CN1456574A - Preparing method for extracting polypeptide antibiotics from silkworm - Google Patents
Preparing method for extracting polypeptide antibiotics from silkworm Download PDFInfo
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- CN1456574A CN1456574A CN 03128069 CN03128069A CN1456574A CN 1456574 A CN1456574 A CN 1456574A CN 03128069 CN03128069 CN 03128069 CN 03128069 A CN03128069 A CN 03128069A CN 1456574 A CN1456574 A CN 1456574A
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Abstract
A process for extracting polypeptide antibiotic from bombxy mori includes such steps as including polypeptide antibiotic in young silkworm of pupa, preparing extracting liquid, separating and purifying. Its advantages are simple process, low cost, broad antibacterial spectrum, and high purity.
Description
Technical field
The present invention relates to fields such as insect immunology, biological chemistry, microbiology, more specifically relate to a kind of preparation method who from the silkworm body, extracts peptide antibiotics.
Background technology
In the world first found antibacterial peptide be 1980 by people such as Sweden scientist G.Boman through injection cloaca through rod bacterium and intestinal bacteria (Escherichia coli K
12D
31) induce and cherish the polypeptide with anti-microbial activity that guppy sky silkworm chrysalis produces, name and be Cecropins.After this between the several years, people find from bacterium, fungi, batrachians, insect, higher plant, Mammals and even the mankind in succession and separate the polypeptide that obtains to have anti-microbial activity.Because it is found that this class active polypeptide has the broad-spectrum high efficacy fungicidal activity to bacterium at first, thereby called after " antibactetial peptides (ABP) ", Chinese is translated into antibacterial peptide, its former antibacterium peptide that means.Along with carrying out in a deep going way of people's research work, find that some antibacterium peptide all has strong lethal effect to part fungi, protozoon, virus and cancer cells etc., thereby at present this class active polypeptide is tended to be referred to as " peptideantibiotics "----peptide antibiotics.
Because the natural polypeptides microbiotic has bacterium, fungi, protozoon, virus and cancer cells etc. is all had strong lethal effect, more and more is subjected to people's attention.At present Chinese scholars is separated to have purified successively from various biologies and is had the polypeptide of anti-microbial activity, representative is from mammalian tissues, isolated in barley and the wheat groat and contained 29-34 amino acid whose defensins class peptide antibiotics, and, apply on the anti-system of plant pest by the synthetic commercialization; From the nervous tissue of the skin of the toad and the frog, Mammals (rabbit, ox, pig) and intestinal tissue, isolated and contained 28-35 amino acid whose maganins class peptide antibiotics; Isolated from the hemolymph system of sky silkworm chrysalis, silkworm, tussah, fruit bat, sarcophagid and contained 37-39 amino acid whose cecropins class peptide antibiotics, these prove fully that all peptide antibiotics exists widely at occurring in nature.In order to obtain the natural polypeptides microbiotic that activity is higher, output is bigger, the researcher studies the following 3 kinds of methods that obtained in succession:
1, direct extraction method: generally be to induce insect as the source of inducing, immune blood lymphocyte is heat-treated, except that purifying behind the deproteinize obtains with the injection intestinal bacteria.Though this method separating effect is better, it is induced, and effect is not good enough, insect source is limited, troublesome poeration, cost height, and yield is few, speed is slow, and is not suitable for industrial extraction and purification.
2, chemical synthesis: by the primary structure of artificial design polypeptide, add special acid, obtain through chemosynthesis.But expensive cost is the biggest obstacle that limits this method industrial applications.
3, genetically engineered synthesis method: utilizing engineered method to produce peptide antibiotics is an effective way that reduces production costs.But peptide antibiotics may cause the host microorganism suicide and can not obtain expression product the toxicity of prokaryotic cell prokaryocyte (as microorganism), has limited its application in prokaryotic expression system to a certain extent.And the low expression efficiency of eukaryotic expression system also is an obstacle of its suitability for industrialized production.With the formal representation peptide antibiotics gene of fusion rotein,, still have the few problem of expression product though can overcome this shortcoming.
Summary of the invention
The objective of the invention is to be to provide the preparation method who extracts peptide antibiotics in a kind of silkworm body, it is simple that it has technology, and raw material sources are extensive, and are with low cost, has a broad antifungal spectrum, purity height.
In order to achieve the above object the present invention by the following technical solutions, peptide antibiotics involved in the present invention is to utilize the silkworm chrysalis behind silkworm larva, pupa, the fresh cocoon reeling to be the preparation method who extracts purifying of raw material, the steps include:
Inducing of A, silkworm
1, inducing of larva: in the first five day that larva is extracted, the silkworm of feeding for 4～5 length of times behind microbial culture medium (mainly being intestinal bacteria or deactivation caryogram polyhedron solution) the sprinkling mulberry leaf with 2%～10% concentration, every day 1 time, continuous 4 days.Simultaneously, adopt ultrasonic wave to induce, condition is 10～20 kilo hertzs to be handled 2～5 hours, and superinduce in 20～24 hours is 3 times at interval, and the young silkworm larva after inducing is stored down in 0～12 ℃ of low temperature, extracts the length of time at 4-5.
2, inducing of silkworm chrysalis: the microbial culture medium (mainly being intestinal bacteria or deactivation caryogram polyhedron solution) with 2%～10% concentration sprays the silkworm of feeding for 4～5 length of times behind the mulberry leaf, and every day 1 time, silkworms spin silk until family; Waited to cocoon back 3-5 days, and adopted 10～20 kilo hertzs of ultrasonic wave to handle, every day 1 time, each 2～5 hours, superinduce in 20～24 hours was 3 times at interval.Silk cocoon after inducing is stored down in 0～12 ℃ of low temperature.Silk cocoon can carry out fresh cocoon reeling earlier, and then extracts peptide antibiotics.
The preparation of B, silkworm peptide antibiotics crude extract
(1) silkworm chrysalis behind the larva that will induce, bright pupa or the fresh cocoon reeling adds the 0.2mol/L phosphate buffered saline buffer of 2～3 times of volumes, and the pH value is 5-7, puts into tissue and smashs refiner to pieces, homogenate 10min.
(2) the gained homogenate is in 4 ℃, and the centrifugal 15min of 12000r/min gets supernatant liquor, places 12 hours down in 4 ℃.
(3) extract is handled 25min in 40～80 ℃ of water-baths, put very low temperature (20--60 ℃) refrigerator and cooled rapidly but behind the 10min, 4 ℃, the centrifugal 10min of 12000r/min gets supernatant.
(4) supernatant liquor carries out fractionation precipitation with 25%, 80% sulfate of ammoniac, sedimentaryly transfers pH9.5-10.5 with yellow soda ash simultaneously in second step, leave standstill 10min after, 4 ℃, the centrifugal 15min of 15000r/min, the gained precipitation is dissolved with the 2-6ml damping fluid, centrifugal according to the method described above again, get supernatant liquor.
(5) supernatant liquor dialysis desalination is 24 hours, and dialysis tubing changed water once every three hours.
The separation and purification of C, silkworm peptide antibiotics
(1) molecular sieve column chromatography
Supernatant liquor is after dialysis treatment, and last dextran G-75 molecular sieve column, balance liquid are the 0.2mol/L phosphate buffered saline buffer, and the pH value is 5-7, and elutriant is 0.5～1.0mol/L phosphate buffered saline buffer gradient elution, pH5-7.Collection vigor peak; The solution of collecting is carried out-20--80 ℃ vacuum-drying gets the active dry powder of silkworm peptide antibiotics.
(2) go up the preparative high performance liquid chromatography post, 10～50% acetonitrile (0.1% trifluoroacetic acid) wash-out is collected active peak.
(3) vigor peak freeze-drying (antibacterial peptide that extracts is carried out lyophilize) in subzero 20 ℃ of preservations, gets the said peptide antibiotics for preparing of the present invention from the silkworm body.
The present invention utilizes the characteristics that its thermostability is strong, molecular weight is little, adopt later the method for 40-80 ℃ of water bath processing in extracting, can remove the foreign protein more than 60%, inactivating oxidase in can also making in the silkworm blood after the heating simultaneously, so further separation and purification work just can be carried out at normal temperatures.Utilize (NH then
4)
2SO
4Carry out fractionation precipitation and regulate the method precipitation peptide antibiotics of iso-electric point, the peptide antibiotics crude product that obtains thus can improve the separating effect of column chromatography.Adopt molecular sieve column chromatography to obtain 3 peaks, peak 2 is an activeconstituents, and strong bacteriostatic action is arranged.25ml peptide antibiotics crude product liquid has been gathered in the crops 10mg dry powder, and yield rate is higher.Carry out HPLC then and be further purified, obtain pure product.The preparation-obtained peptide antibiotics of present method has following performance: (one) biochemical characteristic:
1, thermostability
This peptide antibiotics heats 30min down for 100 ℃ still can keep active.
2, pH value stabilization
This peptide antibiotics vigor in pH3～13 scopes is stable, and optimum pH is 5～7;
3, iso-electric point
Adopt pH gradient electrofocusing's method (IPGIEF) to measure silkworm peptide antibiotics pI value, the pI value is between 9.8～10.7.
4, molecular weight
Adopt the Tricine-SDS-PAGE electrophoretic method, the molecular weight that records this peptide antibiotics is 3～5KD;
5, amino acid is formed
This peptide antibiotics contains the amino acid kind: tryptophane (Trp); Aspartic acid (Asn); Proline(Pro) (Pro); Phenylalanine (Phe); Methionin (Lys); Leucine (Leu); L-glutamic acid (Glu); Glycine (Gly); Xie Ansuan (Val); Glutamine (Gln); Isoleucine (Ile); Methionine(Met) (Met); Arginine (Arg); Threonine (Thr); L-asparagine (Asn); Serine (Ser).(2) physiologically active
1, the mensuration of antibacterial vigor: adopt improved inhibition zone method to take the peptide antibiotics aqueous solution 1ul that present method prepares and be coated with on the culture dish of bacterium liquid with vertical the dropping in of liquid-transfering gun, 24 hours observationss are measured antibacterial vigor with the inhibition zone size.(ten thousand units/ml) aqueous solution compares with Vetstrep 20.The result shows that antimicrobial substance is very big to the living bacterium circle of producing bacillus subtilis.And to streptococcus aureus and intestinal bacteria take second place (seeing Table 1).
Table 1 silkworm peptide antibiotics is to the antibacterial vitality test (5 times average) of bacterium
Bacterial classification antibacterial circle diameter (mm) intestinal bacteria Escherichia coli JM-109 21.3 subtilis Bacillus sublilis 25.0 streptococcus aureus Staphlococus aureus. 19.8
2, antimicrobial spectrum experiment
Method is with 1.Experimental result shows that the silkworm peptide antibiotics all has tangible bacteriostatic activity to intestinal bacteria (Escherichia coliJM-109), subtilis (Bacillus sublilis), streptococcus aureus (Staphlococusaureus).
3, minimum inhibitory concentration
The silkworm peptide antibiotics aqueous solution is made 2 times of serial dilutions, observes the inhibition zone of different concns peptide antibiotics respectively.Following table has provided the minimum inhibitory concentration of peptide antibiotics to several bacteriums.As can be seen, the peptide antibiotics that the present invention obtains has strong bacteriolyze activity to intestinal bacteria from this table.Be diluted to 2
5Doubly, still show strong bacteriolyze activity.But total trend is along with dilution raising, antibiotic vigor decline (seeing Table 2).
Table 2 silkworm peptide antibiotics is measured (5 times average) (ug/m1) to the bacterium minimum inhibitory concentration
Bacterial classification peptide antibiotics intestinal bacteria Escherichia coli JM-109 4.5 subtilis Bacillus sublilis 5 streptococcus aureus Staphlococus aureus. 3.4
4, antibacterial time detecting
Method is with 1.After being added dropwise to peptide antibiotics, culture dish is exposed in the air detects, get its 5 mean values, experimental result shows, behind 5h on the inhibition zone that has dripped Vetstrep, grown assorted bacterium, still keep transparent and have on the inhibition zone of peptide antibiotics, behind 72h, just grow assorted bacterium in dropping.The peptide antibiotics that the present invention of this some proof obtains has the characteristic of permanent bacteria growing inhibiting.
The peptide antibiotics preparation is applied to antibacterium, virus, has organism toxicological harmless, strong, the effective advantage of sterilizing ability.Deriving from the body of silkworm and extract the peptide antibiotics that obtains with the present invention, is a kind of micromolecular peptide class, is crude substance, and good water solubility, high temperature resistant.Bacterium, fungi, protozoon, virus had lethal effect efficiently, as to subtilis, the also height of its specific activity Vetstrep, can be used for plant pest control, animal bacteria or virus type treatment of diseases, fresh flower is fresh-keeping and animal feedstuff additive, also can after further processing is modified, be prepared into the diagnosis, prevention, the medicine that are used for human viral infection, infectation of bacteria, fungi infestation.
The major advantage and the positively effect of invention are:
1, the present invention adopts lower concentration intestinal bacteria solution and the ultrasonic wave induction phase bonded mode of feeding, and has overcome the former injection intestinal bacteria mode inductive limitation of using, and is beneficial to industrially scalable and extracts.
2, utilize the high temperature resistant character of peptide antibiotics, the silk cocoon after inducing is carried out filature, be used for extracting peptide antibiotics again, opened up the new way that improves the silkworm economic worth.
3, the peptide antibiotics preparation method who adopts among the present invention has easy to operate, and plant and instrument is simple, and preparation technology is simple and feasible, fast efficient.
4, extract peptide antibiotics with present method from young silkworm or silkworm chrysalis, raw material sources are extensive, and it is strong that the peptide antibiotics that obtains has an antibacterial ability, has a broad antifungal spectrum.
5, adopt the present invention to extract, per 1000 gram silkworm bodies can be gathered in the crops the pure product of 8mg peptide antibiotics, and yield is higher.
6, the present invention has improved the utilising efficiency of silkworm chrysalis greatly, has important economic benefit, social benefit and ecological, environmental protective benefit.
Embodiment
Embodiment 1:
1, get 4 or 5 age silkworm larva add the mulberry leaf of intestinal bacteria solution that food is sprayed with 2% or 3% or 5% or 7% or 8.5% or 9.5% concentration, regularly freshen food once every day, continuous four days.
2, when freshening food, adopt 11 or 14 or 16 or 17 or 19 kilo hertzs of ultrasonic wave that larva is handled, induce every day 1 time, each 4 hours, superinduce in 21 or 22 or 23 hours was 3 times at interval.Silkworm larva after inducing is in store under 1 ℃ or 3 ℃ or 5 ℃ or 7 ℃ or 9 ℃ or 11 ℃ of low temperature.
3, the larva that will induce is taken out, and adds the 0.2mol/L phosphate buffered saline buffer of 2～3 times of volumes, and pH6.2 puts into tissue and smashs refiner to pieces, homogenate 10min.The gained homogenate is got supernatant liquor in centrifugal 15min, places 12 hours down in 4 ℃.
4, extract is handled 25min in 40 ℃ or 45 ℃ or 50 ℃ or 55 ℃ or 60 ℃ or 65 ℃ or 75 ℃ or 80 ℃ of water-baths, put rapidly in the Ultralow Temperature Freezer behind the cooling 10min, 4 ℃, the centrifugal 10min of 12000r/min gets supernatant.
5, supernatant liquor carries out fractionation precipitation with 25%, 80% sulfate of ammoniac, and sedimentary simultaneously with yellow soda ash accent pH to 10 in second step, centrifugal then, the gained precipitation is dissolved with the 3ml damping fluid, and is once centrifugal again, gets supernatant liquor.
6, supernatant liquor dialysis desalination is 24 hours, and dialysis tubing changed water once every three hours.
7, supernatant liquor is after dialysis treatment, last dextran (Sephadex) G-75 molecular sieve column, and balance liquid is the 0.2mol/L phosphate buffered saline buffer, and pH6.2, elutriant are 0.5～1.0mol/L phosphate buffered saline buffer gradient elution, and pH6.2 collects active peak.
8, with behind freeze-drying of vigor part or the vacuum concentration, last preparative high performance liquid chromatography post, 10～50% acetonitrile (0.1% trifluoroacetic acid) wash-out is collected active peak.
9, vigor peak freeze-drying is in subzero 20 ℃ of preservations, the peptide antibiotics that must prepare from silkworm.Productive rate: 5.2mg/kg silkworm larva.
Embodiment 2:
1, add the mulberry leaf that food is coated with the caryogram polyhedron deactivation solution that is sprayed with 3% or 4.5% or 5.5% or 7.5% or 8% or 9% concentration in 5 length of times of silkworm, regularly freshen food once every day, until silkworms spin silk.
2, treat that silkworm larva is formed cocoon after, with 10 or 10.5 or 13 or 15 or 17.5 or 18 kilo hertzs ultrasonication 4 hours, induce every day 1 time, superinduce in 20～24 hours is 3 times at interval.Silkworm chrysalis after inducing is in store under 2 ℃ or 4 ℃ or 6 ℃ or 8 ℃ or 10 ℃ or 12 ℃ of low temperature.
3, extracting method is identical with embodiment 1.
Productive rate: 8mg/kg silkworm chrysalis.
Claims (1)
1, extract the preparation method of peptide antibiotics in a kind of silkworm body, it comprises the following steps:
Inducing of A, silkworm, at first be inducing of larva: in the first five day that larva is extracted, microbial culture medium with 2%～10% concentration sprays the silkworm of feeding for 4～5 length of times behind the mulberry leaf, every day 1 time, continuous 4 days, simultaneously, the employing ultrasonic wave is induced, condition is 10～20 kilo hertzs to be handled 2～5 hours, and superinduce in 20～24 hours is 3 times at interval, and the young silkworm larva after inducing is stored down in 0～12 ℃ of low temperature; Next is inducing of silkworm chrysalis, the silkworm of feeding for 4～5 length of times behind the microbial culture medium sprinkling mulberry leaf with 2%～10% concentration, and every day 1 time, silkworms spin silk until family; Waited to cocoon back 3-5 days, and adopted 10～20 kilo hertzs of ultrasonic wave to handle, every day 1 time, each 2～5 hours, superinduce in 20～24 hours was 3 times at interval, and the silk cocoon after inducing is stored down in 0～12 ℃;
The preparation of B, silkworm peptide antibiotics crude extract at first is the silkworm chrysalis behind the larva that will induce, bright pupa or the fresh cocoon reeling, adds the 0.2mol/L phosphate buffered saline buffer of 2～3 times of volumes, and pH5-7 puts into tissue and smashs refiner to pieces, homogenate 10min; Next be with the gained homogenate in 4 ℃, the centrifugal 15min of 12000r/min gets supernatant liquor, places 12 hours down in 4 ℃; The 3rd is that extract is handled 25min in 40～80 ℃ of water-baths, put in the Ultralow Temperature Freezer cooling 10min after, 4 ℃, the centrifugal 10min of 12000r/min gets supernatant; The 4th is that supernatant liquor is carried out fractionation precipitation with 25%, 80% sulfate of ammoniac, transfers pH9.5-10.5 with yellow soda ash simultaneously sedimentary, after leaving standstill 10min, 4 ℃, the centrifugal 15min of 15000r/min, the gained precipitation is dissolved with damping fluid, and is centrifugal according to the method described above again, gets supernatant liquor; The 5th is that dialysis tubing changed water once every three hours with dialyse desalination 24 hours of supernatant liquor;
The separation and purification of C, silkworm peptide antibiotics at first is a molecular sieve column chromatography, and supernatant liquor is after dialysis treatment, last dextran G-75 molecular sieve column, balance liquid are the 0.2mol/L phosphate buffered saline buffer, pH5-7, elutriant is 0.5～1.0mol/L phosphate buffered saline buffer gradient elution, pH5-7; Next is collection vigor peak, and the solution of collecting is carried out-20 ℃--80 ℃ of vacuum-dryings get the active dry powder of silkworm peptide antibiotics; The 3rd is to go up the preparative high performance liquid chromatography post, and 10～50% acetonitrile wash-out is collected active peak; The 4th is the freeze-drying of vigor peak, in subzero 20 ℃ of preservations, obtains peptide antibiotics.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101530434B (en) * | 2009-04-10 | 2012-05-09 | 广州暨南大学医药生物技术研究开发中心 | Method for preparing silkworm powder, pupa powder or pupa serum powder containing antibacterial peptide |
| CN105085610A (en) * | 2015-08-17 | 2015-11-25 | 张卫民 | Graded secondary ultrafiltration purification method for antibacterial peptide |
| CN113024630A (en) * | 2021-03-04 | 2021-06-25 | 杭州华玮生物科技有限公司 | Preparation method of whitening and repairing polypeptide and whitening and repairing polypeptide |
-
2003
- 2003-05-30 CN CN 03128069 patent/CN1456574A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101530434B (en) * | 2009-04-10 | 2012-05-09 | 广州暨南大学医药生物技术研究开发中心 | Method for preparing silkworm powder, pupa powder or pupa serum powder containing antibacterial peptide |
| CN105085610A (en) * | 2015-08-17 | 2015-11-25 | 张卫民 | Graded secondary ultrafiltration purification method for antibacterial peptide |
| CN113024630A (en) * | 2021-03-04 | 2021-06-25 | 杭州华玮生物科技有限公司 | Preparation method of whitening and repairing polypeptide and whitening and repairing polypeptide |
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