CN110016456B - Multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis and preparation method thereof - Google Patents
Multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a multifunctional composite microecological preparation nano selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and a preparation method thereof, which can convert toxic sodium selenite into nontoxic red elemental nano selenium and enrich the synthesized nano selenium in intracellular probiotics, Lactococcus lactis NZ 9000. The invention also discloses a method for preparing the multifunctional composite microecological preparation SenPs-rVIP-L.lactis NZ9000 based on Lactococcus lactis NZ9000, which solves the problems of strong toxic effect, low bioavailability, easy environmental pollution, complex flow, long period, high production cost, antibiotic residue, drug resistance and the like of the existing selenium supplement additive.
Description
Technical Field
The invention belongs to the technical field of bioengineering and nano material preparation, and relates to a multifunctional composite microecological preparation nano selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis and a preparation method thereof.
Background
The overuse of feed antibiotics has led to serious problems of drug residues and resistance, and it has therefore been an ongoing endeavor to reduce or even ban the use of antibiotics and to find suitable alternatives. In recent years, many countries in the world have come to have corresponding policies to control the use of antibiotics. However, due to the rapid development of the breeding industry, the breeding density is increased, the risk of disease attack of bred animals is increased, and a novel green feed additive product capable of replacing antibiotics is urgently needed. Nowadays, a novel green, safe and residue-free feed additive, namely a probiotic micro-ecological preparation, is gradually ascending in the ranks of growth-promoting feed additives to replace antibiotics by improving the balance of host intestinal flora, promoting digestion and absorption, enhancing immunity and other effects. With the development of microecological preparation products for feeding, the modern biotechnology plays an important role in promoting the theory and application research of the microecological preparation.
The animal-derived antibacterial peptide has the advantages of no toxic or side effect, difficulty in generating drug resistance, no residue, no pollution and the like, meets the requirement of safe production of animal products, is suitable for being used in feed production, and has potential as a new generation feed additive. The preliminary study of the subject group shows that the neuro-immunoregulation peptide VIP (vasoactive intracellular peptide) belonging to the antibacterial peptide family member has the advantages and potential of being developed into a disease-preventing health feed additive: (1) the structure is simple and clear; (2) has antibacterial, antiinflammatory, and immunoregulatory biological activities; (3) as endogenous gastrointestinal hormone antibacterial peptide, the polypeptide can exert stronger biological activity with smaller action concentration (nmoL grade). The VIP has the characteristics of having the potential and wide application prospect of being developed into a high-efficiency and safe disease-prevention health-care feed additive. However, the extraction of VIP from tissues is expensive, low in yield, complicated in process, and expensive in chemical synthesis, and thus it is difficult to apply the method to production. Therefore, a new technology for biosynthesizing the antibacterial peptide VIP by using the probiotic lactococcus lactis is explored and established, so that the technology becomes a more realistic approach of emerging feeds and feed additives, and the prospect is good.
Selenium is a necessary trace element for human and animals, and is closely related to the health of organisms. However, in actual production, such as livestock and poultry feed, inorganic selenium-sodium selenite is mainly used as an additive form, and the inorganic selenium-sodium selenite has strong toxic effect and low bioavailability and is easy to cause environmental pollution. In addition, the dosage of inorganic selenium is difficult to master, and young animals are sensitive to selenium and are easy to cause poisoning. Therefore, the development of selenium-containing biological agents as efficient and green feeds is urgent. Nano selenium (SeNPs) is a new bioactive substance prepared by nano technology. The nano-selenium has extremely fine granularity, is easy to be directly absorbed and fully utilized by the gastrointestinal tract of animals, and can play the function to a greater extent. The nano selenium is the selenium supplement preparation which has the lowest acute toxicity and less environmental pollution. The characteristics and advantages of nano-selenium make it have wide development and application prospects. The synthesis of nano-selenium (SenPs) with biological function by taking probiotics as a carrier is a green and efficient biological path.
Disclosure of Invention
Technical problem to be solved
In order to avoid the defects of the prior art, the invention provides a multifunctional composite microecological preparation of nano selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis and a preparation method thereof, wherein probiotics, antibacterial peptide and nano selenium are organically combined, and the preparation method of the multifunctional composite microecological preparation of SenPs-rVIP-L.lactic NZ9000 is established. The problems of strong toxicity, poor water solubility, low bioavailability and great environmental pollution of the selenium additive in the existing food and feed, complex flow of the existing production and preparation technology, high production cost, unsafe factors and the like are solved.
Technical scheme
A multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis is characterized in that: is named as: Senps-rVIP-Lactococcus lactis NZ9000, abbreviated: serps-rVIP-l.lactis NZ 9000; the biological expression is as follows: the probiotics L.lactis NZ9000 can convert toxic sodium selenite into nontoxic red elemental nano-selenium SeNPs and enrich the nano-selenium in the bacteria; the lactococcus lactis microecological preparation contains extracellular recombinant secretory expression antibacterial peptide VIP and intracellular enrichment nano-selenium SeNPs.
The nano selenium is enriched in L.lactis NZ9000 thallus cells, and the particle size of the nano selenium is 50nm-180 nm.
A preparation method of the multifunctional composite microecological preparation nano selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis is characterized by comprising the following steps:
design of VIP gene sequence: introduction of the Signal peptide sequence SP usp45 His tag and restriction enzyme recognition site; the endonuclease is Nco I and Kpn I;
constructing a recombinant secretion expression vector of the antibacterial peptide VIP: performing double enzyme digestion on the antibacterial peptide VIP gene and a vector pNZ8148 respectively, and then connecting the plasmid pNZ8148 subjected to enzyme digestion with the VIP gene to construct a target gene on an expression vector, and transforming the recombinant expression plasmid rVIP-pNZ8148 of VIP into MC1061 competent cells after obtaining the recombinant expression plasmid rVIP-pNZ 8148;
the construction steps are as follows: when a recombinant secretion expression vector is constructed by using a double enzyme digestion ligation method, mixing exogenous genes and plasmids according to the molar ratio of 3:1, wherein the reaction system is 10 mu L, stays overnight at 16 ℃, and is transformed into MC1061 competent cells: the process is as follows: rapidly placing the prepared MC1061 competent cells in an ice-water bath; add 10. mu.L of the linker system to the competent cells and ice-wash for 20 min; pulsing in 42 deg.C water bath for 30s, rapidly transferring to ice water bath, standing for 3min, adding 1mL LB liquid culture medium, shaking at 37 deg.C and 200 + -10 rpm for 45 min; taking part of the bacterial liquid and coating the bacterial liquid on an LB flat plate; performing inverted culture at 37 deg.C for 12-16 h; the LB plate contains 34. mu.g/ml of chloramphenicol;
preparation of the MC1061 competent cells: selecting 1 single clone, adding 5mL of SOB culture medium, culturing at 37 ℃ and 200 +/-10 rpm to the later exponential growth stage; adding 2mL of the bacterial solution into 100mL of SOB medium, and culturing at 18 deg.C and 200rpm to OD 600nm 0.55, placing on ice for 10 min; centrifuging at 2500 + -100 g at 4 deg.C for 10min, collecting thallus, removing supernatant, completely removing water drop, adding 1/3 volume of precooled working solution, and suspending the bacteria solution with a pipette; centrifuging at 4 deg.C for 10min at 2500 + -100 g, collecting thallus, removing supernatant, and completely removing water drop; adding 1/12 volumes of working solution to suspend the thalli; gradually adding DMSO and mixing, and iceStanding for 10 min; subpackaging into 1.5ml sterile centrifuge tubes, standing with liquid nitrogen for 1h, and performing subsequent transformation; the addition amount of the DMSO is as follows: 0.3mL/50mL of the bacterial solution;
step 2, selenium-rich induced expression of the antibacterial peptide VIP:
the recombinant plasmid rVIP-pNZ8148 is electrically transferred to an L.lactis NZ9000 competent cell to obtain the VIP-pNZ8148/L.lactis NZ9000 recombinant bacterium, and the specific electric transfer method is as follows: mixing the recombinant plasmid rVIP-pNZ8148 with the competent cells of L.lactis NZ9000, and carrying out ice bath for 5 min; transferring the mixture into a sterile pre-cooled 2mm electric conversion cup; electric shock is carried out under 2.0kV and 186 omega; then adding 1ml of M17 culture medium which is pre-cooled by ice and contains 3% of glycerol, 5% of sucrose, 20mM of magnesium chloride and 2mM of calcium chloride, and standing and culturing for 2h at 30 ℃; spreading 100 μ L of the bacterial solution on M17 agar medium containing 10 μ g/mL chloramphenicol, and culturing at 30 deg.C for 48 h; inoculating the recombinant bacteria to 20mL of M17 culture medium, standing and culturing overnight at 30 ℃, adding sodium selenite Na2SeO3, and culturing for 11-13 h; inoculating part of the culture solution into 50mL of fresh M17 culture medium at a ratio of 4%, standing at 30 deg.C, and allowing OD to reach absorbance 600nm When the concentration reaches about 0.4, adding an inducer nisin into the culture medium for induction for 2.5 h; after the induction expression is finished, the whole fermentation liquor is frozen and dried, and the microecological preparation SeNPs-rVIP-L.lactis NZ9000 is obtained; the sodium selenite Na is added 2 SeO 3 The final concentration of (2) is 200 mug/mL; the final concentration of the added inducer nisin is 100 ng/mL;
centrifuging the culture solution after induction expression at 12000 +/-1000 rpm for 5min at 4 ℃, collecting supernatant A, washing the thalli with 20mmol/L PBS, centrifuging at 12000 +/-1000 rpm for 5min at 4 ℃, and repeatedly washing for 1-2 times; adding lysozyme into the thallus, fully and uniformly mixing, incubating in a water bath at 37 ℃ for 1h, reversing the reaction process, uniformly mixing for several times, and centrifuging to collect supernatant B; mixing the supernatant A and the supernatant B fully, separating and purifying by His-Trap HP affinity chromatography, eluting by eluent containing 500mmol/L imidazole, desalting by desalting column Sephadex G25 to obtain purified recombinant expression antibacterial peptide VIP; pH7.0 of the PBS; the final concentration of the lysozyme is 10 mg/mL.
The determination and characterization of the content of nano selenium in the microecological preparation SenPs-rVIP-L.lactis NZ9000 are as follows: and (3) measuring the selenium content in the nano-selenium microspheres by adopting an atomic absorption spectrum-flame spectrophotometer method. And (3) accurately weighing the standard solution, taking the solution containing no selenium as a blank control, taking the absorbance as a ordinate, and taking the concentration as an abscissa, and drawing a standard curve. Accurately weighing a certain amount of sample, adding mixed acid HClO4+ HNO3, nitrifying overnight until the sample is clear and transparent, and transferring the sample into a volumetric flask. And calculating the corresponding selenium content from the standard curve according to the absorbance of the sample. Cleaning L.lactis NZ9000 enriched with SenPs with PBS, pre-embedding, fixing with 1% starved acid, performing gradient dehydration with alcohol, processing with embedding agent, heating at 70 deg.C, slicing, dyeing, and observing the distribution and particle size of nanoparticles in the cells under a transmission electron microscope; the volume and the proportion of the mixed acid are as follows: HClO 4: HNO3 is 1: 4, and the total volume is 10 ml.
And detecting the recombinant expression level of the antibacterial peptide VIP by using a pig VIP ELISA kit.
Advantageous effects
The invention provides a multifunctional composite microecological preparation nano selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis and a preparation method thereof, wherein the probiotic L.lactis NZ9000 has the effects of improving the health of host intestinal tracts, promoting growth, reducing diarrhea, regulating immunity and the like. Secondly, the bacteria have the characteristics of rapid growth, rapid propagation, strong metabolic capability, strong adaptability and the like, the selenium is converted by utilizing the microorganisms without being influenced by seasons and climate, the production period is short, and the preparation method is pollution-free, and is a simpler, more convenient, more economic, more green and more environment-friendly preparation technology. And the nano-selenium biosynthesized based on L.lactis NZ9000 has the advantages of numerous effects, lowest acute toxicity, less environmental pollution, high bioavailability and the like. Therefore, the invention organically combines the probiotics, the antibacterial peptide and the nano-selenium to prepare the multifunctional composite microecological preparation SenPs-rVIP-L.lactic NZ9000 which not only has the probiotics of the lactic acid bacteria, but also can play the biological functions of the antibacterial peptide and the selenium, directly feeds in the form of the viable bacteria preparation, can simplify the production process,reducing production cost, and reducing the amount of sodium selenite (Na) directly added into the feed 2 SeO 3 ) The product has wide application prospect and good economic benefit due to the brought negative effect.
Drawings
FIG. 1 is a freeze-dried powder of a composite microecological preparation SeNPs-rVIP-L.lactis NZ 9000;
fig. 2 is a Transmission Electron Microscope (TEM) of l.lactic NZ9000 of lactobacillus lactis enriched with nano-selenium;
fig. 3. the antibacterial activity of the complex probiotics SeNPs-rVIP-l.lactis NZ 9000;
figure 4. antioxidant activity of composite microecological formulation SeNPs-rVIP-l.lactis NZ 9000.
Detailed Description
The invention will now be further described with reference to the following examples and drawings:
the invention provides an engineering bacterium rVIP-pNZ8148/L.lactis NZ9000 which can biologically convert virulent sodium selenite into nontoxic red elemental nano-selenium, enrich the red elemental nano-selenium in a bacterium body and express an antibacterial peptide VIP extracellularly.
The technical scheme adopted by the invention is that a method for preparing a composite microecological preparation SenPs-rVIP-L.lactic NZ9000 by extracellularly secreting and expressing an antibacterial peptide VIP and biosynthesizing intracellular nano selenium is implemented according to the following steps:
in the present invention, recombinant expression of the antibacterial peptide VIP is performed
A lactococcus lactis expression system. The pNZ8148 plasmid is not a secretory expression plasmid per se, and in order to realize the secretory expression of VIP, a signal peptide sequence SP is introduced when a VIP gene sequence is designed usp45 Performing double enzyme digestion on an antibacterial peptide VIP gene with a His tag and recognition sites of restriction enzymes (Nco I and Kpn I) and a vector pNZ8148 respectively, and then connecting the digested plasmid pNZ8148 with the VIP gene to construct a target geneAnd constructing the expression vector to obtain a recombinant expression plasmid rVIP-pNZ8148 of VIP. When a recombinant secretion expression vector is constructed by using a double enzyme digestion ligation method, the exogenous gene and the plasmid are mixed according to the molar ratio of 3:1, the reaction system is 10 mu L, the temperature is 16 ℃ overnight, and the mixture is transformed into the prepared MC1061 competent cell.
The MC1061 competent cell was prepared as follows: selecting 1 single clone from a plate, adding 5ml of SOB culture medium, and culturing at 37 ℃ and 200 +/-10 rpm to the later exponential growth stage; adding 2ml of the bacterial liquid into 100ml of SOB medium, culturing at 18 deg.C and 200rpm to OD 600nm 0.55, placing on ice for 10 min; centrifuging at 2500 + -100 g at 4 deg.C for 10min, collecting thallus, removing supernatant, completely removing water drop, adding 1/3 volume of precooled working solution, and suspending the bacteria solution with a pipette; centrifuging at 4 deg.C for 10min at 2500 + -100 g, collecting thallus, removing supernatant, and completely removing water drop; 1/12 volumes of working solution were added to suspend the cells. Gradually adding DMSO (0.3ml/50ml bacterial solution), mixing, and standing on ice for 10 min; subpackaging into 1.5ml EP tube, standing with liquid nitrogen for 1h, and performing subsequent transformation.
The method for transforming rVIP-pNZ8148 into MC1061 competent cells is as follows: rapidly placing the prepared MC1061 competent cells in an ice-water bath; adding 10 μ L of the ligation system into the competent cells, and carrying out ice bath for 20 min; pulsing in 42 deg.C water bath for 30s, rapidly transferring to ice water bath, standing for 3min, adding 1mL LB liquid culture medium, shaking at 37 deg.C and 200 + -10 rpm for 45 min; taking part of the bacterial liquid and coating the part of the bacterial liquid on an LB plate (containing 34 mu g/ml chloramphenicol); culturing at 37 deg.C for 12-16 h.
Identification of recombinant expression plasmids: selecting a monoclonal growing on an LB fixed culture medium (containing 34 mu g/ml chloramphenicol), inoculating the monoclonal to an LB liquid culture medium (containing 34 mu g/ml chloramphenicol), performing shake cultivation at 37 ℃ and 200 +/-10 rpm overnight, extracting and purifying plasmids, and performing Nco I and Kpn I double enzyme digestion and DNA sequencing identification to prove that the construction of the expression vector of the antimicrobial peptide VIP is successful;
step 2, selenium-rich induced expression of the antibacterial peptide VIP:
electrically transforming the recombinant plasmid rVIP-pNZ8148 to an L.lactis NZ9000 competent cell to obtain an rVIP-pNZ8148/L.lactis NZ9000 recombinant bacterium, and specifically electrically transformingThe method comprises the following steps: mixing pNZ8148 empty plasmid (as blank control) and recombinant plasmid rVIP-pNZ8148 with L.lactis NZ9000 competent cell, and ice-cooling for 5 min; transferring the mixture into a sterile pre-cooled 2mm electric conversion cup; under 2.0kV and 186 omega, immediately shocking; then quickly adding 1ml of M17 culture medium which is pre-cooled by ice and contains 3% of glycerol, 5% of sucrose, 20mM of magnesium chloride and 2mM of calcium chloride, and standing and culturing for 2h at 30 ℃; 100. mu.L of the bacterial suspension was applied to M17 agar medium containing 10. mu.g/mL chloramphenicol, and cultured at 30 ℃ for 48 hours. Inoculating the recombinant bacteria to 20mL of M17 culture medium, standing at 30 deg.C for overnight culture, adding sodium selenite Na 2 SeO 3 The cells were cultured (final concentration 200. mu.g/mL) for about 12 hours, while using L.lactis NZ9000 and L.lactis NZ9000 transformed with an empty plasmid pNZ8148 as controls. Inoculating part of the culture solution at a ratio of 4% into 50mL fresh M17 culture medium, standing at 30 deg.C, and culturing until the absorbance OD 600nm When the concentration reaches about 0.4, an inducer nisin (final concentration of 100ng/mL) is added into the culture medium to induce the secretion for 0,2, 4,6 and 8 hours.
Centrifuging the culture solution after induction expression at 12000 +/-1000 rpm for 5min at 4 ℃, collecting supernatant A, washing the thalli with 20mmol/L PBS (pH7.0), centrifuging at 12000 +/-100 rpm for 5min at 4 ℃, and repeatedly washing for 1-2 times; adding lysozyme (with final concentration of 10mg/mL, dissolved in 10mM Tris-HCl), mixing well, incubating in 37 deg.C water bath for 1h, reversing reaction process, mixing well for several times, centrifuging, and collecting supernatant B; and (3) fully mixing the supernatant A and the supernatant B, separating and purifying by His-Trap HP affinity chromatography, eluting by using an eluent containing 500mmol/L imidazole, and desalting by using a desalting column Sephadex G25 to obtain the purified recombinant expression antibacterial peptide VIP.
And 4, detecting the expression level of the recombinant expression VIP:
the recombinant expression level of the antibacterial peptide VIP is detected by using a pig VIP ELISA kit.
Step 5, determination and characterization of nano selenium content in microecological preparation SenPs-rVIP-L.lactis NZ9000
By atomic absorption spectroscopy-flame spectroscopyAnd measuring the selenium content in the nano-selenium microspheres by a photometer method. And accurately weighing the standard solution, taking the solution containing no selenium as a blank control, taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate, and drawing a standard curve. Accurately weighing a certain amount of sample, adding mixed acid HClO 4 +HNO 3 (1: 4)10ml, nitrated overnight until the sample was clear and transparent, and transferred to a volumetric flask. And calculating the corresponding selenium content from the standard curve according to the absorbance of the sample. And cleaning L.lactis NZ9000 enriched with SeNPs with PBS, pre-embedding, fixing with 1% starved acid, performing gradient dehydration with alcohol, processing with embedding agent, heating at 70 deg.C, slicing, dyeing, and observing distribution and particle size of nanoparticles in thallus under transmission electron microscope.
The invention provides a microecological preparation prepared from lactococcus lactis NZ9000, which is characterized in that nano selenium is enriched in L.lactis NZ9000 somatic cells, and the particle size of the nano selenium is 50nm-180 nm.
The invention provides a microecological preparation SeNPs-rVIP-L.lactis NZ9000 prepared from the lactococcus lactis L.NZ 9000, which is characterized by containing an antibacterial peptide VIP with antibacterial activity.
The invention provides a microecological preparation SeNPs-rVIP-L.lactis NZ9000 prepared from lactococcus lactis L.NZ 9000, which is characterized by having stronger antibacterial activity and antioxidant activity.
Example (b):
construction method of plasmid for secretory recombinant expression of antibacterial peptide VIP
(1) Designing and optimizing an antibacterial peptide VIP gene sequence:
by using
In the lactococcus lactis expression system, because the pNZ8148 plasmid is not a secretory expression plasmid, in order to realize the secretory expression of the antibacterial peptide VIP in the lactococcus lactis, a signal peptide sequence SP is introduced when a VIP gene sequence is designed usp45 In order to facilitate the subsequent separation and purification of the antibacterial peptide, a His tag identification sequence (HHHHH) is added into the nucleotide sequence of the antibacterial peptide; make itThe KpnI and NcoI restriction endonucleases are used for constructing recombinant plasmids, sequences which are the same as the enzyme cutting sites of KpnI and NcoI in a pNZ8148 sequence are respectively designed at the two ends of the gene, and the nucleotide sequence of the newly designed recombinant antibacterial peptide VIP is 200 bp.
In addition, in order to efficiently express l.lactic NZ9000 from lactococcus lactis, codons were optimized to improve its expression ability, provided that the amino acid sequence was not changed.
The recombinant antibacterial peptide VIP gene sequences before and after optimization are as follows:
before optimization, I:
and (5) after optimization II:
in the above sequence: boxes are shown for Nco I and Kpn I cleavage sites, respectively, and the signal peptide SP is in bold italic bold usp45 The sequence, the italic part is His label sequence, and the black bold part is VIP gene sequence.
(2) Construction and identification of recombinant secretory expression plasmid of antibacterial peptide VIP
Synthesizing optimized VIP gene with signal peptide and His label to pUC57 vector, performing double enzyme digestion (Nco I and Kpn I) on the antibacterial peptide VIP gene and the vector pNZ8148, and connecting the plasmid pNZ8148 subjected to enzyme digestion with the VIP gene to construct a target gene to an expression vector so as to obtain the VIP recombinant expression plasmid rVIP-pNZ 8148. When a recombinant secretion expression vector is constructed by using a double enzyme digestion ligation method, mixing exogenous genes and plasmids according to the mol ratio of 3:1, wherein the reaction system is 10 mu L, stays overnight at 16 ℃, and is transformed into prepared MC1061 competent cells, and the specific method comprises the following steps: rapidly placing the prepared MC1061 competent cells in an ice-water bath; adding 10 μ L of the ligation system into the competent cells, and carrying out ice bath for 20 min; pulsing in 42 deg.C water bath for 30s, rapidly transferring into ice water bath, standing for 3min, adding 1mL LB liquid culture medium, shaking at 37 deg.C and 200 + -10 rpm for 45 min; taking part of the bacterial liquid and coating the part of the bacterial liquid on an LB plate (containing 34 mu g/ml chloramphenicol); culturing at 37 deg.C for 12-16 h. A single clone growing on an LB fixed culture medium (containing 34 mu g/ml chloramphenicol) is picked up and inoculated into an LB liquid culture medium (containing 34 mu g/ml chloramphenicol), after shaking culture at 37 ℃ and 200 +/-10 rpm overnight, a purified plasmid is extracted and identified by Nco I and Kpn I double enzyme digestion and DNA sequencing to prove that the expression vector of the antimicrobial peptide VIP is successfully constructed.
Selenium-rich induced expression of rVIP-pNZ8148/L.lactis NZ9000 engineering bacteria
Transferring the recombinant plasmid rVIP-pNZ8148 to an L.lactis NZ9000 competent cell to obtain an rVIP-pNZ8148/L.lactis NZ9000 recombinant bacterium, wherein the specific transferring method comprises the following steps: mixing pNZ8148 empty plasmid (as blank control) and recombinant plasmid rVIP-pNZ8148 with L.lactis NZ9000 competent cell, and ice-cooling for 5 min; transferring the mixture into a sterile pre-cooled 2mm electric conversion cup; under 2.0kV and 186 omega, immediately shocking; then quickly adding 1ml of M17 culture medium which is pre-cooled by ice and contains 3% of glycerol, 5% of sucrose, 20mM of magnesium chloride and 2mM of calcium chloride, and standing and culturing for 2h at 30 ℃; 100. mu.L of the bacterial suspension was applied to M17 agar medium containing 10. mu.g/mL chloramphenicol, and cultured at 30 ℃ for 48 hours. Inoculating the recombinant bacteria to 20mL of M17 culture medium, standing at 30 deg.C for overnight culture, adding sodium selenite Na 2 SeO 3 The cells were cultured (final concentration 200. mu.g/mL) for about 12 hours, while using L.lactis NZ9000 and L.lactis NZ9000 transformed with an empty plasmid pNZ8148 as controls. Inoculating part of the culture solution at a ratio of 4% into 50mL fresh M17 culture medium, standing at 30 deg.C, and culturing until the absorbance OD 600nm When the concentration reached about 0.4, the medium was induced for 2.5 hours by adding nisin (final concentration 100 ng/mL). After the induction expression is finished, the whole fermentation liquor is frozen and dried, as shown in figure 1, and the microecological preparation SeNPs-rVIP-L.lactis NZ9000 is obtained.
Separation, purification and identification of antibacterial peptide VIP
Centrifuging the culture solution after induction expression at 4 ℃ at 12000 +/-1000 rpm for 5min, collecting supernatant A, washing the thalli with 20mmol/L PBS (pH7.0), centrifuging at 12000 +/-1000 rpm for 5min at 4 ℃ and repeating washing for 1-2 times; adding lysozyme (the final concentration is 10mg/mL) into the thalli, fully and uniformly mixing, incubating in water bath at 37 ℃ for 1h, reversing the reaction process, uniformly mixing for a plurality of times, and centrifuging to collect supernatant B; and (3) fully mixing the supernatant A and the supernatant B, separating and purifying by His-Trap HP affinity chromatography, eluting by using an eluent containing 500mmol/L imidazole, and desalting by using a desalting column Sephadex G25 to obtain the purified recombinant expression antibacterial peptide VIP. And detecting the expression level of the recombinant VIP in the fermentation liquor by using a pig VIP ELISA kit.
Determination and characterization of nano-selenium content in microecological preparation
And (3) measuring the selenium content in the nano-selenium microspheres by adopting an atomic absorption spectrum-flame spectrophotometer method. And accurately weighing the standard solution, taking the solution containing no selenium as a blank control, taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate, and drawing a standard curve. Accurately weighing a certain amount of sample, adding mixed acid HClO 4 +HNO 3 (1: 4)10ml, nitrated overnight until the sample was clear and transparent, and transferred to a volumetric flask. And calculating the corresponding selenium content from the standard curve according to the absorbance of the sample. And cleaning L.lactis NZ9000 enriched with SeNPs by PBS, pre-embedding, fixing with 1% starved acid, performing gradient dehydration with alcohol, processing with embedding agent, heating at 70 ℃, slicing, dyeing, and observing distribution and particle size of nanoparticles in the cells under a transmission electron microscope, wherein the result is shown in figure 1, and sodium selenite is reduced to nano selenium with particle size of 50-180nm by L.lactis NZ 9000.
Determination of the antibacterial Activity of recombinantly expressed VIP
Streaking enterotoxigenic Escherichia coli K88 in MH solid culture medium, and culturing at 37 deg.C overnight; selecting a monoclonal colony, inoculating the colony in 5mL MH liquid culture medium, and culturing at 37 ℃ for 18 h; transferring 50 μ L of the culture medium into 5mL MH liquid medium, culturing at 37 deg.C and 250rpm to OD 600nm Is 0.6; diluting the bacterial liquid 1000 times to make the bacterial count at 1 × 10 5 -5×10 5 CFUs/mL, diluted bacterial solution was added to a 96-well plate at 100. mu.L per well, 100. mu.L of VIP solution diluted by media at a double ratio was added, and the blank control group was 100. mu.L of bacterial solution and 100 μ L of the antimicrobial peptide-free medium, and 200 μ L of MH medium as a negative control group; the positive control group is artificially synthesized VIP solution with the same concentration; placing a 96-well culture plate in a wet box, and standing and culturing at 37 ℃ overnight; and taking out the 96-hole culture plate, and measuring the absorbance value at 630nm by using a multifunctional microplate reader. As shown in FIG. 3, it is demonstrated that the antimicrobial peptide VIP contained in the probiotic has a strong antimicrobial activity.
Example 6: antioxidant activity of lactococcus lactis NZ9000 enriched with nano-selenium
By establishing 500 μ M H 2 O 2 An induced porcine intestinal epithelial cell IPEC-J2 cell oxidative damage model researches the antioxidant activity of the microecological preparation prepared by the invention. IPEC-J2 cells in logarithmic growth phase were co-cultured with L.lactis NZ9000 (selenium content of 4. mu.g/mL) enriched in nano-selenium for 8h, after which the cells were exposed to a solution containing 500. mu. M H 2 O 2 After the culture is continued for 12 hours in the serum-free culture medium, live cells are stained by a Hoechst 33342 staining reagent kit, and the apoptosis condition is detected by an Annexin V-FITC PI staining reagent kit. The level of Malondialdehyde (MDA) and the activity of total superoxide dismutase (T-SOD) in the cell culture broth were determined using corresponding kits. The results are shown in figure 4, and the nano-selenium enriched lactococcus lactis L.lactis NZ9000 remarkably inhibits H 2 O 2 The induced IPEC-J2 cell apoptosis plays an effective antioxidation role.
The invention organically combines probiotics (lactococcus lactis), essential trace element selenium and antibacterial peptide-VIP and utilizes
The lactococcus lactis expression system efficiently recombines and expresses the antibacterial peptide-VIP for the feed, and simultaneously, lactococcus lactis NZ9000 is utilized to carry out biological conversion on sodium selenite to synthesize biological nano-selenium (SeNPs), so that a preparation method of a multifunctional composite microecological preparation SeNPs-rVIP-L.lactis NZ9000 is automatically designed and established, and the antibacterial and antioxidant activities of the preparation method are evaluated. Through selenium-rich induced expression of the constructed engineering bacteria rVIP-pNZ8148/L.lactis NZ9000, and then spray dryingThe fermented selenium-rich thallus is separated and purified to obtain nano selenium microsphere, and the nano selenium microsphere is prepared into antioxidant preparation, such as powder, liquid, etc. and the recombinant VIP in the fermented liquid is separated and purified to obtain the antibacterial agent. The novel microecological preparation with antibacterial and antioxidant activities, which is prepared by bioengineering based on L.lactis NZ9000, can be used as a feed additive for livestock and poultry for preventing and treating diseases. The invention solves the problems of drug residue, drug resistance and the like existing in the use of the existing antibiotics, the problems of strong toxic effect, low bioavailability, easy environmental pollution, complex flow, long period and high production cost of the existing selenium supplement additive.
Claims (5)
1. A multifunctional composite microecological preparation nano selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis is characterized in that: is named as: Senps-rVIP-Lactococcus lactis NZ9000, abbreviated: serps-rVIP-l.lactis NZ 9000; the biological expression is: the probiotics L.lactis NZ9000 can convert toxic sodium selenite into nontoxic red elemental nano-selenium SeNPs and enrich the nano-selenium in the bacteria; the lactococcus lactis microecological preparation contains extracellular recombinant secretory expression antibacterial peptide VIP and intracellular enrichment nano-selenium SeNPs.
2. The multifunctional composite microecological preparation of claim 1, which is selected from the group consisting of nanoselenium, recombinant expressed vasoactive intestinal peptide, lactococcus lactis, wherein the amino acid sequence of the nanoselenium-recombinant expressed vasoactive intestinal peptide is as follows: the nano-selenium is enriched in L.lactis NZ9000 thallus cells, and the particle size of the nano-selenium is 50nm-180 nm.
3. A method for preparing the multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis of claim 1 or 2, which is characterized by comprising the following steps:
step 1, constructing a recombinant secretion expression vector of the antibacterial peptide VIP:
design of VIP gene sequence: introduction of the Signal peptide sequence SP usp45 His tag and restriction enzyme recognitionDistinguishing sites; the endonuclease is Nco I and Kpn I;
constructing a recombinant secretion expression vector of the antibacterial peptide VIP: performing double enzyme digestion on the antibacterial peptide VIP gene and a vector pNZ8148 respectively, and then connecting the plasmid pNZ8148 subjected to enzyme digestion with the VIP gene to construct a target gene on an expression vector, so as to obtain a recombinant expression plasmid rVIP-pNZ8148 of VIP and then transforming the recombinant expression plasmid rVIP-pNZ8148 into MC1061 competent cells;
the construction steps are as follows: when a recombinant secretion expression vector is constructed by using a double enzyme digestion ligation method, mixing exogenous genes and plasmids according to the molar ratio of 3:1, wherein the reaction system is 10 mu L, stays overnight at 16 ℃, and is transformed into MC1061 competent cells: the process is as follows: rapidly placing the prepared MC1061 competent cells in an ice-water bath; adding 10 mu L of the connection system into the competent cells, and carrying out ice bath for 20 min; pulsing in 42 deg.C water bath for 30s, rapidly transferring to ice water bath, standing for 3min, adding 1mL LB liquid culture medium, shaking at 37 deg.C and 200 + -10 rpm for 45 min; taking part of the bacterial liquid and coating the bacterial liquid on an LB flat plate; performing inverted culture at 37 deg.C for 12-16 h; the LB plate contains 34 mug/ml of chloramphenicol;
preparation of the MC1061 competent cells: selecting 1 single clone, adding 5mL of SOB culture medium, culturing at 37 ℃ and 200 +/-10 rpm to the later exponential growth stage; adding 2mL of the bacterial solution into 100mL of SOB medium, culturing at 18 deg.C and 200rpm to OD 600nm 0.55, placing on ice for 10 min; centrifuging at 2500 + -100 g at 4 deg.C for 10min, collecting thallus, removing supernatant, completely removing water drop, adding 1/3 volume of precooled working solution, and suspending the bacteria solution with a pipette; centrifuging at 4 deg.C for 10min at 2500 + -100 g, collecting thallus, removing supernatant, and completely removing water drop; 1/12 volumes of working solution are added to suspend the thalli; gradually adding DMSO, mixing, and standing on ice for 10 min; subpackaging into 1.5ml sterile centrifuge tubes, standing with liquid nitrogen for 1h, and performing subsequent transformation; the addition amount of the DMSO is as follows: 0.3mL/50mL of bacterial liquid;
step 2, selenium-rich induced expression of the antibacterial peptide VIP:
the recombinant plasmid rVIP-pNZ8148 is electrically transferred to an L.lactis NZ9000 competent cell to obtain the VIP-pNZ8148/L.lactis NZ9000 recombinant bacterium, and the specific electric transfer method is as follows: mixing the recombinant plasmid rVIP-pNZ8148 with the competent cells of L.lactis NZ9000Mixing, and performing ice bath for 5 min; transferring the mixture into a sterile pre-cooled 2mm electric conversion cup; electric shock is carried out under 2.0kV and 186 omega; then adding 1ml of M17 culture medium which is pre-cooled by ice and contains 3% of glycerol, 5% of sucrose, 20mM of magnesium chloride and 2mM of calcium chloride, and standing and culturing for 2h at 30 ℃; spreading 100 μ L of the bacterial solution on M17 agar medium containing 10 μ g/mL chloramphenicol, and culturing at 30 deg.C for 48 h; inoculating the recombinant bacteria to 20mL of M17 culture medium, standing and culturing overnight at 30 ℃, adding sodium selenite Na2SeO3, and culturing for 11-13 h; inoculating part of the culture solution at a ratio of 4% into 50mL fresh M17 culture medium, standing at 30 deg.C, and allowing OD to reach absorbance 600nm When the concentration reaches about 0.4, adding an inducer nisin into the culture medium for induction for 2.5 h; after the induction expression is finished, the whole fermentation liquor is frozen and dried, and the microecological preparation SeNPs-rVIP-L.lactis NZ9000 is obtained; the sodium selenite Na is added 2 SeO 3 The final concentration of (2) is 200 mug/mL; the final concentration of the added inducer nisin is 100 ng/mL;
step 3, separating and purifying recombinant expression antibacterial peptide VIP:
centrifuging the culture solution after induction expression at 12000 +/-1000 rpm for 5min at 4 ℃, collecting supernatant A, washing the thalli with 20mmol/L PBS, centrifuging at 12000 +/-1000 rpm for 5min at 4 ℃, and repeatedly washing for 1-2 times; adding lysozyme into the thalli, fully and uniformly mixing, incubating in a water bath at 37 ℃ for 1h, reversing the reaction process, uniformly mixing for several times, and centrifuging to obtain a supernatant B; mixing the supernatant A and the supernatant B fully, separating and purifying by His-Trap HP affinity chromatography, eluting by eluent containing 500mmol/L imidazole, and desalting by desalting column Sephadex G25 to obtain purified recombinant expression antibacterial peptide VIP; pH7.0 of the PBS; the final concentration of the lysozyme is 10 mg/mL.
4. The method for preparing the multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis according to claim 3, wherein the method comprises the following steps: the determination and characterization of the content of nano selenium in the microecological preparation SenPs-rVIP-L.lactis NZ9000 are as follows: measuring the selenium content in the nano-selenium microspheres by adopting an atomic absorption spectrum-flame spectrophotometer method; accurately weighing a standard solution, taking a solution containing no selenium as a blank control, taking absorbance as a vertical coordinate and taking concentration as a horizontal coordinate, and drawing a standard curve; accurately weighing a certain amount of sample, adding mixed acid HClO4+ HNO3, nitrifying overnight until the sample is clear and transparent, and transferring the sample into a volumetric flask; calculating the corresponding selenium content from the standard curve according to the absorbance of the sample; cleaning L.lactis NZ9000 enriched with Senps with PBS, pre-embedding, fixing with 1% osmic acid, dehydrating with alcohol gradient, processing with embedding agent, heating at 70 deg.C, slicing, dyeing, and observing the distribution and particle size of nanoparticles in thallus under transmission electron microscope; the volume and the proportion of the mixed acid are as follows: HClO 4: HNO3 is 1: 4, and the total volume is 10 ml.
5. The method for preparing the multifunctional composite microecological preparation nano-selenium-recombinant expression vasoactive intestinal peptide-lactococcus lactis according to claim 3, wherein the method comprises the following steps: and detecting the recombinant expression level of the antibacterial peptide VIP by using a pig VIP ELISA kit.
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| KR20210129516A (en) | 2020-04-20 | 2021-10-28 | 주식회사 리비옴 | Microorganism expressing vasoactive intestinal peptide, and use thereof |
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