CN104087606A - Bile salt hydrolase gene BSH and recombinant prokaryotic expression vector thereof - Google Patents
Bile salt hydrolase gene BSH and recombinant prokaryotic expression vector thereof Download PDFInfo
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- CN104087606A CN104087606A CN201310576384.7A CN201310576384A CN104087606A CN 104087606 A CN104087606 A CN 104087606A CN 201310576384 A CN201310576384 A CN 201310576384A CN 104087606 A CN104087606 A CN 104087606A
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Abstract
The invention discloses a bile salt hydrolase gene BSH and a recombinant prokaryotic expression vector. The bile salt hydrolase gene BSH has a preservation number of CGMCC NO.6540 and contains two BSH genes, i.e. BSH1 and BSH2 from enterococcus faecium, and through comparison, the two sequences have a similarity of 68.10%. The recombinant prokaryotic expression vector is obtained by inserting the enterococcus faecium bile salt hydrolase gene BSH1 and BSH2 into multiple cloning sites of a pET-32a vector. The pET-32a vector has a high expression quantity, and is also equipped with Trx.Tag, His.Tag, and S.Tag. The bile salt hydrolase gene provided by the invention undergoes high efficiency expression in E.coliBL21 (DE3) host bacteria, which also provides theoretical support for working out gene engineered strains able to efficiently degrade cholesterol, and lays the foundation for research of efficient expression and expression regulation of BSH gene in different expression systems.
Description
Technical field
The invention belongs to gene engineering field, relate in particular to a kind of bile salt hydrolase gene BSH and recombinant prokaryotic expression vector thereof.
Background technology
Bile salt hydrolase (bile salt hydrolase, BSH) is a kind of meta-bolites producing in microorganism growth, reproductive process.This enzyme can be hydrolyzed combined taurine cholate and glycine cholate, finally be converted into amino acid and free cholic acid, there is function (the KOCIUBINSKI G that reduces serum cholesterol, PEREZ P, ANTONI G.Screening of Bile Resistance and Bile Peicipitation in LacticAcid Bacteria and Bifidobacteria[J] .Food Prot, 1999,62:905～912, Coleman JP, Hudson LL.Cloning and characterization of a conjugated bile acid hydrolase gene fromClostridium perfringens.Appl.Environ.Microbiol, 1995, 61:2518～2520.) BSH is generally intracellular enzyme, optimum pH under micro-acid environment is generally between 5 and 6, insensitive to oxygen, this enzyme from multiple-microorganism, obtained separate and qualification (Li Guijie. the comprehensive evaluation of decreasing cholesterol Bacterium lacticum and bile salt hydrolase activity research. Shanghai: the .2008 of Shanghai Communications University, 2, Niu Zhixia, Liu Enmei. bile salt hydrolase in probiotic bacterium (BSHs) progress. China Dairy Industry, 2007,35 (9): 35～40, BinderH.J, B.Filburn, M.Floch.Bileacidinhibition of intestinal anaerobic organism.Am.J.Clin.Nutr, 1975,28 (10): 119～125, KIM G B, LEE B H.Biochemical and Molecular Insights Into Bile Salt Hydrolase in the Gastrointestinal Microflora a Review Asian-Aust[J] .AnitaSci, 2005,18:1505～1512).At present, the bacterial strain of the product bile salt hydrolase of discovery is all gram-positive microorganism, comprising the probiotic strain of part, as: milk-acid bacteria, bifidus bacillus etc.; A part is to parasitize the bacterial strain in enteron aisle with host with the form of symbiosis in addition, as: genera bacillus, clostridium, faecalis etc., be conventionally grown in the BSH enzyme producing without bacterial strain in the environment of cholate and all do not have activity.
Relatively less about the research report of bile salt hydrolase up to now, domestic research mainly concentrates on the aspect such as screening, the purifying of BSH enzyme of decreasing cholesterol mechanism, high yield BSH enzyme strain excellent; Also carrying out the research about aspects such as BSH enzyme microorganism molecular biology abroad, but also so far there are no breakthrough.
Summary of the invention
For the problems referred to above, the object of the embodiment of the present invention is to provide a kind of bile salt hydrolase gene BSH and recombinant prokaryotic expression vector thereof.
The embodiment of the present invention is to realize like this, a kind of bile salt hydrolase gene BSH, this bile salt hydrolase gene BSH preserving number is CGMCCNo.6540, and two BSH gene BSH1, BSH2 containing from faecium, be 68.10% through comparing two sequence similarities.
Another object of the present invention is to provide a kind of restructuring High level prokaryotic expression carrier that contains above-mentioned faecium bile salt hydrolase gene BSH1, BSH2, it is characterized in that: described recombinant prokaryotic expression vector is in the multiple clone site of pET-32a carrier, insert faecium bile salt hydrolase gene BSH1, BSH2 and obtain; Described pET-32a vector expression amount is high, and with Trx.Tag, His.Tag, S.Tag.
The invention provides two different sequences of a kind of faecium bile salt hydrolase gene BSH, called after gene BSHI respectively, BSH2, be 68.10% through comparing two sequence similarities, there is the nucleotide sequence as shown in SEQIDNo.1, the recombinant prokaryotic expression vector that contains this gene is also disclosed, experimental result shows that bile salt hydrolase gene has carried out high efficient expression in E.coli BL21 (DE3) Host Strains, the engineering strain also for working out with efficient degradation cholesterol provides theoretical support, simultaneously for the high efficient expression of BSH gene in different expression systems and the research of expression regulation lay the foundation.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis qualification of gene BSH1 thermograde pcr amplification product, and 1 swimming lane is DL2,000DNA Marker, and 2～9 swimming lanes are followed successively by 65 DEG C, and 64.2 DEG C, 63 DEG C, 61.1 DEG C, 58.7 DEG C, 56.9 DEG C, 55.7 DEG C, 55 DEG C of thermograde amplified productions;
Fig. 2 is the agarose gel electrophoresis qualification of gene BSH2 thermograde pcr amplification product, and 1 swimming lane is DL2,000DNA Marker, and 2～9 swimming lanes are followed successively by 65 DEG C, and 64.2 DEG C, 63 DEG C, 61.1 DEG C, 58.7 DEG C, 56.9 DEG C, 55.7 DEG C, 55 DEG C of thermograde amplified productions;
PCR, the enzyme of the positive clone of Fig. 3 pMD19T-BSH are cut agarose gel electrophoresis qualification, 1 swimming lane is DL2,000DNA Marker, 2 swimming lanes are that gene BSH1 reclaims product (contrast), 3-5 swimming lane is pMD19T-BSH1 bacterium colony PCR qualification, 6～8 swimming lanes are the EcoRI of pMD19T-BSH1, the qualification of HindIII double digestion, 9 swimming lanes are DL2,000DNA Marker, 10 swimming lanes are that gene BSH2 reclaims product (contrast), 11～13 swimming lanes are pMD19T-BSH2 bacterium colony PCR qualification, 14～16 swimming lanes are the BamHI of pMD19T-BSH2, the qualification of HindIII double digestion;
The agarose gel electrophoresis qualification of the positive clone of Fig. 4 pET-32a-BSH; 1 swimming lane is DL2; 000DNA Marker, 2～6 swimming lanes are pET-32a-BSH1 EcoRI, the qualification of HindIII double digestion; 7 swimming lanes are that BSH1 reclaims product (contrast); 8 swimming lanes are DL2,000DNA Marker, the BamHI that 9～13 swimming lanes are pET-32a-BSH2; the qualification of HindIII double digestion, 14 swimming lanes are that BSH2 reclaims product (contrast);
Fig. 5 is that BL21 (DE3)-pET-32a-BSH1 expression product SDS-PAGE analyzes, 1 swimming lane is albumen maker, 2 swimming lanes are that BL21 (DE3)-pET-32aIPTG induces (negative control), 3 swimming lanes are BL21 (the DE3)-pET-32a-BSH1 recombinant bacterium (negative control) without IPTG induction, 4～8 swimming lanes are BL21 (DE3)-pET-32a-BSH1 IPTG induction 2,4,6,8,10h result;
Fig. 6 is for analyzing for BL21 (DE3)-pET-32a-BSH2 expression product SDS-PAGE, 1 swimming lane is albumen maker, 2 swimming lanes are that BL21 (DE3)-pET-32aIPTG induces (negative control), 3 swimming lanes are BL21 (the DE3)-pET-32a-BSH2 recombinant bacterium (negative control) without IPTG induction, 4～8 swimming lanes are BL21 (DE3)-pET-32a-BSH2IPTG induction 2,4,6,8,10h result;
Fig. 7 is the Western-blot detected result of gene BSH1 prokaryotic expression protein;
Fig. 8 is the Western-blot detected result of gene BSH2 prokaryotic expression protein;
Fig. 9 is that after fusion protein purification, SDS-PAGE detects, 1 swimming lane is albumen maker, 2 swimming lanes are BL21 (DE3)-pET-32a-BSH1 expression product purification result, 3 swimming lanes are the not purifying contrast of BL21 (DE3)-pET-32a-BSH1 expression product, 4 swimming lanes are BL21 (DE3)-pET-32a-BSH2 expression product purification result, and 5 swimming lanes are the not purifying contrast of BL21 (DE3)-pET-32a-BSH2 expression product;
Figure 10 is that the Western-blot of purified product detects; 1 is that BL21 (DE3)-pET-32a-BSH1 purification result detects, and 2 is that BL21 (DE3)-pET-32a-BSH2 purification result detects.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition, for example molecular cloning experiment guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.
The material using in preferred embodiment: faecium screening is preserved; High-fidelity DNA polymerase PFU, Taq archaeal dna polymerase, T4DNA ligase enzyme, restriction enzyme, pMD19-T carrier, DNA gel reclaim test kit, plasmid extraction kit, protein molecular weight standard and double-colored Marker, the DNA Marker III of dying in advance of protein purchased from TIANGEN Biotech (Beijing) Co., Ltd.; Penbritin (Ampicillin, Amp) and kalamycin (Kanamycin, Kan) are Sigma company product; Primer is synthetic to be completed by the handsome Bioisystech Co., Ltd in Shanghai with DNA sequencing; Other chemical reagent is purchased from Beijing Ding Guo biotechnology limited liability company; E. coli jm109, BL21 (DE3) are preserved by this laboratory.
The present invention, on the basis of early-stage Study, carries out pcr amplification according to having announced BSH gene order design primer on the net, and clone obtains two BSH genes, and through comparison, two sequence similarities are 68.10%.And respectively two genes have been carried out to prokaryotic expression, realize the high efficient expression of this gene.
One, the clone of BSH gene and order-checking
The BSH gene order of having announced according to GenBank, utilizes Primer5.0 and Oligo6.0 software design to design respectively special primer: upstream primer BSH1F:5 '-CCG
gAATTCcGGATGTGTACATCCATTGTTTATG-3 ' (SEQ ID No.2), underscore part is EcoRI restriction enzyme site; Downstream primer BSH1R:5 '-CCC
aAGCTTgGGTTATTTGTTTAAATAATGTATTTGT-3 ' (SEQ ID No.3), underscore part is Hind III restriction enzyme site; Upstream primer BSH2F:5 '-CGC
gGATCCgCGATGTGTACGTCTATTACTTA
(SEQIDNo.2), underscore part is BamHI restriction enzyme site to TGTAAC-3 '; Downstream primer BSH2R:5 '-CCC
aAGCTT(SEQIDNo.3), underscore part is Hind III restriction enzyme site to GGGCTAATTTATATATTTAATTTGTTGTTT-3 '.Extract faecium genomic dna with reference to CTAB bacterium genome DNA extracting method.Taking genomic dna as template, carry out pcr amplification with Auele Specific Primer, amplification system and condition are as follows: PCR reaction conditions: 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, 55 DEG C-65 DEG C annealing 30s, 72 DEG C are extended 1min, 35,72 DEG C of abundant 10min that extend of cycle number.PCR reaction system is 25 μ L: upstream and downstream mix primer 1 μ L, and template 1 μ L, Mix(is enzyme-added) and 12.5 μ L, ddH
2o10.5 μ L.Adopt thermograde PCR method amplification amplification to be all single specificity band at about 1000bp place, conform to the gene BSH size of GenBank report, as shown in Figure 1 and Figure 2.
PCR product is carried out to 1.0% (g/mL) agarose gel electrophoresis and detect, then reclaim test kit specification sheets according to DNA gel and cut glue recovery purifying; The DNA fragmentation of purifying is connected and spends the night in 16 DEG C under the effect of T4DNA ligase enzyme with pMD19-T carrier, connect product and transform escherichia coli jm109 competent cell, with the LB plate screening positive colony that contains penbritin, extract plasmid, enzyme checks order after cutting qualification clip size, obtains recombinant vectors pMD19T-BSH1, pMD19T-BSH2.
Bacterium colony PCR qualification and the enzyme of recombinant vectors pMD19T-BSH1, pMD19T-BSH2 are cut qualification result as shown in Figure 3, visible pMD19T-BSH1 and pMD19T-BSH2 all can obtain the object fragment of about 1000bp with PCR qualification and EcoRI/HindIII, BamHI/HindIII double digestion, conform to expected results, confirmation object fragment has been connected into pMD19-T carrier and has been that forward connects.
Sequencing result demonstration, bile salt hydrolase gene BSH1 total length is 978bp, BSH2 total length 975bp, 325,324 amino acid of encoding respectively, are 67.69% through the amino acid sequence similarity of comparison BSH1, BSH2 coding.
Two, the structure of BSH gene recombination prokaryotic expression carrier
High according to prokaryotic expression carrier pET-32a expression amount, and with Trx.Tag, His.Tag, S.Tag, be conducive to the features such as the purifying of expression product, introduce bile salt hydrolase gene BSH1, BSH2 by restriction enzyme EcoRI/HindIII, BamHI/HindIII respectively at its multiple clone site place, realize the recombined pronucleus expression of gene.
To contain gene BSH1, the recombinant vectors pMD19T-BSH1 of BSH2, pMD19T-BSH2 uses respectively EcoRI/HindIII, BamHI/HindIII double digestion, reclaim gene BSH1, BSH2 fragment, again with same through EcoRI/HindIII, the pET-32a carrier of BamHI/HindIII double digestion spends the night in 16 DEG C of connections under the effect of T4DNA ligase enzyme, connect product and transform escherichia coli jm109 competent cell, with the LB plate screening positive colony that contains kalamycin, extract plasmid, PCR and enzyme are cut qualification, obtain gene BSH1, BSH2 recombinant prokaryotic expression vector pET-32a-BSH1, pET-32a-BSH2.Subsequently pET-32a-BSH1, pET-32a-BSH2 are transformed to e. coli bl21 (DE3) ,-80 DEG C save backup.
PCR qualification and the enzyme of gene BSH1, BSH2 recombinant prokaryotic expression vector pET-32a-BSH1, pET-32a-BSH2 are cut qualification result as shown in Figure 4, result shows that object fragment has been connected in pET-32a carrier, and recon has been carried out to order-checking qualification, ensure to be connected into the accuracy of fragment.Sequencing result, with sequencing result is identical in earlier stage, can be used for expressing.
Three, the abduction delivering of recombinant protein and qualification
If empty carrier pET-32a transforms BL21 (DE3) IPTG induction group, not induction group of recombinant bacterium as negative control.The e. coli bl21 (DE3) that contains recombinant plasmid through qualification is inoculated in to 5mLLB(containing penbritin) in, 37 DEG C of overnight incubation, be transferred to fresh LB(containing penbritin in the ratio of 1:100) in, cultivate between 2-3h to OD600=0.5-0.8 for 37 DEG C, add IPTG to final concentration 1mM in 30 DEG C of continuation cultivations, grope the best inducing culture time, collect thalline.SDS-PAGE detects protein expression situation, and result as shown in Figure 5, Figure 6.The recombinant protein size that prediction N end has merged expression vector label is about 45KD.Compare group and recombinant bacterium induction group have a band in desired location as seen, and obviously increase with the lengthening induced product of induction time, and 6h is best induction time.Bibliographical information, amalgamation and expression produces two kinds of expression products sometimes, and one is amalgamation and expression target protein, and a kind of is the target protein of fracture, has lost exactly the target protein that merges part.Therefore infer that by detected result target gene is to express with the form of fusion rotein, do not have the target protein of fracture to express.
Recombinant bacterium, after IPTG inducing culture, by the centrifugal 10min of 5000r/min at 4 DEG C of cultures, is collected crude enzyme liquid.In order to determine the expression-form of target protein matter, adopt the method for ultrasonic wave binding lysozyme smudge cells respectively cleer and peaceful precipitation in the cracking of expression thalline to be carried out to SDS-PAGE analysis.Result shows, target protein is mainly distributed in cracking precipitation, shows that the recombinant protein of expressing mainly exists with inclusion body form.
Fusion rotein Western blot detects: the fusion rotein warp after induction after tropina and purifying
After the separation gel SDS-PAGE electrophoresis of 120g/L, electrotransfer, to film on nitrocellulose filter, carries out
Western blot engram analysis.Primary antibodie is mouse-anti people Anti-6 × His(1:1000 dilution), two resist the sheep anti-mouse igg antibody (1:5000 dilution) for horseradish peroxidase, adopt ECL chemoluminescence method to develop, after exposure, locate visible clear band in expection size, there is black stripe in the about 45KD of molecular weight, conform to 6 × His fusion protein molecule amount size to be expressed of expection, show that target protein obtains correct expression.Western-blot detects as shown in Figure 7, Figure 8.
The purifying of fusion rotein: undertaken by the Ni-NTAAgarose inclusion body purification step of QIAGEN company, purification result is carried out SDS-PAGE detection, and carries out Western-blot detection, target protein detected in tram, in the same size.Detected result is as Fig. 9, Figure 10.The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (2)
1. a bile salt hydrolase gene BSH, is characterized in that, this bile salt hydrolase gene BSH preserving number is CGMCCNo.6540, and two BSH gene BSH1, BSH2 containing from faecium, be 68.10% through comparing two sequence similarities.
2. a restructuring High level prokaryotic expression carrier that contains faecium bile salt hydrolase gene BSH1, BSH2 described in claim 1, is characterized in that: described recombinant prokaryotic expression vector is in the multiple clone site of pET-32a carrier, insert faecium bile salt hydrolase gene BSH1, BSH2 and obtain; Described pET-32a vector expression amount is high, and with Trx.Tag, His.Tag, S.Tag.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105567619A (en) * | 2016-01-15 | 2016-05-11 | 江南大学 | Genetically engineered bacterium for producing bile salt hydrolase variant and preparation method thereof |
| US11291693B2 (en) | 2015-06-25 | 2022-04-05 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
| CN118421542A (en) * | 2024-07-05 | 2024-08-02 | 云南大学 | Enterococcus faecium IOBRA9746 and application thereof |
-
2013
- 2013-11-18 CN CN201310576384.7A patent/CN104087606A/en active Pending
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| Title |
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| AGUS WIJAYA,ET AL: "Cloning of the Bile Salt Hydrolase(bsh) Gene from Enterococcus faecium FAIR-E 345 and Chromosomal Location of bsh Genes in Food Enterococci", 《JOURNAL OF FOOD PROTECTION》 * |
| WIJAYA,A.,ET AL: "Enterococcus faecium bile salt hydrolase (bsh) gene，complete cds", 《GENBANK：AY260046.1》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11291693B2 (en) | 2015-06-25 | 2022-04-05 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
| US11896627B2 (en) | 2015-06-25 | 2024-02-13 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
| CN105567619A (en) * | 2016-01-15 | 2016-05-11 | 江南大学 | Genetically engineered bacterium for producing bile salt hydrolase variant and preparation method thereof |
| CN118421542A (en) * | 2024-07-05 | 2024-08-02 | 云南大学 | Enterococcus faecium IOBRA9746 and application thereof |
| CN118421542B (en) * | 2024-07-05 | 2024-09-17 | 云南大学 | Enterococcus faecium IOBRA9746 and application thereof |
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Application publication date: 20141008 |