CN104593404B - A kind of recombinant plant lactobacillus and preparation method thereof of secretion colicine V - Google Patents
A kind of recombinant plant lactobacillus and preparation method thereof of secretion colicine V Download PDFInfo
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- CN104593404B CN104593404B CN201410856189.4A CN201410856189A CN104593404B CN 104593404 B CN104593404 B CN 104593404B CN 201410856189 A CN201410856189 A CN 201410856189A CN 104593404 B CN104593404 B CN 104593404B
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Abstract
The present invention relates to a kind of recombinant plant lactobacillus and preparation method thereof of secretion colicine V, belong to microbial molecules field of biology, recombinant plant lactobacillus of the invention carries the sequestered expression vector that peptide gene and ripe colicine V fusions are led containing pediocin.The recombinant plant lactobacillus is to be connected into expression vector by the way that pediocin PA-1 to be led to the fusion of peptide and colicine V mature peptides, connection product is transferred to Lactococcus lactis by the method for electrotransformation, recombinant plasmid is extracted, then electricity is transferred in lactobacillus plantarum BM-1 and obtains.The recombinant plant lactobacillus of the present invention can secrete pediocin PA-1 and colicine V simultaneously, good inhibiting effect is all had to Colibacter and single increasing Listeria pathogenic bacteria, it not will produce toxic side effect and residual after animal edible, environment and food security are not threatened, had a good application prospect in feed industry and field of food industry.
Description
Technical field
The present invention relates to microbial molecules field of biology, and in particular to a kind of recombinant plant of secretion colicine V
Lactobacillus and preparation method thereof.
Background technology
In the past 50 years, antibiotic, which is used as, applies most common prophyiaxis and promoting growth class feed addictive, is brought to aquaculture
While interests, fatal drawback is also brought：Microbial population of animal intestinal tract imbalance, suprainfection and bacterial drug resistance etc. are bad anti-
It should gradually highlight, bring and seriously threaten to aquaculture and feed industry.Therefore, exploitation have no toxic side effect, pollution-free and noresidue
Substitutes For Antibiotic, become the task of top priority of researcher.Since 20th century the eighties, micro- life based on lactic acid bacteria
State preparation starts to attract attention, and promotes the growth of intestinal beneficial bacterium, inhibits the growth of harmful bacteria, improves animal health level,
The function of the excitation potential growth performance of animal is gradually reported, in addition again due to environmentally safe, noresidue and nothing
The advantage of toxic side effect, it is considered to be the optimal substitute of feeding antibiotic.
Lactic acid bacteria is most commonly used probiotics in animal microecological formulation, belongs to normal intestinal flora.Lactobacillus plantarum BM-
1(Isolation and partial characterization of a bacteriocin produced by
Lactobacillus plantarum BM-1 isolated from a traditionally fermented Chinese
Meat product.Zhang H et al.2013) belong to lactobacillus in Lactobacillaceae, anaerobism or amphimicrobian, belong to same
Type fermentative lactobacillus, it can be colonized in enteron aisle, be the chief component of physiology barrier.The bacterial strain also secretory piece simultaneously
Coccus element PA-1, pediocin PA-1 are a kind of II a class bacteriocins, it is bacterial metabolism in the process by one kind of Ribosome biogenesis
Biologically active polypeptides matter has pathogenic bacteria Listeria Monocytogenes strong inhibiting effect, because of it
Efficiently, nontoxic, high temperature resistant, noresidue, without drug resistance and the advantages that good biocompatibility, obtain the pass of domestic and foreign scholars
Note.Pediocin PA-1 biological feed additive, biological veterinary research and development in have a wide range of applications and value and wide answer
Use foreground.
Currently, probiotics research and development just develop towards the direction of efficient, single-minded preparation, pass through technique for gene engineering
The engineered strain of structure has the characteristics that be readily produced, preserves, is colonized, breeding or can be directed to certain disease of some stage,
Its effect is more notable, it has also become the research hotspot of biotechnology.In the prior art, the piece ball secreted by lactobacillus plantarum
Rhzomorph PA-1 has Listeria Monocytogenes strong inhibiting effect, but for gram negative pathogenic bacteria unrestraint
Effect, and with its be all II a classes bacteriocin colicine V by Gram-negative bacteria secrete and to nearly source coli strain
There is inhibiting effect, its synthesis is similar with pediocin PA-1 with mechanism of secretion, is controlled by four genes, is separately encoded bacteriocin
Precursor peptide, the dedicated ABC excretory systems of bacteriocin and protection secretion bacterial strain immune protein.Before wherein II a class bacteriocins
The N sections of body peptide have one section of double-glycine type to lead peptide, play an important role in bacteriocin secretion process, it can guide precursor peptide quilt
Abc transport albumen identifies, and is cut in secretion process.
Invention content
The object of the present invention is to provide a kind of lactic acid bacteria recombinant bacterial strain that can inhibit listeria spp and Escherichia coli simultaneously,
The pediocin PA-1 excretory systems of lactobacillus plantarum BM-1 are utilized to secrete colicine V in the bacterial strain so that two kinds thin
Rhzomorph is expressed simultaneously in lactobacillus plantarum, provides antimicrobial spectrum wider engineering strain for food industry.
Present invention firstly provides a kind of recombinant vector, which carries pediocin and leads peptide gene and ripe large intestine
The fusion that bacillin V genes are formed by connecting.
The nucleotide sequence of the fusion such as SEQ ID NO.1 show.
The present invention provides the methods for preparing above-mentioned recombinant vector, include the following steps：
(1) pediocin is led peptide gene to connect with the gene of encoding colicin V maturation proteins, synthesis fusion base
Restriction enzyme site is added at cause, both ends, builds on carrier；
(2) carrier for the carrying synthetic gene that double digestion expression vector and step (1) obtain respectively, after digestion, by the two
Connection；
(3) connection product Electroporation-competent cells extract plasmid, obtain that correct plasmid is sequenced.
In the above method, step (1) described pediocin leads the sequence of peptide gene as shown in SEQ ID NO.2；Large intestine bar
The gene order of rhzomorph V maturation proteins is as shown in SEQ ID NO.3；The sequence of the fusion of synthesis such as SEQ ID NO.1 institutes
Show；The restriction enzyme site of fusion both ends addition is Nco I and Eco R I, carrier pUCK.
In the method for above-mentioned Prepare restructuring carrier, the expression vector of step (2) is p118148, is by expression vector
Promoter PnisA on pNZ8148 replaces with P11 acquisitions, which also includes chloramphenicol resistance gene.Wherein, step (2)
Double digestion uses Nco I and Eco R I.
Wherein, the competent cell of step (3) is the competent cell of Lactococcus lactis NZ9000.
In an embodiment of the present invention, the pediocin provided by the invention that carries leads peptide gene and ripe colicine
The recombinant vector for the fusion that V genes are formed by connecting is named as p118148-ColV.It is through the following steps that structure obtains：
(1) pediocin is led peptide gene to connect with ripe colicin V gene, synthesizes fusion, both ends addition
Restriction enzyme site Nco I and Eco R I are built on pUC carriers；The fusion gene sequence is as shown in SEQ ID NO.1；
(2) carrying for using Nco I and Eco R I double digestion expression vector p118148 and step (1) to obtain respectively synthesizes base
After digestion, the two is connected for the carrier of cause；
(3) competent cell of connection product electrotransformation Lactococcus lactis NZ9000 extracts plasmid, and it is correct to obtain sequencing
Plasmid p118148-ColV.
The present invention also provides the host cells containing above-mentioned recombinant vector.
Further, the present invention provides a kind of recombinant plant lactobacillus of secretion colicine V, contains the above-mentioned of the present invention
Recombinant vector.
The present invention provides the recombinant plant lactobacillus, is prepared by following steps：
(1) fusion for carrying that pediocin leads peptide gene and ripe colicin V gene is formed by connecting is obtained
Recombinant vector；
(2) the recombinant vector electrotransformation of step (1) is entered in lactobacillus plantarum BM-1.
Lactobacillus plantarum BM-1 is the gene that wild-type strain contains pediocin synthesis and secretion, but is free of Escherichia coli
The plain relevant genes of V.
The present invention also provides the application of above-mentioned recombinant vector or recombinant plant lactobacillus in preparing probiotics.
The present invention provides the application of recombinant plant lactobacillus in the food industry.
The probiotics or feed of recombinant plant lactobacillus containing the present invention also belong to protection scope of the present invention.
The pediocin PA-1 of lactobacillus plantarum BM-1 secretions is a kind of II a class bacteriocins, to monocyte hyperplasia Lee
This special Salmonella has strong inhibiting effect, and does not have inhibiting effect to gram negative pathogenic bacteria.Since its antimicrobial spectrum is relatively narrow
Cause it that there is certain limitation in the application of feed and field of food.The present invention has selected to be all II a class bacteriocins, and
Leading peptide sequence and the similar colicine V of pediocin, gene is expressed in lactobacillus plantarum BM-1 as a purpose, is made
It obtains the bacterial strain and secretes two kinds of bacteriocins simultaneously.After testing, which increases listeria spp and large intestine to single
Bacillus all has the good inhibiting effect recombinant bacterial strain and applies the inflammatory reaction that can inhibit host in feed industry, reduces
The diarrhea rate of piglet, and toxic side effect and residual are not will produce, environment and food security are not threatened.Due to lactobacillus plantarum
It can be colonized in enteron aisle, while it plays prebiotic effect so that two kinds of bacteriocins can use in situ, and bacteriocin
Compound use also widened antimicrobial spectrum, so that fungistatic effect is enhanced so that the application field of the recombinant bacterial strain will more extensively.
Description of the drawings
Original PnisA promoters are substituted in the structure schematic diagram of Fig. 1 recombinant plasmids p118148-ColV, P11 promoters,
Pediocin LP lead peptide for pediocin.
Fig. 2 recombinant plant lactobacillus ferment supernatants are to the inhibiting effect of bacillus coli DH 5 alpha, A：Recombinant bacterial strain supernatant pair
Bacillus coli DH 5 alpha has inhibiting effect, shows colicine V successes；Express B：The control group of empty carrier is transferred to Escherichia coli
DH5 α do not have inhibiting effect.
Fig. 3 is to recombinate lactobacillus plantarum fermented supernatant fluid to single inhibiting effect for increasing listeria spp.A：On recombinant bacterial strain
Clear liquid has inhibiting effect to single listeria spp that increases, and shows that the expression of colicine V does not influence bacterial strain pediocin PA-
1 expression；B：The control group for being transferred to empty carrier has inhibiting effect to single listeria spp that increases.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case of essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The Lactococcus lactis NZ9000 and plasmid pNZ8148 of the present invention is the NICE series of products of Nizo research companies, in MoBi Tec
It can be bought on the website of company, detailed description is《Expression System for Lactococcus
lactis.The effective&easy-to-operate Nisin Controlled gene Expression
system》。
Embodiment 1 carries the structure of the lactic acid bacteria expression vectors of fusion segment
Sequence is obtained from GenBank：Pediocin leads the sequence of peptide gene and ripe colicin V gene, passes through
Codon is transformed and optimization, pediocin of the invention lead peptide gene sequence as shown in SEQ ID NO.2, ripe colicine
V gene sequence is as shown in SEQ ID NO.3.Pediocin is led peptide gene to connect with ripe colicin V gene, after connection
Fusion following sequence is synthesized by biotech firm, total 338bp, (both ends are added to Nco respectively as shown in SEQ ID NO.1
I and Eco R I restriction enzyme sites and protection base) synthesis segment build on pUCK carriers.Use Nco I and Eco R I points
Then other double digestion expression vector p118148 and the pUCK carriers for carrying synthesis segment use DNA ligase to connect overnight, will
Connection product electrotransformation Lactococcus lactis NZ9000 obtains recombinant plasmid by extracting plasmid and sequencing, is named as
p118148-ColV。
Embodiment 2 builds recombinant plant lactobacillus strain
1, Lactococcus lactis NZ9000 competent cells are prepared
With SGM17 fluid nutrient medium culture Lactococcus lactis NZ9000, culture is to OD values to 0.2-0.7 at 30 DEG C.It takes
10ml bacterium solutions collect thalline in 6000rpm, 4 DEG C of centrifugation 8min；The 0.5mol/L sucrose solutions that 2ml precoolings are added (contain 10%
Glycerine) it washes twice, 6000rpm, 4 DEG C of centrifugation 8min are discarded supernatant；With 1% precooling of corresponding cell culture fluid volume
0.5mol/L sucrose solutions (containing 10% glycerine) suspension cell, 40 μ l packing are spare.
2, recombinant plasmid electrotransformation Lactococcus lactis NZ9000
40 μ l of extracting lactic acid galactococcus NZ9000 competent cells, the connection product obtained with 5 μ l embodiments 1 mix, and shift
Into 2mm electricity revolving cups, Bio-Rad Gene Pulser Xcell are usedTM1ml recovery cultures are added after electric shock for type electrotransformation instrument
Base SGM17MC (SGM17,0.5mol/L sucrose, the MgCl of 20mmol/L2, the CaCl of 2mmol/L2) in 30 DEG C of culture 2h, it takes suitable
Amount bacterium solution is coated on the MRS tablets containing chloramphenicol (2.5 μ g/ml), and recon is screened after cultivating 48h in 30 DEG C.
3, the competent cell of lactobacillus plantarum is prepared
With MRSS (MRS, 0.3M sucrose, 1% glycine) medium culture lactobacillus plantarum BM-1, OD600=is grown to
0.4~0.6,10ml bacterium solutions are taken, thalline is collected in 6000rpm, 4 DEG C of centrifugation 8min；2ml Washing buffer are added
2 times, 6000rpm, 4 DEG C centrifugation 8min are resuspended in (0.3M sucrose, 1mM MgCl2), discard supernatant；2ml30%PEG-1500 is added
Thalline, 6000rpm is resuspended, 4 DEG C of centrifugation 10min are discarded supernatant, are resuspended with 200 μ l 30%PEG-1500, and 40 μ l packing are spare.
4, recombinant plasmid electrotransformation lactobacillus plantarum
40 μ l of lactobacillus plantarum BM-1 competent cells are taken, mixes, is transferred to 2 μ l recombinant plasmids p118148-ColV
In 2mm electricity revolving cups, Bio-Rad Gene Pulser Xcell are usedTMType electrotransformation instrument, electrotransformation condition are：1.5kv/cm
9ms.1ml recovery mediums (MRS, 0.3M sucrose, 0.1M MgCl are added after electric shock2) in 37 DEG C of culture 2h, take appropriate bacterium solution to apply
The MRS tablets containing chloramphenicol (5 μ g/ml) are distributed in, recon is screened after cultivating 48h in 37 DEG C.Electrotransformation p118148 simultaneously,
As a control group.
The activity test method of the recombinant plant lactobacillus production colicine V of 3 present invention of embodiment
Recombinant plant lactobacillus made from embodiment 2 and control group bacterial strain (bacterial strain that electrotransformation p118148 is obtained) are existed
Culture takes supernatant to 48h, 6000rpm, 4 DEG C of centrifugations in MRS fluid nutrient mediums.After adjusting supernatant pH to 7.0, TCA methods are utilized
Supernatant is concentrated, is concentrated into 100 times.The use of bacillus coli DH 5 alpha and single listeria spp (ATCC54003) that increases is to refer to
Show bacterium, indicator bacteria was cultivated to logarithmic phase early stage, 100 μ l is taken to be added to the solid of 10ml heating and meltings postcooling to 55 DEG C or so
(listeria spp is TYPE culture mediums in culture medium；Escherichia coli are LB culture mediums), it pours into tablet, waits for after mixing
Oxford cup is put after cooled and solidified on culture medium.Into Oxford cup be added concentration after recombinant bacterial strain and control group bacterial strain it is upper
Clear 100 μ l are stood overnight under the conditions of 4 DEG C, are then placed in 37 DEG C of incubator cultures, are observed the formation of inhibition zone.The result shows that turning
The control group for entering empty carrier does not have inhibiting effect to DH5 α, and recombinant bacterial strain supernatant has inhibiting effect (Fig. 2), antibacterial knot to DH5 α
Fruit shows that colicine V is successfully expressed in lactobacillus plantarum.Recombinant bacterial strain supernatant control strain supernatant pair simultaneously
Single listeria spp that increases has similar inhibiting effect, shows that the expression of colicine V does not influence bacterial strain pediocin PA-
1 expression (Fig. 3).
The present invention has initially picked out 5 antibacterial peptides, respectively pBD-1, Prophenin- from the database of antibacterial peptide
1, PMAP-23, Porcine NK-Lysin, colicinV (colicine V).The above antibacterial peptide is obtained from GenBank
Sequence, codon optimization and pediocin PA-1 lead peptide sequence fusion, design restriction enzyme site, company is transferred to synthesize.Remaining
Experimental procedure is as the above method.Finally pass through the verification of bacteriostatic test, only colicinV successful secretions.Because of antibacterial
The protein structure of peptide can influence its whether can be transported in secretion process albumen transport it is extracellular.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of recombinant vector, which is characterized in that carry pediocin and lead peptide gene and connected with ripe colicin V gene
Made of fusion, the nucleotide sequence of the fusion is as shown in SEQ ID NO.1.
2. the method for preparing recombinant vector described in claim 1, which is characterized in that include the following steps：
(1) pediocin is led peptide gene to connect with the gene of encoding colicin V maturation proteins, synthesis fusion, two
End addition restriction enzyme site, builds on carrier；
(2) carrier for the carrying synthetic gene that double digestion expression vector and step (1) obtain respectively, after digestion, the two is connected；
(3) connection product Electroporation-competent cells extract plasmid, obtain that correct plasmid is sequenced.
3. according to the method described in claim 2, it is characterized in that, step (1) described pediocin leads the sequence of peptide gene such as
Shown in SEQ ID NO.2；The gene order of colicine V maturation proteins is as shown in SEQ ID NO.3；The fusion of synthesis
Sequence as shown in SEQ ID NO.1；The restriction enzyme site of fusion both ends addition is Nco I and Eco R I, and carrier is
pUCK。
4. according to any method of claim 2~3, which is characterized in that the competent cell of step (3) is Lactococcus lactis
The competent cell of bacterium NZ9000.
5. the host cell containing recombinant vector described in claim 1.
6. a kind of recombinant plant lactobacillus of secretion colicine V, which is characterized in that contain recombination described in claim 1
Carrier.
7. recombinant plant lactobacillus as claimed in claim 6, which is characterized in that be prepared by following steps：
(1) it obtains and carries the weight that pediocin leads the fusion that peptide gene is formed by connecting with ripe colicin V gene
Group carrier；
(2) the recombinant vector electrotransformation of step (1) is entered in lactobacillus plantarum BM-1.
8. recombinant vector described in claim 1 or any recombinant plant lactobacillus of claim 6~7 are preparing micro- life
Application in state preparation.
9. the application containing any recombinant plant lactobacillus of claim 6~7 in the food industry, the application is not
Diagnostic and therapeutic method including disease.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101273133A (en) * | 2005-08-30 | 2008-09-24 | 联邦科学工业研究组织 | Bacterial delivery of biologically active polypeptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101273133A (en) * | 2005-08-30 | 2008-09-24 | 联邦科学工业研究组织 | Bacterial delivery of biologically active polypeptides |
Non-Patent Citations (4)
| Title |
|---|
| Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis;Marco J. van Belkum et al.;《Molecular Microbiology》;19971231;第23卷(第6期);全文 * |
| J.M. Rodrıguez et al..Heterologous production of bacteriocins by lactic acid bacteria.《International Journal of Food Microbiology》.2003,全文. * |
| Nisin-Controlled Production of Pediocin PA-1 and Colicin V in Nisin- and Non-Nisin-Producing Lactococcus lactis Strains;Nikki Horn et al.;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20040831;第70卷(第8期);全文 * |
| The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by Gram-positive bacteria;Leiv Sigve Hharstein et al.;《Microbiology》;19941231;第140卷;全文 * |
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