CN103405749B - A kind of skin containing toad turns the anti-tumor Chinese herbal preparation and application thereof of glue protein -2 - Google Patents
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation and application thereof of glue protein -2 Download PDFInfo
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- CN103405749B CN103405749B CN201310355387.8A CN201310355387A CN103405749B CN 103405749 B CN103405749 B CN 103405749B CN 201310355387 A CN201310355387 A CN 201310355387A CN 103405749 B CN103405749 B CN 103405749B
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Abstract
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation and application thereof of glue protein 2, belongs to biomedicine technical field.It is characterized in that：Toad skin turns glue protein 2 17 36%, auxiliary material 2 11%, stabilizer 5 19%, solvent 41 70%.A kind of above-mentioned skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein 2, turn glue protein 2 with natural toad skin as active constituent and carrier, add a certain amount of protein stabiliser, the stabilization of the effect of capable of ensureing the medicament well and performance, adjunct ingredient containing figuration and synergistic effect simultaneously, the term of validity of drug can be extended and stablize row, the Chinese medicine preparation has the effects that swelling and pain relieving, antitumor and prevention human body, animal, poultry undesired cell proliferation, and production is easy, it is safe and non-toxic, it is easy to use, it is without any side effects using process.
Description
Technical field
The invention belongs to biomedicine technical field, specially a kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2
And application thereof.
Background technology
The dried venom of toads, toad-cortex, toad skin etc. are with a long history as Chinese medicine in China, have detumescence, analgesic, removing toxic substances, inducing resuscitation of having one's ideas straightened out,
The curative effects such as antitumor.Verified total water carries egg during carrying out preliminary immunocompetence screening to dried venom of toads different solvents extract
Bai Zufen has the function of certain immune-enhancing activity and promotes apoptosis of tumor cells in vitro.Where Zhejiang A ＆ F University
Team's development Bufo japonicus (Bufo japonicus formosus) skin cDNA plasmid library screening operations, it is cloned into scarce
It loses part 5 '-end open reading frame (open reading frame, ORF) base sequence and turns glue protein -2
(transgelin-2, TAGLN2, also known as smooth muscle 22, SM22 β) cDNA.The albumen is initially flat from chicken gizzard
It finds and detaches, 22 kD of molecular weight in sliding flesh, be a kind of cytoskeletal protein combined with actin (actin),
Cell Proliferation, differentiation and the development of vascular system during animal development are participated in, it is also big in kinds cancer generating process
Amount expression.Therefore, TAGLN2 is provided as tumorigenic potential marker for early diagnosis, treatment and the monitoring etc. of tumour
Reference frame.To whether there is in analysis toad skinTAGLN2The expression of full-length gene, by the experimental design of two-wheeled PCR,
The cDNA clone that multiple and different degree lack 5 '-end base sequences is obtained, is disclosedTAGLN2Gene diversity.To research and analyse this
Albumen lays the foundation in the dried venom of toads, toad-cortex, the content of toad skin and its in the effect of anti-tumor aspect.
Invention content
For various problems existing in the prior art, it is an object of the invention to design a kind of skin containing toad of offer to turn glue egg
The technical solution of -2 anti-tumor Chinese herbal preparation and application thereof in vain, gained formulation preparation method is simple, safely and effectively, production cost
It is low, significant in efficacy, using simplicity.
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, it is characterised in that contains weight percentage
Following substance：Toad skin turns -2 17-36% of glue protein, auxiliary material 2-11%, stabilizer 5-19%, solvent 41-70%；The auxiliary material
For one or more kinds of mixtures in polysorbate, Emulsifier EL-60, polylactic acid, glucan；The stabilizer
For one or more kinds of mixtures in polyethylene glycol, histidine, glycine, glycerine；The solvent is distilled water, steams again
One or more kinds of mixtures in water, sterile water, ultra-pure water, deionized water；
The toad skin is turned glue protein -2 and is prepared using following methods：
1）It is screened from toad skin cDNA plasmid libraries, is cloned into lack part 5 '-end open reading frame base sequence
Toad skin turns glue protein -2cDNA；
2）Toad skin is expressed and produced in prokaryotic expression system one by one colibacillus turns glue protein -2:Engineering is built first
Bacterium and induced expression, then high density fermentation engineering bacteria and induced expression answer target protein after inclusion body separation and washing
Property, purifying.
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, it is characterised in that toad skin turns glue protein -2
Content is 21-33%, preferably 23-29%.
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, it is characterised in that the content of auxiliary material is 3-
10%, preferably 4-9%.
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, it is characterised in that the content of stabilizer is 7-
17%, preferably 9-15%.
A kind of skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, it is characterised in that the content of solvent is 45-
68%, preferably 47-64%.
The toad skin turns glue protein -2 and is preparing for undesired cell proliferations systems such as human body, animal, the anti-curing oncomas of poultry
Application in agent.
A kind of above-mentioned skin containing toad turns the anti-tumor Chinese herbal preparation of glue protein -2, and it is activity to turn glue protein -2 with natural toad skin
Ingredient and carrier add a certain amount of protein stabiliser, and the stabilization of the effect of capable of ensureing the medicament well and performance contain simultaneously
Have figuration and the adjunct ingredient of synergistic effect, can extend drug the term of validity and stablize row, the Chinese medicine preparation have swelling and pain relieving,
The effects that antitumor and prevention human body, animal, poultry undesired cell proliferation, and simplicity is produced, it is safe and non-toxic, it is easy to use, make
It is without any side effects with process.
Unless otherwise stated, others are that the weight percent of pure material contains to percentage composition involved in present specification
Amount.
Specific implementation mode
In conjunction with the embodiment of the present invention, the advantageous effect that further illustrates the present invention.
Embodiment 1
64g distilled water is added into the clean container after stringent sterilization, is then slowly added into 23g toad skins and turns glue protein -2,
Add 2g polysorbates and 2g Emulsifier EL-60s after mixing, stir half an hour, then slowly plus people 3g polyethylene glycol,
3g histidines, 2g glycine and 1g glycerine, are finally sufficiently stirred and are uniformly dissolved, and refrigerator cold-storage preserves up to final products.
Embodiment 2
47g redistilled waters are added into the clean container after stringent sterilization, is then slowly added into 29g toad skins and turns glue protein -2,
5g polylactic acid and 4g glucans are added after mixing, is stirred half an hour, then slowly add people's 15g polyethylene glycol, it is last abundant
Stirring and dissolving is uniform, and refrigerator cold-storage preserves up to final products.
Embodiment 3
58g sterile waters are added into the clean container after stringent sterilization, is then slowly added into 25g toad skins and turns glue protein -2,
1g polysorbates and 5g glucans are added after mixing, is stirred half an hour, then slowly adds people's 11g glycerine, are finally fully stirred
It mixes and is uniformly dissolved, refrigerator cold-storage preserves up to final products.
Embodiment 4
52g ultra-pure waters are added into the clean container after stringent sterilization, is then slowly added into 27g toad skins and turns glue protein -2,
2g polysorbates, 2g Emulsifier EL-60s, 2g polylactic acid and 2g glucans are added after mixing, are stirred half an hour, then
Slowly plus people 10g histidines and 3g glycine, it is finally sufficiently stirred and is uniformly dissolved, refrigerator cold-storage preserves up to final products.
Embodiment 5
61g deionized waters are added into the clean container after stringent sterilization, is then slowly added into 24g toad skins and turns glue protein-
2,5g Emulsifier EL-60s are added after mixing, are stirred half an hour, then slowly add people 2.5g polyethylene glycol, 2.5g group ammonia
Acid, 2.5g glycine and 2.5g glycerine, are finally sufficiently stirred and are uniformly dissolved, and refrigerator cold-storage preserves up to final products.
Embodiment 6
Be added into the clean container after stringent sterilization 11g distilled water, 10g redistilled waters, 10g sterile waters, 10g ultra-pure waters and
10g deionized waters are then slowly added into 28g toad skins and turn glue protein -2, add 4g polysorbates, 2g polyoxies after mixing
Ethylene castor oil and 1g polylactic acid stir half an hour, then slowly add people 7g histidines and 7g glycine, are finally sufficiently stirred molten
Solution is uniform, and refrigerator cold-storage preserves up to final products.
The toad skin is turned glue protein -2 and is prepared using following methods：
1）It is screened from toad skin cDNA plasmid libraries, is cloned into lack part 5 '-end open reading frame base sequence
Toad skin turns -2 cDNA of glue protein；
2）Toad skin is expressed and produced in prokaryotic expression system one by one colibacillus turns glue protein -2:Engineering is built first
Bacterium and induced expression, then high density fermentation engineering bacteria and induced expression answer target protein after inclusion body separation and washing
Property, purifying.
The beneficial test result further illustrated the present invention below by way of experiment.
Experiment one：
Each processing selects healthy male mice 30,7 week old, weight respectively（19±2）G, by Disease Control and Prevention Center of Zhejiang Province
It provides, it is all to have credit number for examination animal.Experiment tumor strain used：Mouse With Lewis Lung Cancer cell line, is immunized by Zhejiang University
Laboratory provides.Experiment anti-tumor agent used is that Zhejiang A ＆ F University's above-described embodiment is made.By lung cancer tumor strain when experiment
It is 1 × 10 that concentration of cell suspension, which is made,6/ ml, is inoculated in that mouse right lower extremity armpit is subcutaneous, and every 0.2ml monitors Mice Body daily
The variation of quality, food ration, active state, hair etc..Mouse starts nape part rough coat, obfuscation occur after 12nd day, living
It is dynamic slow, it becomes thin, 15% or more weight loss, ingests, amount of drinking water reduction, mouse enters tumour constitution status.Then at random will
Mouse is divided into control group and each 15 of processing group is tested.Start within 11st day control group and normally raises without any processing, place
Reason group gives the biological agent in injection embodiment, and each group is injected 1 time, each 2ml, coprocessing 15d daily.Period all animals
Freer diet, daily timing observation mouse reactivity, hair color, defecation, food-intake and changes of weight.The estimation of gross tumor volume
Using following formula：V=ab2/2（A, b is respectively the length and width of tumor mass）.Each group puts to death mouse and completely strips tumor after 12d
Body is impregnated after weighing with formaldehyde.Using spss11.5 statistics softwares, measurement data is indicated with mean ± standard deviation, is compared and is adopted
Use one-way analysis of variance.Test result is shown in Table 1.
Mouse is administered the influence of front and back weight in 1 embodiment 1-6 of table
| Processing | Mouse number（Only） | Before administration | After administration |
| Embodiment 1 | 15 | 22.78±2.14 | 21.35±0.86 |
| Embodiment 2 | 15 | 22.14±2.26 | 21.27±1.89 |
| Embodiment 3 | 15 | 22.25±1.57 | 21.04±1.43 |
| Embodiment 4 | 15 | 22.45±2.18 | 21.46±2.07 |
| Embodiment 5 | 15 | 22.67±1.96 | 21.12±1.61 |
| Embodiment 6 | 15 | 22.41±0.74 | 21.23±0.28 |
| Control group | 15 | 22.55±1.87 | 23.56±1.75 |
Test result shows：Embodiment 1-6 has good inhibiting effect, embodiment 1-6 processing to mouse tumor cell
Mouse weight is substantially reduced afterwards, and knurl is obviously reduced, and control group weight, knurl obviously increase, and wherein embodiment 1 is better than implementation
Example 2, embodiment 3 are better than embodiment 4, and embodiment 5 is better than embodiment 6.
Claims (3)
1. the anti-tumor Chinese herbal preparation that a kind of skin containing toad turns glue protein -2 is preparing prevention human body, animal, poultry lung carcinoma cell system
Application in agent, it is characterised in that said preparation is made of following methods：64g is added into the clean container after stringent sterilization to steam
Distilled water is then slowly added into 23g toad skins and turns glue protein -2, adds 2g polysorbates and 2g polyoxyethylene castors after mixing
Sesame oil stirs half an hour, then slowly adds people 3g polyethylene glycol, 3g histidines, 2g glycine and 1g glycerine, is finally sufficiently stirred
It is uniformly dissolved, refrigerator cold-storage preserves up to final products；
The toad skin is turned glue protein -2 and is prepared using following methods：
1）It is screened from toad skin cDNA plasmid libraries, is cloned into the toad skin of lack part 5 '-end open reading frame base sequence
The GenBank accession number for turning glue protein -2 cDNA, cDNA is JX197456；
2）Toad skin is expressed and produced in prokaryotic expression system one by one colibacillus turns glue protein -2:Build engineering bacteria simultaneously first
Induced expression, then high density fermentation engineering bacteria and induced expression, after inclusion body separation and washing, by target protein renaturation,
Purifying.
2. the anti-tumor Chinese herbal preparation that a kind of skin containing toad turns glue protein -2 is preparing prevention human body, animal, poultry lung carcinoma cell system
Application in agent, it is characterised in that said preparation is made of following methods：Into the clean container after stringent sterilization be added 58g without
Bacterium water is then slowly added into 25g toad skins and turns glue protein -2, adds 1g polysorbates and 5g glucans after mixing, stirring
Half an hour, then slowly add people's 11g glycerine, it is finally sufficiently stirred and is uniformly dissolved, refrigerator cold-storage preserves up to final products；
The toad skin is turned glue protein -2 and is prepared using following methods：
1）It is screened from toad skin cDNA plasmid libraries, is cloned into the toad skin of lack part 5 '-end open reading frame base sequence
The GenBank accession number for turning glue protein -2 cDNA, cDNA is JX197456；
2）Toad skin is expressed and produced in prokaryotic expression system one by one colibacillus turns glue protein -2:Build engineering bacteria simultaneously first
Induced expression, then high density fermentation engineering bacteria and induced expression, after inclusion body separation and washing, by target protein renaturation,
Purifying.
3. the anti-tumor Chinese herbal preparation that a kind of skin containing toad turns glue protein -2 is preparing prevention human body, animal, poultry lung carcinoma cell system
Application in agent, it is characterised in that said preparation is made of following methods：61g is added into the clean container after stringent sterilization to go
Ionized water is then slowly added into 24g toad skins and turns glue protein -2, adds 5g Emulsifier EL-60s, stirring half after mixing
Hour, then slowly add people 2.5g polyethylene glycol, 2.5g histidines, 2.5g glycine and 2.5g glycerine, finally it is sufficiently stirred dissolving
Uniformly, refrigerator cold-storage preserves up to final products；
The toad skin is turned glue protein -2 and is prepared using following methods：
1）It is screened from toad skin cDNA plasmid libraries, is cloned into the toad skin of lack part 5 '-end open reading frame base sequence
The GenBank accession number for turning glue protein -2 cDNA, cDNA is JX197456；
2）Toad skin is expressed and produced in prokaryotic expression system one by one colibacillus turns glue protein -2:Build engineering bacteria simultaneously first
Induced expression, then high density fermentation engineering bacteria and induced expression, after inclusion body separation and washing, by target protein renaturation,
Purifying.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310355387.8A CN103405749B (en) | 2013-08-15 | 2013-08-15 | A kind of skin containing toad turns the anti-tumor Chinese herbal preparation and application thereof of glue protein -2 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310355387.8A CN103405749B (en) | 2013-08-15 | 2013-08-15 | A kind of skin containing toad turns the anti-tumor Chinese herbal preparation and application thereof of glue protein -2 |
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| CN103405749A CN103405749A (en) | 2013-11-27 |
| CN103405749B true CN103405749B (en) | 2018-09-11 |
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| CN113559256B (en) * | 2021-08-09 | 2024-03-29 | 广州科力生物科技有限公司 | Nocardia immunopotentiator and application thereof in preparation of pig vaccine |
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Non-Patent Citations (3)
| Title |
|---|
| A novel interplay between oncogenic PFTK1 protein kinase and tumor suppressor TAGLN2 in the control of liver cancer cell motility;WKC Leung等;《Oncogene》;20110516;第30卷;第4464–4475页 * |
| Frequent inactivation of RASSF1A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer;Masao Ito等;《Cancer Letters》;20051231;第225卷;第131–139页 * |
| 日本蟾蜍TAGLN2 cDNA的克隆与生物信息学分析;诸葛慧等;《药学学报》;20130228;第48卷(第2期);第250-254页 * |
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