CN101993878B

CN101993878B - RRNA mosaic promoter and expression vector containing same

Info

Publication number: CN101993878B
Application number: CN2010105063564A
Authority: CN
Inventors: 崔中利; 曹慧; 赵晓丽; 刘娟
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2010-10-14
Filing date: 2010-10-14
Publication date: 2012-04-18
Anticipated expiration: 2030-10-14
Also published as: CN101993878A

Abstract

The invention belongs to the field of molecular biology, and discloses an rRNA mosaic promoter and an expression vector containing the same. The mosaic promoter forms an annular DNA structure by inserting two lacO1 promoters of a lac operon into the upstream and downstream of an escherichia coli rRNArrnB operon promoter P1 respectively or inserting araI1 and araO2 promoters of an arabinose operoninto the upstream and downstream of an escherichia coli rRNA operon promoter P1 respectively to realize control of the promoter P1. The induced expression vector is obtained by substituting the rRNArrnBP1 mosaic promoter for a promoter T7 of a vector pET29a. The mosaic promoter and the expression vectors pRNP2 and pRBB can conveniently realize efficient expression of exogenous genes in multiple escherichia coli strains, wherein the pRBB also can be used for directed evolution of genes.

Description

A kind of rRNA chimeric promoters and contain the expression vector of this chimeric promoters

Technical field

The invention belongs to biology field, relate to a kind of rRNA chimeric promoters and contain the expression vector of this chimeric promoters, particularly a kind of rRNA that contains the dna circle controlling element RrnBThe P1 chimeric promoters, and contain the expression vector of this chimeric promoters.

Background technology

In intestinal bacteria, ribosomal quantity is to be controlled by the transcriptional level of the rRNA manipulator of seven copies on the karyomit(e).When intestinal bacteria for the time when being 20 min, the rrna quantity in each cell has 70,000 approximately, when growth velocity is slow, in each cell also nearly 20,000.Rrna googol amount like this in cell has been explained the superpower ability of rRNA manipulator promotor.

E.coliPromotor P1 in the rRNA manipulator by the core promoter that comprises-10 districts and-35 districts, be positioned at the UP element that is rich in the AT base at the upper reaches ,-35 district and be positioned at that the binding site of 3 to 5 transcription factors (Fis) at the UP element upper reaches forms.Have superpower ability although proved rRNA promotor P1, it is not used to E.coli yet and carries out high level expression, and its major cause is the synthetic control that is subordinated to the cell speed of growth in the body of rRNA.In the cell fast breeding phase, P1 is an activatory, and when cell is in the quiescent stage of growth, then P1 is regulated by negative.So, the rRNA promotor the pre-induction phase with continuous activation, so be difficult to it is regulated and control.That therefore, realizes rRNA promotor P1 adjustablely will carry out high level expression to foreign gene and have great importance.

In expression vector commonly used at present, most of escherichia coli promoter of use is all from corresponding manipulator, and they all have can be regional with aporepressor specificity bonded operon.

Lac3 operons are arranged, except main operon in the manipulator LacO1(O1), also have two auxiliary LacO, they are LacO2(O2) and LacO3(O3), O2 is centered close to 401 bp places, O1 downstream LacThe inside of Z gene, O3 is centered close to 92 bp places, the O1 upper reaches, and they are all with to check effect relevant, thus the Lac aporepressor can be combined in simultaneously on two operon sites and to form dna circle, and stable dna circle makes that checking effect reaches maximum.

Ara4 operons are arranged in the manipulator, be respectively AraI1, AraI2, AraO1With AraO2When not having pectinose to exist, regulate albumen and be combined in simultaneously AraI1With AraO2On, form stable dna circle, P _BADTranscribing of promotor is suppressed.

Summary of the invention

The objective of the invention is to be difficult to deficiency that intestinal bacteria rRNA promotor P1 is regulated and control, according to the special rRNA of the adjusted and controlled design of dna circle shape in the genetic expression to prior art RrnBThe P1 chimeric promoters is in the hope of realizing RrnBThe controlled expression of P1 promotor.

Another object of the present invention provides the expression vector that contains this chimeric promoters.

Another purpose of the present invention provides a kind of β-1,4-glucose incision enzyme gene recombinant expression vector and utilize this recombinant expression vector to express β-1, the method for 4-endoglucanase.

The object of the invention can be realized through following technical scheme:

A kind of rRNA RrnBP1 chimeric promoters, this chimeric promoters are at intestinal bacteria rRNA RrnBThe upstream and downstream of manipulator promotor P1 is inserted two of lactose manipulator respectively LacO1Promotor; Perhaps insert the pectinose manipulator respectively in the upstream and downstream of intestinal bacteria rRNA manipulator promotor P1 AraI1With AraO2Promotor forms the cyclic DNA structure and realizes the control to promotor P1.

Wherein, the upstream and downstream of described intestinal bacteria rRNA manipulator promotor P1 is inserted two of lactose manipulator respectively LacO1Promotor, rRNA RrnBP1 chimeric promoters sequence is SEQ ID NO. 1, called after P _16S-2

Described upstream and downstream at intestinal bacteria rRNA manipulator promotor P1 is inserted the pectinose manipulator respectively AraI1With AraO2Promotor, rRNA RrnBP1 chimeric promoters sequence is SEQ ID NO. 2, called after P _16S-B

A kind of intestinal bacteria inducible expression vector, this inducible expression vector by the T7 promotor of carrier pET29a by rRNA of the present invention RrnBThe P1 chimeric promoters replaces gained.

Described inducible expression vector is the rRNA of SEQ ID NO. 1 by sequence by the T7 promotor of carrier pET29a preferably RrnBThe P1 chimeric promoters replaces gained, called after pRNP2.

Described inducible expression vector is the rRNA of SEQ ID NO. 2 by sequence by the T7 promotor of carrier pET29a preferably RrnBThe P1 chimeric promoters replaces gained, called after pRBB.

A kind of β-1,4-glucose incision enzyme gene recombinant expression vector, this recombinant expression vector are with β-1, the 4-glucose incision enzyme gene Cel5G is inserted into intestinal bacteria inducible expression vector according to the invention XhoI with NdeGained between the I site.

Described β-1,4-glucose incision enzyme gene recombinant expression vector be preferably with β-1, the 4-glucose incision enzyme gene Cel5G is inserted into intestinal bacteria inducible expression vector pRNP2's XhoI with NdeGained between the I site, called after pRNP2- Cel5G.

Described β-1,4-glucose incision enzyme gene recombinant expression vector be preferably with β-1, the 4-glucose incision enzyme gene Cel5G is inserted into intestinal bacteria inducible expression vector pRBB's XhoI with NdeGained between the I site, called after pRBB- Cel5G

A kind of pRBB-that utilizes Cel5GRecombinant expression vector is expressed β-1, and the method for 4-endoglucanase comprises the steps:

(1) make up to regulate albumin A raC recombinant expression vector: be the carrier that sets out with carrier pET22b, usefulness contains self promotor AraCT7 promotor among the gene substitution carrier pET22b obtains described adjusting albumin A raC recombinant expression vector;

(2) make up pRBB- Cel5GRecombinant expression vector;

(3) the recombinant expression vector cotransformation intestinal bacteria TOP10 competent cell that utilizes step (1) and step (2) to make up contains two resistance LB flat board top sieve menu bacterium colonies of penbritin and kantlex at the same time;

(4) culturing step (3) screens the positive strain that obtains and expresses β-1,4-endoglucanase.

Expression vector pRNP2 of the present invention and pRBB construction process may further comprise the steps:

1) according to a pair of overlapping primer of chimeric promoters sequences Design to be synthesized, wherein upstream primer comprises an adjusting protein binding site and rRNA RrnB core promoter sequence, UP element and transcription factor (Fis) binding site; Downstream primer comprises one and regulates protein binding site; The promotor upstream and downstream that makes amplification obtain respectively has one to regulate protein loci; One is positioned at downstream, core promoter-10 district, and another one is positioned at the Fis binding site upper reaches.

2) with round pcr or overlap extension pcr, carry out pcr amplification with archaeal dna polymerase, obtain chimeric promoters fragment P _16S-2And P _16S-B

3) the chimeric promoters fragment that obtains that will increase is mixed with commercial pMD18-T carrier; Method according to introducing on the T carrier working instructions is operated; Through the transformant colony colour, filter out the positive colony that bacterium colony is white in color, extract plasmid; Enzyme is cut and is checked order and identify positive colony, obtains containing the segmental reorganization of purpose T carrier p16S-2 and p16S-B.

4) the segmental reorganization of the correct purpose T-carrier that contains that obtains in the step 3) is used restriction enzymes double zyme cutting; Reclaim the purpose fragment; PET29a with reclaiming behind T4 dna ligase and the same double digestion is connected transformed clone host bacterium, extraction transformant plasmid; Positive recombinant is identified in order-checking, obtains expression vector pRNP2 and pRBB.

Beneficial effect of the present invention:

1) regulate and control the activity of promotor through dna circle, realized the MC of rrnB P1 strong promoter and be used for genetic expression, being formed on of dna circle improved the level that checks to a great extent, and it has been eliminated before inducing undesirable background widely and has transcribed.

2) expression vector pRNP2 can utilize number of different types intestinal bacteria as the host bacterium, such as E. coliDH10B, DH5a and BL21 (DE3) are preferably E. coliDH10B, the regulation and control preciseness is preferably E. coliBL21 (DE3).In BL21 (DE3), do not having under the inductor inductive situation, foreign gene is suppressed to express fully, therefore can not cause the instability of plasmid, the reduction of the decline of the cell speed of growth and recombinant protein output.In addition when expression has toxalbumin, can make goal gene be in silence state fully and do not transcribe, thereby avoid of the influence of goal gene toxicity host cell and plasmid stability.Expression vector pRBB can be used for expression of exogenous gene efficiently, and in the coli strain that AraC lacks, this carrier is controlled expression not, and host range widely, therefore can be used for the orthogenesis of foreign gene.

3) in the pAC+pRBB coexpression system through the high expression level of pAC, realized that the rigorous control of pRBB is expressed.

4) chimeric promoters according to the invention and expression vector pRNP2 and pRBB can be implemented in easily and carry out efficiently expressing of foreign gene in the multiple coli strain, and wherein pRBB can also be used for the orthogenesis of gene.

Description of drawings

Fig. 1 E.coliRRNA promotor P1 structure iron

Fig. 2 chimeric promoters P _16S-2Structure iron

Fig. 3 chimeric promoters P _16S-2Regulation and control figure

Fig. 4 expression vector pRNP2 makes up synoptic diagram

Fig. 5 chimeric promoters P _16S-BStructure iron

Fig. 6 chimeric promoters P _16S-2Regulation and control figure

Fig. 7 expression vector pRBB makes up synoptic diagram

Fig. 8 recombinant plasmid pAC makes up synoptic diagram.

Embodiment

Embodiment 1: promoter fragment P _16S-2Amplification and in the structure of interstitial granules p16S-2

According to E. coliRRNA RrnBThe P1 promoter sequence reaches LacOperon LacOSequences Design PCR primer is specially:

Upstream primer S1:SEQ ID NO. 3 (contains SphThe I restriction enzyme site), downstream primer S2:SEQ ID NO. 4.With overlapping long primer to S1/S2 each other template amplification obtain P _16S-2Promoter fragment.The PCR condition is: 10 μ L, 5 * PrimeSTAR Buffer (Mg ²⁺Plus), 50 μ M dNTP Mixture, 0.5 μ M S1,0.5 μ M S2,1.25 U PrimeSTAR HS archaeal dna polymerases (TAKARA) are transferred reaction volume to 50 μ L with sterilized water.The pcr amplification reaction program is: 98 ℃, and 10 s, 55 ℃, 15 s, 72 ℃, 30 s circulate 29 times; 72 ℃, 10 min.

Agarose gel electrophoresis purification PCR product with 2%, and utilize TaqEnzyme can make the amplified fragments end add the characteristic of Desoxyadenosine, carries out PCR once more and makes each segmental sticky end all put down and add a dA by benefit.The PCR condition is: 2.5 μ L 10 * TaqBuffer (Mg ²⁺Plus), 50 μ M dNTP Mixture, 10 ng DNA reclaim fragment, 1 U TaqArchaeal dna polymerase is transferred reaction volume to 25 μ L with sterilized water.The pcr amplification reaction program is: 72 ℃, and 20 min; 10 ℃, 10 min.

The T/A clone is a kind of method that the PCR product is directly cloned; Utilize the Taq enzyme to have elongase activity; Add an A residue to 3 ' end of the PCR product of extension fully, utilize linearized vector that contains single thymus pyrimidine (T) 3 ' overhang and the insertion fragment that has single adenylic acid(AMP) (A) 3 ' overhang to carry out then.PMD18-T simple carrier is formed by reconstruction, has with the identical function of pUC18 carrier.Therefore the polylinker zone of this carrier is positioned at gene inside, can screen with indigo plant/hickie and identify to contain and insert segmental positive colony.Carrier contains ampicillin resistance gene, can be used for screening carrier-containing bacterium.

Dna fragmentation and pMD18-T Vector after PCR adds " A " end reaction press the 2:1 mixed, are connecting under the liquid effect, and 16 ℃ of water-baths are spent the night.Enzyme connects product transformed into escherichia coli DH10B competent cell, on penbritin (100 μ gmL-1) LB flat board in 37 ℃ of overnight cultures.Interstitial granules p16S-2 during transformant obtains after PCR and order-checking evaluation.Enzyme connects the enzyme adopt Takara company and connects test kit and carry out, and its reaction system does

PMD18-T simple carrier	0.5 μL
		The PCR product	4.0 μL
Connect solution I	5 μL
		The total reaction system	10 μL

Following all enzymes connect process and all adopt identical enzyme to connect reaction system in this patent.

Embodiment 2: the structure of expression vector pRNP2

P16S-2 and pET29a plasmid are obtained the double digestion fragment of promoter fragment P16S-2 and the pET29a that removes the T7 promotor with NdeI and SphI double digestion, connect according to 2:1 mixed enzyme after the purifying and recovering.Enzyme connects product Transformed E .coli DH10B competent cell, and coating contains the LB flat board of 50 μ gmL-1 kantlex.Transformant obtains expression vector pRNP2 after PCR and order-checking evaluation.

Embodiment 3: the structure that contains β-1,4 glucose incision enzyme gene (cel5G) recombinant plasmid pRNP2-cel5G and expression strain

The recombinant plasmid pET29a-that pRNP2 and this laboratory have been built Cel5G (Cui Zhongli etc., one Chinese patent application number: 201010018298.0) use respectively XhoI with NdeI carries out double digestion.The enzyme system of cutting is:

NdeI enzyme (10 U μ L ^-1)	1.0 μL
		XhoI enzyme (10 U μ L ^-1)	1.0 μL
10 * general damping fluid	5.0 μL
		pET29a- cel5G	20.0 μL
Distilled water	24.0 μL
		TV	50.0 μL

In 37 ℃ of water-baths more than reaction 3 h, enzyme is cut product and is carried out 0.75% agarose gel electrophoresis and cut glue and reclaim, and method is the same.

Enzyme is cut product and after the agarose gel electrophoresis separation and purification, is reclaimed the test kit recovery with dna gel.Then double digestion is obtained Cel5G gene fragment and pRNP2 double digestion fragment connect the back of spending the night in 16 ℃ of enzymes and transform E.coliThe fresh competent cell of DH10B, coating contains 50 μ gmL ^-1The LB of kantlex is dull and stereotyped.Select positive colony, obtain recombinant strains pRNP2- Cel5G (DH10B).

Again with recombinant expression plasmid pRNP2- Cel5G transforms respectively E.coliDH5a, the fresh competent cell of BL21 (DE3) obtains expression strain pRNP2- Cel5G (DH5a), pRNP2- Cel5G [BL21 (DE3)].

Embodiment 4: β-1,4 endoglucanase Cel5G vitality test

With the expression strain pRNP2-that builds Cel5G (DH10B), pRNP2- Cel5G (DH5a), pRNP2 -cel5G [BL21 (DE3)] and pET29a- Cel5G [BL21 (DE3)] inserts in the LB test tube that contains kantlex with fixed rotating speed 200 rmin ^-1Shaking culture is spent the night, and is forwarded in the fresh LB substratum that contains kantlex of 100 mL by 1% inoculum size then, and 37 ℃ of shaking culture are to OD ₆₀₀Be about 0.6; Add IPTG to final concentration 0.1 mM, 20 ℃ of inducing culture 20 h; Simultaneously not add three expression strains of IPTG inductive as contrast.6000 rmin ^-1, 4 ℃ of centrifugal 10 min, collect thalline.According to 10 mLg ^-1The ratio of wet cell is with 50 mM citrate buffer solution (pH4.8) suspension bacterial sediments, and add proteinase inhibitor PMSF to final concentration be 0 1 mM is with the broken somatic cells of supersound process, 12000 rmin ^-1, behind 4 ℃ of centrifugal 15 min, the gained supernatant is the crude enzyme liquid that contains recombinase Cel5G.Measure the expression amount and the activity thereof of β-1,4 endoglucanase, result's demonstration, Cel5The G gene can be at P _16S-2Well express in-the expression system.At P _16S-2In-the expression system; Target protein enzyme work after IPTG induces reaches 50% in the T7-expression system and is merely 2% after inducing without the enzyme work of inductive target protein, and the enzyme of the target protein that background is expressed in the T7-system is lived in also having reached and induced 25% of back protease activity.This shows that dna circle plays an important role in transcriptional control really, being formed on of dna circle improved the level of checking to a great extent.In addition, be structured on the pRNP2 expression vector Cel5The G gene can be in different host bacterium good abduction delivering, but expression effect is not quite similar.The expression level of Cel5G in E.coli DH10B is the highest, and the expression level in E.coli BL21 (DE3) is minimum, and under the non-inductive condition in E.coli BL21 (DE3), the cel5G gene is in silence state fully and does not transcribe.

Embodiment 5: promoter fragment P _16S-BAmplification and in the structure of interstitial granules p16S-B

According to E. coli rRNA rrnB P1 promoter sequence and ara operon araI1 and araO2 sequences Design PCR primer; Be specially: upstream primer S1:SEQ ID NO. 5 (containing the SphI restriction enzyme site), downstream primer S2:SEQ ID NO. 5 (containing the XbaI enzyme cutting site).Use primer that S1/S2 is obtained the P16S-B promoter fragment with bacillus coli gene group DNA as template (ordinary method extraction) amplification.The PCR condition is: 10 μ L, 5 * PrimeSTAR Buffer (Mg2+ plus), 50 μ M dNTP Mixture, 0.5 μ M S1; 0.5 μ M S2; 1 μ L dna profiling, 1.25 U PrimeSTAR HS archaeal dna polymerases (TAKARA) are transferred reaction volume to 50 μ L with sterilized water.The pcr amplification reaction program is: 98 ℃, and 10 s, 55 ℃, 15 s, 72 ℃, 30 s circulate 29 times; 72 ℃, 10 min.

Dna fragmentation after PCR adds " A " end reaction and pMD18-T Vector proportional mixing are connecting under the liquid effect, and 16 ℃ of water-baths are spent the night.Enzyme connects product transformed into escherichia coli DH10B competent cell, at penbritin (100 μ gmL ^-1) on the LB flat board in 37 ℃ of overnight cultures.Interstitial granules p16S-B during transformant obtains after PCR and order-checking evaluation.

Embodiment 6: the structure of expression vector pRBB

P16S-B and pET29a plasmid are used XbaI with SphThe I double digestion obtains promoter fragment P _16S-BWith the double digestion fragment of the pET29a that removes the T7 promotor, press 2:1 example mixed enzyme after the purifying and recovering and connect.Enzyme connects product and transforms E.coliThe TOP10 competent cell, coating contains 50 μ gmL ^-1The LB of kantlex is dull and stereotyped.Transformant obtains expression vector pRBB after PCR and order-checking evaluation.

Embodiment 7: the structure of recombinant plasmid pAC

With amplification obtain with self promotor AraCGene fragment reclaim purifying after NdeIWith SphThe I double digestion is removed the purifying fragment of T7 promotor and is pressed the 2:1 mixed in molar ratio with the pET22b double digestion, connecting under the liquid effect, and 16 ℃ of water-baths are spent the night.Enzyme connects product and transforms E.coliThe TOP10 competent cell, coating contains 100 μ gmL ^-1The LB of penbritin is dull and stereotyped.Transformant is cut and is checked order through enzyme and obtains recombinant plasmid pAC after identifying.The enzyme system of cutting is:

NdeI enzyme (10 U μ L ^-1)	1.0 μL
		SphI enzyme (10 U μ L ^-1)	1.0 μL
10 * general damping fluid	5.0 μL
		PET22b or PCR product	20.0 μL
Distilled water	24.0 μL
		TV	50.0 μL

Embodiment 8: contain β-1,4 glucose incision enzyme gene ( Cel5G) recombinant plasmid pRBB- Cel5GThe structure of expression strain

The recombinant plasmid pET29a-that pRBB and this laboratory have been built Cel5G (Cui Zhongli etc., one Chinese patent application number: 201010018298.0) use respectively XhoI with NdeI carries out double digestion.

The enzyme system of cutting is:

Enzyme is cut product and after the agarose gel electrophoresis separation and purification, is reclaimed the test kit recovery with dna gel.Then double digestion is obtained Cel5G gene fragment and pRBB double digestion fragment connect the back of spending the night in 16 ℃ of enzymes and transform E.coliThe fresh competent cell of TOP10, coating contains 50 μ gmL ^-1The LB of kantlex is dull and stereotyped.Select positive colony, obtain recombinant strains pRBB- Cel5G (TOP10).

Embodiment 9: β-1,4 endoglucanase Cel5G vitality test

PAC and pRBB-with equimolar amount Cel5G plasmid cotransformation intestinal bacteria TOP10 competent cell contains 100 mgL at the same time ^-1Penbritin and 50 mgL ^-1The dull and stereotyped top sieve menu of the two resistance LB bacterium colony of kantlex.

Picking list colony inoculation is in containing 100 mgL from two resistance LB flat boards ^-1Penbritin and 50 mgL ^-1In two resistance LB liquid nutrient mediums of kantlex, 37 ℃ of shaking culture are to OD ₆₀₀Be about 0.6, adding L-arabinose to final concentration is 0.2 gL ^-1, centrifugal collection thalline behind 20 ℃ of continuation shaking culture 20 h is not to add L-arabinose inductive expression strain as contrast.The ultrasonication somatic cells, behind the centrifugal removal cell debris, the gained supernatant is the crude enzyme liquid that contains recombinase Cel5G.Measure the expression amount and the activity thereof of β-1,4 endoglucanase, the result shows, coexpression Cel5The G gene with AraCBehind the gene, the inductive enzyme activity is not merely and induces 5% of back enzyme activity, explanation AraCGene product can check P effectively _16S-BThe transcriptional activity of promotor further specifies being formed on of dna circle and has improved the level that checks to a certain extent.

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Claims

1. a rRNA rrnB P1 chimeric promoters is characterized in that this chimeric promoters sequence is SEQ ID NO.1.

2. an intestinal bacteria inducible expression vector is characterized in that this inducible expression vector is replaced gained by the T7 promotor of carrier pET29a by the described rRNA rrnB of claim 1 P1 chimeric promoters.

3. β-1; 4-glucose incision enzyme gene recombinant expression vector; It is characterized in that this recombinant expression vector is with β-1,4-glucose incision enzyme gene cel5G is inserted into gained between XhoI and the NdeI site of the said intestinal bacteria inducible expression vector of claim 2.