CN107523510A - A kind of preparation method of antiviral inactive yeast engineering bacteria - Google Patents
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Abstract
This application discloses a kind of preparation method of antiviral inactive yeast engineering bacteria.This method comprises the following steps：Induced expression is gone out to recombinate the engineering bacteria of chicken interferon, after being handled by 71 DEG C of 30min complete inactivations, it is inhibited to NDV that the rChIFN α and rChIFN γ contained in inactivation engineering bacteria are demonstrated using MTT experiment, show to have the inactivation engineering bacteria of ecological security still to remain its antiviral biological activity.The method of the present invention is simple and easy, and cost is cheap, has obvious effect to gal virus disease preventing and treating.
Description
Technical field
The application belongs to biological technical field, specifically, is related to a kind of preparation side of antiviral inactive yeast engineering bacteria
Method.
Background technology
Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a kind of green, safety probiotic feed additive,
Such feed addictive plays an important role in enhancing animal body immunity, improvement intestinal microbial balance etc..
Chicken interferon (ChIFN) is a kind of with efficient, the zooblast factor of broad-spectrum disease resistance toxic action, is to treat and prevent chicken new city
The important drugs of a variety of birds infectious viral diseases such as epidemic disease, infective bronchitis, infectious bursa of Fabricius and egg drop syndrome.
At present, the production to Avian Interferon both at home and abroad, which is mainly produced and purified by technique for gene engineering, is prepared into note
Liquid is penetrated to be applied, exist production cost it is high and easily occur stress the defects of, so as to limit Avian Interferon in aquaculture
Extensive use.There is important meaning to the prebiotic engineering bacteria research of Interferon of Poultry transgenosis for being not required to purifying and directly orally utilizing
Justice, it is the important channel for ensureing Green Husbandry.But transgene activity engineering bacteria is due to there may be potential ecology
The hidden danger such as safety, and constrained by the countries in the world related transgenic safety management regulation including China, therefore, it is difficult to direct
Applied to poultry cultivation.
The content of the invention
In view of this, the application is directed to the problem of above-mentioned, there is provided a kind of preparation side of antiviral inactive yeast engineering bacteria
Method, a kind of safe antiviral activity Yeast engineering bacteria is obtained, it is inhibited to the virus such as newcastle disease, can conduct
Probiotics anti-virus feed additive application, to change the Land use systems of existing Avian Interferon, reduce the utilization of interferon into
This.
In order to solve the above-mentioned technical problem, this application discloses a kind of preparation method of antiviral inactive yeast engineering bacteria,
Comprise the following steps：
1) the genetic stability identification of engineered strain：
Engineering bacteria is screened using SC Selective agar mediums, engineered strain is inoculated on SC solid selection mediums,
Culture is formed to single bacterium colony under the conditions of being placed in 30 DEG C, is chosen the single bacterium colony and is inoculated in shaken cultivation in YPD culture mediums, extracts gene
Group enters performing PCR identification, and qualification result is incubated overnight for positive inoculation in YPD fluid nutrient mediums；Take the appropriate bacterium solution
It is coated on brewer's wort solid medium and is inoculated in malt juice liquid medium, the form of culture 24h observation recombinant strains；
Take above-mentioned bacterium solution to be inoculated in right amount in the inducing culture for adding galactolipin simultaneously and carry out Fiber differentiation, detected using ELISA
rChIFN；
2) inactivation of engineered strain：Using containing 2% galactolipin inducing culture Fiber differentiation to exponential phase
Zymocyte liquid, directly through water-bath inactivation treatment, antiviral inactive yeast engineering bacteria is prepared；
3) inactivated bacteria inactivating efficacy detects：After inactivation engineering bacteria is resuspended in into 2ml sterile salines, 200 μ l are taken to be coated with
Into YPD solid mediums, after putting 30 DEG C of inversion culture 48h, observation whether there is the formation of single bacterium colony；
4) inactivated bacteria antiviral activity detects：The antiviral activity of inactivated bacteria is detected by MTT experiment.
Further, the morphological requirements of the culture 24h recombinant strains in step 1) are：The form of bacterium colony is big and has light
Pool, and the smooth moistening in surface, engineering bacteria stands a period of time close-packed arrays in bottom of bottle in malt juice liquid medium.
Further, the inactivation condition of the engineered strain in step 2)：Fiber differentiation is to exponential phase OD600For 0.4
Zymocyte liquid, inactivation mode inactivate for water-bath, and inactivation temperature is 61-71 DEG C, inactivation time 15min-45min.
Further, it is specially by the antiviral activity of MTT experiment detection inactivated bacteria in the step 4)：By chicken embryo into
Fibrocyte is with 5.0 × 104Individual/ml density is inoculated in 96 orifice plates, and 100 μ L are inoculated with per hole, put 37 DEG C of 5%CO2Trained in incubator
Support, cultivate to 80% cell attachment, 100 μ L mixed liquors are inoculated with per hole；After cultivating 10h, liquid in 96 orifice plates is discarded, with
100TCID50Virus liquid is inoculated with 100 μ L per hole, puts and 2h is acted in incubator, abandon virus liquid and cleaned 2 times with hanks liquid, and inoculation is new
Maintaining liquid, continue after cultivating 48h, 20 μ L volume 2mg/ml MTT liquid are added per hole, continue to cultivate 4h；Suctioned out after culture terminates
Liquid in hole, DMSO solution is added with 100 μ L/ holes, under room temperature condition, slight concussion acts on 10min, makes crystal fully molten
Solution；Determine OD490Numerical value, viral suppression is calculated according to equation below：
Viral suppression (%)=(the average OD- virus control group OD of experimental group)/(Normal group OD- virus control groups
OD)*100。
Further, the composition of the mixed liquor is 10%FBS the μ L of DMEM 50 and the μ L of lysate 50 with 1:1 ratio
Mix.
Further, the engineered strain is INVSC1/pYEIFNG and INVSC1/pYEIFNR engineering bacterias.
Compared with prior art, the application can be obtained including following technique effect：
1) present invention comprehensive utilization SC screening and culturing mediums, malt extract medium, Genomic PCR and ELISA are to laboratory
Early stage, the genetic stability for turning ChIFN saccharomyces cerevisiae engineered yeasts of structure research was analyzed.As a result show, engineering bacteria form is just
Often, target ChIFN- α, ChIFN- γ genes can be stable in the presence of in host genome, and obtain correct accurate translation, rChIFN
Accumulate in yeast cells.Water-bath inactivation research shows that 71 DEG C of processing 15-60min can be by engineering bacteria complete inactivation.According to going out
Viable bacteria crude protein detects discovery, two kinds of ChIFN- α, ChIFN- γ engineerings to the inhibitory action of NDV using MTT experiment
The optimal water-bath inactivation condition of bacterium is 71 DEG C of 30min.
2) saccharomyces cerevisiae was embodied in feed addictive catalogue in 2013, was one using the micro-organisms pharmaceutical protein
Bar high effective way, it is significant in animal epidemic preventing and treating.Compared with the Land use systems of traditional transgenic engineering body, go out
Land use systems living have obvious advantage, and it breaches the limitation that transgene activity engineering bacteria utilizes, and effectively eliminate transgenosis life
The potential source biomolecule potential safety hazard of thing.ChIFN inactivation engineering bacterias are directly applied to poultry cultivation, Ke Yizeng as feed addictive
Strong immunity of poultry, the generation of virosis is reduced, reduce husbandry sector economic loss caused by epidemic disease, ChIFN inactivation wine brewing
Yeast engineering bacteria has a good application prospect.
Certainly, implementing any product of the application must be not necessarily required to reach all the above technique effect simultaneously.
Brief description of the drawings
Accompanying drawing described herein is used for providing further understanding of the present application, forms the part of the application, this Shen
Schematic description and description please is used to explain the application, does not form the improper restriction to the application.In the accompanying drawings：
Fig. 1 is that the application ChIFN- α detect with ChIFN- γ Genomic PCRs；Wherein, A：ChIFN- α engineering bacteria genomes
PCR testing results；M：LD2000DNA Marker；1：ddH2O；2：INVSC1(WT)；3：pYEIFNR Plasmid；4-7：
ChIFN-α；B:ChIFN- γ engineering bacteria Genomic PCR testing results；M：LD2000DNA Marker；1：ddH2O；2：INVSC1
(WT)；3：pYEIFNG Plasmid；4-7：ChIFN-γ；
Fig. 2 is inhibitory action of the application ChIFN- α saccharomyces cerevisiae engineered yeast crude protein extract solutions to NDV；
Fig. 3 is inhibitory action of the application ChIFN- γ saccharomyces cerevisiae engineered yeast crude protein extract solutions to NDV.
Embodiment
Presently filed embodiment is described in detail below in conjunction with drawings and Examples, and thereby how the application is applied
Technological means can fully understand and implement according to this to solve technical problem and reach the implementation process of technical effect.
The structure of the preparation method of the antiviral inactive yeast engineering bacteria of embodiment 1
1) bacterial strain
INVSC1/pYEIFNG and INVSC1/pYEIFNR recombinant Saccharomyces cerevisiaes engineering bacteria contains ChIFN- γ respectively
(U27465.1) and ChIFN- α genes (U07868.1), formulated by agro-biological engineering research institute of Guizhou University and be stored in -80
DEG C refrigerator.
2) screening and culturing of engineered strain and identification
2.1) engineering bacteria is screened using SC Selective agar mediums, engineered strain is inoculated in SC solid selection mediums
On, cultivate under the conditions of being placed in 30 DEG C and formed to single bacterium colony, -80 DEG C save backup；
2.2) -80 DEG C of INVSC1/pYEIFNG for preserving or turning out immediately are taken to be applied with INVSC1/pYEIFNR engineering bacterias
It is distributed in YPD solid mediums, puts 30 DEG C of 24~48h of culture；The engineering bacteria single bacterium colony of picking activation is inoculated in 5ml YPD liquid
In culture medium, after 30 DEG C of culture about 16h, the genome for extracting the engineering bacteria enters performing PCR identification, chooses qualification result and is shown as sun
Property engineering bacteria be inoculated in 15ml YPD fluid nutrient mediums, put 30 DEG C, 200r/min overnight shaking cultures, measure bacterium solution OD600
Value.
The Genomic PCR testing result of INVSC1/pYEIFNG and INVSC1/pYEIFNR engineering bacterias shows, two kinds of engineerings
Bacterium can amplify and convert the basically identical target stripe of plasmid amplification stripe size (Fig. 1), illustrate ChIFN- α and ChIFN-
γ genes are stable in the presence of in saccharomyces cerevisiae engineered yeast, and the gene has preferable stability.
3) induced expression of Yeast engineering bacteria
3.1) bacterium solution in screening and culturing medium is inoculated in the YPD culture mediums of 50mL addition galactolipin derivants with 1% amount
In, it is placed in 30 DEG C, 200r/min overnight shaking cultures 16h.
3.2) OD for being incubated overnight bacterium solution is determined600After numerical value, through 1500 × g, 4 DEG C of centrifugation 5min, supernatant is abandoned.
3.3) cell is resuspended using 1.0ml sterile salines, 10000r/min centrifugation 1min, supernatant is abandoned, by the thalline
Save backup.
4) detection of interferon recombinant protein
4.1) prepared by engineering bacteria crude protein extract solution
A takes above-mentioned thalline, is resuspended in 500 μ L lysis buffer, 1500 × g, 4 DEG C of centrifugation 5min, abandons supernatant.
B adds appropriate lysate into step a thalline and thalline is resuspended and makes resuspended bacterium solution OD600=50~100.
C adds isometric pickling glass pearl, ice bath 40s after vortex oscillation 40s, is repeated 6 times so that yeast cells is abundant
Cracking.
D cell lysis is taken supernatant to be placed in 80 DEG C of ﹣ and saved backup with maximum velocity centrifugation 10min.
4.2) recombinant C hIFN ELISA quantitative analyses
Above-mentioned 4.1) methods described preparation engineering Yeast protein lysate is taken, bio tech ltd is ground with reference to Shanghai one
Kit specification method (green skies Bioisystech Co., Ltd) detection recombination yeast chicken α, interferon content.
To after Fiber differentiation 16h recombination engineering carry out ELISA test and analyze to obtain, INVSC1/pYEIFNR with
Two kinds of engineering bacterias of INVSC1/pYEIFNG can give expression to rChIFN, and the expression quantity of two kinds of restructuring chicken interferons is respectively
42.13ng/L and 45.49ng/L.
5) screening and optimization of engineering bacteria water-bath inactivation condition
Learn from else's experience Fiber differentiation 16h ChIFN engineering bacteria bacterium solutions it is appropriate, with 1500 × g centrifuge 5min, collect thalline, with nothing
Bacterium PBS wash 3 times, be resuspended in 2ml PBS, by re-suspension liquid be placed in treatment temperature be 56-71 DEG C, processing time 10-60min
Under conditions of carry out inactivation condition research.After taking the μ l of bacterium solution 100 after processing to be coated on YPD culture mediums, 30 DEG C of cultures are placed in
48h, observe and single bacterium colony appearance is whether there is on culture medium, analyze inactivating efficacy, and go out using cell phenotype conversion detection is specific
The antiviral biological activity of same batch processing sample under the conditions of work.
As can be drawn from Table 2：Inactivation temperature and the way crossover study design result of time two obtain, and 3 kinds of bacterial strains are at 71 DEG C
Can effectively it be inactivated under conditions of 0.25h.
The recombination engineering water-bath of table 1 inactivates orthogonal experiment and result
Note：+ represent not to be inactivated；- represent by complete inactivation
Inhibitory action of the crude protein extract solution of embodiment 2 to NDV
By chicken embryo fibroblasts with 5.0 × 104Individual/ml density is inoculated in 96 orifice plates, and 100 μ L are inoculated with per hole, put 37
DEG C 5%CO2Cultivate, cultivated to 80% cell attachment in incubator, 100 μ L mixed liquors are inoculated with per hole, and (composition of mixed liquor is
The 10%FBS μ L of DMEM 50 and the μ L of lysate 50 is with 1:1 ratio mixes).After cultivating 10h, liquid in 96 orifice plates is discarded, with
100TCID50Virus liquid is inoculated with 100 μ L per hole, puts and 2h is acted in incubator, abandon virus liquid and cleaned 2 times with hanks liquid, and inoculation is new
Maintaining liquid, continue after cultivating 48h, 20 μ L volume 2mg/ml MTT liquid are added per hole, continue to cultivate 4h.Suctioned out after culture terminates
Liquid in hole, DMSO solution is added with 100 μ L/ holes, under room temperature condition, slight concussion acts on 10min, makes crystal fully molten
Solution.Determine OD490Numerical value, calculate action effect of the inactivation engineering crude protein extract to virus.
Viral suppression is calculated according to equation below：
Viral suppression (%)=(the average OD- virus control group OD of experimental group)/(Normal group OD- virus control groups
OD)*100。
The engineering bacteria crude protein extract solution under the conditions of different inactivation treatments is detected by MTT experiment to NDV inhibitions.Grind
Study carefully result to show (Fig. 2, Fig. 3), rChIFN activity gradually reduces with the rise for the treatment of temperature, ChIFN- α and ChIFN- γ
Crude protein extract solution of the saccharomyces cerevisiae engineered yeast under the conditions of 71 DEG C of inactivation 0.5h reaches maximum to NDV inhibition, suppresses
Rate is respectively 35.75% and 33.71%.
Present invention research finds that on the premise of biological activity is ensured inactivation is to eliminate life caused by transgenic organism
The optimal path of thing safety, the water-bath inactivation mode that use is easy and effective, safe and reliable and inexpensive, research have particular organisms
The ChIFN inactivation engineering bacterias of activity are learned, the inactivation engineering bacteria of initiative has the securities such as ecology and antiviral activity, and this is gene
The direct application of engineering recombination engineering opens a safe and reliable new way, and inactivates engineering bacteria as fowl for ChIFN
The commercial application of class feed addition is laid a good foundation.
Some vocabulary has such as been used to censure special component or method among specification and claim.Art technology
Personnel are, it is to be appreciated that different regions may call same composition with different nouns.This specification and claims are not
In a manner of the difference of title is used as and distinguishes composition.As the "comprising" of the specification in the whole text and claim mentioned in is
One open language, therefore " include but be not limited to " should be construed to." substantially " refer in receivable error range, this area
Technical staff can solve the technical problem within a certain error range, basically reach the technique effect.Specification is follow-up
It is described as implementing the better embodiment of the application, so description is for the purpose of the rule for illustrating the application, not
To limit scope of the present application.The protection domain of the application is worked as to be defined depending on appended claims institute defender.
It should also be noted that, term " comprising ", "comprising" or its any other variant are intended to nonexcludability
Comprising, so that commodity or system including a series of elements not only include those key elements, but also including without clear and definite
The other element listed, or also include for this commodity or the intrinsic key element of system.In the feelings not limited more
Under condition, the key element that is limited by sentence "including a ...", it is not excluded that in the commodity including the key element or system also
Other identical element be present.
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification
And environment, and can be carried out in the scope of the invention is set forth herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in power appended by invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit requires.
Claims (6)
1. a kind of preparation method of antiviral inactive yeast engineering bacteria, it is characterised in that comprise the following steps：
1) the genetic stability identification of engineered strain：
Engineering bacteria is screened using SC Selective agar mediums, engineered strain is inoculated on SC solid selection mediums, is placed in
Culture is formed to single bacterium colony under the conditions of 30 DEG C, is chosen the single bacterium colony and is inoculated in shaken cultivation in YPD culture mediums, extraction genome enters
Performing PCR identifies that qualification result is incubated overnight for positive inoculation in YPD fluid nutrient mediums；Take appropriate bacterium solution coating
In brewer's wort solid medium and malt juice liquid medium is inoculated in, the form of culture 24h observation recombinant strains；Simultaneously
Take above-mentioned bacterium solution to be inoculated in right amount in the inducing culture for adding galactolipin and carry out Fiber differentiation, utilize ELISA to detect rChIFN；
2) inactivation of engineered strain：Utilize the fermentation containing 2% galactolipin inducing culture Fiber differentiation to exponential phase
Bacterium solution, directly through water-bath inactivation treatment, antiviral inactive yeast engineering bacteria is prepared；
3) inactivated bacteria inactivating efficacy detects：After inactivation engineering bacteria is resuspended in into 2ml sterile salines, 200 μ l are taken to be applied to YPD
In solid medium, after putting 30 DEG C of inversion culture 48h, observation whether there is the formation of single bacterium colony；
4) inactivated bacteria antiviral activity detects：The antiviral activity of inactivated bacteria is detected by MTT experiment.
2. the preparation method of antiviral inactive yeast engineering bacteria according to claim 1, it is characterised in that in step 1)
Cultivating the morphological requirements of 24h recombinant strains is：The form of bacterium colony is big and glossy, and the smooth moistening in surface, in brewer's wort
Engineering bacteria stands a period of time close-packed arrays in bottom of bottle in fluid nutrient medium.
3. the preparation method of antiviral inactive yeast engineering bacteria according to claim 1, it is characterised in that in step 2)
The inactivation condition of engineered strain：Fiber differentiation is to exponential phase OD600For 0.4 zymocyte liquid, inactivation mode is gone out for water-bath
Living, inactivation temperature is 61-71 DEG C, inactivation time 15min-45min.
4. the preparation method of antiviral inactive yeast engineering bacteria according to claim 1, it is characterised in that the step 4)
In by MTT experiment detect inactivated bacteria antiviral activity be specially：By chicken embryo fibroblasts with 5.0 × 104Individual/ml density
It is inoculated in 96 orifice plates, 100 μ L is inoculated with per hole, put 37 DEG C of 5%CO2Cultivate, cultivate to 80% cell attachment, per hole in incubator
It is inoculated with 100 μ L mixed liquors；After cultivating 10h, liquid in 96 orifice plates is discarded, with 100TCID50Virus liquid is inoculated with 100 μ L per hole, puts training
Support in case and act on 2h, abandon virus liquid and cleaned 2 times with hanks liquid, be inoculated with new maintaining liquid, continue after cultivating 48h, 20 μ L are added per hole
Volume 2mg/ml MTT liquid, continue to cultivate 4h；Liquid in hole is suctioned out after culture terminates, DMSO solution is added with 100 μ L/ holes,
Under room temperature condition, slight concussion acts on 10min, crystal is fully dissolved；Determine OD490Numerical value, disease is calculated according to equation below
Malicious inhibiting rate：
Viral suppression (%)=(the average OD- virus control group OD of experimental group)/(Normal group OD- virus control group OD) *
100。
5. the preparation method of antiviral inactive yeast engineering bacteria according to claim 4, it is characterised in that the mixed liquor
Composition for 10%FBS the μ L of DMEM 50 and the μ L of lysate 50 with 1:1 ratio mixes.
6. the preparation method of the antiviral inactive yeast engineering bacteria according to any claim in claim 1-5, it is special
Sign is that the engineered strain is INVSC1/pYEIFNG and INVSC1/pYEIFNR engineering bacterias.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781372A (en) * | 2010-03-11 | 2010-07-21 | 南京农业大学 | Sulphation modification method of codonopsis pilosula polysaccharide |
| CN102321631A (en) * | 2011-08-30 | 2012-01-18 | 贵州大学 | Construction and application of artificially-synthesized chicken interferon gene sequence and recombinant engineering bacterium thereof |
| CN104830690A (en) * | 2015-05-27 | 2015-08-12 | 贵州大学 | Preparation method of active engineering yeast |
-
2017
- 2017-10-16 CN CN201710959634.3A patent/CN107523510A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781372A (en) * | 2010-03-11 | 2010-07-21 | 南京农业大学 | Sulphation modification method of codonopsis pilosula polysaccharide |
| CN102321631A (en) * | 2011-08-30 | 2012-01-18 | 贵州大学 | Construction and application of artificially-synthesized chicken interferon gene sequence and recombinant engineering bacterium thereof |
| CN104830690A (en) * | 2015-05-27 | 2015-08-12 | 贵州大学 | Preparation method of active engineering yeast |
Non-Patent Citations (4)
| Title |
|---|
| FENGXIANG HOU等: "Antiviral activity of rChIFN-α against vesicular stomatitis virus and Newcastle disease virus: a novel recombinant chicken interferon-α showed high antiviral activity", 《RESEARCH IN VETERINARY SCIENCE》 * |
| 布莱尔: "《有机禽营养与饲养》", 28 February 2013 * |
| 张斌: "富硒酵母抗氧化活性的初步研究", 《蚌埠学院学报》 * |
| 贾洪锋: "《食品微生物》", 31 August 2015 * |
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