CN103421844A - Silkworm bioreactor for producing fodder antimicrobial peptides and construction method thereof - Google Patents
Silkworm bioreactor for producing fodder antimicrobial peptides and construction method thereof Download PDFInfo
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Abstract
The invention relates to a construction method of a silkworm bioreactor for the coexpression of temporin-1Sd and cecropin AD. The construction method comprises the following steps: after fusing gene sequences of the temporin-1Sd and the cecropin AD or the gene sequences of the temporin-1Sd and the cecropin AD the gene of the GFP (Green Fluorescent Protein), cloning in a baculovirus transfer vector to construct a transfer expression vector; performing the transposition recombination on the constructed transfer expression vector and baculovirus DNA in escherichia coli to obtain recombination baculoviruses; enabling the recombination baculoviruses to be infected with silkworms; cultivating the infected silkworms and expressing relevant antibacterial peptide proteins and fusion proteins; gaining and drying the antibacterial peptides. The temporin-1Sd and the cecropin AD can be produced safely and efficiently in the silkworm bioreactor by adopting a baculovirus expression system, and are used as animal feed additives, and the silkworm bioreactor and the construction method have the advantages of safety, no side effect, high efficiency, low energy consumption, low cost, no drug resistance due to antibiotics and the like.
Description
Technical field
The invention belongs to the genetically engineered field, the silkworm biological reactor construction process that relates to a kind of coexpression temporin-Lb temporin-1Sd and cecropin cecropin AD, relate in particular to and utilize the method for recombinant baculovirus at insect expression in vivo temporin-Lb temporin-1Sd and cecropin cecropin AD, can be used for preparing feeding antibacterial peptide additive.
Background technology
Along with the continuous appearance of resistant organism and bacterium multidrug resistant phenomenon, seeking safely and effectively microbiotic or its substitute becomes the pharmaceutical industries problem demanding prompt solution.Antibacterial peptide, because having the advantages such as has a broad antifungal spectrum, Heat stability is good, unique, the difficult generation resistance of antibacterial mechanisms, is expected to become the effective ways that solve this difficult problem.Antibacterial peptide be a kind of by the secretion of multiple organism, there is wide spectrum antibacterium, fungi, virus, parasite, antitumor and promote the effects such as wound healing and angiogenesis, and do not destroy eukaryotic cell, non-immunogenicity, very likely becoming the new source of antibiotic, antiviral and antitumor drug, is the important component part of a lot of biological congenital nonspecific defense systems.
Cecropin (cecropins) is that Sweden scientist Boman in 1980 etc. separate and obtain first antibacterial peptide from silkworm chrysalis.At present, found more than 1700 kind of antibacterial peptide, and cecropin is that research at present is the clearest, the most obvious antibacterial peptide of effect.Cecropin generally contains 37～39 amino-acid residues, not containing halfcystine, its N end regions has strong basicity, can form parents' spirane structure of almost Perfect, and can form hydrophobic spiral at the C end regions, the hinge area that has glycine and proline(Pro) to form between the two, the C end of most polypeptide is amidated, and amidation has vital role to its anti-microbial activity.Cecropin (cecropins) has very strong lethality to gram-positive microorganism, part Gram-negative bacteria, and fungi and eukaryotic cell are not had to toxicity.In addition, research shows that temporin-Lb temporin-1Sd produces obvious restraining effect to various bacteria, has application prospect preferably.Temporin-Lb mainly exists in the body of gland of frog body skin histology, thereby is secreted into parcel natural cover for defense with the skin appearance face forms together with when being subject to the invasion and attack of pathogenic micro-organism, at present from 8 genus, approximately the frog skin of kind more than 40, has extracted antibacterial peptide.
Due to antibacterial peptide, content is atomic in animal body, in animal body, extracts low, the time-consuming length of yield of antibacterial peptides, complex process, somewhat expensive, can't realize scale operation, and this becomes the biggest obstacle that the restriction antibacterial peptide enters practical application.Therefore, people start to turn to the production problem of utilizing gene engineering method to study and solve antibacterial peptide.At present, the genetically engineered drug majority that has entered clinical application is to adopt prokaryotic expression system to produce, but due to the lethal effect of antibacterial peptide to bacterium, can not directly express and there is bioactive antibacterial peptide with prokaryotic expression system, if, and the form of employing fusion rotein is expressed, will bring very burden to the aftertreatment of expression product.Therefore, investigator both domestic and external adopts eukaryotic expression system to carry out the antibacterial peptide gene engineering research more.In recent years, the yeast of take causes people's attention as the research of genetically engineered recipient bacterium, yeast has the gene expression regulation more complete than intestinal bacteria mechanism and to processing modification and the secretion capacity of expression product, and can not produce intracellular toxin, be eukaryotic gene recipient bacterium good in genetically engineered, but, because the kind of yeast generation intrinsic protein product is many, content is high, bring a lot of problems to the purifying of antigen peptide, still need and further improve and solve.
Modern study thinks that silkworm biological reactor is expected to solve many production problems of antigen peptide based on characteristics own.In brief, " silkworm biological reactor " refers to and will be cultivated in the silkworm chrysalis body of specific recombinant baculovirus (gene) implantation silkworm, silkworm chrysalis can initiatively be transcribed and be translated implanting gene, the biologically active substance that Nature creating is useful to the mankind, with the demands such as prevention, treatment and health care that meet disease.
In recent years, silkworm biological reactor is widely used on medical science, veterinary science and agricultural.Used at present the antigen protein of the multiple mankind of silkworm biological reactor successful expression and animal virus, wherein quite a few is carrying out commercialized development.As utilizing silkworm biological reactor to express not containing virulent genetic material virus like particle (VLPs), the unique texture had, the ability of showing epi-position and energy effective stimulus produce immune response, for the research prevention of various diseases provides good method.
The relevant technologies of baculovirus expression vector system is relatively ripe, and this system expression foreign gene is not only economical, efficient, and a new technological approaches can be provided.Utilize silkworm biological reactor coexpression temporin-Lb temporin-1Sd and cecropin cecropin AD, guarantee that it has normal protein structure and biological activity; Afterwards, its silkworm is processed and adds in feed, improve the nutritive value of feed and the resistance against diseases of animal.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide utilize baculovirus expression vector system in insect body safety, express efficiently the method for temporin-Lb temporin-1Sd and cecropin cecropin AD.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method of expressing temporin-Lb temporin-1Sd and cecropin cecropin AD, comprise: temporin-Lb temporin-1Sd and cecropin cecropin AD gene order or by after its fusion rotein, they are cloned in baculovirus transfer vector, build and obtain shifting expression vector; The transfer expression vector that structure is obtained and baculovirus DNA carry out the swivel base restructuring in intestinal bacteria, obtain recombinant baculovirus; By the recombinate shape virus infection insect host; Cultivate infected insect host and express corresponding antibacterial peptide; Results expressed temporin-Lb temporin-1Sd and the cecropin cecropin AD of drying treatment.
Described antibacterial peptide gene sequence and the sequence that merges green fluorescent protein are as the sequence 1 in SEQ ID NO1(sequence table), the sequence 2 in SEQ ID NO2(sequence table) as shown in nucleotide sequence and the sequence 3 in SEQ ID NO3(sequence table) sequence 4 in SEQ ID NO4(sequence table) corresponding aminoacid sequence.Described baculovirus delivery carrier is selected from the pfastbac-dul double-promoter, can express two copies of two kinds of antibacterial peptides or same antibacterial peptide simultaneously.
Constructed transfer expression vector is the carrier pfastbac-temporin-1Sd-cecropin AD with the antibacterial peptide gene original series, and with the carrier pfastbac-dul-temporin-1Sd-gfp-cecropin AD of antibacterial peptide fusion protein sequence
Described baculovirus DNA is BmNPV.
Described recombinant baculovirus is selected from following any one recombinant virus: (1) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-temporin-1Sd-cecropin AD; (2) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-temporin-1Sd-gfp-cecropin AD; (3) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-gfp temporin-1Sd-cecropin AD.
Described insect host comprises silkworm (Bombyx mori), Semen Ricini silkworm (Philosamia cynthia ricim), Philosamia cynthia (Philosamia cynthia pryeri), wild silkworm (Dictyoplca japanica), yamama (Antheraea yamamai), tussah (Antheraea pernyi), wild silkworm (Bombyx mandarina), wild giant silkworm (Antheraea polyphymus) etc.
Described insect host is silkworm larva and pupa (Bombyx mori); Described infection refers to that recombinant baculovirus infects 1-5 insect larvae or the pupal cell in age by eating or seeing through epidermis, be preferably: by silkworm larva or the pupa in recombinant Bombyx mori baculovirus infected silkworm cell or percutaneous puncture-inoculation 1-5 age, body fluid or the tissue homogenate of collector's silkworm larva or pupa after infecting 3-6 days; The early stage tender pupa that described optimum pupal cell is 1-2 days.
The present invention adopts gene recombination technology, temporin-Lb temporin-1Sd and cecropin cecropin AD gene and fusion rotein gfp are cloned into baculovirus delivery carrier pfastbac-dul, under polyhedron promotor and the control of p10 promotor strong promoter, pass through vitro recombination, the original series of antibacterial peptide and the sequence after fusion are incorporated on the genome of baculovirus, obtain recombinant virus; Recombinant virus can infect by epidermis insect larvae or the pupal cell (the early stage tender pupa that optimal time is 1-2 days) of 1-5 age (optimal time was four or five ages), Expression product temporin-Lb temporin-1Sd and cecropin cecropin AD.
The inventive method adopt baculovirus expression system in silkworm biological reactor safety, produce temporin-Lb temporin-1Sd and cecropin cecropin AD efficiently, the ecosystem energy consumption is few.Because silkworm is approved as food medicine dual-purpose insect by China Ministry of Health, thus by the inventive method prepared antibacterial peptide, security is high, can directly make to animal edible.
The inventive method can decrease temporin-Lb temporin-1Sd and the production cost of cecropin cecropin AD, and can obtain two kinds of different antibacterial peptides, has the plurality of advantages such as safety, efficient, less energy consumption, cost are low simultaneously.
The accompanying drawing explanation
Fig. 1: gene clone conceptual scheme
Fig. 2: recombinant vectors pMD-cecropin AD and pMD-temporin-1Sd double digestion are identified figure:
Fig. 3: the single endonuclease digestion of pMD-gfp recombinant plasmid vector is identified figure
Fig. 4: the double digestion qualification result figure of recombinant vectors
Fig. 5: the blue hickie screening figure of recombination to construct
Fig. 6: the pathology figure after Bmn cell infection baculovirus:
Fig. 7: the fluorescent microscope qualification result figure of reconstitution cell
Fig. 8: spray-dired preparation is to the escherichia coli (resistance) from hospital's clinical case separation and the bacteriostatic experiment result of staphylococcus haemolyticus
Embodiment
Below provide a series of concrete experimental techniques, further set forth the present invention, this part experimental technique is not to further restriction of the present invention.
1. about the configuration of solution and substratum
Ethidium bromide (EB) mother liquor: it is 10 mg/mL that EB is made into to concentration, wraps up in containers store with aluminium foil or black paper bag and gets final product in room temperature.
The IPTG liquid storage: 1 mol/L, cross filtering with 0.2 μ m filter after the ultrapure water preparation.
The LB substratum: Tryptones 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, adjust the pH value and contain 1.5% agar to the 7.0(solid medium), 121 ℃ of high temperature and high pressure steam sterilizing 15 min.
2. the clone of gene and order-checking
2.1 antibacterial peptide gene is synthetic by the handsome biotech firm in Shanghai, enzyme-added site BamH I and the Xba I of cutting in both sides, and SphI and XhoI.
2.2 the detection design of primers of goal gene, capitalization is partly restriction enzyme site
cecropin-f: GGATCCatgaatttcgcaaagatcctatc (BamHI)
cecropin-r: TCTAGAtcattttcctatggctttagctg (XbalI)
temporin-f: CTCGAGttccttgggaccatcaacttat (XhoI)
temporin-r: GCATGCgacatctgttgagcatttagcca (SphI)
gfp-cec-f: GGATCCatggtgagcaagcagatcctg (BamHI)
gfp-cec-r: GGATCCcacccactcgtgcaggctgc (BamHI)
gfp-tem-f: CTCGAGatggtgagcaagcagatcctg (XhoI)
gfp-tem-r: CTCGAGcacccactcgtgcaggctgc (XhoI)
2.3 pcr amplification reaction synthetic gene sequence
After pcr amplification finishes, get 5.0 μ L reaction product and carry out electrophoresis in 1.0% TAE sepharose, the ethidium bromide that contains 0.5 μ g/mL in gel, electrophoretic buffer is 0.5 * TAE, 100V, approximately after 20min, in the ultraviolet gel imaging system, observe clip size, compare with the DNA Marker of standard, result shows that the antibacterial peptide gene size that successfully increases is consistent with expection.
2.4 gene fragment reclaims test kit and reclaims DNA fragmentation
PCR product or enzyme are cut product and are carried out agarose gel electrophoresis, cut the target DNA band; The operating basis specification sheets.DNA solution is stored in-20 ℃ standby.Detected through gel electrophoresis reclaims successfully.
2.5 DNA fragmentation is connected with cloning vector
By specification requires to carry out.Linked system: 1 μ L pMD18-T vector, 4 μ L purpose fragments, 5 μ L Ligation solution I, more than 16 ℃ of connection 30min.
2.6 a small amount of of E.coli heat shock competent cell preparation
The recovery of ruling on the LB flat board of the E. coli glycerol stock of-70 ℃ of cold storage; Picking list bacterium colony, be inoculated into 4 mL not containing in antibiotic LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night; The bacterium liquid of getting 1/100 volume overnight incubation is inoculated into fresh containing in antibiotic LB liquid nutrient medium, and 37 ℃ of shaking culture 2～3h, make OD
600Value reaches 0.6 left and right; Simultaneously by CaCl
2(75mmol/L) be placed in precooling on ice with the EP pipe; 1mL bacterium liquid is collected in the aseptic 1.5mL EP pipe that precooling is good, placed 5min on ice, 4 ℃, centrifugal 5 min of 4000r/min, abandon supernatant; Thalline is resuspended in the 75mmol/L CaCl of 800 μ l precoolings
2In solution, ice bath 30min; 4 ℃, the centrifugal 5min of 4000 r/min, abandon supernatant; The 75mmol/L CaCl containing 10% glycerine that adds 200 μ L precoolings in precipitation
2Solution, blow afloat thalline gently with the rifle head, makes to mix; Put on ice after 3～4 h for transforming.
2.7 connecting the heat shock of product transforms
Get the connection product and add in competent cell, ice bath 30 min; 42 ℃ of heat shock 90s, put rapidly 2min on ice; Add the LB liquid nutrient medium of 1mL balance to room temperature, 37 ℃ of incubations are cultivated 1h; The centrifugal 3min of 5000r/min, (stay 150～200 μ L) after removing the part supernatant and coat containing on suitable antibiotic LB flat board; Be inverted overnight incubation for 37 ℃.
2.8 bacterium liquid PCR identifies recombinant plasmid
A plurality of single colony transformation of picking are inoculated in 1mL containing in the LB substratum of 100 μ g/mL Amp respectively, and 37 ℃ of shaking culture are spent the night; Get 1 μ L bacterium liquid in the Eppendorf pipe, unloaded plasmid, as negative control, adds PCR mix and specific detection primer, and pcr amplification 20ul reminds, the agarose gel electrophoresis observations.
2.9 a small amount of extracting plasmid DNA fast of alkaline process
Collect 3mL bacterium liquid in the Ep pipe, the centrifugal 5min of 5000rpm collects thalline; Operation, according to Xygen plasmid corpusculum test kit specification sheets, is dissolved plasmid DNA with 100 μ L 0.1 * TE (pH8.0) in detail, and-20 ℃ save backup.
2.10 the enzyme of recombinant plasmid is cut evaluation
It is as follows that enzyme is cut system:
| ddH 2O | 15μl |
| 10×Buffer E | 2μl |
| Recombinant plasmid dna | 2μl |
| BamHⅠ | 0.5μl |
| XbaⅠ | 0.5μl |
| Cumulative volume | 20μl |
| ddH 2O | 15μl |
| 10×Buffer E | 2μl |
| Recombinant plasmid dna | 2μl |
| SphI | 0.5μl |
| XhoI | 0.5μl |
| Cumulative volume | 20μl |
37 ℃ of reaction 1～2h, agarose gel electrophoresis detects and cuts out purpose band gfp, cecropin AD and temporin-1Sd, and as shown in Figure 2 and Figure 3, in Fig. 2, M is DNA Marker; 1 is: the BamHI of pMD-cecropin AD and XbalI double digestion product; 2 are: the XhoI of pMD-temporin-1Sd and SphI double digestion product.In Fig. 3, M is DNA Marker DL2000; The BamHI single endonuclease digestion product of swimming lane 1:pMD-gfp; The XhoI single endonuclease digestion product of swimming lane 2:pMD-gfp.By Fig. 2 and Fig. 3 presentation of results goal gene, successfully be connected on the pMD-18T carrier.
2.11 the order-checking of recombinant plasmid is identified and is analyzed
Taking a morsel is accredited as positive colony bacterium liquid and delivers to Shanghai order-checking section of handsome Bioisystech Co., Ltd and checked order, and for sequencing result, the software such as DNAStar, DNAMAN compares to sequence.Result shows that the sequence of pcr amplification is consistent with theoretical sequence, there is no frameshit or sudden change.
3. the structure of recombinant baculovirus transfer vector pfast-dul
3.1 the acquisition of purpose fragment and carrier
BamH I and Xba I double digestion for correct recombinant plasmid pMD-cecropin AD identified in order-checking.It is as follows that enzyme is cut system:
| ddH 2O | 23μl |
| 10×Buffer B | 5μl |
| Recombinant plasmid dna | 20μl |
| BamHI | 1μl |
| XbaⅠ | 1μl |
| Cumulative volume | 50μl |
37 ℃ of reaction 2 ~ 4h, agarose gel electrophoresis reclaims the purpose fragment.
Eukaryotic expression plasmid pfast-dul makes same enzyme and cuts processing, and the amount of plasmid is reduced to 4 μ l, and after reaction finishes, in 65 ℃ of deactivations 10 minutes ,-20 ℃ saved backup.
3.2 enzyme is cut the recovery of product
With 2.4.
3.3 the purpose fragment is connected with carrier
The cecropin AD purpose fragment that enzyme cuts back to close is connected with the transfer vector pfast-dul after enzyme is cut.Linked system is as follows:
| The T4 DNA ligase | 1μl |
| 10 * connection Buffer | 1μl |
| The purpose fragment reclaimed | 6μl |
| Carrier | 2μl |
| Cumulative volume | 10μl |
16 ℃ are reacted after 8 hours for transforming.
3.4 the conversion of recombinant plasmid and evaluation.
To connect product transformed competence colibacillus cell with reference to the methods involving in 2.1, screen containing on the agar plate of amicillin resistance, after selected clone, extract plasmid.Recombinant plasmid is cut with gene sequencing and is identified through enzyme, and as shown in Figure 4, in figure, M is DNA Marker; 1 swimming lane: the BamHI of pastbac-gfp-cecropin AD and PstI double digestion product; The XhoI of 4 swimming lanes: pastbac-gfp-temporin-1Sd and KpnI double digestion product.Identify correct recombinant plasmid called after pastbac-temporin-cecropin, obtain in an identical manner recombinant plasmid pastbac-gfp-temporin--cecropin, pastbac-temporin-gfp-cecropin.
4. the preparation of the acquisition of recombinating silkworm nucleus multiple the body of angle virus and viral DNA
4.1 donor plasmid and the restructuring of baculovirus BmNPV genome swivel base
Ice bath 30min, 42 ℃ of heat shock 45s, be transferred to rapidly ice bath 2-3min in mixture of ice and water, add 1ml LB substratum, 37 ℃, 200rpm are hatched 4h, do 10-1,10-2,10-3 serial dilution, respectively get 100 μ l coated plates (containing microbiotic, X-gal) from 10-2,10-3 diluent, cultivate 36-48h for 37 ℃.
As shown in Figure 5, wherein hickie is restructuring positive bacteria, 1 negative contrast in figure to result; The 2nd, pfast-temporin-1Sd
-cecropin AD; The 3rd, pfast-temporin-1Sd-gfp-cecropin AD; The 4th, pfast-gfp-temporin-1Sd-cecropin AD transforms DH10-bac.
4.2 the group extracting of recombinant baculovirus BmNPV genomic gene and PCR identify
On blue hickie flat board, picking hickie bacterium colony shakes cultivation to the LB substratum that contains 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tetracyclines, cultivate 12h for 37 ℃, extract Bacmid DNA with solution I, II, III method, as template, whether M13 upstream and downstream primer PCR identifies positive clone.
4.3 cell transfecting and Identification of recombinant baculovirus
Extract restructuring Bacmid, transfection insect cell: (1) 1 μ g restructuring Bacmid DNA adds 100 μ l two without in (serum-free, unparalleled anti-) TC-100 nutrient solution; (2) 6 μ l liposomes are joined in the two nothings of 100 μ l (serum-free, unparalleled anti-) TC-100 nutrient solution and mix; (3) (1) and (2) solution is mixed, room temperature is placed 15-45min; (4) by insect cell (cover 70-80% area) with two without nutrient solution, be three times, liposome and DNA mixture add 800 μ l two without the TC-100 nutrient solution again, mixing, add in cell, 27 ℃, standing 5h; (5) remove liposome, DNA mixture liquid, the TC-100 nutrient solution continues to cultivate 3-4d to add 2ml (to contain two anti-, serum) fully, collects supernatant.Recombinant virus called after rBmNPV-temporin-1Sd-cecropin AD; RBmNPV-temporin-1Sd-gfp-cecropin AD; RBmNPV-gfp temporin-1Sd-cecropin B.As shown in Figure 6, in figure, 1 is normal Bmn cell to the pathology photo of recombinant virus on cell; 2 for having infected recombinant bombyx mori nuclear polyhedrosis virus rBmNPV(temporin-1Sd-cecropin AD); 3 for having infected recombinant bombyx mori nuclear polyhedrosis virus rBmNPV(temporin-1Sd-gfp-cecropin AD); 4 for having infected recombinant bombyx mori nuclear polyhedrosis virus rBmNPV(gfp temporin-1Sd-cecropin AD) pathology figure after 48h.
4.4 the evaluation of recombinant virus
Utilize PCR method to analyze exogenous origin gene integrator.The extracting method of free virus genomic dna is as follows: get viral supernatant 150 μ l, add 150 μ l NaOH(0.5 mol/L) after mix, add again 20 μ l(8 mol/L) ammonium acetate, mix the isopyknic phenol of rear use and chloroform respectively extracting once, after alcohol precipitation with the TE dissolving DNA of 20 μ l.
Get above-mentioned virus genom DNA 1 μ l and carry out pcr amplification, reaction system and reaction conditions are with 2.3.Get 15 μ l reaction product electrophoretic analysiss, the result proof has obtained recombinant virus.
5.
The expression of recombinant virus foreign protein detects
Prepare insect cell Bmn(and cover the 70-80% area), by obtain recombinant baculovirus rBmNPV-temporin-1Sd-cecropin AD; RBmNPV-temporin-1Sd-gfp-cecropin AD; RBmNPV-gfp temporin-1Sd-cecropin B infects respectively the Bmn cell, after 48h, observes the fluorescence result as shown in Figure 7, and in figure, 1 is the contrast of Bmn cell; 2 is recombinant bombyx mori nuclear polyhedrosis virus rBmNPV(temporin-1Sd-gfp-cecropin AD) infection Bmn cell 48h fluorogram; 3 is recombinant bombyx mori nuclear polyhedrosis virus rBmNPV(gfp-temporin-1Sd-cecropin AD) infection Bmn cell 48h fluorogram.
The above results explanation gfp fusion protein is expressed normal, can illustrate that thus the temporin-1Sd and the cecropin AD that clone on same position can smooth high efficient expressions.
6. recombinant virus high efficient expression in silkworm pupa and silkworm body
Silkworm pupa used is that the high expression level kind is that JY1(is preserved by this laboratory).JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.After first feeding 48h select the silkworm that mean body weight is identical and cocoon seven days after 15 identical silkworm chrysalises of mean body weight, every silkworm chrysalis and silkworm inoculate approximately 1.0 * 10
5RBmNPV, collect morbidity silkworm blood and silkworm chrysalis after 4-5 days ,-20 ℃ frozen for detection of.
7. spraying drying
The silkworm or the silkworm chrysalis that produce antibacterial peptide are made to homogenate with the 10000rpm/min homogenizer, homogenate is inputted to spray-dryer, material flow is 0.5kg/min; Spraying drying condition: hot air temperature 130-135 ℃, exhaust temperature 60-65 ℃, material 15-40 in spraying drying completes drying in second, filter 40 eye mesh screens, obtain silkworm powder or dried silkworm chrysalis meal containing antibacterial peptide, use as fodder additives, check its anti-microbial activity as shown in Figure 8, in figure: 1 means the anti-microbial activity of penbritin; 2 negative contrast ddH
2O; 3,4,5,6,7 anti-microbial activities that are antibacterial peptide in spray-dired preparation.This product all has obvious anti-microbial activity to pathogenic bacterium escherichia coli (resistance) and the staphylococcus haemolyticus from clinical separation.
Sequence table
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CAA AGA GTT AGA GAC GCT GTC ATC TCT GCT GGT CCT GCT 78
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15 20 25
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40 45 50
A AA AAC CCA AAA ATG TTG AAA CTT TGG AAA TGG AAT TGG 195
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80
Claims (10)
1. a method of expressing temporin-Lb temporin-1Sd and cecropin cecropin AD, comprise: temporin-Lb temporin-1Sd and cecropin AD gene order or itself and green fluorescent protein are merged after, they are cloned in baculovirus transfer vector, build and obtain shifting expression vector; The transfer expression vector of structure and baculovirus DNA are carried out to the swivel base restructuring in intestinal bacteria, obtain recombinant baculovirus; By the recombinate shape virus infection insect host; Cultivate infected insect host and express corresponding temporin-Lb temporin-1Sd and cecropin AD; Results the expressed antibacterial peptide of drying treatment.
2. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that the nucleotide sequence of described antibacterial peptide temporin-Lb temporin-1Sd and cecropin AD is as shown in sequence 1 and sequence 2 in sequence table, corresponding aminoacid sequence is as shown in sequence 3 and sequence 4 in sequence table.
3. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that described baculovirus delivery carrier is selected from the pfastbac-dul double-promoter, can express two copies of two kinds of antibacterial peptides or same antibacterial peptide simultaneously.
4. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that the transfer expression vector that described structure obtains is the carrier pfast-temporin-1Sd-AD with the antibacterial peptide gene original series, pfast-cecropin AD-AD, with the carrier pfast-temporin-1Sd-gfp-AD with the antibacterial peptide fusion protein sequence, pfast-cecropin AD-gfp-AD.
5. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that described baculovirus DNA is BmNPV.
6. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that described recombinant baculovirus is selected from following any one recombinant virus: recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-temporin-1Sd-cecropinAD; Recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-temporin-1Sd-gfp-cecropin AD; Recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-gfp temporin-1Sd-cecropin AD.
7. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, be characterised in that described insect host comprises silkworm, Semen Ricini silkworm, Philosamia cynthia, wild silkworm, yamama, tussah, wild silkworm, wild giant silkworm and corresponding insect cell.
8. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 1 and cecropin cecropin AD, it is characterized in that described insect host is silkworm larva and pupa; Described infection refers to that recombinant baculovirus infects 1-5 insect larvae or the pupal cell in age by eating or seeing through epidermis.
9. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 8 and cecropin cecropin AD, it is characterized in that, the insect larvae in described infection 1-5 age or pupal cell are by silkworm larva or the pupa in recombinant Bombyx mori baculovirus infected silkworm cell or percutaneous puncture-inoculation 1-5 age, body fluid or the tissue homogenate of collector's silkworm larva or pupa after infecting 3-6 days.
10. according to the method for expression temporin-Lb temporin-1Sd claimed in claim 9 and cecropin cecropin AD, it is characterized in that the early stage tender pupa that described pupal cell is 1-2 days.
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