CN104962566A - Recombinant bacteriocin and preparation method and application thereof - Google Patents

Abstract

The invention relates to a recombinant bacteriocin and a preparation method and an application thereof, and belongs to the technical field of biology. A nucleotide sequence of a LacAB gene of the recombinant bacteriocin is shown in an SEQ ID NO:1, and a recombinant expression vector pGEX-4T-1-LacAB comprises the nucleotide sequence shown in the SEQ ID NO:1. Escherichia coli BL21 containing the recombinant expression vector pGEX-4T-1-LacAB is induced by IPTG to express objective protein, and a bacterium solution is collected, recombinant protein in the bacterium solution is extracted; an obtained recombinant protein solution passes through a GST purification column, a GST label is combined with GST rabphilin rab on the column, and purified GST-LacAB protein is obtained through an eluant; the GST label is cut and removed by thrombin, purification is conducted through a GST purification kit, and the single recombinant bacteriocin LacAB is obtained. According to the recombinant bacteriocin and the preparation method and the application thereof, the antimicrobial activity and the stability of the recombinant bacteriocin are good, the antimicrobial spectrum is wide, and the recombinant bacteriocin can serve as an ideal antibiotic succedaneum, a human and animal biological drug, a feed supplement, an antiseptic substance, a disinfection agent and the like.

Description

A kind of recombinant bacteria element and its preparation method and application

Technical field

The present invention relates to a kind of recombinant bacteria element and its preparation method and application, particularly a kind of recombinant bacteria element LacAB and its preparation method and application, belongs to biological technical field.

Background technology

The definition of bacteriocin is propose (Jack RW in nineteen fifty-three by Jocob the earliest, Tagg JR, Ray B.Bacteriocins of gram-positive bacteria.Microbiol Rev, 1995, 59:171-200.), at present, the definition of bacteriocin that document and research institution adopt is: bacterium in metabolic process by the polypeptide with anti-microbial activity that rrna produces, protein or protein mixture, the bacterium nearer to close source relation has restraining effect, can kill or suppress the microorganism in habitat similarly, the bacterial strain producing bacteriocin has self specific immunity and can not murder producing strains (Datta V, Myskowski SM, Kwinn LA, Chiem DN, Varki N, Kansal RG, Kotb M, Nizet V.Mutational analysis of the group A streptococcal operon enc oding streptolysin Sand its virulence role in invasive infection.Mol Microbiol, 2005, 56:681-695, Bowdish, D.M., Davidson, D.J.Hancock, R.E.A re-evaluation of the role of host defencepeptides in mammalian immunity.Curr.Protein Pept.Sci., 2005,6:35 ~ 51, Deegan L.H., Cotter P.D., Hill C., et al.Bacteriocins:Biological tools for bio-preservation andshelf-life extension.International Dairy Journal.4th NIZO Dairy Conference-Prospectsfor Health, Well-being and Safety, 2006,16:1058-1071.).Bacteriocin has diversity, current research is ClassII a and ClassII b (Cotter more widely, P.D., Hill, C., Ross, R.P..Bacteriocins:Developing innate immunity for food.Nature Reviews Microbiology, 2005,3:777-788.), the wherein bacteriocin that is made up of two different peptides of ClassII b bacteriocin, best bacteriostatic activity is in two simultaneous situations of peptide.The pre-synthesis N-end of these two peptides comprises the double-glycine model of 15-30 amino acid leader sequence, and is held two glycine to split by C-, makes bacteriocin transmembrane transport (Ha by specific ABC-translocator ^οvarstein LS, Diep DB, Nes IF.A family of bacteriocin ABC transporterscarry out proteolytic processing of their substrates concomitant with export.MolMicrobiol 1995,16:229-240.).Leader sequence promotes specific ABC-translocator, also has the function keeping bacteriocin activity until bacteriocin secretion.Encode the gene of two preformed genes of peptide and encoding immune albumen at identical operon, and the bacterial strain that immune protein protection produces bacteriocin is not killed (Nissen-MeyerJ, Rogne P, Oppega by non-self bacterial element ^οrd C, Haugen HS, Kristiansen PE.Structure-function relationships ofnon-lanthionine containing peptide (class II) bacteriocins produced by grampositivebacteria.Curr Pharm Biotechnol.2009,10:19-37; Oppega ^οrd C, Rogne P, Emanuelsen L, Kristiansen PE, Fimland G, Nissen-Meyer J.The two-peptide class IIbacteriocins:structure, production, and mode of action.J Mol MicrobiolBiotechnol.2007,13:210-219.).For some bacteriocins, operon also comprises the specific ABC-transporter gene of coding and is called accessory protein.Coding ClassII b bacteriocin gene at least needs 4 different genes: encoding leader sequence gene, specific immunogene, the gene of coding ABC-translocator, accessory protein gene (the Klaenhammer of the apparent formation of bacteriocin, T.R.Genetics of bacteriocins produced by lactic acidbacteria.FEMS Microbiol Rev.1993,12,39-85.).

Because the anti-microbial activity of bacteriocin can suppress foodborne pathogenic bacterium and food spoilage, therefore bacteriocin lab is extensively studied.What the past, we mainly studied is gene, molecular characterization, biosynthetic adjustment, antimicrobial spectrum etc.Find that the output of bacteriocin is a problem merited attention under study for action, can antibiotic substitute as an alternative at medical bacteriocin, can be used as natural antiseptic agent in the food industry.But the output of bacteriocin is very low, study more deep nisin and pediocin, through the optimization of fermenting process, output maximum value is at 100-200mg/L.The abstraction and purification bacteriocin come from fermented supernatant fluid needs a lot of complex steps, loses time, and result protein yield is also very low.Therefore, to study and the bacteriocin recombinant expression system of setting up high stable becomes inevitable choice.In recent years, improved output and the purifying of bacteriocin by some heterologous expression systems, need different hosts to obtain the recombinant protein product of high yield, such as milk-acid bacteria, E.coli and yeast.Escherichia expression system is easy to be stable, but incomplete for the posttranslational modification of recombinant bacteria fibroin, therefore can affect the activity of recombinant bacteria element.

Form existence in the bacteriocin gene major part of expression in escherichia coli with fusion rotein and strengthen its stability.The expression method of fusion rotein holds connection one section of protein sequence be easy at expression in escherichia coli by the N-at bacteriocin gene, and the fusion rotein given expression to can not produce injury to Host Strains, effectively improves the expression level of goal gene.The expression vector of widespread use has the series such as pGEX, pQE and pET, pGEX carrier has the ability of high expression fusion rotein, by the regulation and control of native gene laclq, the expression product of laclq is combined with promotor tac and stops protein expression, and the Insert Fragment therefore in pGEX carrier controls by promotor tac.Thiadiazolidine isomerase (GST) is the conventional fusion protein expression vector that pGEX carrier connects, and molecular weight larger needs, with zymoplasm or the excision of the Xa-factor, obtains target protein.GST can protect recombinant protein not by the degraded of extraneous proteolytic enzyme as albumen identification tag, increases the solubility of target protein, improves the stability expressed.Realize recombinant protein high expression in E. coli system, suitable culture condition (as culture temperature, IPTG concentration, pH, incubation time etc.) is significant for its potential activity of performance.Research finds, intestinal bacteria optimum growth temperature is 37 DEG C, very easily forms inclusion body at this temperature, reduces the expression amount that temperature can increase recombinant protein.Medium component can suppress the too fast growth of thalline, thus reduces the formation volume of inclusion body.But the recombinant protein of non-activity is obtained through escherichia expression system, its reason is the disappearance of the disulfide linkage of recombinant protein in escherichia expression system on the one hand, affect the correct folding of albumen, recombinant protein is lost activity, Zhao Aizhen (2011) expresses Enterocin A recombinant mutant by escherichia expression system, proves that disulfide linkage is the important factor in order of Enterocin A activity.Its activity is affected on the other hand for recombinant bacteria fibroin in E. coli system cannot obtain amidation modification after translation.

Summary of the invention

First object of the present invention is to provide a kind of recombinant bacteria element LacAB gene;

Second object of the present invention is to provide a kind of the recombinant expression vector pGEX-4T-1-LacAB and the construction process thereof that comprise recombinant bacteria element LacAB gene;

3rd object of the present invention is the preparation method providing a kind of recombinant bacteria element LacAB;

4th object of the present invention is the purposes providing a kind of recombinant bacteria element LacAB.

Technical problem to be solved by this invention is achieved through the following technical solutions:

A kind of recombinant bacteria element LacAB gene, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.

Recombinant expression vector pGEX-4T-1-LacAB containing this nucleotide sequence also belongs within protection category of the present invention certainly.And

Host cell containing this recombinant expression vector pGEX-4T-1-LacAB also belongs within protection category of the present invention certainly, and described host cell is intestinal bacteria TOP10, DH5 α and BL21.

Build the method for described recombinant expression vector pGEX-4T-1-LacAB, it is characterized in that, comprise the following steps:

(1) with lactobacterium casei genomic dna for template, design primer amplified obtains two sections of fragments of ClassIIb bacteriocin gene, is respectively ClassIIb-LacA fragment and ClassIIb-LacB fragment;

(2) the bacteriocin gene ClassIIb-LacA sequence obtained according to step (1) and ClassIIb-LacB sequence, the special primer designed with restriction enzyme site adopts overlapping PCR method amplification LacAB recombination sequence, and the LacAB gene product obtained that increases connects with the pMD18-T carrier that same enzyme is cut;

(3) connect product conversion competent escherichia coli cell E.coli TOP10, bacterium colony PCR identifies positive colony, and positive colony, through digestion verification and order-checking, obtains pMD18-T-LacAB recombinant cloning vector;

(4) by pMD18-T-LacAB recombinant cloning vector and expression vector pGEX-4T-1 EcoR I and SalI restriction enzymes double zyme cutting, the digestion products LacAB of pMD18-T-LacAB recombinant cloning vector is cloned on pGEX-4T-1 carrier, build recombinant expression vector pGEX-4T-1-LacAB, recombinant expression vector transformation of E. coli competent cell E.coliTOP10, bacterium colony PCR identifies positive colony, positive colony, through digestion verification and order-checking, obtains pGEX-4T-1-LacAB recombinant expression vector.

In the present invention, preferably, the nucleotide sequence of the Auele Specific Primer of the LacA fragment that increases in step (1) is as shown in SEQ ID NO:2 and SEQ ID NO:3; The nucleotide sequence of the Auele Specific Primer of amplification LacB fragment is as shown in SEQ ID NO:4 and SEQ ID NO:5; Described digestion products LacAB mixes according to mol ratio 3: 1 with pGEX-4T-1 carrier, and 16 DEG C of metal baths spend the night connection.

In the present invention, preferably, the nucleotide sequence of the Auele Specific Primer P1/P2 of the LacA fragment that increases in step (2) is respectively as shown in SEQ ID NO:6 and SEQ ID NO:7; The nucleotide sequence of the Auele Specific Primer P3/P4 of amplification LacB fragment is as shown in SEQ ID NO:8 and SEQ ID NO:9, and 5 ' of the upstream primer P3 of 3 ' of LacA downstream primer P2 end and LacB holds 15 base pair complementarity; The concrete steps of described overlapping PCR method: the first step, respectively with P1/P2, P3/P4 for primer, ClassIIb-LacA gene and ClassIIb-LacB gene are template, carry out LacA and LacB that pcr amplification obtains the sticky end of pairing complimentary to one another; Second step, take P1/P4 as primer, LacA/LacB template, carries out pcr amplification, obtains recombinant bacteria plain gene LacAB.

A kind of method preparing recombinant bacteria element LacAB, it is characterized in that, target protein is induced to express through IPTG the e. coli bl21 containing recombinant expression vector pGEX-4T-1-LacAB, collect bacterium liquid, extract the recombinant protein in bacterium liquid, the recombinant protein solution obtained is by GST purification column, GST rabphilin Rab on GST label and pillar combines, the GST-LacAB albumen of purifying is obtained by elutriant, after zymoplasm excision GST label, use GST Purification Kit, obtain single recombinant bacteria element LacAB.

In the present invention, preferably, specifically comprise the following steps:

(1) abduction delivering of pGEX-4T-1-LacAB recombinant expression vector

By the e. coli bl21 containing recombinant expression vector pGEX-4T-1-LacAB in containing the LB substratum of 100mg/L Amp after incubated overnight, be transferred in the LB liquid nutrient medium containing 100mg/L Amp according to the ratio of 1: 50 (V/V), 2 ~ 3h is cultivated with 200rpm rotating speed, when bacterium liquid OD600 reaches 0.6 ~ 1.0, add IPTG by concentration 0.2 ~ 1.2mmol/L and induce 1 ~ 5h, the centrifugal 10min of 5000rpm, obtains yeast culture thing;

(2) extraction of recombinant protein

Bacterial lysate is added in the thalline collected.The centrifugal 5min of 4 DEG C of cracking 10min, 12000rpm, gets supernatant liquor, and supernatant liquor mixes with 2 × SDS PAGE loading buffer1: 1.With whirlpool device thermal agitation, guarantee precipitation at the bottom of pipe to shake loose, uncap sample on 100 DEG C of constent temperature heaters heating 10min.After sample is cool, the centrifugal 3min of 12000r/min, stand-by or direct loading preserved by-80 DEG C, sample;

Described bacterial lysate contains 50mMTris-HCl (pH8.5 ~ 9.0), 2mMEDTA, 100mMNaCl, 0.5%TritonX-100,1mg/mL N,O-Diacetylmuramidase;

(3) purifying of recombinant protein

Mixed with GSH-Sepharose 4B suspension by sample, room temperature effect 10min, the GST rabphilin Rab on GST label and pillar combines, and collects elutriant, obtains the GST-LacAB albumen of purifying;

(4) enzyme of recombinant protein is cut

Add the zymoplasm normal-temperature reaction 16h of 1 unit by the GST-LacAB albumen of 100ug, make the enzyme degree of cutting reach more than 90%;

(5) purifying of target protein

After recombination fusion protein is cut by zymoplasm, use GST Purification Kit, obtain single recombinant bacteria element LacAB.

In the present invention, preferably, it is characterized in that, IPTG abduction delivering condition is: inducing temperature is 30 DEG C, IPTG concentration is 0.8mmol/L, and the abduction delivering time is 3h, and optimum expression bacterial strain is competence e. coli bl21 expression strain.

The present invention adopts BCA determination of protein concentration kit measurement recombinant bacteria element GST-LacAB protein concn, and obtaining GST-LacAB protein content according to typical curve Equation for Calculating is 2.47mg/mL.The present invention adopts BCA determination of protein concentration kit measurement recombinant bacteria element LacAB protein concn, and target protein content is 1.85mg/mL.

Bacteriostatic activity is analyzed, after purifying, recombinant bacteria element shows the analysis of different sources indicator bacteriostatic activity, lactobacillus casei bacteriocin antimicrobial spectrum is wide, not only to gram-positive microorganism pathogenic bacterium streptococcus aureus, micrococcus luteus, listeria bacteria, there is obvious restraining effect, to part gram negative pathogenic bacteria intestinal bacteria, Salmonellas etc., also there is obvious restraining effect.The stability study of purification of Recombinant bacteriocin shows, recombinant bacteria element has stronger thermostability, acid and alkali-resistance stability, freeze-thawing resistant stability, tensio-active agent (SDS, tween 20, tween-80, urea, triton x-100) are on bacteriocin bacteriostatic activity without impact, and EDTA can strengthen bacteriostatic action.Bacteriocin after purifying is respectively 62.5 μ g/ml and 125 μ g/ml to streptococcus aureus and colibacillary minimal inhibitory concentration.

Therefore, present invention also offers the application of recombinant bacteria element LacAB in preparation antibacterials, antiviral, fodder additives, sanitas or elimination agent that described method prepares.

Compared with prior art, beneficial effect of the present invention is embodied in:

The present invention builds the genetic engineering bacterium of high expression recombinant bacteria element LacAB by genetic engineering technique, improves the output of bacteriocin LacAB; The present invention produces bacteriocin LacAB by gene engineering research, and cost is lower than chemical synthesis; And only need enzyme to cut can to obtain recombinant bacteria element LacAB, purification step is simple, and yield is high; Recombinant bacteria element anti-microbial activity of the present invention and good stability, antimicrobial spectrum is wide, can be used as desirable Substitutes For Antibiotic and people and animals' biomedicinal, fodder additives, sanitas, elimination agent etc.

Accompanying drawing explanation

Fig. 1 is expression vector pGEX-4T-1-LacAB design of graphics;

Fig. 2 is lactobacillus casei bacteriocin gene PCR result figure, wherein, and M:DL2000Marker, 5-negative control, 1,2-lactobacterium casei;

Fig. 3 is LacAB fusion gene PCR schematic diagram;

Fig. 4 is lactobacterium casei LacAB recombinant bacteria plain gene PCR result figure, wherein, and M:DL2000Marker, 1-negative control, 2-lactobacterium casei LacAB gene;

Fig. 5 is that bacterium colony PCR identifies positive colony result figure;

Fig. 6 is that positive colony enzyme cuts qualification result figure, wherein, and the EcroR I of M:DNA Marker, 1:pMD18-T-lacAB recombinant plasmid, Sal I double digestion, the EcroR I single endonuclease digestion of 2:pMD18-T-lacAB recombinant plasmid;

Fig. 7 is that bacterium colony PCR identifies positive colony result figure, wherein, and M:DL2000Marker, 1,2-lactobacillus casei bacteriocin;

Fig. 8 is that positive colony enzyme cuts qualification result figure, M:DL5000Marker, 1, the EcroR I of 2-pGEX-4t-1-LacAB recombinant plasmid, Sal I double digestion, 3, the EcroR I single endonuclease digestion of 4-pGEX-4t-1-LacAB recombinant plasmid;

Fig. 9 is expression product SDS-PAGE qualification result figure, wherein, and M-standard protein, the empty carrier that 1-does not induce, the empty carrier of 2-induction;

Figure 10 is the selection result figure of the best induced concentration of IPTG, wherein, and M-standard protein; 1-is without the sample of induction; 2,3,4,5,6,7-is respectively the sample of inducing through the IPTG of 0.2mmol/L, 0.4mmo/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L concentration;

Figure 11 is the selection result figure of best inducing temperature, wherein, and M-standard protein; 1-is without the sample of induction; 2,3,4,5-is respectively the samples of 21 DEG C, 25 DEG C, 30 DEG C, 37 DEG C inductions.

Figure 12 is the selection result figure of best induction time, wherein, and M-standard protein; 1-is without the sample of induction; 2,3,4,5,6-is respectively the sample of induction 1.0h, 2.0h, 3h, 4h, 5h;

Figure 13 is optimum expression bacteria selection result figure, wherein, and M-standard protein; 1,3,5-is competence e. coli bl21 respectively, and DH5 α and TOP10 is without the sample of induction; 2,4,6-is respectively competence e. coli bl21, and DH5 α and TOP10 is through the sample of induction;

Figure 14 is the purification result figure of restructuring bacteriocin, wherein, and M-standard protein; The induction empty carrier of 1-purifying; 2-purifying do not induce empty carrier; The induction recombinant protein of 3-purifying; 4-purifying do not induce recombinant protein;

Figure 15 is protein concentration canonical plotting;

Figure 16 is LacAB purifying protein SDS-PAGE electrophorogram, wherein, and M-standard protein; The GST-LacAB albumen of 1-purifying; 2-zymoplasm enzyme cuts GST-LacAB albumen; The LacAB of 3-purifying;

Figure 17 is the bacteriostatic activity histogram of restructuring bacteriocin albumen;

To be treatment of different temperature suppress the anti-microbial activity of streptococcus aureus to bacteriocin to Figure 18 affects histogram, and wherein, bore dia is 6.0 ± 0.2mm, CK is untreated bacteriocin;

Figure 19 is that bacteriocin affects histogram on colibacillary bacteriostatic activity after treatment of different temperature, and wherein, bore dia is 6.0 ± 0.2mm, CK is untreated bacteriocin;

Figure 20 is that bacteriocin affects histogram on the bacteriostatic activity of streptococcus aureus after different pH process, and wherein, bore dia is 6.0 ± 0.2mm, CK is untreated bacteriocin;

Figure 21 is that bacteriocin affects histogram on intestinal bacteria bacteriostatic activity under condition of different pH, and wherein, bore dia is 6.0 ± 0.2mm, CK is untreated bacteriocin;

Figure 22 is that bacteriocin affects histogram on streptococcus aureus bacteriostatic activity after the process of different surfaces promoting agent, wherein, C1-SDS, C2-tween 20, C3-tween-80, C4-urea, C5-triton x-100, C6-EDTA, CK-do not add the blank of tensio-active agent;

Figure 23 is that bacteriocin affects histogram on intestinal bacteria antibacterial circle diameter after the process of different surfaces promoting agent, wherein, C1-SDS, C2-tween 20, C3-tween-80, C4-urea, C5-triton x-100, C6-EDTA, CK-do not add the blank of tensio-active agent.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.

The clone of embodiment 1 bacteriocin LacAB gene and Prokaryotic expression vector construction

1 materials and methods

1.1 materials and reagent

PMD18-T carrier for building cloning vector, purchased from precious bio tech ltd, Dalian; PGEX-4T-1 is used for construction of expression vector, purchased from precious bio tech ltd, Dalian.DNA of bacteria extracts test kit, sepharose DNA reclaims test kit all purchased from Beijing Tian Gen biochemical technology company limited; ExTaq, 10 × ExTaqBuffer, DNA Marker and dNTP MTxture are all purchased from the precious biotech firm in Dalian.

1.2 solution preparation

Penbritin: 1g penbritin dry powder is dissolved in 20ml ddH ₂in O, through 0.22m membrane filtration, be sub-packed in sterilizing EP pipe, frozen in-20 DEG C.

Primer dilutes: the sterilizing DEPC water of primer nM/10mL, in the synthesis of invitrogen company, according to the nM number of primer, dilutes by primer, and namely gained primer concentration can be used for PCR reaction.

DEPC water: 1mLDEPC is added 999mL ddH ₂in O, sealing incubator overnight, then 121 DEG C, moist heat sterilization 30min, cool rear 4 DEG C of preservations are stand-by.

1.3 experimental technique

1.3.1 the pcr amplification of lactobacillus casei bacteriocin (classII b) gene

With lactobacterium casei genomic dna for template, according to the bacteriocin gene ClassIIb of the lactobacterium casei of login (accession number of LacA and LacB is respectively wp_003596087.1 and wp_003596088.1) in document and GenBank, application Primer5.0 software designs specificity amplification primer (table 1) to bacteriocin gene ClassIIb, carries out pcr amplification.Primer is synthesized by the Shanghai biological company limited of raw work.After PCR reaction terminates, the PCR primer sepharose of 1% carries out electrophoresis, reclaim test kit with the centrifugal cylindricality plain agar sugar gel DNA of Tian Gen biological reagent company and reclaim object fragment, served the order-checking of marine life engineering company limited, sequencing result carries out blast analysis in NCBI.

Table 1 bacteriocin PCR primer

1.3.2 the clone of bacteriocin gene and Prokaryotic expression vector construction

The present invention obtains bacteriocin gene LacAB sequence by overlapping PCR method, builds cloning vector pMD18-T-LacAB, and then construction of expression vector pGEX-4T-1-LacAB, and Fig. 1 is shown in by carrier cloning collection of illustrative plates.

1.3.2.1 the amplification of bacteriocin recombination LacAB

Overlap extension pcr (gene splicing by overlap extension PCR, be called for short SOE PCR) owing to adopting the primer with spacer end, PCR primer is made to define overlapping chain, thus by the extension of overlapping chain in amplified reaction subsequently, the amplified fragments lap splice of different sources is got up.

According to bacteriocin gene LacA, LacB sequence that PCR reaction obtains, utilize software Primer5.0 to design and adopt overlapping PCR method amplification LacAB recombination sequence with restriction enzyme site special primer.Concrete steps are as follows: in two steps reaction obtain lactobacillus casei bacteriocin (classII b) gene complete sequence.The first step respectively with P1/P2, P3/P4 for primer, ClassIIb-LacA gene and ClassIIb-LacB gene are template, carry out pcr amplification obtain product be Y1 and Y2, carry out glue recovery; Second step, take P1/P4 as primer, Y1/Y2 template, carries out pcr amplification, obtains recombinant bacteria plain gene LacAB.

1) design of primers

According to the restriction enzyme site in multiple clone site on carrier, and object fragment sequence feature, design the primer with restriction enzyme site.Concrete primer is as table 2:

Table 2 lactobacillus casei bacteriocin (class II b) gene PCR amplimer

2) PCR reaction system and PCR reaction conditions as follows:

PCR reaction system is as follows:

The first step PCR reacts

Second step PCR reacts

After PCR reaction terminates, the PCR primer sepharose of 1% carries out electrophoresis, reclaims test kit reclaim object fragment with the centrifugal cylindricality plain agar sugar gel DNA of Tian Gen biological reagent company.

1.3.2.2 the structure of bacteriocin gene cloning vector

The PCR primer reclaimed by purifying and plasmid pMD18-T simple spend the night at 16 DEG C of metal baths and are connected, connect product conversion competent escherichia coli cell (E.coli TOP10), select positive colony and cultivate 16-24h to 5mL liquid state containing 37 DEG C of shaking tables in the LB nutrient solution of Amp, bacterium liquid carries out bacterium liquid PCR to be identified, or upgrading grain carries out enzyme and cuts qualification.

1) bacterium liquid PCR identifies: take P1/P4 as primer, and appropriate bacterium liquid is template, carries out bacterium colony PCR and identifies positive bacterium colony.PCR reaction conditions: 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, and 30 circulations, extend 10min after 72 DEG C.After PCR reaction terminates, the PCR primer sepharose of 1% carries out electrophoresis, detects whether there is object band.

2) enzyme cuts qualification: above walk the bacterium liquid obtained and extract recombinant plasmid, carry out single, double enzyme cut qualification with EcoRI, SalI.

Bacterium liquid PCR qualification and enzyme being cut the qualification positive is sent to Hua Da and carries out DNA sequencing.Check order correct bacterium liquid, enlarged culturing, for subsequent use and positive bacterial classification of preservation for upgrading grain.

1.3.2.3 the structure of bacteriocin gene expression vector

1) recombinant plasmid pMD18-T-LacAB double digestion

The correct recombinant plasmid pMD18-T-LacAB of order-checking is used EcoR I, Sal I restriction enzymes double zyme cutting respectively, carries out agargel electrophoresis, object fragment adopted Agarose Gel DNA Purifica to reclaim.Endonuclease reaction condition is 2h in 37 DEG C of water-baths.

2) expression vector pGEX-4T-1 double digestion

TaKaRa plasmid extraction kit is used to extract plasmid pGEX-4T-1, through EcoR I, SalI restriction enzymes double zyme cutting.

3) connect

Goal gene T4DNA ligase enzyme after being cut by enzyme is connected between EcoR I, SalI restriction enzyme site of expression vector pGEX-4T-1, builds recombinant expression vector, called after pGEX-4T-1-LacAB.The goal gene of double digestion and expression vector to be mixed at 3: 1 in molar ratio, reaction conditions is that 16 DEG C of metal baths spend the night.

4) screening of the conversion of recombinant expression vector, positive recombinant, qualification and order-checking

The conversion of recombinant expression vector, the screening of positive recombinant, qualification and order-checking are with the screening of the conversion of cloning vector, positive recombinant, qualification and order-checking.

2 results

The amplification of 2.1 lactobacillus casei bacteriocins (classII b) gene

With lactobacterium casei genomic dna for template, pcr amplification is carried out according to the primer that the bacteriocin gene ClassIIb of the lactobacterium casei logged in document and GenBank designs, amplify two sections of fragments of ClassIIb bacteriocin gene, clip size is about 234bp, 216bp (Fig. 2) respectively, reclaims test kit reclaim and check order with sepharose DNA.Sequencing result is carried out BLAST analysis on NCBI, and the homology of 71a and 77a of this bacterial strain and Lactobacillus casei is 100%.

The clone of 2.2 bacteriocin gene and the structure of cloning vector

2.2.1 the amplification of bacteriocin recombination LacAB

Utilize overlapping PCR method amplification LacAB recombination sequence.Design the Auele Specific Primer P1/P2 of LacA respectively, the Auele Specific Primer P3/P4 of LacB, wherein the downstream primer of LacA upstream primer P1 and LacB all adds suitable restriction enzyme site, and the 3 ' end of LacA downstream primer P2 holds 15 base pair complementarity with 5 ' of the upstream primer P3 of LacB.PCR process is as follows: the first step, amplifies LacA and LacB of the sticky end being with pairing complimentary to one another with primer P1/P2 and P3/P4 respectively; Second step, dilutes 100 times by the first step PCR primer, and the rear 0.5ul that respectively gets mixes, and with this mixture for template, carries out pcr amplification with primer P1/P4, obtains recombinant bacteria plain gene LacAB sequence (Fig. 3).Pcr amplification result 1% agarose gel electrophoresis detects, as shown in Figure 4, result shows result, and PCR primer has obvious product band at molecules of interest amount place, what this illustrated this Success in Experiment has amplified recombinant bacteria plain gene LacAB, and its nucleotide sequence is as shown in SEQ ID NO:1.

2.2.2 bacterium colony PCR identifies cloning vector pMD18-T-LacAB

By increasing, the fragment obtained is connected on pMD18-T carrier, is transformed into E.coli TOP10.Through restructuring bacterium colony PCR qualification, 1% agarose gel electrophoresis detects, as shown in Figure 5.5, the fusion gene clip size of the fragment that amplifies of 6,9, No. 10 recombinant bacterium liquid and clone is basically identical, is tentatively defined as positive colony.

2.2.3pMD18-T-LacAB digestion verification

Bacterium colony PCR is identified that positive bacterium liquid extracts plasmid, recombinant plasmid pMD18-T-LacAB uses EcroRI single endonuclease digestion and EcroR I and SalI double digestion to verify respectively, result is as Fig. 6, during with EcroR I single endonuclease digestion, band is had at ~ 3200bp (pMD18-T-LacAB carrier lengths adds goal gene length) place, during with EcroR I and SalI double digestion, all there is band at ~ 2700bp (carrier lengths) and ~ 500bp place.Result illustrates, clone is carried pMD18-T-LacAB and successfully constructs.

2.2.4 positive colony order-checking

Positive colony is delivered to the order-checking of Shanghai Sheng Gong biotechnology company limited, sequencing result blast analyzes, and result shows, and the sequence of the sequence that pcr amplification goes out and recombination LacAB is completely the same.This illustrates, the recombinant bacteria element plain gene LacAB amplified without sudden change of this Success in Experiment.

The structure of 2.3 bacteriocin expression vectors

2.3.1 bacterium liquid PCR identifies

By bacteriocin cloning vector pMD18-T-LacAB and expression vector pGEX-4T-1 double digestion, recombinant plasmid pMD18-T-LacAB digestion products is cloned on pGEX-4T-1 carrier, builds recombinant expression vector pGEX-4T-1-LacAB.By recombinant plasmid transformed to E.coli TOP10, coat AMP resistant panel 37 DEG C of incubated overnight, picking list bacterium colony, cultivate in the liquid LB medium adding AMP, 37 DEG C of incubator overnight, identify positive colony with bacterium colony PCR, result shows, and colony PCR amplification goes out about 500bp size fragment (Fig. 7).This illustrates, containing object fragment LacAB gene in the plasmid of proposition.

2.3.2 enzyme cuts qualification

Bacterium colony PCR is identified positive bacterium liquid upgrading grain, by recombinant plasmid EcroR I single endonuclease digestion and EcroR I and the checking of Sal I double digestion, digestion products detects through 1% agarose gel electrophoresis, during with EcroR I single endonuclease digestion, band is had at ~ 4500bp (carrier lengths adds goal gene length) place, with the two enzyme of EcroR I and SalI, there is band (Fig. 8) at ~ 500bp and ~ 4000bp (carrier segments) place, tentatively determine to build recombinant expression vector success.

2.3.3 the order-checking of recombinant plasmid

PCR is verified as the recombinant bacterium liquid of positive colony, delivers to the raw work order-checking in Shanghai.Adopt pGEX-4T-1 carrier universal primer, check order.Sequencing result is carried out blast analysis, and the sequence of sequencing result and recombinant bacteria plain gene LacAB is completely the same.Result confirms, in goal gene success insertion vector, illustrates that expression vector establishment is correct.

The abduction delivering of embodiment 2 bacteriocin and purifying

1 materials and methods

1.1 materials and solution preparation

Competence intestinal bacteria (E.coli BL21), competence intestinal bacteria (E.coli TOP10), competence intestinal bacteria (E.coli DH5 α) are all purchased from precious biological (Dalian) company limited.

Bacterial lysate: 50mMTris-HCl (pH8.5 ~ 9.0), 2mMEDTA, 100mMNaCl, 0.5%TritonX-100,1mg/mL N,O-Diacetylmuramidase.

1.2 method

1.2.1 the abduction delivering of RT-PCR expression vector pGEX-4T-1-LacAB

The RT-PCR expression vector pGEX-4T-1-LacAB transform competent E. coli cell that embodiment 1 builds, (E.coliBL21), through double digestion, picking identifies that correct mono-clonal bacterium colony is in 700ul LB substratum, add 0.7ulAmp (100mg/mL), 37 DEG C of 200r/min shaking tables are cultivated, activated overnight.With 1: 50 ratio (200ul), the overnight culture of activation is added in 10mL LB liquid nutrient medium, add 10uLAmp (100mg/ml), 37 DEG C of 200r/min shaking table enlarged culturing 2h-3h, the OD value of period sampling monitoring bacterium liquid, control bacterium liquid OD600 between 0.6-1.0, with the growth shape state making intestinal bacteria be in the most applicable expression foreign protein.From 10ml enlarged culturing thing, get 3ml bacterium liquid as the blank not adding IPTG, all the other 7ml bacterium liquid add 7ul IPTG (storage concentration is 0.5mol/l).With the rotating speed of 200r/min, 3h cultivated by 37 DEG C of shaking tables.With 5000r/min, 4 DEG C of centrifugal 10min collect thalline, topple over supernatant, and each centrifuge tube collects 3ml culture.Add 1ml dH2O, broken up fully to wash by precipitation vibrator at the bottom of pipe, the centrifugal 2min of 8000r/min, topples over supernatant.Repeat above-mentioned steps.Water in centrifuge tube is clean, and thalline-20 DEG C preserves stand-by or cracking at once, extracts recombinant protein.

1.2.2 the extraction of recombinant protein

1, in the thalline collected, bacterial lysate (thalline of general 1ml bacterium liquid collection adds 50ul bacterial lysate) is added, 4 DEG C of cracking 10min.

2 ,-80 DEG C of multigelations of the sample after cracking three times, ensure the complete cracking of thalline.The centrifugal 5min of 12000r/min, gets supernatant liquor.

3, supernatant liquor mixes with 2 × SDS PAGE loading buffer 1: 1, (can increase according to the amount of precipitation or reduce the amount of loading buffer, general 200ul be proper).With whirlpool device thermal agitation, guarantee precipitation at the bottom of pipe to shake loose.

4, sample is uncapped on 100 DEG C of constent temperature heaters heating 10min (Marker also will heat).After sample is cool, the centrifugal 3min of 12000r/min, stand-by or direct loading preserved by-80 DEG C, sample.The albumen extracted carries out SDS-PAGE and analyzes and coomassie brilliant blue staining analysis.

1.2.3 the optimization of abduction delivering condition

1.2.3.1IPTG the screening of best induced concentration

Fix 37 DEG C, 3h induction time and bacterium liquid OD ₆₀₀the condition of=0.6, respectively at 0.2mmol/L, 0.4mmo/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L IPTG concentration abduction delivering bacterial strain, by the difference of SDS-PAGE electroresis appraisal target protein expression amount, determine best induced concentration.

1.2.3.2 the screening of best inducing temperature

The best IPTG induced concentration selecting back to filter out, fixing 3h induction time and bacterium liquid OD ₆₀₀the condition of=0.6, respectively at 21 DEG C, 25 DEG C, 30 DEG C, 37 DEG C abduction delivering bacterial strains, by the difference of SDS-PAGE electroresis appraisal target protein expression amount, determines best inducing temperature.

1.2.3.3 the screening of best induction time

Select the best IPTG induced concentration filtered out, best inducing temperature and bacterium liquid OD above ₆₀₀the condition of=0.6, respectively at 1h, 2h, 3h, 4h, 5h abduction delivering bacterial strain, by the difference of SDS-PAGE electroresis appraisal target protein expression amount, determines best induction time.

1.2.3.4 the screening of optimum expression bacterial strain

Recombinant expression vector pGEX-4T-1-LacAB is transformed into respectively competence intestinal bacteria TOP10, in DH5 α and BL21, carries out the abduction delivering of recombination fusion protein above with the best IPTG induced concentration, best inducing temperature and the best induction time that screen.By the difference of SDS-PAGE electroresis appraisal target protein expression amount, determine best expression strain.

1.2.4 the purifying of recombinant protein

1, filler GSH-Sepharose 4B is the suspension of 50%, and front overhang is even.Get 90-100 μ L GSH-Sepharose4B suspension in centrifuge tube, low-speed centrifugal, removes supernatant liquor, and it is resuspended then to add PBS.This step is the damping fluid in order to upgrade filler.

2, combine: sample is mixed with GSH-Sepharose 4B suspension, room temperature effect 10min, then centrifugal.

3, loading: suspension is all added on centrifugal column, the centrifugal 1min of 3000rpm.

4, rinsing: add 500 μ L 1X PBS in centrichromatography post, the centrifugal 1min of 3000rpm, repeats once.

5, wash-out: centrichromatography column sleeve enters the 1.5mL centrifuge tube of sterilizing, adds 100 μ L reduced form GSH solution, room temperature effect 10 minutes in chromatography column.Centrifugal 1 minute of 3000rpm.The liquid collecting of centrifuge tube adduction is the sample of wash-out.Repeat elution step 2-3 time, add 100 microlitre reduced form GSH solution at every turn, can yield be increased.

1.2.4.1 recombinant protein is quantitative

The BCA determination of protein concentration test kit of green skies company is adopted to carry out quantitatively the protein after purifying.Micropore microplate reader method is adopted to detect.

1.2.4.2 the enzyme of fusion rotein is cut

In order to improve the stability of exogenous protein expression, usually need to add an extra label construction of fusion protein at the amino of the target protein of expressing or carboxyl terminal, the label that this experiment adds is GST.Employing zymoplasm cut, this is because this enzyme can carry out site-specific cutting to the fusion rotein with come-at-able Thrombin recognition sequence, for digest comprise Thrombin recognition sequence pGEX carrier prepared by GST label protein.At 22 DEG C, in 1 × PBS, can cut 100 μ g GST label protein to be measured during the enzyme 16h of a unit, cutting efficiency is more than 90%.Enzyme cut after solution carry out SDS-PAGE electrophoresis, upper strata glue is configured to 12% separation gel.

1.2.4.3 the purifying of target protein

After recombination fusion protein is cut by zymoplasm, still can use GST Purification Kit, obtain single LacAB albumen.By SDS-PAGE electrophoresis detection purification effect.

1.2.5 target protein anti-microbial activity detects

Recombinant bacteria element anti-microbial activity detects and adopts Oxford cup agar diffusion method to detect recombinant bacteria element bacteriostatic activity, with streptococcus aureus and intestinal bacteria for indicator, this method can rapidly, the anti-microbial activity of visual inspection object product.

2 results

2.1 expression of recombinant plasmid pGEX-4T-1-LacAB in E.coli

Carry out abduction delivering by recombinant plasmid pGEX-4T-1-LacAB Transformed E .coliBL21, make inductor with IPTG, identified by SDS-PAGE gel.Result as shown in Figure 9, the empty carrier of induction increases the protein band of a treaty 27ku, the recombinant protein of induction increases the protein band of a treaty 47ku, just in time empty carrier and target protein sum, and corresponding protein band does not all appear in the empty plasmid of not inducing and recombinant protein, illustrate that fusion rotein GST-LacAB can normally express in E.coli BL21.

The screening of 2.2 abduction delivering conditions

2.2.1IPTG the screening of best induced concentration

Inductor IPTG has genotoxic potential, and expensive, directly affects the expression of protein content in Induction Process, and excessive concentration will produce toxicity to Host Strains, and concentration is too low will affect the expression amount of product.In order to fusion rotein can in intestinal bacteria stably express, need to determine that the best induced concentration of IPTG is on the impact of expressing quantity.As seen from Figure 10, have the protein band of a treaty 47ku to be GST-LacAB through IPTG induction, and do not add IPTG induction not there is corresponding band.When IPTG concentration changes between 0.2mmol/L to 0.8mmol/L, expression of recombinant proteins amount increases gradually, and IPTG concentration is when 0.8mmol/L, and expressing quantity reaches the highest; When IPTG concentration is at 0.8mmol/L to 1.0mmol/L, expressing quantity has a small amount of falling, and when IPTG concentration is at 1.2mmol/L, expressing quantity significantly falls after rise, and therefore the present invention selects best induced concentration to be 0.8mmol/L.

2.2.2 the screening of best inducing temperature

Temperature is the important factor affecting protein expression, can be seen by Figure 11, all occurs expression of recombinant proteins through 0.8mmol/L IPTG induction, does not have corresponding target protein band without what induce.When inducing temperature is 21 DEG C to 30 DEG C, the expression amount of target protein raises with temperature and increases, along with temperature be elevated to 30 DEG C time, target protein expression amount reaches the highest, but along with the continuation of temperature raises, expression amount changes, so determine that best inducing temperature is 30 DEG C in reducing.

2.2.3 the screening of best induction time

After bacteria-induction, the conservative control of induction time can make foreign protein be expressed fully and can not produce excessively induction and cause bacteriolyze.As seen from Figure 12, that does not induce does not have corresponding target protein band, induces recombinant protein all to have expressed, add the object band of a treaty 47ku through 0.8mmol/L IPTG.At first 3 hours of induction, expressing quantity prolongation in time and increasing; When being 3h to induction time, expressing quantity reaches the highest, but again along with the prolongation of induction time, expression amount is on a declining curve, and therefore the present invention determines that the best induction time of IPTG is 3h.

2.2.4 the screening of optimum expression bacterial strain

Object carrier is proceeded to respectively competence intestinal bacteria TOP10, in DH5 α and BL21, carry out the abduction delivering of recombinant protein above with the best inductive condition filtered out, SDS-PAGE electrophoresis detection expression of recombinant proteins amount.As can be seen from Figure 13, in these three kinds of expression strains, the expression all not having obvious recombinant protein of not inducing, the recombinant protein amount expressed in the BL21 bacterial strain after induction is the highest, apparently higher than the inducible protein expression amount of TOP10 and DH5 α.Result illustrates, competence e. coli bl21 is best suited for the expression strain of the recombinant protein abduction delivering in the present invention, and in the present invention, the expression of subsequent experimental recombinant protein is all selected to express in competence e. coli bl21.

To sum up, best IPTG abduction delivering condition is: inducing temperature is 30 DEG C, IPTG concentration is 0.8mmol/L, and the abduction delivering time is 3h, and optimum expression bacterial strain is competence e. coli bl21.

The purifying of 2.3 bacteriocins is with quantitative

2.3.1 the purifying of recombinant bacteria element GST-LacAB

Target protein is induced to express by IPTG, collect bacterium liquid, by solution by GST purification column, GST rabphilin Rab on GST label and pillar combines, the GST-LacAB albumen of purifying is obtained by elutriant, target protein is in the same size is about 47ku with expection, and GST correction (as shown in figure 14) is described.

2.3.2 recombinant bacteria element GST-LacAB's is quantitative

Adopt BCA determination of protein concentration kit measurement protein concn, typical curve equation is: y=1.0834x+0.226, R ²=0.9918 (Figure 15).Purifying protein dilutes 10 times, records OD ₅₆₂=0.2698, obtaining GST-LacAB protein content according to typical curve Equation for Calculating is 2.47mg/mL.

2.3.3 the purifying of target protein LacAB is with quantitative

In order to excise GST label, after GST-LacAB albumen is combined with purification column, add zymoplasm excision GST label, GST continues to be combined on pillar, is eluted by target protein by elutriant from purification column, as shown in figure 16.Swimming lane 2 demonstrates three protein bands after zymoplasm cutting, molecular weight is that the band of 47ku and recombination fusion protein size are basically identical, this may cut not exclusively due to zymoplasm enzyme, there is GST-fusion rotein, illustrates that recombination fusion protein is not cut open completely.Swimming lane 3 cuts the purified target protein band of GST label, and size is about 20ku.Through BCA determination of protein concentration kit measurement protein concn, target protein content 1.85mg/mL.

2.4 target protein activation analysiss

Recombinant bacteria element anti-microbial activity detects and adopts Oxford cup agar diffusion method, and with streptococcus aureus and intestinal bacteria for indicator, result is as shown in table 3.The recombinant bacteria element of purifying is 12.15mm to the antibacterial circle diameter of streptococcus aureus, be 10.57mm to colibacillary antibacterial circle diameter, illustrate that recombinant expressed recombinant bacteria element has obvious bacteriostatic activity, and compare with the bacteriostatic activity of strain fermentation supernatant liquor, there was no significant difference, illustrates that bacteriostatic activity is mainly produced by bacteriocin.

The bacteriostatic activity of table 3 recombinant bacteria fibroin

Adopt blood cell counting plate counting process calculate viable count number show bacteriostatic activity.Experimental result as shown in figure 17, in the lacAB solution group adding purifying bacterium number with add strain fermentation supernatant liquor group without significant difference (p > 0.05), and significantly lower than the control group (p < 0.01) adding sterilized water, this explanation, the recombinant protein solution group adding purifying with add lactobacterium casei fermented supernatant fluid group and all have obvious fungistatic effect, this result is consistent with Oxford cup agar diffusion method result, illustrates that the recombinant expressed bacteriocin of the present invention has good bacteriostatic activity.

Embodiment 3 recombinant bacteria element Antibacterial Property

1 experimental technique

The antimicrobial spectrum of 1.1 bacteriocins detects

1) Oxford cup agar diffusion method

Choosing typical gram positive bacterium-streptococcus aureus, micrococcus luteus, Listeria Monocytogenes, genus bacillus, Lactococcus lactis, faecalis, Bacterium lacticum and gram negative bacterium-intestinal bacteria, pseudomonas and Salmonellas etc. is indicator.Adopt Oxford cup agar diffusion method to observe expression product to the restraining effect (the recombinant bacteria cellulose solution concentration added is 1mg/mL) of bacterium on flat board, measure its anti-microbial activity by antibacterial circle diameter, test parallel 3 times.

2) blood cell counting plate counting process

By indicator 37 DEG C of cultivations in the substratum be applicable to, be cultured to colony number and reach 10 ⁶cfu/mL.Get the indicator suspension of 1ml, adding appropriate is 1mg/mL containing recombinant bacteria cellulose solution to bacteriocin final concentration, and co-cultivation 6h in 37 DEG C of incubators, suitably dilutes bacterium liquid with physiological saline, calculate viable count with blood cell counting plate, tests parallel 3 times.

The minimum inhibitory concentration (MIC) of 1.2 bacteriocins measures

By measuring the concentration (OD of inoculum ₆₀₀) and antibacterial circle diameter determination minimum inhibitory concentration (MIC).Minimum bacteriostatic activity concentration (MIC) refers to the minimum active concentration that indicator can be suppressed to grow.

1.3 temperature are on the impact of bacteriocin activity

Choose streptococcus aureus and intestinal bacteria are indicator.Indicator to be inoculated in fresh NB liquid nutrient medium 37 DEG C by 1%, and 200rpm is cultured to OD ₆₀₀be about 0.5-0.6, get the indicator suspension of 1ml, bacteriocin solution to the bacteriocin final concentration adding purifying is 1mg/mL, respectively 8 DEG C, 15 DEG C, 25 DEG C, 30 DEG C, 37 DEG C cultivations, and 6h, 12h, 18h sampling after cultivation.

With blood cell counting plate counting process bacterial detection element bacteriostatic activity.With do not add bacteriocin for contrast, often group experiment need in triplicate.

The repeated pruning of 1.4 bacteriocins

Test is with streptococcus aureus ATCC25923 and intestinal bacteria ATCC25922 for indicator carries out bacteriostatic test, and with Oxford cup agar diffusion method bacterial detection element bacteriostatic activity, each test need repeat 3 times [142].

1.4.1 temperature is to bacteriocin stability test

(1) prepare 18 aseptic Ep pipes, add the bacteriocin solution after 1mL purifying respectively;

(2) Ep pipe is divided into 3 groups, often organize Ep pipe and be heated to 60 DEG C, 80 DEG C, 100 DEG C, 121 DEG C respectively, heat-up time is respectively 5min, 10min, 20min, 30min, carries out bacteriostatic experiment after cooling, in bacteriostatic test, bacteriocin final concentration is 1mg/mL; Contrast with untreated purification of bacterial element.

1.4.2pH to bacteriocin stability test

(1) with stroke-physiological saline solution and 1mol/L HCl or 1mol/L NaOH prepare pH3.0,4.0,5.0,6.0,7.0,8.0, the diluent of 9.0 and 10.0;

(2) the bacteriocin solution after purifying will be added in the diluent of different pH value, 37 DEG C of incubations 30min, 1h, 2h, 6h, be 6.5 do bacteriostatic test by aseptic HCl solution and NaOH solution adjustment sample pH value, in bacteriostatic test, bacteriocin final concentration is 1mg/mL; With untreated purification of bacterial element for contrast.

1.4.3 tensio-active agent is to bacteriocin stability test

(1) get the bacteriocin (1mL) of equivalent purifying, add SDS, tween 20, tween-80, urea, triton x-100, EDTA that final concentration is 1mg/mL respectively;

(2) by add tensio-active agent bacteriocin 37 DEG C at incubation 2h, do bacteriostatic test, in bacteriostatic test, bacteriocin final concentration is 1mg/mL; Contrast with the bacteriocin not adding tensio-active agent.

1.4.4 multigelation is to bacteriocin stability test

Recombinant bacteria cellulose solution after purifying is melted sampling after-80 DEG C of cryopreservation 10min, gets 100 μ L samples, suitably dilute, carry out bacteriostatic test, remaining sample melts sampling after continuing freezen protective 10min, still gets 100 μ L samples, suitably dilute, carry out bacteriostatic test; As stated above after multigelation 6 times sampling and measuring number of freezing and thawing on the impact of bacteriocin bacteriostatic activity.In bacteriostatic test, bacteriocin final concentration is 1mg/mL.

2 results

The antimicrobial spectrum of 2.1 bacteriocins

Lactobacillus casei bacteriocin LacAB anti-microbial activity after employing agar diffusion method detection purifying.The present invention lists the indicator of 20 kinds of different strains altogether, comprises gram-positive microorganism, Gram-negative bacteria, and do bacteriostatic test with the bacteriocin protein solution after purifying, test-results is in table 4.

Bacteriocin after result shows purifying can effectively suppress multiple Gram-positive pathogenic bacterium and food spoilage, comprises streptococcus aureus, Listeria monocytogenes, micrococcus luteus etc.Lactobacillus casei bacteriocin has restraining effect in various degree to 18 strains in 20 strain indicators, not only has stronger restraining effect to pathogenic gram-positive bacterial, also has certain fungistatic effect to the gram negative bacterium such as intestinal bacteria, Salmonellas.Antimicrobial spectrum comprises most of gram-positive microorganism, part Gram-negative bacteria, belongs to broad spectrum bacteriocin.More weak to the restraining effect of milk-acid bacteria, obvious inhibition zone is not produced to Lactobacterium acidophilum, short lactobacillus.The most obvious to gram-positive microorganism streptococcus aureus and Gram-negative bacteria intestinal bacteria fungistatic effect, antibacterial circle diameter reaches more than 15mm.

Table 4 bacteriocin LacAB antimicrobial spectrum

Note: bore dia is that 6.0 ± 0.2mm adds 100 μ L LacAB, ++ represent fungistatic effect obvious, antibacterial circle diameter is greater than 15mm; + representative has fungistatic effect, antibacterial circle diameter 10-15mm;-represent without obvious antibacterial circle diameter, without fungistatic effect.

Adopt the bacteriostatic activity of blood cell counting plate bacterium counting number testing inspection recombinant bacteria element.Joined respectively by the bacteriocin of purifying in the bacterial suspension of above-mentioned 20 kinds of indicators, bacteriocin final concentration is 1mg/mL, after 37 DEG C of effect 6h, suitably dilutes bacterial suspension, blood cell counting plate count detection viable count.The results are shown in Table 5 after effect.

Table 5 bacteriocin LacAB is to the anti-microbial effect of bacterium

Note: Jun Shuo unit is CFU/mL.

As can be seen from Table 5, after process 6h, staphylococcus aureus colony number compared with control group decreases about 91%, intestinal bacteria colony number compared with control group decreases about 90%, except Lactobacterium acidophilum and short lactobacillus, other bacterial strain colony numbers also have remarkable minimizing, and reduction is all greater than 70%, and Lactobacterium acidophilum and short lactobacillus colony number compared with control group does not have noticeable change, cytometry is consistent with Oxford cup agar diffusion test result.

The minimum inhibitory concentration (MIC) of 2.2 bacteriocins

The mensuration of purification of bacterial element minimum inhibitory concentration is with streptococcus aureus and intestinal bacteria for indicator, detects the bacterium liquid OD of different bacterium element concentration ₆₀₀, see the following form shown in (table 6).

When gram-positive microorganism streptococcus aureus is indicator, bacteriocin concentration changes between 1000-62.5 μ g/mL, the OD of bacterium liquid ₆₀₀with the OD of control group ₆₀₀without considerable change, difference remarkable (p > 0.05), when concentration continues to reduce, the OD of bacterium liquid ₆₀₀change obviously compared with control group, (p < 0.01) significant difference.When showing that streptococcus aureus is indicator, bacteriocin minimum inhibitory concentration (MIC) is 62.5 μ g/ml.

When Gram-negative bacteria intestinal bacteria are indicator, bacteriocin concentration changes between 1000-125 μ g/ml, the OD of bacterium liquid ₆₀₀with the OD of control group ₆₀₀without considerable change (p > 0.05), when concentration continues to reduce, the OD of bacterium liquid ₆₀₀obviously (p < 0.01), when showing that intestinal bacteria are indicator, bacteriocin minimum inhibitory concentration (MIC) is 125 μ g/mL in change compared with control group.

Table 6 bacteriocin is to streptococcus aureus and intestinal bacteria minimum inhibitory concentration

Note: different letters represents significant difference (p < 0.01), identical letter represents difference not significantly (p > 0.05).

2.3 temperature are on the impact of bacteriostatic activity

As shown in Table 7, under 8 DEG C, 15 DEG C, 25 DEG C, 30 DEG C conditions, experimental group bacterium digital display work compared with control group reduces, and illustrates that the element of the recombinant bacteria after purifying has obvious bacteriostatic action to streptococcus aureus.In the identical treatment time, the experimental group bacterium number difference of differing temps not significantly (p > 0.05), shows that recombinant bacteria element bacteriostatic activity is not by the impact of experimental temperature; The different treatment time, at identical temperature, experimental group is compared with control group, and bacterium colony significant difference (p < 0.05), illustrates that recombinant bacteria element suppresses the growth of streptococcus aureus.Experimental result shows, recombinant bacteria element all has obvious bacteriostatic activity when differing temps, is temperature-insensitive type bacteriocin.

2.4 bacteriocin stability studies

2.4.1 temperature is to bacteriocin stability test

The bacteriocin of purifying keeps 5min, 10min, 20min, 30min at 60 DEG C, 80 DEG C, 100 DEG C, 121 DEG C respectively, carries out bacteriostatic test, by Oxford cup agar diffusion test determination temperature on the impact of bacteriocin activity after cooling.With the bacteriocin do not heated for contrast.

As seen from Figure 18, during with gram positive bacterium streptococcus aureus for indicator, the bacteriocin of purifying within 60 DEG C of heating 30min, bacteriostatic activity compared with control group without considerable change; Almost unchanged at 80 DEG C of heating 5min, when being heated to 30min, bacteriostatic activity decreases, but difference is not obvious; Along with the continuation of temperature raises, the continuous prolongation in treatment time, the bacteriostatic activity of recombinant bacteria element reduces, at 100 DEG C and 121 DEG C of heating 5min, bacteriostatic activity change is without significant difference (p > 0.05), but at 121 DEG C of heating 10min, bacteriostatic activity significantly reduces (p < 0.01), when being heated to 30min, bacteriostatic activity have lost 28% and 35% respectively, but still there is certain bacteriostatic activity, illustrate that recombinant bacteria element has good thermotolerance.

Figure 19 can find out, during with Gram-negative bacteria intestinal bacteria for indicator, the bacteriocin of purifying within 60 DEG C of heating 30min, experimental group compared with control group bacteriostatic activity without considerable change; Almost unchanged at 80 DEG C of heating 5min, when being heated to 30min, bacteriostatic activity decreases, but difference is not significantly (p > 0.05); At 100 DEG C and 121 DEG C of heating 5min, bacteriostatic activity change is without significant difference, and along with the prolongation of heat-up time, bacteriostatic activity significantly reduces, and during heating 30min, bacteriostatic activity have lost 32% and 38% (p < 0.01) respectively.Bacteriocin is consistent with to the variation tendency of streptococcus aureus bacteriostatic activity to the variation tendency of intestinal bacteria bacteriostatic activity.

The result explanation of comprehensive Figure 18 and 19, purification of bacterial element has good thermostability, through differing temps and the process of heat-up time, certain bacteriostatic activity is still kept to gram positive bacterium streptococcus aureus and gram negative bacterium intestinal bacteria, and after 100 DEG C of process 30min, antibacterial circle diameter all arrives more than 11mm, illustrate that bacteriocin still has bacteriostatic action, after 121 DEG C of process 30min, antibacterial circle diameter is significant difference (p < 0.01) compared with control group, and fungistatic effect is affected.

2.4.2pH to bacteriocin stability test

With 1mol/L HCl, 1mol/L NaOH, the bacteriocin solution of purifying is adjusted pH3 ~ 10, adjust pH 6.5 to do bacteriostatic experiment at 37 DEG C after incubation 30min, 1h, 2h, 6h, determine the impact of pH on bacteriostatic activity.

Figure 20 result shows, during using streptococcus aureus as indicator, purification of bacterial bacteriocin processes 2h under the condition of pH3 ~ 7, bacteriostatic activity is without significantly sacrificing compared with control group, and under pH > 7 condition, bacteriostatic activity is along with pH value increase and the prolongation in treatment time, bacteriostatic activity is obvious downtrending, as pH10 condition process 6h, bacteriostatic activity loss 36%.Illustrate thus, the bacteriocin of purifying is unstable in the basic conditions, alkaline purification suppresses the fungistatic effect impact of streptococcus aureus significantly to it, relatively stable under acidic or neutral conditions, and acid or neutral solution process suppress the fungistatic effect of streptococcus aureus not make significant difference to it.

Figure 21 result shows, during with intestinal bacteria as indicator, purification of bacterial element processes 1h under the condition of pH3 ~ 7, bacteriostatic activity is without considerable change compared with control group, when after process 6h, bacteriostatic activity slightly loses, but on the impact remarkable (p > 0.05) of bacteriostatic activity, and under pH > 7 condition, bacteriostatic activity significantly reduces (p < 0.01), under pH10 condition, process 30min, 1h, 2h, 6h bacteriostatic activity have lost 31% respectively, 41%, 46%, 49%, fungistatic effect is lost obviously, make bacteriocin inactivation.Illustrate thus, the bacteriocin of purifying is unstable in the basic conditions, alkaline purification suppresses colibacillary fungistatic effect to affect obviously on it, and relatively stable under acidic or neutral conditions, acid or neutral solution process suppress colibacillary fungistatic effect not make significant difference to it.

Shown by Figure 20 and Figure 21, through different pH process, purification of bacterial element has a certain impact to suppression streptococcus aureus and colibacillary bacteriostatic activity, wherein, acid and neutral pH process does not make significant difference to its bacteriostatic activity, and alkaline pH process can reduce its bacteriostatic activity to a certain extent.This illustrates, the bacteriocin of purifying is more relatively stable than in the basic conditions under acidity and alkaline condition.

2.4.3 tensio-active agent is to bacteriocin stability test

The bacteriocin of purifying adds SDS, tween 20, tween-80, urea, triton x-100, EDTA that final concentration is 1mg/mL respectively, incubation 2h process under 37 DEG C of conditions, then do Oxford cup agar diffusion test, determine the impact of various tensio-active agent on bacteriocin bacteriostatic activity.

As seen from Figure 22, after purification of bacterial element adds tween 20, tween-80, urea, triton x-100 process, to the bacteriostatic activity of streptococcus aureus without considerable change (p > 0.05), after adding SDS process, the antibacterial circle diameter of bacteriocin becomes large, but without significant difference, after adding EDTA process, bacteriostatic activity obviously strengthens (p < 0.01), illustrates that EDTA can strengthen the bacteriostatic activity of bacteriocin.

As seen from Figure 23, purification of bacterial element to add after SDS, tween 20, tween-80, urea, triton x-100 process compared with control group, to intestinal bacteria bacteriostatic activity without considerable change (p > 0.05), after adding EDTA process, bacteriostatic activity obviously strengthens (p < 0.01), illustrate that EDTA has the effect strengthening bacteriostatic activity, especially particularly remarkable to intestinal bacteria effect, this may be that gram negative bacterium is relevant with intestinal bacteria.The lipopolysaccharides of EDTA release can effectively penetrate Gram-negative bacteria adventitia, and release bacteriocin arrives cytolemma, can effectively suppress or kill Gram-negative bacteria.

2.4.4 multigelation is to bacteriocin stability test

By the bacteriocin of purifying under-80 DEG C of conditions, multigelation does bacteriostatic experiment 6 times, to determine the impact of multigelation on bacteriocin bacteriostatic activity.Not add bacteriocin group for negative control.As can be seen from Table 8, purification of bacterial element freeze thawing first 4 times almost free of losses is changed to colibacillary bacteriostatic activity, loss is had a little to the bacteriostatic activity of streptococcus aureus, but do not affect fungistatic effect, when number of freezing and thawing being increased to 6 times, bacteriocin declines to some extent with intestinal bacteria bacteriostatic activity to streptococcus aureus compared with control group, but is not all a greater impact.Illustrate that the bacteriocin multigelation after purifying is to bacteriostatic activity no significant difference (p > 0.05).

Table 8 multigelation is on the impact of bacteriocin activity

Note: bore dia is 6.0 ± 0.2; CK represents unprocessed bacteriocin.

Last it is noted that the foregoing is only preferred embodiment of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. a recombinant bacteria element LacAB gene, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.

2. a recombinant expression vector pGEX-4T-1-LacAB, is characterized in that, it comprises nucleotide sequence according to claim 1.

3. build the method for recombinant expression vector pGEX-4T-1-LacAB described in claim 2, it is characterized in that, comprise the following steps:

(1) with lactobacterium casei genomic dna for template, design primer amplified obtains two sections of fragments of ClassIIb bacteriocin gene, is respectively ClassIIb-LacA fragment and ClassIIb-LacB fragment;

(2) the bacteriocin gene ClassIIb-LacA sequence obtained according to step (1) and ClassIIb-LacB sequence, the special primer designed with restriction enzyme site adopts overlapping PCR method amplification LacAB recombination sequence, and the LacAB gene product obtained that increases connects with the pMD18-T carrier that same enzyme is cut;

(3) connect product conversion competent escherichia coli cell E.coli TOP10, bacterium colony PCR identifies positive colony, and positive colony, through digestion verification and order-checking, obtains pMD18-T-LacAB recombinant cloning vector;

(4) by pMD18-T-LacAB recombinant cloning vector and expression vector pGEX-4T-1 EcoR I and Sal I restriction enzymes double zyme cutting, the digestion products LacAB of pMD18-T-LacAB recombinant cloning vector is cloned on pGEX-4T-1 carrier, build recombinant expression vector pGEX-4T-1-LacAB, recombinant expression vector transformation of E. coli competent cell E.coli TOP10, bacterium colony PCR identifies positive colony, positive colony, through digestion verification and order-checking, obtains pGEX-4T-1-LacAB recombinant expression vector.

4. method according to claim 3, is characterized in that, in step (1), the nucleotide sequence of the Auele Specific Primer of amplification LacA fragment is as shown in SEQ ID NO:2 and SEQ ID NO:3; The nucleotide sequence of the Auele Specific Primer of amplification LacB fragment is as shown in SEQ ID NO:4 and SEQ ID NO:5; Described digestion products LacAB mixes according to mol ratio 3:1 with pGEX-4T-1 carrier, and 16 DEG C of metal baths spend the night connection.

5. method according to claim 3, is characterized in that, in step (2), the nucleotide sequence of the Auele Specific Primer P1/P2 of amplification LacA fragment is respectively as shown in SEQ ID NO:6 and SEQ ID NO:7; The nucleotide sequence of the Auele Specific Primer P3/P4 of amplification LacB fragment is as shown in SEQ ID NO:8 and SEQ ID NO:9, and 5 ' of the upstream primer P3 of 3 ' of LacA downstream primer P2 end and LacB holds 15 base pair complementarity; The concrete steps of described overlapping PCR method: the first step, respectively with P1/P2, P3/P4 for primer, ClassIIb-LacA gene and ClassIIb-LacB gene are template, carry out LacA and LacB that pcr amplification obtains the sticky end of pairing complimentary to one another; Second step, take P1/P4 as primer, LacA/LacB template, carries out pcr amplification, obtains recombinant bacteria plain gene LacAB.

6. the host cell containing recombinant expression vector pGEX-4T-1-LacAB, it is characterized in that, described host cell is intestinal bacteria TOP10, DH5 α and BL21.

7. prepare the method for recombinant bacteria element LacAB for one kind, it is characterized in that, induce target protein to express through IPTG e. coli bl21 described in claim 6, collect bacterium liquid, extract the recombinant protein in bacterium liquid, the recombinant protein solution obtained is by GST purification column, GST rabphilin Rab on GST label and pillar combines, and is obtained the GST-LacAB albumen of purifying by elutriant, after zymoplasm excision GST label, use GST Purification Kit, obtain single recombinant bacteria element LacAB.

8. method according to claim 7, is characterized in that, specifically comprises the following steps:

(1) abduction delivering of pGEX-4T-1-LacAB recombinant expression vector

By e. coli bl21 described in claim 6 in containing the LB substratum of 100mg/L Amp after incubated overnight, be transferred in the LB liquid nutrient medium containing 100mg/L Amp according to the ratio of 1:50 (V/V), 2 ~ 3h is cultivated with 200rpm rotating speed, when bacterium liquid OD600 reaches 0.6 ~ 1.0, add IPTG by concentration 0.2 ~ 1.2mmol/L and induce 1 ~ 5h, the centrifugal 10min of 5000rpm, obtains yeast culture thing;

(2) extraction of recombinant protein

Bacterial lysate is added in the thalline collected.The centrifugal 5min of 4 DEG C of cracking 10min, 12000rpm, gets supernatant liquor, and supernatant liquor mixes with 2 × SDS PAGE loading buffer 1:1.With whirlpool device thermal agitation, guarantee precipitation at the bottom of pipe to shake loose, uncap sample on 100 DEG C of constent temperature heaters heating 10min.After sample is cool, the centrifugal 3min of 12000r/min, stand-by or direct loading preserved by-80 DEG C, sample;

Described bacterial lysate contains 50mMTris-HCl (pH8.5 ~ 9.0), 2mMEDTA, 100mMNaCl, 0.5%TritonX-100,1mg/mL N,O-Diacetylmuramidase;

(3) purifying of recombinant protein

Mixed with GSH-Sepharose 4B suspension by sample, room temperature effect 10min, the GST rabphilin Rab on GST label and pillar combines, and collects elutriant, obtains the GST-LacAB albumen of purifying;

(4) enzyme of recombinant protein is cut

Add the zymoplasm normal-temperature reaction 16h of 1 unit by the GST-LacAB albumen of 100ug, make the enzyme degree of cutting reach more than 90%;

(5) purifying of target protein

After recombination fusion protein is cut by zymoplasm, use GST Purification Kit, obtain single recombinant bacteria element LacAB.

9. the method according to claim 7 or 8, is characterized in that, IPTG abduction delivering condition is: inducing temperature is 30 DEG C, IPTG concentration is 0.8mmol/L, and the abduction delivering time is 3h, and optimum expression bacterial strain is competence e. coli bl21 expression strain.

10. the application of recombinant bacteria element LacAB in preparation antibacterials, antiviral, fodder additives, sanitas or elimination agent that described in any one of claim 7-9, method prepares.