CN105524158A - Fly maggot antibacterial peptide, encoding gene, carrier, engineering bacteria and application thereof - Google Patents
Fly maggot antibacterial peptide, encoding gene, carrier, engineering bacteria and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
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Abstract
The invention discloses a fly maggot antibacterial peptide, an encoding gene, a carrier, engineering bacteria and application thereof. An amino acid sequence of the fly maggot antibacterial peptide is shown in SEQ ID No.2. The fly maggot antibacterial peptide has the advantages that because the used expression system and all expression control originals are all from food-level lactobacillus and cheese lactobacillus, the recombinant lactobacillus for finally expressing the antibacterial peptide is a real food-level product; the antibacterial peptide is displayed at the surface of a cell wall of the lactobacillus, the stability in a host intestinal tract is higher, the easiness in degrading in the intestinal tract environment is avoided, and the long-time function is conveniently realized; a common induced type promoter for expressing the lactobacillus is not used, but a constitutive promoter is used, and the inducers, such as Nisin, are not needed in the fermentation process, so that the production technology is simple and convenient; compared with the antibacterial peptide which is directly extracted from fly maggot, the recombinant lactobacillus for expressing the antibacterial peptide is safer, and the cost of scaled production is lower.
Description
(1) technical field
The present invention relates to a kind of antibacterial peptide and application thereof, particularly a kind of fly maggot antibacterial peptide, the food-grade recombination lactic acid bacterium of expressing antibacterial peptide and the application prepared in antimicrobial additive thereof.
(2) background technology
Antibacterial peptide (antibacterialpeptides) is that the class that Immune System produces resists the polypeptide of extraneous pathogenic infection, it is except having the pathogenic agent such as nonspecific opposing bacterium, fungi, virus, also has the effect of antitumor cell, lethal effect is had especially to multi-drug resistant bacteria and tumour cell, and do not destroy the normal cell of human body, be the novel antibacterial medicine that a class has great potential.This kind of active polypeptide of antibacterial peptide has the advantages such as immunogenicity is little, Heat stability is good, good water solubility, has a broad antifungal spectrum.There are some researches show, in animal diets, add that antibacterial peptide can improve the production performance of animal and efficiency of feed utilization, improves animal intestinal micro-ecology environment, the cell promoting animal and humoral immunoresponse(HI), improve immunity function and the disease resistance of animal.In addition, in feed, add antibacterial peptide, can microbiotic be replaced, eliminate antibiotic excessive use and cause pathogenic bacterium to produce the problem of resistance and agricultural-food antibiotic remains.
Fly and larva maggot primary growth are in the severe environment containing a large amount of bacterium such as rotten thing, ight soil and rubbish, its body surface is with many pathogenic bacterias, various diseases can be propagated, but himself can not cause death because of infection pathogen, this illustrates that fly should have powerful immunizing power to pathogenic micro-organism, and can produce potent antibacterial material.This material is exactly fly maggot antibacterial peptide.
Fly maggot antibacterial peptide is a series of slightly acidic peptide materials with anti-microbial activity that fly maggot produces after pathogenic bacterium inducing, and thermally-stabilised height, its molecular weight is usually at 5-10kDa.Fly maggot antibacterial peptide not only has the function of kill bacteria, and also have the function of the microorganisms such as opposing fungi, even virus, fungi, algae, protozoon and parasite, even tumour etc. all have suppression or killing action.
Fly maggot antibacterial peptide is the mixture of a series of multiple antibacterial peptide, and independent purifying wherein a kind of antibacterial peptide is extremely difficult.It is produced and extracts main dependence in natural fly larvina and extracts total antibacterial peptide at present, and productive rate is low, and anti-microbial activity is unstable.Simultaneously because maggot grow environment contains a large amount of pathogenetic bacteria, therefore antibacterial peptide extracts production process and wants the rigorous concentration healthy prevention, otherwise product easily causes disease spread and diffusion for feed interpolation purposes.The large-scale production of fly maggot antibacterial peptide distance, also has longer technological gap.
Enteric pathogenic bacteria is the murderous one of the main reasons of livestock and poultry aquaculture.Aquaculture, to antibiotic life-time service, causes spreading unchecked of resistant organism.According to statistics, the 56 strain enteric pathogenic bacterias be separated from chitling road, the bacterial strain of 85% all has resistance to norfloxicin, Ciprofloxacin, penicillin, Ampicillin Trihydrate and cefoxitin.Antibiotic overuse also causes the serious problems that in fishery products, antibiotic residual quantity is too high, causes serious threat to the health of human consumer.Terramycin, the common aquatic products such as paraxin microbiotic detects repeatly in commercially available fish shrimp crab series products, and even exported product is also detected repeatly.
Antibacterial peptide, can the use of substitute antibiotics completely as fodder additives, and antibacterial range is wide, has no drug resistance and the problem of antibiotic remains.But the natural origin of antibacterial peptide is deficient and extraction cost too high be the major obstacle limiting its use.
Adopt genetic engineering technique, utilize Microbial cell factories, import antibacterial peptide gene, obtain the engineering bacteria of recombinant expressed antibacterial peptide.Utilize industrial fermentation technology, large-scale production recombinant antibacterial peptide is feasible technological line.A lot of recombinant pharmaceutical proteins uses intestinal bacteria, and pichia spp, Bacillusexpression system achieves large-scale production.
Milk-acid bacteria is the safe microorganisms that can be used for foodstuff production of U.S. FDA certification, and the probiotic agent that one of most common use is added for feed, has and improve intestinal microecology system, maintains enteron aisle balance and function, improves effect of host immunity.Milk-acid bacteria is the natural fungal component of host intestine, can stick and field planting, play prebiotic effect for a long time in digestive tube.Utilize milk-acid bacteria as host, express restructuring fly maggot antibacterial peptide, antibacterial peptide is secreted or is showed in the cell wall of recombinant lactic acid bacteria.Restructuring living lactic acid bacteria is added into feed as probiotic bacterium, feeding animals.Recombinant lactic acid bacteria is grown host intestine is decided at the higher level but not officially announced, constantly secretes antibacterial peptide, kills enteric pathogenic bacteria, promotes host immune response, has concurrently and plays antibacterial and probiotic effect, have huge applications potentiality.
Milk-acid bacteria recombinant expression vector is one of the gordian technique in milk-acid bacteria application, but as shuttle vectors, a lot of all with antibiotics resistance gene as selection markers, if these resistant genes are followed thalline and are entered host, host intestine flora may be entered in the mode of Horizontal transfer to propagate, accelerate the development of drug resistance of flora.Develop a kind of not containing the lactic acid bacteria expression vectors of antibiotic marker be its at food, the prerequisite of field of medicaments expansive approach.
Relative to intracellular expression, engineering bacteria cell wall is shown and secreting, expressing recombinant protein, is more conducive to the release of albumen and plays effectiveness.And the protein stability that the protein ratio being illustrated in cell wall dissociates is better, can play effectiveness for a long time in host.
Milk-acid bacteria is expressed and presenting and expressing exogenous antigen albumen, has application repeatly, achieve good effect at live bacterial vaccines product scope.But milk-acid bacteria is as the cell factory of expressing outer derived antimicrobial peptide, particularly showing at cell wall, is but the blank field of research so far.
In recent years, along with the development of milk-acid bacteria genomics and enriching constantly of genetic manipulation instrument, the drug delivery being delivery vector with live body milk-acid bacteria becomes a focus of milk-acid bacteria applied research gradually.Milk-acid bacteria is a very good delivery vector because it is probiotic bacterium, adapt to intestinal environment, the more important thing is, it determine grow the epithelial cell that site is small intestine inwall, the site, space of much virus can be competed, thus play antiviral effect.Meanwhile, milk-acid bacteria itself is again natural immunological adjuvant, can secrete polysaccharide, has good immunologic enhancement to human body.
Food grade lactic acid bacterium exploitation fly maggot antibacterial peptide is used also to have following advantage:
Recombinant antibacterial peptide can viable bacteria form use, without the need to purifying, with low cost, is easy to a large amount of production.Milk-acid bacteria inherently can produce the antimicrobial substances such as bacteriocin, can play and combine antibacterial effect with fly maggot antibacterial peptide.
Domestic patent (publication number CN103908668A) discloses fly maggot antibacterial peptide with immunoadjuvant function and preparation method thereof and the vaccine preparation containing this antibacterial peptide adjuvant, and they are preparing the application in vaccine preparation.This patent is relevant with extracting directly antibacterial peptide, does not use the technique means of recombinant antibacterial peptide.
Milk-acid bacteria live vector has been widely used in the vaccine development of mucosa-immune.
European Union's patent (EP1066375B1) discloses a kind of lactobacillus reuteri cell wall and shows the method for antigen, and United States Patent (USP) live (US2009/0324638A1) discloses a kind of method for milk-acid bacteria secretion or cell walls display protein matter or polypeptide.But all there is no the patent disclosure of expressing in milk-acid bacteria about fly maggot antibacterial peptide or showing.
So far, the total solution of the recombinant expressed fly maggot antibacterial peptide of a kind of food grade lactic acid bacterium is not also had.
(3) summary of the invention
The object of the invention is to provide a kind of food grade and expresses fly maggot antibacterial peptide recombinant lactic acid bacteria and application thereof, namely provides a kind of fly maggot antibacterial peptide, encoding gene, carrier, engineering bacteria and the application as antiseptic-germicide thereof.
The technical solution used in the present invention is:
The invention provides a kind of fly maggot antibacterial peptide, described fly maggot antibacterial peptide aminoacid sequence is for shown in SEQIDNO.2.
The present invention also provides a kind of described fly maggot antibacterial peptide encoding gene, and the nucleotides sequence of described encoding gene is classified as shown in SEQIDNO.1.
The present invention relates to a kind of recombinant vectors built by described fly maggot antibacterial peptide encoding gene.
The invention still further relates to a kind of food grade recombination engineering bacteria being transformed preparation by described recombinant vectors.
In addition, the present invention also provides a kind of described fly maggot antibacterial peptide encoding gene preparing the application in antibacterial peptide, and described is applied as: be converted in milk-acid bacteria by the recombinant vectors containing fly maggot antibacterial peptide encoding gene, be seeded to MRS substratum, cultivate 48 hours, as thalline OD for 30 DEG C
600nmbe greater than 20 and pH value lower than 4 time reaction terminate, by nutrient solution at 4 DEG C, the centrifugal 30min of 6000rpm, collect thalline, namely obtain containing the somatic cells of fly maggot antibacterial peptide.
The invention still further relates to a kind of described fly maggot antibacterial peptide and prepare the application in antiseptic-germicide, described antiseptic-germicide is the antiseptic-germicide of gram-positive microorganism or Gram-negative bacteria, preferred described gram-positive microorganism is subtilis, streptococcus aureus or bacillus cereus, and Gram-negative bacteria is Listeria monocytogenes or intestinal bacteria.Described antiseptic-germicide is the culture obtained through fermentation culture by the recombination engineering bacteria (preferred recombinant lactic acid bacteria) containing fly maggot antibacterial peptide encoding gene, and the consumption of described antiseptic-germicide is 100 milliliters of bacterium liquid (10
7~ 10
9cFU/mL) per kilogram feed.Concrete described antiseptic-germicide is prepared as follows: the recombination engineering bacteria (preferred recombinant lactic acid bacteria) containing fly maggot antibacterial peptide encoding gene is inoculated MRS substratum, cultivates 48 hours for 30 DEG C, detects cell density and culture system pH value, thalline OD
600nmbe greater than 20 and pH value can think fermentation ends lower than 4,4 DEG C, the centrifugal 30min of 6000rpm, collect thalline, be fungistat, during use, thalline equal-volume sterilizing PBS damping fluid suspended bacteria body can be prepared bacteria suspension.
Recombination engineering bacteria containing fly maggot antibacterial peptide encoding gene of the present invention obtains as follows: (1) peptide expression frame is: LDH promotor (SEQIDNO.4)-signal peptide (SEQIDNO.5)-anchorin (SEQIDNO.6)-fly maggot antibacterial peptide (SEQIDNO.3);
(2) alr gene (alanine racemase) expression cassette: with plant lactobacillus genomic dna for template, use primer alrF (SEQIDNO.10) and alrR (SEQIDNO.11), pcr amplification, obtain alr (alanine racemase) gene (SEQIDNO.9), by P
gADPHthe pcr amplification product (SEQIDNO.9) of gene fragment (SEQIDNO.8) and alr is template, adds primer P
gADPHf (SEQIDNO.12) and alrR (SEQIDNO.11), by promotor P
gADPHwith alr gene fusion, build alr gene expression frame.
(3) the Lactococcus lactis bacterial strain of alr gene (alanine racemase) defective type antibiotic-free mark: by DNA fragmentation: alr upstream region of gene 500bp homology arm (SEQIDNO.17)-lox site sequence (SEQIDNO.13)-chloramphenicol resistance gene expression cassette (SEQIDNO.16)-lox site sequence (SEQIDNO.15)-alr downstream of gene 500bp homology arm (SEQIDNO.18) is electroporated to Lactococcus lactis bacterial strain, the Lactococcus lactis bacterial strain of screening alr gene (alanine racemase) defective type antibiotic-free mark.
The recombinant vectors that the present invention is built by described fly maggot antibacterial peptide encoding gene is by IDH promotor, USp45 signal peptide and GSA grappling combinationally use structure recombinant vectors, again recombinant vectors is converted in milk-acid bacteria, the recombination engineering bacteria obtained makes the secreted expression of antibacterial peptide to extracellular, or is illustrated in the surface of cell walls.Expression vector does not carry exogenous microbiotic, the alanine racemase carried by carrier, cultivates, screening positive clone in the MRS substratum not adding D-alanine.
The expression of antibacterial peptide is controlled by constitutive promoter, and after vector linearization, remove antibiotics resistance gene, by the control of signal peptide and anchorin, antibacterial peptide is finally secreted into outside expressive host lactic-acid bacteria cells, or grappling is illustrated on cell walls.
Milk-acid bacteria of the present invention refers to all bacterial strains of all genus lactubacillus that " can be used for the bacterial classification list of food " (defend to do to supervise and send out (2010) No. 65) that the Ministry of Health issues comprises: i.e. Lactobacterium acidophilum (Lactobacillusacidophilus), lactobacterium casei (Lactobacilluscasei), lactobacillus crispatus (Lactobacilluscrispatus), lactobacillus bulgaricus (Lactobacillusbulgaricus), lactobacillus delbruckii (Lactobacillusdelbrueckii), lactobacillus fermentum (Lactobacillusfermentum), lactobacillus gasseri (Lactobacillusgasseri), lactobacterium helveticus (Lactobacillushelveticus), Lactobacillus johnsonii (Lactobacillusjohnsonii), lactobacillus paraceasi (Lactobacillusparacasei), plant lactobacillus (Lactobacillusplantarum), lactobacillus reuteri (Lactobacillusreuteri), lactobacillus rhamnosus (Lactobacillusrhamnosus), lactobacillus salivarius (Lactobacillussalivarius), and Lactococcus lactis subsp.lactis (Lactococcuslactissubspecieslactis) etc.
Antibacterial peptide of the present invention and anchorin are all optimized nucleotide sequence according to the use Preference of milk-acid bacteria to codon, are beneficial to express at lactic-acid bacteria cells.Its optimization principles is: TTCTTT; GTAGTT; TCCTCT; GAGGAA; TGCTGT; AGACGT; AGGCGT.
LDH promotor of the present invention refers to constitutive promoter, and this constitutive promoter is the similar sequence of lactobacterium casei LDH promotor.
Compared with existing antibacterial peptide production technology, the present invention has following advantage:
(1) because used expression system and all expression regulation original papers all come from food grade lactic acid bacterium, cheese milk-acid bacteria self, so final recombinant lactic acid bacteria of expressing antibacterial peptide is real food grade products;
(2) antibacterial peptide is illustrated in lactic-acid bacteria cells wall surface, has higher stability in host intestine, is not easy to degrade in intestinal environment, is convenient to long-term role.
(3) do not use milk-acid bacteria to express conventional inducible promoter, and use constitutive promoter, do not need to use the inductors such as Nisin during fermentation, thus its production technique is very easy.
(4) compared with the antibacterial peptide from fly maggot extracting directly, the recombinant lactic acid bacteria of expressing antibacterial peptide is safer, and large-scale production cost is lower.
(4) accompanying drawing explanation
Fig. 1 is the expression cassette schematic diagram of antibacterial peptide and anchorin.
Fig. 2 is that recombinant lactic acid bacteria suppresses gram-positive microorganism Bactericidal test figure, and a is 5 microlitre bacteria suspensions, and b is 10 microlitre bacteria suspensions, c is 20 microlitre bacteria suspensions, and d is 50 microlitre bacteria suspensions, and CK is sterilized water contrast, 1 is 1 microlitre bacteria suspension, and 25 is 25 microlitre bacteria suspensions, and 5 is 5 microlitre bacteria suspensions.
Fig. 3 is that recombinant lactic acid bacteria suppresses Gram-negative bacteria Bactericidal test figure, and a is 5 microlitre bacteria suspensions, and b is 10 microlitre bacteria suspensions, and c is 20 microlitre bacteria suspensions, and d is 50 microlitre bacteria suspensions, and CK is sterilized water contrast.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: antibacterial peptide and anchorin encoding gene codon optimized
Because milk-acid bacteria has stronger codon preference.Therefore in order to the antibacterial peptide gene (nucleotides sequence is classified as shown in SEQIDNO.1, and aminoacid sequence is SEQIDNO.2) that insect can be made to originate is smoothly at lactic-acid bacteria cells high expression level, need to be optimized its nucleotide series, remove rare codon.Its optimization principles is: TTC → TTT; GTA → GTT; TCC → TCT; GAG → GAA; TGC → TGT; AGA → CGT; AGG → CGT;
Nucleotides sequence after the optimization of fly maggot antibacterial peptide is classified as shown in SEQIDNO.3.
Embodiment 2: the structure of antibacterial peptide and anchorin expression cassette
The expression of antibacterial peptide and location are realized by multiple combinationally using of expression regulation original paper.These expression regulation original papers comprise promotor, signal peptide sequence, antibacterial peptide gene, anchorin sequence, and the catenation sequence (see Fig. 1) between each expression regulation original paper, peptide expression frame is: LDH promotor (SEQIDNO.4)-signal peptide (SEQIDNO.5)-anchorin (SEQIDNO.6)-fly maggot antibacterial peptide (SEQIDNO.3).
In the present invention, the expression of antibacterial peptide finally controls with constitutive promoter, and this constitutive promoter derives from lactobacterium casei serum lactic dehydrogenase (LDH) promotor, and nucleotides sequence is classified as shown in SEQIDNO.4.
Signal peptide sequence is Bacterium lacticum USP45 signal peptide, and nucleotides sequence is classified as shown in SEQIDNO.5.
Anchorin nucleotides sequence is classified as shown in SEQIDNO.6.
Anchorin aminoacid sequence is for shown in SEQIDNO.7.
The structure of embodiment 3:alr gene (alanine racemase) expression cassette
The glyceraldehyde 3-phosphate dehydrogenase promotor P of full genome synthesizing lactic acid bacterium
gADPH(nucleotides sequence is classified as shown in SEQIDNO.8), with plant lactobacillus genomic dna for template, use primer alrF (SEQIDNO.10) and alrR (SEQIDNO.11), pcr amplification, obtains alr (alanine racemase) gene (nucleotides sequence is classified as shown in SEQIDNO.9).
With above-mentioned P
gADPHthe pcr amplification product (SEQIDNO.9) of gene fragment (SEQIDNO.8) and alr is template, adds primer P
gADPHf (SEQIDNO.12) and alrR, by promotor P
gADPHwith alr gene fusion, successfully construct alr gene expression frame.
Fusion DNA vaccine condition: 94 degree of denaturation 5min, 94 degree of sex change 1min, 58 degree of annealing 30 seconds, 72 degree of extensions 2 minutes, 32 circulations.
The structure of embodiment 4:alr gene (alanine racemase) defective type Lactococcus lactis bacterial strain
(1) the chlorampenicol resistant expression cassette in loxp site is with to build
Adopt OverlappingPCR technology, will with lox site DNA fragmentation (SEQIDNO.13), chloramphenicol resistance gene expression cassette (SEQIDNO.14), with the DNA fragmentation (SEQIDNO.15) in lox site, permeate a DNA fragmentation (SEQIDNO.16), is lox site sequence-chloramphenicol resistance gene expression cassette-lox site sequence.
(2) alr knocks out box structure
Clone's lactobacillus acidophilus strains alr upstream region of gene flank 500bpDNA sequence (SEQIDNO.17), with the chloramphenicol resistance gene expression cassette (SEQIDNO.16) of lox site sequence, downstream flank 500bpDNA sequence (SEQIDNO.18), by above-mentioned sequence according to following order, be fused into a DNA fragmentation: alr upstream region of gene 500bp homology arm (SEQIDNO.17)-lox site sequence-chloramphenicol resistance gene expression cassette-lox site sequence (SEQIDNO.16)-alr downstream of gene 500bp homology arm (SEQIDNO.18), DNA fragmentation after this fusion is alr and knocks out box fragment.The primer that cloned upstream flanking sequence is used is UparmF and UparmR (SEQIDNO.19 and SEQIDNO.20), and the primer that cloned downstream flanking sequence is used is DownarmF and DownarmR (SEQIDNO.21 and SEQIDNO.22).
(3) alr that step (2) builds is knocked out box fragment by electroporated to lactococcal strain, coating resistance/L-Ala is dull and stereotyped.Resistance/L-Ala flat board M17 nutrient agar configuration (buying in Shanghai Ding Guo biotechnology company limited), adds chloramphenicol concentration 10 mcg/ml, D-alanine 10 mg/ml.Configuration MRS agar plate (substratum is bought in Shanghai Ding Guo biotechnology company limited).Screen the bacterium colony at resistance/L-Ala plated growth, and the bacterium colony that can not grow in MRS nutrient agar, be tentatively defined as positive colony.
Verify further by bacterium colony PCR, to obtain double exchange recon.Bacterium colony PCR the primer is UparmF and DownarmR (SEQIDNO.19 and SEQIDNO.22).
(4) containing the double exchange recon (need in advance prepare competence) of temperature-sensitive plasmid pFED760 (Erythromycinresistant) step of converting (3) by demonstrating of Cre recombinase encoding gene, 37 DEG C of MRS be incubated at containing 10 mcg/ml paraxin and 10 mg/ml D L-Ala are dull and stereotyped, after bacterium colony occurs, parallel switching dibbling is in the MRS Agar Plating containing 30 mcg/ml erythromycin and 10 mcg/ml paraxin respectively, screening is to Chloramphenicol-sensitive but have the recon of Erythromycinresistant, use the excision of colony polymerase chain reaction (PCR) method checking chloramphenicol resistance gene.
(5) recon step (4) obtained does not contain any antibiotic MRS (adding 10 mg/ml D L-Ala) substratum in 10ml, 37 DEG C of incubated overnight, to make the plasmid loss containing Cre recombinase encoding gene, nutrient solution is coated with MRS flat board, 37 DEG C of incubated overnight, get the parallel coated plate of bacterium colony dull and stereotyped and not dull and stereotyped containing any antibiotic MRS in the MRS containing 30 mcg/ml erythromycin, with the recon of screening to erythromycin-sensitive, verified the loss of the plasmid containing Cre recombinase encoding gene by bacterium colony PCR.Confirm as the lactococcal strain of alr gene (alanine racemase) defective type antibiotic-free mark.
Embodiment 5: the structure of expressing fly maggot antibacterial peptide food-grade recombination lactic acid bacterium, transforms and screening
(1) the 16srDNA gene order of Lactococcus lactis (Lactococcuslactis) is cloned.
(2) alr gene expression frame is cloned, with the plasmid in embodiment 3 for template.
(3) OverlappingPCR technology is by first fragment of 16srDNA (SEQIDNO.23), peptide expression frame (embodiment 2), alr gene expression frame (embodiment 3), second fragment of 16srDNA (SEQIDNO.24), permeate a DNA fragmentation.
(4) by the defective type Lactococcus lactis of the DNA fragmentation of fusion by electroporated acquisition to embodiment 4, the MRS culture medium flat plate of D L-Ala is not added in coating.Choose the bacterium colony that can grow, verify further by bacterium colony PCR, to obtain double exchange recombinant bacterium, namely containing the recombinant lactic acid bacteria of fly maggot antibacterial peptide encoding gene.
Embodiment 6: the fermentation expressing fly maggot antibacterial peptide food-grade recombination lactic acid bacterium
The double exchange recombinant bacterial strain inoculation MRS substratum that embodiment 5 is obtained, 30 degree cultivations 48 hours, detection cell density and culture system pH value, thalline OD
600nmbe greater than 20 simultaneously pH value can think fermentation ends lower than 4.4 degree of centrifugal 30min of 6000rpm, collect thalline, equal-volume sterilizing PBS damping fluid suspended bacteria body prepares bacteria suspension, and bacterium number reaches 10
9cFU/ml, as next step experiment material.
Embodiment 7: recombinant lactic acid bacteria suppresses pathogenic bacteria experiment
Recombinant lactic acid bacteria suppresses pathogenic bacteria experimental implementation:
(1) aseptic filter paper sheet, cutting diameter is the circle of 7mm.
(2) draw 5 microlitres, 10 microlitres, 20 microlitres, 50 microlitre different volumes embodiment 6 prepare bacteria suspension (being numbered a, b, c, d), instill filter paper respectively, blower cold wind dries, and volume can instill greatly several times, repeatedly dries up.Numbering CK is sterilized water contrast.
(3) (bacterium number reaches 10 to indicate pathogenic bacteria to be cultured to OD value=1.0 with nutrient broth medium
9individual/ml), draw 100 microlitres, coating nutrient broth agar is dull and stereotyped, and super clean bench dries 30min.
(4) filter paper is placed on nutrient broth agar flat board by aseptic technique respectively, 37 degree of incubated overnight, observes inhibition zone.
Inhibition zone the results are shown in Figure 2 and Fig. 3, and used pathogenic bacteria title is as shown in table 1, table 2:
Table 1 gram positive bacterium
| Streptococcus aureus ATCC6538 | Enterococcus faecalis ATCC29212 |
| Subtilis ATCC23857 | Bacillus cereus CICC10040 |
| Tetrads ATCC35098 | Streptococcus aureus |
Table 2 gram negative bacterium
| Swine escherichia coli | Pig Listeria monocytogenes |
| Bacillus foecalis alkaligenes ATCC8750 | Vibrio harveyi |
| Aeromonas hydrophila |
This experiment conclusion is the ability that recombinant lactic acid bacteria has the multiple pathogenetic bacteria of stronger suppression.
Embodiment 8: recombinant lactic acid bacteria hemolytic experiment
(1) 2% red corpuscle is prepared
Rabbit, extracts blood 5ml from heart, adds 0.2ml antithrombotics EDTA, with phosphoric acid buffer (PBS) washing, centrifugal, the white corpuscle on removing surface, aobvious red to supernatant liquor, gets the PBS that 1ml adds 50ml again after abandoning supernatant liquor.
(2) experimentation
Before each use red corpuscle, wash with PBS, until without obviously red (slightly yellow) in supernatant liquor, draw substrate 200 μ l, be diluted to 10ml, make the solution of red blood cells of 2%.Matching while using, the used time shakes up.
Bacteria suspension prepared by the embodiment 6 of 0.5ml and 0.5ml 2% solution of red blood cells mix, static 3h under room temperature condition, uses the centrifugal 3min of 10050r/min afterwards.Take out 100 μ l on 96 orifice plates, with the wavelength detecting of 545nm.Respectively using water and PBS damping fluid as positive control and negative control.2, each sample is parallel, is result, the results are shown in Table shown in 3 with mean value.
Hemolysis rate (%)=(absorption of sample-negative control absorbs)/(absorption of positive control absorption-negative control) × 100%.Hemolysis rate is considered as haemolysis more than 5%.
Table 3 haemolysis result
| Milk-acid bacteria bacteria suspension volume | Hemolysis rate |
| 5 microlitres | 0.2％ |
| 10 microlitres | 0.3％ |
| 20 microlitres | 0.3％ |
| 50 microlitres | 1％ |
Conclusion: this experimental result illustrates, recombinant lactic acid bacteria does not have haematolysis ability.
Embodiment 9: recombinant lactic acid bacteria infects the application experiment of fodder additives as diarrhea
(1) prepare SPF level C57BL/6 mouse in 10 week age, set up the diarrhea of mouse infection model of the rear gavage campylobacter jejuni of enteroinvasive E．Coli ATCC43893 premonition.
(2) microorganism prepares
Milk-acid bacteria: the frozen thalline (recombinant lactic acid bacteria containing fly maggot antibacterial peptide encoding gene prepared by embodiment 5) in-80 DEG C is seeded in liquid MRS substratum with the inoculum size of volumetric concentration 2%, at 37 DEG C, overnight stand cultivates 18-24h, this is for go down to posterity for the first time, carry out again after end going down to posterity for twice, centrifugal (5000r/min, 10min, 4 DEG C), collect thalline and fermented supernatant fluid.The thalline collected cleans twice by PBS, and resuspended, concentration adjusts to 10
7cFU/mL, 10
8cFU/mL and 10
9cFU/mL, stand-by; 0.22 μm of aseptic millipore filtration crossed by supernatant liquor, is stored in 4 DEG C of refrigerators.
Intestinal bacteria ATCC43893: the frozen thalline in-80 DEG C is seeded in LB liquid medium with the inoculum size of volumetric concentration 2%, at 37 DEG C, overnight stand cultivates 18-24h, this is for go down to posterity for the first time, carries out going down to posterity for twice after completing again, centrifugal (5000r/min, 10min, 4 DEG C), collect thalline, PBS cleaning twice, resuspended, concentration adjusts to 10
8cFU/mL.
Campylobacter jejuni: be seeded in the special BHI brain heart infusion broth substratum (purchased from Ding Guo bio tech ltd, Shanghai) of cultivation campylobacter jejuni by the frozen thalline in-80 DEG C with the inoculum size of volumetric concentration 2%, (gaseous constituent is 5%O to be placed in three gas
2, 10%CO
2and 85%N
2) incubator, 48h is cultivated every other day in 37 DEG C, this is for go down to posterity for the first time, then coats in the Colombia's blood agar (purchased from Ding Guo bio tech ltd, Shanghai) containing 5% aseptic Sheep Blood, again cultivates 48h under the same terms, go down to posterity twice, from planar surface, surperficial thalline is scraped down gently, centrifugal under 5000r/min, 10min, the condition of 4 DEG C, PBS cleaning twice, PBS is resuspended, adjustment concentration to 10
7cFU/mL, 10
8cFU/mL and 10
9cFU/mL, stand-by.
(3) mouse nuclei is set up
Intestinal bacteria bacteria suspension infected group: after 10 week age, SPF level C57BL/6 mouse normally fed one week, and gavage intestinal bacteria bacteria suspension 0.3mL (prepared by step 2, and 10
8cFU/mL), every day uninterrupted gavage.After 8 days, every mouse to increase every day gavage campylobacter jejuni bacteria suspension 0.3mL (prepared by step 2,10
9cFU/mL), colibacillary gavage is still uninterrupted, until terminate after 24 days period.
Non-infected group: after 10 week age, SPF level C57BL/6 mouse normally fed one week, gavage sterilized water, gavage amount and frequency are equal to infected group.
(4) the milk-acid bacteria gavage experiment of infecting mouse
Successful for infection mouse is divided into two groups, and (bacteria concentration is 10 to the milk-acid bacteria bacteria suspension of sample sets gavage step 2 preparation
8cFU/mL) 0.3mL, the sterilized water of the same volume of control group gavage, two groups are uninterrupted gavage every day, altogether gavage 12 days.
Non-infecting mouse is divided into two groups, and (bacteria concentration is 10 to the milk-acid bacteria bacteria suspension 0.3mL of sample sets gavage step 2 preparation
8cFU/mL), the sterilized water of the same volume of control group gavage, two groups are uninterrupted gavage every day, altogether gavage 12 days.
(5) campylobacter jejuni quantitative measurement in enteron aisle
From gavage milk-acid bacteria and sterilized water terminate to it, totally 12 days, on average sampling in every four days, often organize and be sampled as 6 at every turn, dissect mouse and get its colon portion content (ight soil), grind, be inoculated in selective medium-Colombia's blood agar of campylobacter jejuni after gradient dilution, in three gas incubator (5%O
2, 10%CO
2and 85%N
2) middle cultivation 48h, statistics campylobacter jejuni list bacterium colony number, the bacterium number of every sub-sampling is calculated according to the colony counts mean value of 6 mouse.
(6) experimental result is in table 4
Table 4 campylobacter jejuni quantitative measurement result
Conclusion: after recombinant lactic acid bacteria gavage C. jejuni infec-tion mouse, obviously can reduce the quantity of campylobacter jejuni in mouse intestinal, bacterium number have dropped 4 orders of magnitude, reaches the bacterium number level of non-infecting mouse; And the infecting mouse bacterium number of sterilized water gavage rises 1 order of magnitude, this illustrates that recombinant lactic acid bacteria obviously can reduce the quantity of the enteron aisle campylobacter jejuni of infecting mouse, can reach the level of normal uninfected mice.
Claims (9)
1. a fly maggot antibacterial peptide, is characterized in that described fly maggot antibacterial peptide aminoacid sequence is for shown in SEQIDNO.2.
2. a fly maggot antibacterial peptide encoding gene described in claim 1, is characterized in that the nucleotides sequence of described encoding gene is classified as shown in SEQIDNO.1.
3. the recombinant vectors built by fly maggot antibacterial peptide encoding gene described in claim 2.
4. one kind is transformed the recombination engineering bacteria of preparation by recombinant vectors described in claim 3.
5. described in a claim 2, fly maggot antibacterial peptide encoding gene is preparing the application in antibacterial peptide, it is characterized in that described being applied as: be converted in milk-acid bacteria by the recombinant vectors containing fly maggot antibacterial peptide encoding gene, be seeded to MRS substratum, cultivate 48 hours, as thalline OD for 30 DEG C
600nmbe greater than 20 and pH value lower than 4 time reaction terminate, by nutrient solution at 4 DEG C, the centrifugal 30min of 6000rpm, collect thalline, namely obtain containing the somatic cells of fly maggot antibacterial peptide.
6. described in a claim 1, fly maggot antibacterial peptide is preparing the application in antiseptic-germicide.
7. apply as claimed in claim 6, it is characterized in that described antiseptic-germicide is the antiseptic-germicide of gram-positive microorganism or Gram-negative bacteria.
8. apply as claimed in claim 7, it is characterized in that described gram-positive microorganism is subtilis, streptococcus aureus, bacillus cereus, Gram-negative bacteria is Listeria monocytogenes, intestinal bacteria.
9. apply as claimed in claim 6, it is characterized in that described antiseptic-germicide is prepared as follows: by the recombination engineering bacteria inoculation MRS substratum containing fly maggot antibacterial peptide encoding gene, cultivate 48 hours, as thalline OD for 30 DEG C
600nmbe greater than 20 and pH value lower than 4 time fermentation ends, 4 DEG C, the centrifugal 30min of 6000rpm, collect thalline, be fungistat.
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| CN109320596A (en) * | 2018-11-15 | 2019-02-12 | 东北农业大学 | Fly maggot antibacterial peptide FX1 and its application in pannage |
| CN113845584A (en) * | 2021-11-08 | 2021-12-28 | 江苏三仪生物工程有限公司 | Preparation method of recombinant avian epidermal growth factor |
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| CN113845584A (en) * | 2021-11-08 | 2021-12-28 | 江苏三仪生物工程有限公司 | Preparation method of recombinant avian epidermal growth factor |
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