CN105709208A - Preparation technology of bitter gourd protein enteric-coated tablets - Google Patents
Preparation technology of bitter gourd protein enteric-coated tablets Download PDFInfo
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- CN105709208A CN105709208A CN201610067205.0A CN201610067205A CN105709208A CN 105709208 A CN105709208 A CN 105709208A CN 201610067205 A CN201610067205 A CN 201610067205A CN 105709208 A CN105709208 A CN 105709208A
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- A61K9/2806—Coating materials
- A61K9/2833—Organic macromolecular compounds
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Abstract
A preparation technology of bitter gourd protein enteric-coated tablets includes the steps that seeds are hulled and placed in a mortar, and liquid nitrogen is added for grinding; a whole genome is obtained through a CTAB method; an associated primer is designed according to the gene sequence of bitter gourd protein MAP30, and conditions and a system of a PCR are determined; a PCR product is subjected to TA cloning after being recycled, competent cells DH5 alpha are introduced, a positive bacterial colony is screened out, and double-enzyme digestion is conducted after culture; PET-28a is cultured overnight, an expression plasmid is extracted, and enzyme digestion is conducted; a targeted DNA fragment subjected to double-enzyme digestion and the expression plasmid are connected overnight under a solution I system and then introduced into BL21 (DE) competent cells, and a positive bacterial colony is screened out; the positive bacterial colony is amplified and cultured for protein expression under induction of IPTG; the protein is purified and dried; obtained target protein MAP30 serves a main effective component, the target protein MAP30 and a series of auxiliary materials are together prepared into the bitter gourd protein enteric-coated tablets with a health-care function. The bitter gourd tablets are high in content of effective protein MAP30 and can better promote health of human bodies.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of bitter melon protein enteric coatel tablets
Preparation technology.
Background technology
Traditional plant amedica Research Thinking is generally loaded down with trivial details extraction and separates, and finally needs from extraction
Composite parts in isolate active single component.But the relevant of plant amedica effectively becomes
Dividing often content relatively low, separating step is loaded down with trivial details, uses traditional extracting method efficiency the lowest, meeting
Cannot meet the later stage researchs and produces needs.Current molecular biotechnology reaches its maturity, utilizes
Genetic engineering realizes the heterologous production of a certain purpose product and has had become as new study hotspot.Closely
Nian Lai, along with the development of extraction and separation technology, people are separated to multiple have fall blood from Fructus Momordicae charantiae
The ribosome inactivating protein of the effects such as sugar, mutation, antitumor and raising body immunity,
Wherein in these bitter melon protein compositions, molecular weight is the I type ribosome inactivating protein of 30kD
The properties of MAP30 (Momordica anti-HIV protein of 30kD) is superior, tool
There are immunomodulating, antiviral, antitumor, antifungic action, highly beneficial to health,
Can be made into a kind of health care medicine.But as a kind of albumen, make common preparation can under one's belt by
Digestion destroys, and big by utilizing traditional method for extracting MAP30 to pollute from Fructus Momordicae charantiae, efficiency
Low, therefore prepare to produce active bitter gourd first with technique for gene engineering by engineering bacteria large scale fermentation
Albumen MAP30, is finally made a kind of bitter melon protein enteric rich in activated protein MAP30
Sheet.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of Fructus Momordicae charantiae
The preparation technology of albumen enteric coatel tablets, uses genetic engineering to produce high-purity bitter melon protein MAP30,
Make the bitter melon protein enteric coatel tablets of great health care；Bitter melon protein enteric coatel tablets not only avoid biography
System Fructus Momordicae charantiae slice mouthfeel problem poor, that easily decompose under one's belt, more decreases tradition bitter melon protein
MAP30 extracts the pollution brought, and the content of effective albumen MAP30 in this Fructus Momordicae charantiae slice
Height, more can promote the health of human body.
To achieve these goals, the technical solution used in the present invention is:
The preparation technology of a kind of bitter melon protein enteric coatel tablets, comprises the following steps:
Step one: the process of Fructus Momordicae charantiae raw material: take the seed in appropriate ripe Fructus Momordicae charantiae, removes and plants
Sub-shell, is then placed in mortar, and it is powdered to add appropriate liquid nitrogen grinding, and subpackage obtains everywhere
Seed of bitter gourd powder after reason；
Step 2: the process of seed of bitter gourd powder: by the seed of bitter gourd powder after subpackage in step one,
Utilize CTAB method to extract and obtain Fructus Momordicae charantiae full-length genome, as the template of PCR reaction；
The determination of step 3: PCR reaction: first according to the base of relevant bitter melon protein MAP30
Because of the primer that sequential design is relevant, and finally determine condition and the system of PCR；
Step 4: TA clones: PCR primer carries out TA clone after reclaiming, and imports impression
State cell DH5 α, through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony, extracts mesh
Double digestion is carried out after mark DNA fragmentation；
Step 5: the process of expression vector: incubated overnight PET-28a, extracts expression plasmid,
And carry out enzyme action with two kinds of enzymes identical with step 4；
Step 6: connect: by by the fragment of identical enzyme action in step 4 and step 5 at solution
The system of I overnight connects；
Step 7: the product overnight connected in step 6 is imported competent cell BL21
(DE), through blue white macula screening, bacterium colony PCR, positive bacterium colony is filtered out；
Step 8: fall amplification culture luring at IPTG by the positive bacteria filtered out in step 7
Lead down the expression carrying out bitter melon protein MAP30；
Step 9: after fermentation culture through a series of process step 8 is obtained bitter melon protein
MAP30 is purified dry；
Step 10: enteric coatel tablets processed: the bitter melon protein that first will determine in step 9 that fermentation obtains
The ratio of MAP30 and adjuvant, the most size-reduced, sieve, dispensing, mixing, wet granulation,
Particle drying, tabletting, enteric coated, pack, store.
In described step one, Fructus Momordicae charantiae powder is packed as: often in pipe, the Specific amounts of subpackage is 10-15g.
In described step 2, the detailed process of CTAB method is: by water-bath temperature control button adjusting to 60
DEG C, the Fructus Momordicae charantiae powder that preheating extract with CTAB buffer → grinding obtains is transferred to 10mL preheating
In extract with CTAB buffer, at 60 DEG C, frequently rock centrifuge tube, be incubated 1h → cooling for a moment,
Adding 10mL chloroform/isoamyl alcohol (24:1), acutely shake centrifuge tube, 10000r/min is centrifuged
15min → supernatant proceeds to the new centrifuge tube equipped with 25mL 100% ethanol, gently reverse mixed
Even → 10000r/min is centrifuged 15min, abandons supernatant, obtains DNA and precipitates → add 700 μ l 70%
Ethanol, 70 μ l 3mol/L NaAc solution, with rinsing DNA precipitation, then go to 1.5mL
Eppendorf → 10000r/min is centrifuged the 70% ethanol rinse DNA precipitation of 30s → again, from
Heart 30s, abandons the ethanol → add 500 μ l TE of volatilizing under supernatant → room temperature, and 4 DEG C overnight dissolve
DNA → add 4-10 μ l RNase A, reverse mixing, and be incubated at 55 DEG C 30-60min →
Add 1.25mL ethanol, 54 μ l NaAc, precipitation DNA → centrifugal 30s, second of volatilizing under room temperature
Alcohol → DNA resolution of precipitate is in 300 μ l TE.
In described step 3, design of primers is
F15′-CGTCGACCTGTGGTATGCTTACTACTT-3′F2
5 '-GGAATTCTCAATTCACAACAGATTCC-3 ', the restriction enzyme site of introducing is Sal
I and EcoR I.
In described step 3, the system of PCR and condition are specifically respectively as follows: PCR 20 μ l system and are:
ddH2The each 1 μ l of O 7 μ l, primer, DNA profiling 1 μ l, 2 × Taq PCR Master Mix 10 μ l；
PCR reaction condition is: 95 DEG C of 5min, (95 DEG C of 30s, 58 DEG C of 30s, 722min) 35cycle；
72 DEG C of 7min, 4 DEG C of ∞.
Double digestion system condition in described step 4 is: plasmid DNA 25 μ l, Sal I He
EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h.
Expression plasmid double digestion system condition in described step 5 is: plasmid DNA 25 μ l,
Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h.
In described step 6 after same enzyme processes the 10 μ l systems that are attached particularly as follows:
Solution I 5 μ l, target dna 3 μ l, expression plasmid 2 μ l, 16 DEG C overnight connect.
In described step 8, the actual conditions of IPTG abduction delivering is: IPTG concentration is
0.65mol/L, inducing temperature 27 DEG C, induction time 4h.
Albumen MAP30 purification step in described step 9 is particularly as follows: at IPTG abduction delivering
After, collect thalline, then use sterile water wash 3 times, carry out ultrasonic under 80Hz frequency
Broken, until antibacterial all crushes, solution become limpid till, then 12000r/min from
Heart 15min, collects supernatant, then the recycling manual purifying protein of nickel ion pillar, loading
Rear first with 50mmol/L PBS (pH7.4), with 60mL/min flow velocity washing foreign protein to baseline,
The most respectively with 50,100 and 200mmol/L imidazoles eluting, collect each eluting peak and carry out
SDS-PAGE detects, and then collects and obtains net net weight histone, 4 DEG C of preservations after lyophilization
Standby.
Because of in view of the most labile character of bitter melon protein MAP30 in described step 10,
The final tablet prepared is enteric coated, it is ensured that the effect of Fructus Momordicae charantiae slice.
In described step 10, the concrete technology step of enteric coatel tablets processed is: weighs large scale fermentation and obtains
Pure bitter melon protein MAP3060g, with 6g starch mix, the starch slurry adding 10% glues
Soft material is made in mixture stirring, then pelletizes by 14 mesh nylon mesh, wet granular is placed in exsiccator
Middle 40-60 DEG C of aeration-drying, crosses 14 mesh nylon mesh granulate, claims dry particle weight, adds granule
The Pulvis Talci mixing of amount 5%, then obtains label with tabletting machine；The most enteric coated.
Concrete technology step enteric coated in described step 10 is: first by No. II acrylic resin
5% resin solution is made by the dissolving of 1:14 anharmonic ratio example with 95% ethanol, then by phthalic acid
The weight ratio mixing of 10:7:15 pressed by diethylester, Tween-80, Oleum Ricini, adds after grinding
In 5% No. II acrylic resin soln, cross 120 mesh sieves and obtain spare resin coating solution, by hardship
Melon albumen MAP30 label is put in coating pan, by after coating method Bao Fenyi six layers, spray
Entering above-mentioned resin coating solution, pot temperature control, at about 35 DEG C, has been sprayed in 4 hours.
In described step 10, enteric coated prescription is: No. II acrylic resin 2.5kg, Oleum Ricini
0.75kg, the ethanol 35kg of 95%, diethyl phthalate 0.5kg, Tween-80
0.35kg。
Beneficial effects of the present invention:
It is many that the pollution brought in order to avoid traditional extraction process is big, efficiency is low, purity is low etc.
Problem, the present invention uses genetic engineering, and utilizing works bacterium uses the method fermented the most extensive
Ground produces high-purity destination protein MAP30；In order to avoid the every one-tenth in traditional Fructus Momordicae charantiae slice
Being grouped into indefinite, content low, health care is prominent, quality is difficult to the problems such as guarantee,
The present invention have chosen the single-activity bitter melon protein MAP30 of suitable dose as bitter melon protein intestinal
Molten middle effective ingredient；For the chemical nature in view of effective active compositions most of in Fructus Momordicae charantiae
Being all albumen, normal oral Fructus Momordicae charantiae tablet is destroyed the most under one's belt, does not reaches expection health care and makees
With, common Fructus Momordicae charantiae tablet is changed into the enteric coated tablet being difficult to be destroyed by peptic digestion by this invention；In order to
The Fructus Momordicae charantiae slice prepared with tradition Fructus Momordicae charantiae sarcocarp is avoided to there is the problem that bitterness mouthfeel is poor, the present invention
The target protein MAP30 directly using fermentation purification is raw material, makes final Fructus Momordicae charantiae slice with general
Logical Fructus Momordicae charantiae slice is compared, and mouthfeel is more preferable.
Due to the fact that and utilize technique for gene engineering to use engineering bacterium fermentation productive target albumen
MAP30, and as raw material and be made for health care more preferably, absorb more preferably, mouthfeel more
Good enteric coated tablet.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
Step one: the process of Fructus Momordicae charantiae raw material: take the seed in the ripe Fructus Momordicae charantiae of 150g, removes
Seed hulls, is then placed in mortar, adds appropriate liquid nitrogen grinding powdered, by 10g's
Seed of bitter gourd powder after amount subpackage process；
Step 2: the process of seed of bitter gourd powder: by the 10g seed of bitter gourd powder after subpackage, respectively
Utilizing CTAB method to extract and obtain Fructus Momordicae charantiae full-length genome, the detailed process of CTAB method is: by water
Bath temperature control button adjusting, to 60 DEG C, preheats the Fructus Momordicae charantiae powder that extract with CTAB buffer → grinding obtains
End is transferred in 10mL preheating extract with CTAB buffer, frequently rocks centrifuge tube at 60 DEG C,
Insulation 1h → cooling for a moment, adds 10mL chloroform/isoamyl alcohol (24:1), and acutely concussion is centrifugal
Pipe, 10000r/min is centrifuged 15min → supernatant and proceeds to be centrifuged equipped with the new of 25mL 100% ethanol
Pipe, gently reverse mixing → 10000r/min is centrifuged 15min, abandons supernatant, obtain DNA
Precipitating → add 700 μ l 70% ethanol, 70 μ l 3mol/L NaAc solution precipitate with rinsing DNA,
Then go to 1.5mL Eppendorf → 10000r/min and be centrifuged 30s → use 70% ethanol rinse again
DNA precipitates, centrifugal 30s, abandons the ethanol that volatilizees under supernatant → room temperature → add 500 μ l TE, and 4
DEG C overnight dissolving DNA → add 4 μ l RNase A, reverse mixing, and be incubated at 55 DEG C
30min → addition 1.25mL ethanol, 54 μ l NaAc, precipitation DNA → centrifugal 30s, room temperature
Lower volatilization ethanol → DNA resolution of precipitate is in 300 μ l TE, and the final full-length genome obtained will
Template as PCR reaction；
The determination of step 3: PCR reaction: do not have intron in the gene of MAP30, so
Can directly expand from Fructus Momordicae charantiae full-length genome, will be by 20 μ l PCR system: ddH2O
The each 1 μ l of 7 μ l, primer, DNA profiling 1 μ l, 2 × Taq PCR Master Mix 10 μ l prepare,
By PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 30s, 58 DEG C of 30s, 722min) 35cycle；
72 DEG C of 7min, 4 DEG C of ∞, carry out amplification in vitro and obtain the specific fragment of target MAP30；
Design of primers is F15 '-CGTCGACCTGTGGTATGCTTACTACTT-3 ' F2
5 '-GGAATTCTCAATTCACAACAGATTCC-3 ', the restriction enzyme site of introducing is Sal
I and EcoR I；
Step 4: TA clones: PCR primer carries out TA clone after reclaiming, and imports impression
State cell DH5 α, through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony, extracts mesh
Carry out double digestion after mark DNA fragmentation, double digestion system condition particularly as follows: plasmid DNA 25 μ l,
Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h；
Step 5: the process of expression vector: incubated overnight PET-28a bacterial strain, after use test kit
Extract plasmid, and carry out enzyme action, expression plasmid double digestion body with two kinds of enzymes identical with step 4
It is that condition is: plasmid DNA 25 μ l, Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O
5 μ l, mixing, 37 DEG C of 1h；
Step 6: connect: be attached after same enzyme in step 4 with step 5 processes, even
The 10 μ l systems that connect particularly as follows: Solution I 5 μ l, target dna 3 μ l, expression plasmid 2ul,
16 DEG C overnight；
Step 7: the product overnight connected is imported competent cell BL21 (DE), through indigo plant
White macula screening, bacterium colony PCR, filter out positive bacterium colony；
Step 8: the amplification culture that then fallen by positive bacteria also utilizes under the conditions of 27 DEG C
The IPTG induction bitter melon protein MAP30 of 0.65mol/L expresses 4h；
Step 9: after IPTG abduction delivering, collects thalline, then clear with sterilized water
Washing 3 times, carry out ultrasonication under 80Hz frequency, until antibacterial all crushes, solution becomes clear
Till bright, then it is centrifuged 15min at 12000r/min, collects supernatant, then recycle nickel
The manual purifying protein of ion pillar, first uses 50mmol/L PBS (pH7.4) after loading, with
60mL/min flow velocity washing foreign protein is to baseline, the most respectively with 50,100 and
200mmol/L imidazoles eluting, collects each eluting peak and carries out SDS-PAGE detection, then receive
Collection obtains net net weight histone, and after lyophilization, 4 DEG C save backup；
Step 10: enteric coatel tablets processed: first weigh the pure bitter melon protein that large scale fermentation obtains
MAP3060g, mixes with 6g starch, and the starch slurry adding 10% is made binding agent stirring and made soft
Material, then pelletizes by 14 mesh nylon mesh, wet granular is placed in 40 DEG C of aeration-drying in exsiccator,
Cross 14 mesh nylon mesh granulate, claim dry particle weight, add the Pulvis Talci mixing of grain amount 5%,
Then obtain label with tabletting machine, then utilize enteric coated prescription coating solution: No. II
Acrylic resin 2.5kg, Oleum Ricini 0.75kg, the ethanol 35kg of 95%, phthalic acid two
Ethyl ester 0.5kg, Tween-80 0.35kg, first by No. II acrylic resin and 95% ethanol
Dissolve by 1:14 anharmonic ratio example and make 5% resin solution, then by diethyl phthalate, poly-mountain
The weight ratio mixing of 10:7:15 pressed by pear ester-80, Oleum Ricini, adds 5% No. II propylene after grinding
In acid resin solution, cross 120 mesh sieves and finally give spare resin coating solution, then by Fructus Momordicae charantiae egg
White MAP30 label is put in coating pan, by after coating method Bao Fenyi six layers, spray into
Stating resin coating solution, pot temperature control, at about 35 DEG C, has been sprayed in 4 hours and has been obtained rich in MAP30
Enteric Fructus Momordicae charantiae slice, then pack, store, obtain final bitter melon protein enteric coatel tablets.
Embodiment 2
Step one: the process of Fructus Momordicae charantiae raw material: take the seed in the ripe Fructus Momordicae charantiae of 150g, removes
Seed hulls, is then placed in mortar, adds appropriate liquid nitrogen grinding powdered, by 15g's
Seed of bitter gourd powder after amount subpackage process；
Step 2: the process of seed of bitter gourd powder: by the 15g seed of bitter gourd powder after subpackage, respectively
Utilizing CTAB method to extract and obtain Fructus Momordicae charantiae full-length genome, the detailed process of CTAB method is: by water
Bath temperature control button adjusting, to 60 DEG C, preheats the Fructus Momordicae charantiae powder that extract with CTAB buffer → grinding obtains
End is transferred in 10mL preheating extract with CTAB buffer, frequently rocks centrifuge tube at 60 DEG C,
Insulation 1h → cooling for a moment, adds 10mL chloroform/isoamyl alcohol (24:1), and acutely concussion is centrifugal
Pipe, 10000r/min is centrifuged 15min → supernatant and proceeds to be centrifuged equipped with the new of 25mL 100% ethanol
Pipe, gently reverse mixing → 10000r/min is centrifuged 15min, abandons supernatant, obtain DNA
Precipitating → add 700 μ l 70% ethanol, 70 μ l 3mol/L NaAc solution precipitate with rinsing DNA,
Then go to 1.5mL Eppendorf → 10000r/min and be centrifuged 30s → use 70% ethanol rinse again
DNA precipitates, centrifugal 30s, abandons the ethanol that volatilizees under supernatant → room temperature → add 500 μ l TE, and 4
DEG C overnight dissolving DNA → add 6 μ l RNase A, reverse mixing, and be incubated at 55 DEG C
50min → addition 1.25mL ethanol, 54 μ l NaAc, precipitation DNA → centrifugal 30s, room temperature
Lower volatilization ethanol → DNA resolution of precipitate is in 300 μ l TE, and the final full-length genome obtained will
Template as PCR reaction；
The determination of step 3: PCR reaction: do not have intron in the gene of MAP30, so
Can directly expand from Fructus Momordicae charantiae full-length genome, will be by 20 μ l PCR system: ddH2O
The each 1 μ l of 7 μ l, primer, DNA profiling 1 μ l, 2 × Taq PCR Master Mix 10 μ l prepare,
By PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 30s, 58 DEG C of 30s, 722min) 35cycle；
72 DEG C of 7min, 4 DEG C of ∞, carry out amplification in vitro and obtain the specific fragment of target MAP30；
Design of primers is F15 '-CGTCGACCTGTGGTATGCTTACTACTT-3 ' F2
5 '-GGAATTCTCAATTCACAACAGATTCC-3 ', the restriction enzyme site of introducing is Sal
I and EcoR I；
Step 4: TA clones: PCR primer carries out TA clone after reclaiming, and imports impression
State cell DH5 α, through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony, extracts mesh
Carry out double digestion after mark DNA fragmentation, double digestion system condition particularly as follows: plasmid DNA 25 μ l,
Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h；
Step 5: the process of expression vector: incubated overnight PET-28a bacterial strain, after use test kit
Extract plasmid, and carry out enzyme action, expression plasmid double digestion body with two kinds of enzymes identical with step 4
It is that condition is: plasmid DNA 25 μ l, Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O
5 μ l, mixing, 37 DEG C of 1h；
Step 6: connect: be attached after same enzyme in step 4 with step 5 processes, even
The 10 μ l systems that connect particularly as follows: Solution I 5 μ l, target dna 3 μ l, expression plasmid 2ul,
16 DEG C overnight；
Step 7: the product overnight connected is imported competent cell BL21 (DE), through indigo plant
White macula screening, bacterium colony PCR, filter out positive bacterium colony；
Step 8: the amplification culture that then fallen by positive bacteria also utilizes under the conditions of 27 DEG C
The IPTG induction bitter melon protein MAP30 of 0.65mol/L expresses 4h；
Step 9: after IPTG abduction delivering, collects thalline, then clear with sterilized water
Washing 3 times, carry out ultrasonication under 80Hz frequency, until antibacterial all crushes, solution becomes clear
Till bright, then it is centrifuged 15min at 12000r/min, collects supernatant, then recycle nickel
The manual purifying protein of ion pillar, first uses 50mmol/L PBS (pH7.4) after loading, with
60mL/min flow velocity washing foreign protein is to baseline, the most respectively with 50,100 and
200mmol/L imidazoles eluting, collects each eluting peak and carries out SDS-PAGE detection, then receive
Collection obtains net net weight histone, and after lyophilization, 4 DEG C save backup；
Step 10: enteric coatel tablets processed: first weigh the pure bitter melon protein that large scale fermentation obtains
MAP30 60g, mixes with 6g starch, and the starch slurry adding 10% is made binding agent stirring and made soft
Material, then pelletizes by 14 mesh nylon mesh, wet granular is placed in 50 DEG C of aeration-drying in exsiccator,
Cross 14 mesh nylon mesh granulate, claim dry particle weight, add the Pulvis Talci mixing of grain amount 5%,
Then obtain label with tabletting machine, then utilize enteric coated prescription coating solution: No. II
Acrylic resin 2.5kg, Oleum Ricini 0.75kg, the ethanol 35kg of 95%, phthalic acid two
Ethyl ester 0.5kg, Tween-80 0.35kg, first by No. II acrylic resin and 95% ethanol
Dissolve by 1:14 anharmonic ratio example and make 5% resin solution, then by diethyl phthalate, poly-mountain
The weight ratio mixing of 10:7:15 pressed by pear ester-80, Oleum Ricini, adds 5% No. II propylene after grinding
In acid resin solution, cross 120 mesh sieves and finally give spare resin coating solution, then by Fructus Momordicae charantiae egg
White MAP30 label is put in coating pan, by after coating method Bao Fenyi six layers, spray into
Stating resin coating solution, pot temperature control, at about 35 DEG C, has been sprayed in 4 hours and has been obtained rich in MAP30
Enteric Fructus Momordicae charantiae slice, then pack, store, obtain final bitter melon protein enteric coatel tablets.
Embodiment 3
Step one: the process of Fructus Momordicae charantiae raw material: take the seed in the ripe Fructus Momordicae charantiae of 150g, removes
Seed hulls, is then placed in mortar, adds appropriate liquid nitrogen grinding powdered, by 15g's
Seed of bitter gourd powder after amount subpackage process；
Step 2: the process of seed of bitter gourd powder: by the 15g seed of bitter gourd powder after subpackage, respectively
Utilizing CTAB method to extract and obtain Fructus Momordicae charantiae full-length genome, the detailed process of CTAB method is: by water
Bath temperature control button adjusting, to 60 DEG C, preheats the Fructus Momordicae charantiae powder that extract with CTAB buffer → grinding obtains
End is transferred in 10mL preheating extract with CTAB buffer, frequently rocks centrifuge tube at 60 DEG C,
Insulation 1h → cooling for a moment, adds 10mL chloroform/isoamyl alcohol (24:1), and acutely concussion is centrifugal
Pipe, 10000r/min is centrifuged 15min → supernatant and proceeds to be centrifuged equipped with the new of 25mL 100% ethanol
Pipe, gently reverse mixing → 10000r/min is centrifuged 15min, abandons supernatant, obtain DNA
Precipitating → add 700 μ l 70% ethanol, 70 μ l 3mol/L NaAc solution precipitate with rinsing DNA,
Then go to 1.5mL Eppendorf → 10000r/min and be centrifuged 30s → use 70% ethanol rinse again
DNA precipitates, centrifugal 30s, abandons the ethanol that volatilizees under supernatant → room temperature → add 500 μ l TE, 4 DEG C
Overnight dissolving DNA → add 10 μ l RNase A, reverse mixing, and be incubated at 55 DEG C
60min → addition 1.25mL ethanol, 54 μ l NaAc, precipitation DNA → centrifugal 30s, room temperature
Lower volatilization ethanol → DNA resolution of precipitate is in 300 μ l TE, and the final full-length genome obtained will
Template as PCR reaction；
The determination of step 3: PCR reaction: do not have intron in the gene of MAP30, permissible
Directly expand from Fructus Momordicae charantiae full-length genome, will be by 20 μ l PCR system: ddH2O 7μl、
The each 1 μ l of primer, DNA profiling 1 μ l, 2 × Taq PCR Master Mix 10 μ l prepare, by PCR
Reaction condition: 95 DEG C of 5min, (95 DEG C of 30s, 58 DEG C of 30s, 72 2min) 35cycle；
72 DEG C of 7min, 4 DEG C of ∞, carry out amplification in vitro and obtain the specific fragment of target MAP30；
Design of primers is F15 '-CGTCGACCTGTGGTATGCTTACTACTT-3 ' F2
5 '-GGAATTCTCAATTCACAACAGATTCC-3 ', the restriction enzyme site of introducing is Sal
I and EcoR I；
Step 4: TA clones: PCR primer carries out TA clone after reclaiming, and imports impression
State cell DH5 α, through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony, extracts mesh
Carry out double digestion after mark DNA fragmentation, double digestion system condition particularly as follows: plasmid DNA 25 μ l,
Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h；
Step 5: the process of expression vector: incubated overnight PET-28a bacterial strain, after use test kit
Extract plasmid, and carry out enzyme action, expression plasmid double digestion body with two kinds of enzymes identical with step 4
It is that condition is: plasmid DNA 25 μ l, Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O
5 μ l, mixing, 37 DEG C of 1h；
Step 6: connect: be attached after same enzyme in step 4 with step 5 processes, even
The 10 μ l systems that connect particularly as follows: Solution I 5 μ l, target dna 3 μ l, expression plasmid 2ul,
16 DEG C overnight；
Step 7: the product overnight connected is imported competent cell BL21 (DE), through indigo plant
White macula screening, bacterium colony PCR, filter out positive bacterium colony；
Step 8: the amplification culture that then fallen by positive bacteria also utilizes under the conditions of 27 DEG C
The IPTG induced protein of 0.65mol/L expresses 4h；
Step 9: after IPTG abduction delivering, collects thalline, then clear with sterilized water
Washing 3 times, carry out ultrasonication under 80Hz frequency, until antibacterial all crushes, solution becomes clear
Till bright, then it is centrifuged 15min at 12000r/min, collects supernatant, then recycle nickel
The manual purifying protein of ion pillar, first uses 50mmol/L PBS (pH7.4) after loading, with
60mL/min flow velocity washing foreign protein is to baseline, the most respectively with 50,100 and
200mmol/L imidazoles eluting, collects each eluting peak and carries out SDS-PAGE detection, then receive
Collection obtains net net weight histone, and after lyophilization, 4 DEG C save backup；
Step 10: enteric coatel tablets processed: first weigh the pure bitter melon protein that large scale fermentation obtains
MAP3060g, mixes with 6g starch, and the starch slurry adding 10% is made binding agent stirring and made soft
Material, then pelletizes by 14 mesh nylon mesh, wet granular is placed in 60 DEG C of aeration-drying in exsiccator,
Cross 14 mesh nylon mesh granulate, claim dry particle weight, add the Pulvis Talci mixing of grain amount 5%,
Then obtain label with tabletting machine, then utilize enteric coated prescription coating solution: No. II
Acrylic resin 2.5kg, Oleum Ricini 0.75kg, the ethanol 35kg of 95%, phthalic acid two
Ethyl ester 0.5kg, Tween-80 0.35kg, first by No. II acrylic resin and 95% ethanol
Dissolve by 1:14 anharmonic ratio example and make 5% resin solution, then by diethyl phthalate, poly-mountain
The weight ratio mixing of 10:7:15 pressed by pear ester-80, Oleum Ricini, adds 5% No. II propylene after grinding
In acid resin solution, cross 120 mesh sieves and finally give spare resin coating solution, then by Fructus Momordicae charantiae egg
White MAP30 label is put in coating pan, by after coating method Bao Fenyi six layers, spray into
Stating resin coating solution, pot temperature control, at about 35 DEG C, has been sprayed in 4 hours and has been obtained rich in MAP30
Enteric Fructus Momordicae charantiae slice, then pack, store, obtain final bitter melon protein enteric coatel tablets.
Claims (10)
1. the preparation technology of bitter melon protein enteric coatel tablets, it is characterised in that: comprise the following steps:
Step one: the process of Fructus Momordicae charantiae raw material: take the seed in appropriate ripe Fructus Momordicae charantiae, removes seed hulls, is then placed in mortar, and it is powdered to add appropriate liquid nitrogen grinding, subpackage processed after seed of bitter gourd powder；
Step 2: the process of seed of bitter gourd powder: by the seed of bitter gourd powder after subpackage in step one, utilizes CTAB method to extract and obtains Fructus Momordicae charantiae full-length genome, as the template of PCR reaction；
The determination of step 3: PCR reaction: first according to the primer that the gene order design of relevant bitter melon protein MAP30 is relevant, and finally determine condition and the system of PCR；
Step 4: TA clones: PCR primer carries out TA clone after reclaiming, and imports competent cell DH5 α, through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony, carries out double digestion after extracting target DNA fragments；
Step 5: the process of expression vector: incubated overnight PET-28a, extracts expression plasmid, and carries out enzyme action with two kinds of enzymes identical with step 4；
Step 6: connect: be overnight connected in the system of solution I by the fragment of step 4 enzyme action identical with step 5；
Step 7: the product overnight connected in step 6 is imported competent cell BL21 (DE), through blue white macula screening, bacterium colony PCR, filters out positive bacterium colony；
Step 8: the amplification culture that fallen by the positive bacteria filtered out in step 7 also carries out the expression of bitter melon protein MAP30 under the induction of IPTG；
Step 9: after fermentation culture, through a series of process step 8 is obtained bitter melon protein MAP30 be purified dry；
Step 10: enteric coatel tablets processed: first will determine the ratio of the bitter melon protein MAP30 that obtains of fermentation and adjuvant in step 9, the most size-reduced, sieve, dispensing, mixing, wet granulation, particle drying, tabletting, store after enteric coated, packaging.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterised in that in described step one, Fructus Momordicae charantiae powder is packed as: often in pipe, the Specific amounts of subpackage is 10-15g.
nullThe preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1，It is characterized in that，In described step 2, the detailed process of CTAB method is: by water-bath temperature control button adjusting to 60 DEG C，The Fructus Momordicae charantiae powder that preheating extract with CTAB buffer → grinding obtains is transferred in 10mL preheating extract with CTAB buffer，Centrifuge tube is frequently rocked at 60 DEG C，Insulation 1h → cooling is for a moment，Add 10mL chloroform/isoamyl alcohol (24:1)，Acutely shake centrifuge tube，10000r/min is centrifuged 15min → supernatant and proceeds to the new centrifuge tube equipped with 25mL 100% ethanol，Gently reverse mixing → 10000r/min is centrifuged 15min，Abandon supernatant，Obtain DNA and precipitate → add 700 μ l 70% ethanol，70 μ l 3mol/L NaAc solution are with rinsing DNA precipitation，Then go to 1.5mL Eppendorf → 10000r/min and be centrifuged the 70% ethanol rinse DNA precipitation of 30s → again，Centrifugal 30s，Abandon the ethanol → add 500 μ l TE of volatilizing under supernatant → room temperature，4 DEG C of overnight dissolving DNAs → add 4-10 μ l RNase A，Reverse mixing，And at 55 DEG C, it is incubated 30-60min → addition 1.25mL ethanol、54μl NaAc，Precipitation DNA → centrifugal 30s，Volatilize ethanol → DNA resolution of precipitate in 300 μ l TE under room temperature.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterized in that, in described step 3, design of primers is F15 '-CGTCGACCTGTGGTATGCTTACTACTT-3 ' F25 '-GGAATTCTCAATTCACAACAGATTCC-3 ', and the restriction enzyme site of introducing is Sal I and EcoR I；
In described step 3, the system of PCR and condition are specifically respectively as follows: PCR 20 μ l system and are: ddH2The each 1 μ l of O 7 μ l, primer, DNA profiling 1 μ l, 2 × Taq PCR Master Mix 10 μ l；PCR reaction condition is: 95 DEG C of 5min, (95 DEG C of 30s, 58 DEG C of 30s, 72 2min) 35cycle；72 DEG C of 7min, 4 DEG C of ∞.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterised in that the double digestion system condition in described step 4 is: plasmid DNA 25 μ l, Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterised in that the expression plasmid double digestion system condition in described step 5 is: plasmid DNA 25 μ l, Sal I and EcoR I 1 μ l, Buffer 5 μ l, ddH respectively2O 5 μ l, mixing, 37 DEG C of 1h.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterised in that the system being attached after same enzyme processes in described step 6 is specially, 10 μ l systems: Solution I 5 μ l, target dna 3 μ l, expression plasmid 2 μ l, 16 DEG C overnight connect.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterised in that in described step 8, the actual conditions of IPTG abduction delivering is: IPTG concentration is 0.65mol/L, inducing temperature 27 DEG C, induction time 4h.
nullThe preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1，It is characterized in that，Albumen MAP30 purification step in described step 9 is particularly as follows: after IPTG abduction delivering，Collect thalline，Then sterile water wash is used 3 times，Ultrasonication is carried out under 80Hz frequency，Until antibacterial all crushes，Till solution change is limpid，Then it is centrifuged 15min at 12000r/min，Collect supernatant，Then the recycling manual purifying protein of nickel ion pillar，First with 50mmol/L PBS (pH7.4) after loading，With 60mL/min flow velocity washing foreign protein to baseline，The most respectively with 50、100 and 200mmol/L imidazoles eluting，Collect each eluting peak and carry out SDS-PAGE detection，Then collect and obtain net net weight histone，After lyophilization, 4 DEG C save backup.
The preparation technology of a kind of bitter melon protein enteric coatel tablets the most according to claim 1, it is characterized in that, in described step 10, enteric coated prescription is: No. II acrylic resin 2.5kg, Oleum Ricini 0.75kg, the ethanol 35kg of 95%, diethyl phthalate 0.5kg, Tween-80 0.35kg；
In described step 10, the concrete technology step of enteric coatel tablets processed is: weigh the pure bitter melon protein MAP30 60g that large scale fermentation obtains, mix with 6g starch, the starch slurry adding 10% is made binding agent stirring and is made soft material, then pelletize by 14 mesh nylon mesh, wet granular is placed in 40-60 DEG C of aeration-drying in exsiccator, cross 14 mesh nylon mesh granulate, claim dry particle weight, add the Pulvis Talci mixing of grain amount 5%, then obtain label with tabletting machine, the most enteric coated；
Concrete technology step enteric coated in described step 10 is: first by the dissolving of 1:14 anharmonic ratio example, No. II acrylic resin and 95% ethanol are made 5% resin solution, diethyl phthalate, Tween-80, Oleum Ricini are pressed the weight ratio mixing of 10:7:15 again, add after grinding in 5% No. II acrylic resin soln, cross 120 mesh sieves and obtain spare resin coating solution, bitter melon protein MAP30 label is put in coating pan, by after coating method Bao Fenyi six layers, spray into above-mentioned resin coating solution, pot temperature control, at about 35 DEG C, has been sprayed in 4 hours.
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