyme cut after gene fragment; Carrier segments after double digestion is connected with aprE gene fragment, transformation of E. coli DH5 α, be applied to respectively on the LB solid medium flat board containing 50 μ g/mL kalamycins or 20 μ g/mL paraxin and screen, bacterium liquid PCR verifies and sequence verification, clone called after pWEBK15-aprE and pWEBC27-aprE checking order correct.
In contrast, after using primer P31/P33 and P32/P33 (see table 1) amplification aprE, BamH I and Sma I double digestion respectively, pHT01 and pHT43 carrier is connected into respectively, clone called after pHT01-aprE and pHT43-aprE that final order-checking is correct.
Extract pWEBK15-aprE, pWEBC27-aprE, pWEBK15 and pWEBC27 plasmid, electricity is transformed in subtilis WB800N bacterial strain (bacterial strains of 8 Deficient In Extracellular Proteases), is applied to screening positive clone on the LB solid medium flat board containing 50 μ g/mL kalamycins and 20 μ g/mL paraxin respectively.The sub-called after WB800N (pWEBK15-aprE) of positive colony, WB800N (pWEBC27-aprE), WB800N (pWEBK15) and WB800N (pWEBC27).Wherein WB800N (pWEBK15) and WB800N (pWEBC27) is as empty map bacterial strain.
Extract pHT01-aprE and pHT43-aprE, electricity is transformed into subtilis WB800N bacterial strain, be applied to screening positive clone on the LB solid medium flat board containing 10 μ g/mL paraxin, the sub-called after WB800N (pHT01-aprE) of positive colony and WB800N (pHT43-aprE).
Picking mono-clonal WB800N (pWEBK15-aprE) respectively, WB800N (pWEBC27-aprE), activation culture 10h in WB800N (pWEBK15) and WB800N (pWEBC27) to the LB substratum containing 50 μ g/mL kalamycins and 20 μ g/mL chlorampenicol resistants, (albumen 1.5% is inoculated in fresh Superrich substratum again with the inoculum size of 4%, yeast extract 2.5%, K
2hPO
40.3%, glucose 2%, CaCl
20.1%, pH 7.4), cultivate 24h.Collect fermented liquid supernatant and carry out proteinase activity detection and SDS-PAGE analysis (Fig. 6).The inducing culture of WB800N (pHT01-aprE) and WB800N (pHT43-aprE) expresses handbook with reference to the subtilis that MoBiTec company's site provides, inoculate single bacterium colony to fresh 2xYT medium substratum (16g/L peptone, 10g/L acid hydrolysis casein, 5g/L NaCl, 10 μ g/mL paraxin) middle incubated overnight; Be inoculated into according to 1%-4% and cause OD600 in the fresh 2xYT substratum of 60-80mL and be about 0.15; When OD600 reaches 0.7-0.8, being divided equally by substratum is two bottles, and one bottle adds the IPTG abduction delivering of 1mM wherein, and other one bottle is continued to cultivate in contrast; Every 4h sampling, induction 12-16h.The average enzyme activity of the sub-WB800N of positive colony (pWEBK15-aprE) and WB800N (pWEBC27-aprE) shake flask fermentation is respectively 1600U/mL and 1850U/mL, and use business-like pHT01 and pHT43 vector expression amount lower, most high enzymatic activity is respectively 140U/mL and 195U/mL, all enzyme activity determination data are the data of non-optimization of fermentation conditions, measuring method reference literature [the Food science of basic protein enzyme activity, 2009,30 (23): 347-352].It is about 30kDa that aprE gene SDS-PAGE analyzes the molecular weight of Sumizyme MP that display test expresses, with report in document consistent.
Claims (10)
1. the Shuttling expression vector of bacillus coli-bacillus subtilis of a single resistance, comprise following element: the composing type strong promoter of subtilis, the encoding sequence of signal peptide, multiple clone site and replication initiation sequence, colibacillary replication initiation sequence, the terminator sequence of antibiotics resistance gene, it is characterized in that: be also included in intestinal bacteria and subtilis difunctional promotor, a kind of antibiotics resistance gene that all can start antibiotics resistance gene and express, the size of carrier is less than 4kb.
2. the Shuttling expression vector of bacillus coli-bacillus subtilis of single resistance as claimed in claim 1, it is characterized in that: the composing type strong promoter of described subtilis, signal peptide, multiple clone site and replication initiation sequence are cloned from pWB980 carrier together, its strong promoter is P43, signal peptide is SacB, colibacillary replication initiation sequence is from pUC57 carrier, namely the difunctional promotor that all can start antibiotics resistance gene expression in intestinal bacteria and subtilis can start kalamycin tolerant gene expression, also can start chloramphenicol resistance gene to express, antibiotics resistance gene is the kalamycin resistance gene (kan gene) in pET-28a (+) carrier or the chloramphenicol resistance gene (Cat gene) in Bacillus licheniformis ATCC14580 genome, Cat gene has following 1) or 2) any one in DNA sequence dna:
1) nucleotide sequence shown in SEQ ID NO.10;
2) with 1) DNA sequence dna that limits has more than 90% homology.
3. expression vector as claimed in claim 1 or 2, it is characterized in that: described in intestinal bacteria and subtilis, all can start the genome that difunctional promotor that antibiotics resistance gene expresses derives from subtilis (Bacillus subtilis) BS168 or Bacillus licheniformis (Bacillus licheniformis) ATCC14580, have following 1)-21) any one in DNA sequence dna:
1) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.1;
2) under strict conditions with 1) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
3) with 1) or 2) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
4) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.2;
5) under strict conditions with 4) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
6) with 4) or 5) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
7) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.3;
8) under strict conditions with 7) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
9) with 7) or 8) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
10) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.4;
11) under strict conditions with 10) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
12) with 10) or 11) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
13) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.5;
14) under strict conditions with 13) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
15) with 13) or 14) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
16) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.6;
17) under strict conditions with 16) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
18) with 16) or 17) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated;
19) its DNA sequence dna is nucleotide sequence shown in SEQ ID NO.7;
20) under strict conditions with 19) DNA sequence dna that limits can hybridize, and can promote the DNA sequence dna that goal gene is transcribed and translated;
21) with 19) or 20) DNA sequence dna that limits has more than 90% homology, and can promote the DNA sequence dna that goal gene is transcribed and translated.
4. expression vector as claimed in claim 3, is characterized in that: described expression vector has following 22)-27) DNA sequence dna shown in any one:
22) DNA sequence dna is nucleotide sequence shown in SEQ ID NO:8;
23) with 22) DNA sequence dna that limits has more than 90% homology;
24) with 22) sequence in the DNA sequence dna that limits outside antibiotics resistance gene promoter sequence (6-415bp) has more than 90% homology;
25) DNA sequence dna is nucleotide sequence shown in SEQ ID NO:9;
26) with 25) DNA sequence dna that limits has more than 90% homology;
27) with 25) sequence in the DNA sequence dna that limits outside antibiotics resistance gene promoter sequence (6-415bp) has more than 90% homology.
5. the transgenic cell line containing expression vector described in claim 1 or 2.
6. the transgenic cell line containing expression vector described in claim 4.
7. the engineering strain containing expression vector described in claim 1 or 2.
8. the engineering strain containing expression vector described in claim 4.
9. the application of expression vector described in claim 3 in promoting goal gene to transcribe and translate.
10. the application of expression vector described in claim 4 in promoting goal gene to transcribe and translate.
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| CN114015678A (en) * | 2021-09-30 | 2022-02-08 | 中南民族大学 | Aminopeptidase Amp0279 derived from Bacillus sphaericus C3-41 as well as recombinant strain and application thereof |
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