CN110016456A - Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method - Google Patents
Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method Download PDFInfo
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Abstract
The present invention relates to a kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation methods, the sodium selenite of toxicity can be converted to nontoxic red elemental nano-selenium, and the nanometer selenium of synthesis is enriched in intracellular probiotics-Lactococcus lactis Lactococcus lactis NZ9000.The invention also discloses the methods for preparing multi-functional compound micro-ecological preparation SeNPs-rVIP-L.lactis NZ9000 based on Lactococcus lactis Lactococcus lactis NZ9000, solve that existing selenium-supply additive toxic effect is strong, bioavailability is low, easily causes the problems such as environmental pollution and existing production technology process are complicated, the period is long, production cost is high problem and antibiotic residue, drug resistance.
Description
Technical field
The invention belongs to bioengineering and technical field of nanometer material preparation, are related to a kind of multi-functional compound micro-ecological preparation
Nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method.
Background technique
Excessive use of feeding antibiotic derives medicament residue and the big severe problem of drug resistance two, therefore reduces even
Forbid the use of antibiotic and find suitable substitute to have arrived very urgent stage.In recent years, many countries in the world
Corresponding policy has been put into effect to control the use of antibiotic.But it since the fast development of cultivation industry, cultivation density increase, supports
The risk for growing animal diseases morbidity improves, and is badly in need of the novel green feedstuff additive product of alternative antibiotic.Even to this day,
Green, safety and novel green feed addictive --- the probiotics micro-ecological formulation of noresidue improve host intestine bacterium with it
Group's balance promotes digestion and absorption, strengthen immunity and other effects to rank among the ranks of feed additive for promoting growth gradually to replace
For the use of antibiotic.With going deep into for feeding micro-ecological preparation research and development of products, modern biotechnology is promoting probiotics
Theory and application study in terms of play an important role.
Animal derived antibacterial peptide has no toxic side effect, be difficult to generate drug resistance, noresidue and pollution-free etc., numerous advantage,
The needs for meeting livestock product safety production, are suitble to use in Feed Manufacturing, have as the latent of feed addictive of new generation
Matter.This seminar early-stage study shows the neu- roimmunomodulation peptide VIP (Vasoactive for being under the jurisdiction of antibacterial peptide family member
Intestinal peptide) have exploitation for the advantage and potentiality of disease-prevention health feed addictive: (1) structure simply clear；
(2) there is the various biologicals such as antibacterial, anti-inflammatory, immunological regulation activity；(3) it is used as endogenous gastrointestinal hormone class antibacterial peptide, with
Lesser activity (nmoL grades) can play stronger bioactivity.The characteristic that VIP itself has makes it have to develop to be
Efficiently, the potentiality and wide application prospect of safe disease-prevention health feed addictive.However, extracting VIP cost from tissue
Height, pick-up rate is low, process is tedious, and prices are rather stiff for chemical synthesis, it is difficult to be applied to production.Therefore, it explores and establishes utilization
Probiotics-Lactococcus lactis biosynthesis antibacterial peptide VIP new technology, makes the way of emerging feed and feed addictive
Diameter is more real, and shows good prospect.
Selenium is microelement necessary to humans and animals, closely related with body health.However, at present in actual production
As, mainly using inorganic selenium-sodium selenite as addition form, toxic effect is strong, bioavailability is low and easily makes in animal and fowl fodder
At environmental pollution.In addition, the dosage of inorganic selenium is difficult to grasp, young animal is more sensitive to selenium, easily leads to poisoning.Cause
This, the Dietary Selenium class biological agent of exploitation efficiently, green is the task of top priority.Nanometer selenium (SeNPs) is a kind of using nanotechnology
The novel bioactive substance being prepared.The granularity of nanometer selenium is superfine, is easily directly absorbed and makes full use of by animal gastrointestinal tract,
Its function can be played to a greater extent.Nanometer selenium be it has been found that acute toxicity is minimum, the lesser selenium-replenishing preparation of environmental pollution.
Numerous characteristics and advantages of nanometer selenium will make it have wide development and application prospect.There is life by carrier synthesis of probiotics
The nanometer selenium (SeNPs) of object function is a kind of green, efficient biological pathway.
Summary of the invention
Technical problems to be solved
In order to avoid the shortcomings of the prior art, the present invention proposes a kind of multi-functional compound micro-ecological preparation nanometer selenium-
Vasoactive intestinal peptide-Lactococcus lactis and preparation method are recombinantly expressed, by probiotics, antibacterial peptide and the organic knot of nanometer selenium three
It closes, establishes the preparation method of multi-functional compound micro-ecological preparation SeNPs-rVIP-L.lactic NZ9000 a kind of.To solve
That there are toxic effects is strong, water-soluble for selenium additive in the problem of existing antibiotic usage and existing food and feed
Property is poor, bioavailability is low, environmental pollution is big and existing technology for producing process complexity, high production cost, exists not
The problems such as safety factor.
Technical solution
A kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis, feature
It is: name are as follows: SeNPs-rVIP-Lactococcus lactis NZ9000, abbreviation are as follows: SeNPs-rVIP-L.lactis
NZ9000；Biological expression are as follows: probiotics L.lactis NZ9000 can convert the sodium selenite of toxicity to nontoxic red
Elemental nano-selenium SeNPs, and nanometer selenium is enriched in thallus；The extracellular antibacterial peptide VIP containing recombinant secretor expression, it is intracellular
It is enriched with the Lactococcus lactis probiotics of nanometer selenium SeNPs.
The nanometer selenium is enriched in L.lactis NZ9000 somatic cells, and the partial size of nanometer selenium is 50nm-180nm.
A kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis
Preparation method, it is characterised in that steps are as follows:
Step 1, antibacterial peptide VIP recombinant secretor expression vector building:
It designs VIP gene order: introducing signal peptide sequence SPusp45, His label and restriction enzyme enzyme recognition site；Institute
Stating restriction endonuclease is Nco I and Kpn I；
It constructs the recombinant secretor expression vector of antibacterial peptide VIP: antibacterial peptide VIP gene and carrier pNZ8148 is carried out respectively
Then plasmid pNZ8148 and VIP gene after digestion is attached, target gene is building up to expression vector by double digestion
On, it converts after obtaining the recombinant expression plasmid rVIP-pNZ8148 of VIP to MC1061 competent cell；
Construction step: when using double digestion connection method building recombinant secretor expression vector, by foreign gene and plasmid mole
Number is than being that the ratio of 3:1 is mixed, and reaction system is 10 μ L, and 16 DEG C overnight, conversion to MC1061 competent cell: process
Are as follows: the MC1061 competent cell prepared is immediately placed in ice-water bath；10 μ L linked systems are added to competent cell
In, ice bath 20min；42 DEG C of water-bath pulse 30s, are quickly moved in ice-water bath and place 3min, and 1mL LB liquid medium is added,
37 DEG C, 200 ± 10rpm shaking table culture 45min；Part bacterium solution is taken to be coated on LB plate；12-16h is cultivated in 37 DEG C of inversions；It is described
LB plate is upper containing 34 μ g/ml chloramphenicol；
The preparation of the MC1061 competent cell: 5mL SOB culture medium, 37 DEG C, 200 are added in 1 monoclonal of picking
± 10rpm was cultivated to the exponential growth later period；Take 2mL bacterium solution be added 100mL SOB culture medium in, in 18 DEG C, 200rpm cultivate to
OD600nm=0.55, it is placed in 10min on ice；2500 ± 100g is centrifuged 10min under the conditions of 4 DEG C, collects thallus, abandons supernatant, complete
It is complete to remove water droplet, the pre-cooling working solution of 1/3 volume is added, with liquid-transfering gun suspension bacteria liquid；Under the conditions of 4 DEG C 2500 ± 100g from
Heart 10min collects thallus, abandons supernatant, sucks water droplet completely；1/12 volume working solution, suspension thalline is added；It is gradually added into
DMSO is simultaneously mixed, and places 10min on ice；Packing enters in 1.5ml sterile centrifugation tube, and liquid nitrogen carries out subsequent transformation after placing 1h；
The additional amount of the DMSO are as follows: 0.3ml/50mL bacterium solution；
Step 2, the selenium-rich inducing expression of antibacterial peptide VIP:
Recombinant plasmid rVIP-pNZ8148 electricity is gone into L.lactis NZ9000 competent cell, obtains VIP-
PNZ8148/ L.lactis NZ9000 recombinant bacterium, specific electricity shifting method are as follows: by recombinant plasmid rVIP-pNZ8148 with
The competent cell of L.lactis NZ9000 mixes, ice bath 5min；Mixture is transferred in the 2mm electrotransformation cup of sterile pre-cooling；
It shocks by electricity under 2.0kV, 186 Ω；The pre-cooling of 1ml ice is added later contains 3% glycerol, 5% sucrose, 20mM magnesium chloride and 2mM chlorine
Change 30 DEG C of stationary culture 2h in the M17 culture medium of calcium；100 μ L bacterium solutions are taken to be coated on the M17 agar containing 10 μ g/mL chloramphenicol
On culture medium, 30 DEG C of culture 48h；The M17 culture that recombinant bacterium is seeded to 20mL is stayed overnight based on 30 DEG C of stationary cultures, and sub- selenium is added
Sour sodium Na2SeO3 cultivates 11~13h；It takes part culture solution to be inoculated in the fresh M17 of 50mL respectively according to 4% ratio to cultivate
In base, in 30 DEG C of stationary cultures, as absorbance OD600nmWhen reaching 0.4 or so, inducer nisin induction is added into culture medium
2.5h；After inducing expression, entire fermentation liquid is freeze-dried, as probiotics SeNPs-rVIP-L.lactis
NZ9000；The addition sodium selenite Na2SeO3200 μ g/mL of final concentration；The final concentration that inducer nisin is added
100ng/mL；
Step 3, recombinant expression antibacterial peptide VIP are isolated and purified:
Culture solution after inducing expression is collected supernatant A, is used in combination after 4 DEG C, 12000 ± 1000rpm centrifugation 5min
The PBS of 20mmol/L cleans thallus, is centrifuged 5min in 4 DEG C, 12000 ± 1000rpm, and repeated washing 1~2 time；Then to thallus
Middle addition lysozyme is incubated for 1h in 37 DEG C of water-baths after mixing well, and reaction process is mixed by inversion for several times, and supernatant is collected after centrifugation
Liquid B；It separated, purified through His-Trap HP affinity chromatography after supernatant A is sufficiently mixed with B, with miaow containing 500mmol/L
The elution of azoles, then the recombinant expression antibacterial peptide VIP that can be purified after desalting column Sephadex G25 desalination；Institute
State the pH7.0 of PBS；The final concentration of 10mg/mL of lysozyme.
The measurement and its characterization of nanometer Se content in the probiotics SeNPs-rVIP-L.lactis NZ9000: it adopts
With Se content in atomic absorption spectrum-flame spectrophotometer method measurement nanometer selenium microballoon.Standard solution is accurately weighed,
Using the solution without selenium as blank control, using absorbance as ordinate, concentration is abscissa, draws standard curve.It accurately weighs
Nitration mixture HClO4+HNO3 is added in a certain amount of sample, and nitrification is as clear as crystal to sample overnight, is transferred in volumetric flask.According to sample
Absorbance calculates the content of corresponding selenium from standard curve.And the L.lactis NZ9000 PBS that SeNPs will be enriched with
Pre- embedding treatment is carried out after cleaning, 1%, which starves acid, fixes, alcohol serial dehydration, embedding medium processing, 70 DEG C of heating, slice dyeing
Afterwards, the distribution and particle size of nanoparticle in thallus are observed under transmission electron microscope；The nitration mixture addition volume and ratio are as follows:
HClO4: HNO3=1: 4, total volume 10ml.
The recombinant expression level of the antibacterial peptide VIP is detected with pig VIP ELISA kit.
Beneficial effect
A kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-lactic acid proposed by the present invention
Galactococcus and preparation method, probiotics --- Lactococcus lactis L.lactis NZ9000 itself have improvement host intestine strong
Health promotes growth, reduces the effects of diarrhea and immunological regulation.Secondly, bacterium has, growth is rapid, breeding is fast, metabolic capability
By force, the features such as adaptable, and do not influenced by season and weather using microorganism conversion selenium, it is with short production cycle, pollution-free,
It is a kind of more easy, economic, green, environmental protection technology of preparing.And it is closed based on Lactococcus lactis L.lactis NZ9000 biology
At nanometer selenium have many advantages, such as that effect is numerous, acute toxicity is minimum, environmental pollution is smaller, bioavailability is high.Therefore, originally
Invention organically combines probiotics, antibacterial peptide and nanometer selenium three, a kind of multi-functional compound micro-ecological preparation SeNPs- of preparation
RVIP-L.lactic NZ9000 had not only had the probiotic of lactic acid bacteria, but also can play the biological function of antibacterial peptide and selenium,
And it is directly fed in the form of active bacteria formulation, can simplify production procedure, reduced production cost, also reduce simultaneously
Sodium selenite (Na is directly added in feed2SeO3) brought by negative effect, the product will have broad application prospects and
Good economic benefit.
Detailed description of the invention
The freeze-dried powder of Fig. 1 compound micro-ecological preparation SeNPs-rVIP-L.lactis NZ9000；
Fig. 2 is the transmission electron microscope (TEM) for being enriched with the Lactococcus lactis L.lactic NZ9000 of nanometer selenium；
The antibacterial activity of Fig. 3 compound micro-ecological preparation SeNPs-rVIP-L.lactis NZ9000；
The antioxidant activity of Fig. 4 compound micro-ecological preparation SeNPs-rVIP-L.lactis NZ9000.
Specific embodiment
Now in conjunction with embodiment, attached drawing, the invention will be further described:
The sodium selenite of severe toxicity can be biologically converted into nontoxic red elemental nano-selenium the present invention provides a kind of, and will
It is enriched in thallus, while the engineering bacteria rVIP-pNZ8148/L.lactis NZ9000 of exocytosis expression antibacterial peptide VIP.
The technical scheme adopted by the invention is that antibacterial peptide VIP is expressed using L.lactic NZ9000 exocytosis, and
The intracellular nanometer selenium of biosynthesis, the method to prepare compound micro-ecological preparation SeNPs-rVIP-L.lactic NZ9000, is pressed
Implement according to following steps:
Step 1, antibacterial peptide VIP recombinant secretor expression vector building and identification:
In the present invention, the recombinant expression of antibacterial peptide VIP usesLactococcus expression system.PNZ8148 matter
Grain is not secretive expression vector, introduces signal peptide when designing VIP gene order for the secreting, expressing for realizing VIP
Sequence SPusp45, the antibacterial peptide VIP gene and load of His label and restriction enzyme (Nco I and Kpn I) recognition site will be had
Body pNZ8148 carries out double digestion respectively, is then attached plasmid pNZ8148 and the VIP gene after digestion, by purpose
It is gene constructed on expression vector, obtain the recombinant expression plasmid rVIP-pNZ8148 of VIP.It is constructed using double digestion connection method
It when recombinant secretor expression vector, is mixed in the ratio that foreign gene and plasmid mole ratio are 3:1, reaction system is 10 μ
L, 16 DEG C overnight, conversion to the MC1061 competent cell prepared.
The preparation method is as follows: picking from the plate 1 monoclonal, addition 5ml SOB is cultivated MC1061 competent cell
Base, 37 DEG C, 200 ± 10rpm cultivates to the exponential growth later period；Take 2ml bacterium solution be added 100ml SOB culture medium in, in 18 DEG C,
200rpm is cultivated to OD600nm=0.55, it is placed in 10min on ice；2500 ± 100 g are centrifuged 10min under the conditions of 4 DEG C, collect bacterium
Body abandons supernatant, completely removes water droplet, the pre-cooling working solution of 1/3 volume is added, with liquid-transfering gun suspension bacteria liquid；Under the conditions of 4 DEG C
2500 ± 100g is centrifuged 10min, collects thallus, abandons supernatant, sucks water droplet completely；1/12 volume working solution, suspended bacteria is added
Body.DMSO (0.3ml/50ml bacterium solution) is gradually added and mixes, places 10min on ice；Packing enters in 1.5mlEP pipe, and liquid nitrogen is put
Subsequent transformation is carried out after setting 1h.
It is as follows that rVIP-pNZ8148 converts the method to MC1061 competent cell: the MC1061 competence that will be prepared
Cell is immediately placed in ice-water bath；10 μ L linked systems are added in competent cell, ice bath 20min；42 DEG C of water-bath pulses
30s is quickly moved in ice-water bath and places 3min, and 1mL LB liquid medium, 37 DEG C, 200 ± 10 rpm shaking table cultures are added
45min；Take part bacterium solution to be coated on LB plate (containing 34 μ g/ml chloramphenicol)；12-16h is cultivated in 37 DEG C of inversions.
The identification of recombinant expression plasmid: the Dan Ke that picking is grown on (contain 34 μ g/ml chloramphenicol) on LB film solid media
It is grand, be inoculated into LB liquid medium (containing 34 μ g/ml chloramphenicol), 37 DEG C, 200 ± 10rpm shaking table culture overnight after, extracting
Plasmid purification, then through Nco I and the identification of I double digestion of Kpn and DNA sequencing with prove the expression vector establishment of antibacterial peptide VIP at
Function；
Step 2, the selenium-rich inducing expression of antibacterial peptide VIP:
Recombinant plasmid rVIP-pNZ8148 electricity is gone into L.lactis NZ9000 competent cell, obtains rVIP-
PNZ8148/L.lactis NZ9000 recombinant bacterium, specific electricity shifting method are as follows: by pNZ8148 empty plasmid (as blank control)
And the correct recombinant plasmid rVIP-pNZ8148 of identification is mixed with the competent cell of L.lactis NZ9000 respectively, ice bath
5min；Mixture is transferred in the 2mm electrotransformation cup of sterile pre-cooling；Under 2.0kV, 186 Ω, shock by electricity immediately；Add rapidly later
Enter 30 DEG C of standings in the M17 culture medium containing 3% glycerol, 5% sucrose, 20mM magnesium chloride and 2mM calcium chloride of 1ml ice pre-cooling
Cultivate 2h；100 μ L bacterium solutions are taken to be coated on the M17 agar medium containing 10 μ g/mL chloramphenicol, 30 DEG C of culture 48h.Recombination
The M17 culture that bacterium is seeded to 20mL is stayed overnight based on 30 DEG C of stationary cultures, and sodium selenite Na is added2SeO3(200 μ g/mL of final concentration)
12h or so is cultivated, while being pair with the L.lactis NZ9000 that L.lactis NZ9000 and conversion have empty plasmid pNZ8148
According to.Part culture solution is taken to be inoculated in the fresh M17 culture medium of 50mL respectively according to 4% ratio, in 30 DEG C of stationary cultures,
As absorbance OD600nmWhen reaching 0.4 or so, inducer nisin (final concentration 100ng/mL) secretion induction is added into culture medium
0,2,4,6,8h.
Step 3, the separation for recombinantly expressing antibacterial peptide VIP, purifying
By the culture solution after inducing expression after 4 DEG C, 12000 ± 1000rpm centrifugation 5min, supernatant A is collected, is used in combination
20mmol/L PBS (pH7.0) clean thallus, in 4 DEG C, 12000 ± 100rpm be centrifuged 5min, repeated washing 1~2 time；Then
Lysozyme (final concentration of 10mg/mL, be dissolved in 10mMTris-HCl) is added into thallus, is incubated after mixing well in 37 DEG C of water-baths
1h is educated, reaction process is mixed by inversion for several times, and supernatant B is collected after centrifugation；Through His-Trap after supernatant A and B are sufficiently mixed
HP affinity chromatography is separated, is purified, with the elution of the imidazoles containing 500mmol/L, then through desalting column Sephadex G25
The recombinant expression antibacterial peptide VIP that can be purified after desalination.
Step 4, the expression detection for recombinantly expressing VIP:
The recombinant expression level of antibacterial peptide VIP is detected with pig VIP ELISA kit.
The measurement and its characterization of nanometer Se content in step 5, probiotics SeNPs-rVIP-L.lactis NZ9000
Using Se content in atomic absorption spectrum-flame spectrophotometer method measurement nanometer selenium microballoon.Accurately weigh standard
Product solution, using the solution without selenium as blank control, using absorbance as ordinate, concentration is abscissa, draws standard curve.
A certain amount of sample is accurately weighed, nitration mixture HClO is added4+HNO3(1:4) 10ml, nitrification is as clear as crystal to sample overnight, is transferred to
In volumetric flask.According to sample absorbance, the content of corresponding selenium is calculated from standard curve.And it will be enriched with SeNPs's
L.lactis NZ9000 carries out pre- embedding treatment after being cleaned with PBS, 1%, which starves acid, fixes, alcohol serial dehydration, at embedding medium
Reason, 70 DEG C of heating observe the distribution and particle size of nanoparticle in thallus after slice dyeing under transmission electron microscope.
The present invention provides the probiotics prepared by the Lactococcus lactis L.lactis NZ9000, which is characterized in that
Nanometer selenium is enriched in L.lactis NZ9000 somatic cells, and the partial size of the nanometer selenium is 50nm-180nm.
The present invention provides the probiotics SeNPs-rVIP- prepared by the Lactococcus lactis L.lactis NZ9000
L.lactis NZ9000, which is characterized in that contain the antibacterial peptide VIP with antibacterial activity.
The present invention provides the probiotics SeNPs-rVIP- prepared by the Lactococcus lactis L.lactis NZ9000
L.lactis NZ9000, which is characterized in that the probiotics have stronger antibacterial activity and antioxidant activity.
Embodiment:
Secreting type recombinantly expresses the construction method of the plasmid of antibacterial peptide VIP
(1) design and optimization of antibacterial peptide VIP gene order:
UsingLactococcus lactis expression system, since pNZ8148 plasmid is not secretive expression vector,
To realize secreting, expressing of the antibacterial peptide VIP in Lactococcus lactis, when designing VIP gene order, signal peptide sequence is introduced
SPusp45, isolated and purified for convenience of antibacterial peptide is subsequent, His tag recognition sequence be added in antibacterial peptide nucleotide sequence
(HHHHH)；Using KpnI and NcoI restriction enzyme construction recombination plasmid, separately designed and pNZ8148 at gene both ends
The identical sequence of KpnI and NcoI restriction enzyme site in sequence, newly-designed recombinant antibacterial peptide VIP nucleotides sequence are classified as 200bp.
In addition, for the high efficient expression in Lactococcus lactis L.lactic NZ9000, not change before amino acid sequence is
It mentions, codon is optimized, to improve its ability to express.
The recombinant antibacterial peptide VIP gene order for optimizing front and back is as follows:
Optimization preceding I:
II after optimization:
In above-mentioned sequence: box shows that respectively Nco I and Kpn I restriction enzyme site, black italic thickened portion are signal
Peptide SPusp45Sequence, italicized item are His sequence label, and black thickened portion is VIP gene order.
(2) building and identification of the recombinant secretor type expression plasmid of antibacterial peptide VIP
By on the VIP gene chemical synthesis to pUC57 carrier with signal peptide and His label after optimization, then by antibacterial peptide
VIP gene and carrier pNZ8148 carry out double digestion (Nco I and Kpn I) respectively, plasmid pNZ8148 and the VIP gene after digestion
It is attached, target gene is building up on expression vector, obtain the recombinant expression plasmid rVIP-pNZ8148 of VIP.It uses
When double digestion connection method constructs recombinant secretor expression vector, mixed in the ratio that foreign gene and plasmid mole ratio are 3:1
It closes, reaction system is 10 μ L, and 16 DEG C overnight, converts to the MC1061 competent cell prepared, the specific method is as follows: will make
The MC1061 competent cell got ready is immediately placed in ice-water bath；10 μ L linked systems are added in competent cell, ice bath
20min；42 DEG C of water-bath pulse 30s, are quickly moved in ice-water bath and place 3min, and 1mL LB liquid medium, 37 DEG C, 200 are added
± 10rpm shaking table culture 45min；Take part bacterium solution to be coated on LB plate (containing 34 μ g/ml chloramphenicol)；12- is cultivated in 37 DEG C of inversions
16h.The monoclonal that picking is grown on (contain 34 μ g/ml chloramphenicol) on LB film solid media, is inoculated into LB liquid medium
In (contain 34 μ g/ml chloramphenicol), 37 DEG C, 200 ± 10rpm shaking table culture overnight after, extracting and purifying plasmid, then through I He of Nco
I double digestion of Kpn and DNA sequencing identification are successful with the expression vector establishment for proving antibacterial peptide VIP.
The selenium-rich inducing expression of rVIP-pNZ8148/L.lactis NZ9000 engineering bacteria
Recombinant plasmid rVIP-pNZ8148 electricity is gone into L.lactis NZ9000 competent cell, obtains rVIP-
PNZ8148/L.lactis NZ9000 recombinant bacterium, specific electricity shifting method are as follows: by pNZ8148 empty plasmid (as blank control)
And the correct recombinant plasmid rVIP-pNZ8148 of identification is mixed with the competent cell of L.lactis NZ9000 respectively, ice bath
5min；Mixture is transferred in the 2mm electrotransformation cup of sterile pre-cooling；Under 2.0kV, 186 Ω, shock by electricity immediately；Add rapidly later
Enter 30 DEG C of standings in the M17 culture medium containing 3% glycerol, 5% sucrose, 20mM magnesium chloride and 2mM calcium chloride of 1ml ice pre-cooling
Cultivate 2h；100 μ L bacterium solutions are taken to be coated on the M17 agar medium containing 10 μ g/mL chloramphenicol, 30 DEG C of culture 48h.Recombination
The M17 culture that bacterium is seeded to 20mL is stayed overnight based on 30 DEG C of stationary cultures, and sodium selenite Na is added2SeO3(200 μ g/mL of final concentration)
12h or so is cultivated, while being pair with the L.lactis NZ9000 that L.lactis NZ9000 and conversion have empty plasmid pNZ8148
According to.Part culture solution is taken to be inoculated in the fresh M17 culture medium of 50mL respectively according to 4% ratio, in 30 DEG C of stationary cultures,
As absorbance OD600nmWhen reaching 0.4 or so, inducer nisin (final concentration 100ng/mL) induction is added into culture medium
2.5h.After inducing expression, entire fermentation liquid is freeze-dried, as shown in Fig. 1, as probiotics SeNPs-
rVIP-L.lactis NZ9000。
Separation, purifying and the identification of antibacterial peptide VIP
By the culture solution after inducing expression after 4 DEG C, 12000 ± 1000rpm centrifugation 5min, supernatant A is collected, is used in combination
20mmol/L PBS (pH7.0) clean thallus, in 4 DEG C, 12000 ± 1000rpm be centrifuged 5min, repeated washing 1~2 time；Then
Lysozyme (final concentration of 10mg/mL) is added into thallus, is incubated for 1h in 37 DEG C of water-baths after mixing well, reaction process is reverse
Supernatant B is collected in mixing for several times, after centrifugation；Divided after supernatant A and B are sufficiently mixed through His-Trap HP affinity chromatography
From, purifying, with the elution of the imidazoles containing 500mmol/L, then can be purified after desalting column Sephadex G25 desalination
Recombinant expression antibacterial peptide VIP.The expression for recombinating VIP in fermentation liquid is detected with pig VIP ELISA kit.
The measurement and characterization of nanometer Se content in probiotics
Using Se content in atomic absorption spectrum-flame spectrophotometer method measurement nanometer selenium microballoon.Accurately weigh standard
Product solution, using the solution without selenium as blank control, using absorbance as ordinate, concentration is abscissa, draws standard curve.
A certain amount of sample is accurately weighed, nitration mixture HClO is added4+HNO3(1:4) 10ml, nitrification is as clear as crystal to sample overnight, is transferred to
In volumetric flask.According to sample absorbance, the content of corresponding selenium is calculated from standard curve.And it will be enriched with SeNPs's
L.lactis NZ9000 carries out pre- embedding treatment after being cleaned with PBS, 1%, which starves acid, fixes, alcohol serial dehydration, at embedding medium
Reason, 70 DEG C of heating observe the distribution and particle size of nanoparticle in thallus after slice dyeing under transmission electron microscope, as a result as attached
Shown in Fig. 1, sodium selenite is restored the nanometer for generating that particle size is 50-180nm by Lactococcus lactis L.lactis NZ9000
Selenium.
The Determination of Antibacterial Activity of the VIP of recombinant expression
By enterotoxigenic E.coli K88 streak inoculation in MH solid medium, 37 DEG C are incubated overnight；Picking Dan Ke
Grand bacterium colony is inoculated in 5mL MH fluid nutrient medium, 37 DEG C of culture 18h；50 μ L culture solutions are taken to be transferred to 5mL MH fluid nutrient medium
In, 37 DEG C of 250rpm are cultivated to OD600nmIt is 0.6；Above-mentioned bacterium solution is diluted 1000 times, makes bacterial population 1 × 105-5×
105The bacterium solution diluted is added in 96 well culture plates by CFUs/mL, and every 100 μ L of hole adds 100 μ L culture medium multiple proportions
Diluted VIP solution, blank control group are the culture medium that 100 μ L bacterium solutions and 100 μ L are free of antibacterial peptide, negative control group 200
The MH culture medium of μ L；Positive controls are the artificial synthesized VIP solution of same concentrations；96 well culture plates are placed in wet box, 37 DEG C
Stationary culture is stayed overnight；It takes out 96 well culture plate multi-function microplate readers and measures absorbance value at 630nm.As shown in Fig. 3,
Illustrate that the antibacterial peptide VIP contained in probiotics has stronger antibacterial activity.
Embodiment 6: it is enriched with the antioxidant activity of the Lactococcus lactis L.lactis NZ9000 of nanometer selenium
By establishing 500 μM of H2O2The swine intestinal epithelium cell IPEC-J2 cell oxidative damage model of induction has studied this
Invent the antioxidant activity of the probiotics of preparation.By the IPEC-J2 cell in logarithmic growth phase and it is enriched with nanometer selenium
After Lactococcus lactis L.lactis NZ9000 (Se content is 4 μ g/mL) co-cultures 8h, expose cells to containing 500 μM
H2O2Serum free medium in, continue cultivate 12h after, carries out living cells dyeing with 33342 staining kit of Hoechst, use
Annexin V-FITC PI staining kit detects Apoptosis situation.In cell culture fluid malonaldehyde (MDA) level and
The activity of total superoxide dismutase (T-SOD) is detected with corresponding kit.As a result as shown in Fig. 4, it is enriched with nanometer selenium
Lactococcus lactis L.lactis NZ9000 significantly suppress H2O2The IPEC-J2 Apoptosis of induction has played effective anti-
Oxidation.
The present invention organically combines probiotics (Lactococcus lactis), essential trace element selenium and antibacterial peptide-VIP three,
It utilizesLactococcus lactis expression system high efficiency recombinant expressed feed antibacterial peptide-VIP, while utilizing Lactococcus lactis
Bacterium L.lactis NZ9000 carries out biological conversion to sodium selenite and synthesising biological nanometer selenium (SeNPs), designed, designed are built
The preparation method of multi-functional compound micro-ecological preparation SeNPs-rVIP-L.lactis NZ9000 a kind of is found, and to its antibacterial
It is evaluated with antioxidant activity.It is rich by being carried out to the engineering bacteria rVIP-pNZ8148/L.lactis NZ9000 of building
Selenium inducing expression, then spray drying can directly be prepared into the feed addictive rich in nanometer selenium and Antibacterial Constituents, or
Selenium-rich thallus after fermentation is isolated and purified into out nanometer selenium microballoon, and is prepared into the anti-oxidation preparations such as pulvis, liquid, fermentation liquid
In the VIP of recombinant expression isolated and purified, can be prepared into antibacterial agent.Pass through bioengineering system based on L.lactis NZ9000
The standby novel probiotics with antibacterial, antioxidant activity can be used as additive for farm animal feed for the prevention of disease and
Treatment.The invention solves the problems such as medicament residue present in current antibiotic usage, drug resistance and the addition of existing selenium-supply
Agent toxic effect is strong, bioavailability is low, easily cause environmental pollution and existing production technology process are complicated, the period is long, at
Produce problem at high cost.
Claims (5)
1. a kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis, feature exist
In: name are as follows: SeNPs-rVIP-Lactococcus lactis NZ9000, abbreviation are as follows: SeNPs-rVIP-L.lactis
NZ9000；Biological expression are as follows: it is single that probiotics L.lactis NZ9000 can convert the sodium selenite of toxicity to nontoxic red
Matter nanometer selenium SeNPs, and nanometer selenium is enriched in thallus；The extracellular antibacterial peptide VIP containing recombinant secretor expression, enrichment intracellular
The Lactococcus lactis probiotics of nanometer selenium SeNPs.
2. multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-lactic acid cream according to claim 1
Coccus, it is characterised in that: the nanometer selenium is enriched in L.lactis NZ9000 somatic cells, and the partial size of nanometer selenium is
50nm-180nm。
3. a kind of multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-cream as claimed in claim 1 or 2
The preparation method of yogurt coccus, it is characterised in that steps are as follows:
Step 1, antibacterial peptide VIP recombinant secretor expression vector building:
It designs VIP gene order: introducing signal peptide sequence SPusp45, His label and restriction enzyme enzyme recognition site；In described
Enzyme cutting is Nco I and Kpn I；
It constructs the recombinant secretor expression vector of antibacterial peptide VIP: antibacterial peptide VIP gene and carrier pNZ8148 is carried out to double enzymes respectively
It cuts, then plasmid pNZ8148 and the VIP gene after digestion is attached, target gene is building up on expression vector, is obtained
It converts after obtaining the recombinant expression plasmid rVIP-pNZ8148 of VIP to MC1061 competent cell；
Construction step: when using double digestion connection method building recombinant secretor expression vector, by foreign gene and plasmid mole ratio
It is mixed for the ratio of 3:1, reaction system is 10 μ L, and 16 DEG C overnight, conversion to MC1061 competent cell: process are as follows: will
The MC1061 competent cell prepared is immediately placed in ice-water bath；10 μ L linked systems are added in competent cell, ice
Bathe 20min；42 DEG C of water-bath pulse 30s, are quickly moved in ice-water bath and place 3min, addition 1mL LB liquid medium, 37 DEG C,
200 ± 10rpm shaking table culture 45min；Part bacterium solution is taken to be coated on LB plate；12-16h is cultivated in 37 DEG C of inversions；The LB plate
It is upper containing 34 μ g/ml chloramphenicol；
The preparation of the MC1061 competent cell: 1 monoclonal of picking is added 5mL SOB culture medium, 37 DEG C, 200 ±
10rpm was cultivated to the exponential growth later period；Take 2mL bacterium solution be added 100mL SOB culture medium in, in 18 DEG C, 200rpm cultivate to
OD600nm=0.55, it is placed in 10min on ice；2500 ± 100g is centrifuged 10min under the conditions of 4 DEG C, collects thallus, abandons supernatant, completely
Water droplet is removed, the pre-cooling working solution of 1/3 volume is added, with liquid-transfering gun suspension bacteria liquid；2500 ± 100g is centrifuged under the conditions of 4 DEG C
10min collects thallus, abandons supernatant, sucks water droplet completely；1/12 volume working solution, suspension thalline is added；It is gradually added into DMSO simultaneously
It mixes, places 10min on ice；Packing enters in 1.5ml sterile centrifugation tube, and liquid nitrogen carries out subsequent transformation after placing 1h；It is described
The additional amount of DMSO are as follows: 0.3ml/50mL bacterium solution；
Step 2, the selenium-rich inducing expression of antibacterial peptide VIP:
Recombinant plasmid rVIP-pNZ8148 electricity is gone into L.lactis NZ9000 competent cell, obtains VIP-pNZ8148/
L.lactis NZ9000 recombinant bacterium, specific electricity shifting method are as follows: by recombinant plasmid rVIP-pNZ8148 and L.lactis
The competent cell of NZ9000 mixes, ice bath 5min；Mixture is transferred in the 2mm electrotransformation cup of sterile pre-cooling；In 2.0kV,
It shocks by electricity under 186 Ω；The M17 containing 3% glycerol, 5% sucrose, 20mM magnesium chloride and 2mM calcium chloride of 1ml ice pre-cooling is added later
30 DEG C of stationary culture 2h in culture medium；100 μ L bacterium solutions are taken to be coated on the M17 agar medium containing 10 μ g/mL chloramphenicol, 30
DEG C culture 48h；The M17 culture that recombinant bacterium is seeded to 20mL is stayed overnight based on 30 DEG C of stationary cultures, and sodium selenite Na2SeO3 training is added
Support 11~13h；It takes part culture solution to be inoculated in the fresh M17 culture medium of 50mL respectively according to 4% ratio, is stood in 30 DEG C
Culture, as absorbance OD600nmWhen reaching 0.4 or so, inducer nisin is added into culture medium and induces 2.5h；Inducing expression knot
Entire fermentation liquid is freeze-dried by Shu Hou, as probiotics SeNPs-rVIP-L.lactis NZ9000；The addition
Sodium selenite Na2SeO3200 μ g/mL of final concentration；The final concentration 100ng/mL that inducer nisin is added；
Step 3, recombinant expression antibacterial peptide VIP are isolated and purified:
Culture solution after inducing expression collects supernatant A, and use 20mmol/L after 4 DEG C, 12000 ± 1000rpm centrifugation 5min
PBS clean thallus, in 4 DEG C, 12000 ± 1000rpm be centrifuged 5min, repeated washing 1~2 time；Then it is added into thallus molten
Bacterium enzyme is incubated for 1h in 37 DEG C of water-baths after mixing well, and reaction process is mixed by inversion for several times, and supernatant B is collected after centrifugation；By supernatant
Liquid A is separated through His-Trap HP affinity chromatography after being sufficiently mixed with B, is purified, with the eluent of the imidazoles containing 500mmol/L
Elution, then the recombinant expression antibacterial peptide VIP that can be purified after desalting column Sephadex G25 desalination；The PBS's
pH7.0；The final concentration of 10mg/mL of lysozyme.
4. multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-lactic acid cream according to claim 3
The preparation method of coccus, it is characterised in that: nanometer selenium contains in the probiotics SeNPs-rVIP-L.lactis NZ9000
The measurement and its characterization of amount: using Se content in atomic absorption spectrum-flame spectrophotometer method measurement nanometer selenium microballoon.It is quasi-
Standard solution really is weighed, using the solution without selenium as blank control, using absorbance as ordinate, concentration is abscissa, is drawn
Standard curve.A certain amount of sample is accurately weighed, nitration mixture HClO4+HNO3 is added, nitrification is as clear as crystal to sample overnight, is transferred to
In volumetric flask.According to sample absorbance, the content of corresponding selenium is calculated from standard curve.And it will be enriched with SeNPs's
L.lactis NZ9000 carries out pre- embedding treatment after being cleaned with PBS, 1%, which starves acid, fixes, alcohol serial dehydration, embedding medium processing,
70 DEG C of heating observe the distribution and particle size of nanoparticle in thallus after slice dyeing under transmission electron microscope；The nitration mixture addition
Volume and ratio are as follows: HClO4: HNO3=1: 4, total volume 10ml.
5. multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-lactic acid cream according to claim 3
The preparation method of coccus, it is characterised in that: the recombinant expression level of the antibacterial peptide VIP is carried out with pig VIP ELISA kit
Detection.
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| KR102238519B1 (en) * | 2018-12-28 | 2021-04-13 | (주)메디톡스 | Microorganism expressing heterologous protein, and use thereof |
| JP2023523214A (en) * | 2020-04-20 | 2023-06-02 | 株式会社 リビオム | Microorganism expressing vasoactive intestinal peptide and use thereof |
| KR102527953B1 (en) * | 2021-03-29 | 2023-05-03 | 주식회사 리비옴 | Microorganism expressing heterologous protein, and use thereof |
| CN116218711B (en) * | 2022-12-21 | 2024-02-06 | 陕西省微生物研究所 | Lactococcus lactis PZ1 and application thereof in preparation of selenium-enriched oligopeptide |
| CN116121142B (en) * | 2023-01-17 | 2024-06-14 | 西安上甫科技有限公司 | Lactococcus lactis (Lactococcus lactis) SPL018 and application thereof |
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