CN101307292A - Easy and rapid screening method for bioactive substance - Google Patents
Easy and rapid screening method for bioactive substance Download PDFInfo
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- CN101307292A CN101307292A CNA2007100098122A CN200710009812A CN101307292A CN 101307292 A CN101307292 A CN 101307292A CN A2007100098122 A CNA2007100098122 A CN A2007100098122A CN 200710009812 A CN200710009812 A CN 200710009812A CN 101307292 A CN101307292 A CN 101307292A
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Abstract
The invention relates to a set of general, simple and rapid biological active substance screening methods with accuracy and higher flux. The methods belong to the life science field. The screening of a biological active substance sample library needs retaining objective samples with high activity and high yield and washing out valueless non-objective samples. However, in a general case, as the amount of the samples in the library is quite great, a traditional screen method of one-by-one detection wastes time and labor with high workload and low efficiency. Furthermore, a full automatic high-flux screen workstation can not be popularized due to a long price. The invention adopts conventional apparatuses to work out a complete set of screen methods involving culture and detection. The methods are suitable for the screening of most samples under most conditions.
Description
Technical field:
The invention belongs to life science, be specifically related to one group can be easy, fast and accurately and what method of the active size of the general screening biologically active substance of higher flux and amount.
Background technology:
Since 20 beginnings of the century discovery penicillin and since with its realization application, biotechnology and technical field are flourish, up till now, human biologically active substance service society development of having developed thousands of kinds, comprise the many materials of microbiotic, VITAMIN, amino acid, organic acid, zymin, nucleic acid analog, hormone or the like, relate to medicine, food, chemical industry or the like numerous areas.At the initial stage, these materials all come from nature, are found and utilize by human, but a little less than the general activity of these crude substance, yield poorly, and not too be suitable for direct use, have very big side effect or the like as some microbiotic.In the mid-term of technical development, human output and the activity that improves these materials such as methods such as " mutagenesis ", " metabolism control ", " cytogamy " of using, even can transform its characteristic to a certain extent, these technology have been facilitated the foundation of modern biological industry.Along with the development of modern biotechnology, people can pass through to transform such as technique to high-efficiency ground such as " orthogenesiss " characteristic of crude substance in the laboratory, even artificial accelerated evolutionary process, and it is used for us to create the non-existent material of nature.No matter find the still artificial transformation of natural bioactivity substance or create biologically active substance from occurring in nature, all need the natural sample library or the library, transformation back that obtain are screened, the purpose sample that keeps high reactivity and high yield is eliminated unworthy non-purpose sample.But in the ordinary course of things, the quantity of sample is quite huge in the library, therefore, traditional consuming time, consumption power of screening method that detects one by one, workload is big, and efficient is low, though once brought into play vital role in history, has been not suitable for present practical application now.In the recent period, developed countries such as America and Europe develop full-automatic high flux screening workstation, state-of-the-art computer technology and robot technology have been used, can realize efficient high flux screening, but prices are rather stiff for this type of main equipment, even in developed country, the large-scale experiment chamber that also only is financially strong has the ability to purchase, and is difficult to apply in a short time.Therefore, use existing general low-cost plant and instrument, develop a kind of easyly, fast and accurately and the universal bolter selecting technology method of higher flux, have important practical significance.
Summary of the invention:
What the objective of the invention is to design a kind of easy, fast and accurately and method of the active size of higher flux ground screening biologically active substance and amount, be suitable for the screening of microbiotic, VITAMIN, amino acid, organic acid, zymin, nucleic acid analog, hormone or the like many materials, these biologically active substances can be to come from natural zooblast, vegetable cell, microorganism cells, also can be cell or the clone who comes from genetic engineering technique or other technological transformation, prerequisite be that this material to be screened has suitable detection model.The implementation phase that this method having following three:
1. cultivation stage
(1) solid culture
Biology for being fit to solid culture uses this method.The agar (or suitable peptizer of other proper concn) that in waiting to screen the substratum of material, adds 1%-2%, autoclave sterilization is handled, for substratum that can not autoclave sterilization, after its substratum specified otherwise degerming, the agar-agar soln that adds sterilization, agar-agar soln should be cooled to add about 40 ℃ again, and it is 1%-2% that add-on should be controlled its adding back agar final concentration.The substratum that will contain agar is poured in the ware with horizontal bottom under aseptic condition, the sterilization in advance of ware and ware lid is because the bottom volume of ware can measure, therefore, control the volume of the nutrient agar of pouring into, just can control the thickness of the solid medium flat board of formation.Use the sterilization punch tool to prepare duplicate little agar block, it is transferred in the sterilization culture dish with the sterilization tweezers.Use the sterilization toothpick, the sample transfer in the library to be screened is inoculated on the little agar block cultivates, culture condition is decided on waiting the characteristic of screening material.Because the volume and the shape of little agar block are in full accord, therefore can guarantee the nutrition unanimity that each sample obtains, under the situation of culture condition unanimity, the data that obtain have reliable comparability.
(2) liquid culture
Biology for being fit to liquid culture uses this method.Use the porous cell culture plate, as 96 orifice plates, if the disposable plate of having sterilized, need not sterilization, if reuse, can use radioelement sterilization, under the situation that aseptic requirement is not strict, can use that disinfecting substance such as alcohol soaks, the easy mode of uviolizing or the like sterilizes.Use liquid-transfering device (as multichannel pipettor, continuous sample introduction device or the like) that the nutrient solution of prior degerming is added, add-on is decided on the cultivation situation.Use the sterilization toothpick, the sample transfer in the library to be screened is inoculated in the nutrient solution cultivates, culture condition is decided on waiting the characteristic of screening material.If sample needs aerated culture, then in advance the growth plate lid is done following improvement: use awl to cover punching, then at plate lid outer side covers last layer ventilation filtration sterilization layer (can use eight layers of gauze, sponge or the suitable material of other function) at plate.
2. detecting stage
(1) identification plate detects
The material that can react with active substance to be measured and can be detected is laid on transparent level with the solid-state form of gel (as detection material being added in the agar-agar soln of 1%-2%, bed board promptly becomes solid-state after the cooled and solidified) and identifies on the flat board.If test substance is cultivated acquisition in the mode of solid agar block, the little agar block after then will cultivating under aseptic condition is arranged on the identification plate; If test substance obtains in the mode of liquid culture, then use the culture plate reproducer to get the nutrient solution of trace, transfer on the identification plate.After test substance and the detection material reaction, generally can around test substance, form the zone that color change takes place for transparent circle, variable color circle or the like.Use scanner that identification plate is scanned into electronic pictures, the image processing software that uses a computer identification is also distinguished background colour and color change zone, thereby can measure the size and the color in color change zone, the numerical value input data statistics process software with obtaining carries out statistical study.Generally speaking, regional area occupied is big more, and test substance is active and content is high more, and the relation of colour-change and activity and content then will be according to the practical situation judgment processing.
(2) microplate reader detects
For the material of liquid culture, can join in the nutrient solution with the detection material of test substance generation color reaction, after the mixing reaction, use microplate reader to measure its absorbancy at a certain wavelength place.
3. preservation stage
Selected purpose bacterial strain transfer is inoculated into strain inclined plane or the frozen pipe of preparation bacterial classification.
Embodiment:
Embodiment 1
(1) will produce bacterium GZ through the extracellular lipase of ultraviolet radiation mutagenic treatment and be located away from the solid medium flat board (substratum is traditional beef-protein medium), 37 ℃ of constant temperature culture 48 hours.
(2) in the horizontal bottom area is 50 square centimeters sterilization ware, pour volume into and be 25 cubic centimetres sterilising medium A (%): wheat bran 7, soybean cake powder 3, (NH
4)
2SO
40.25, pH7.0, agar 2.Ware is placed on the level table, treat after the culture medium solidifying that the use diameter is that 0.5 centimetre sterilization punch tool is prepared into little agar block with culture medium flat plate, with the sterilization tweezers little agar block is transferred in the sterilization culture dish again, if do not use immediately, should place low temperature environment to preserve.
(3) with the dull and stereotyped taking-up of cultured bacterium GZ, under aseptic condition, use the sterilization toothpick that single bacterium colony is shifted respectively and be inoculated on the little agar block prepared in the step (2), little agar block was placed 37 ℃ of constant temperature culture 48 hours.
(4) in the bottom area of transparent level is 50 square centimeters sterilization ware, pours volume into and be 25 cubic centimetres the evaluation gel B preparation of heating fusion and identify dull and stereotyped.Identify gel B prescription: sweet oil emulsion 5%, agar 2% uses distilled water to supply volume.Ware is placed on the level table, after gel B to be identified solidifies, cultured little agar block in the step (3) is being transferred under the aseptic condition on the evaluation flat board, proper alignment, dull and stereotyped used ware of evaluation and ware lid all will be sterilized in advance.The evaluation flat board that will have little agar block is positioned over 48 ℃ and cultivated 24 hours.
(5) will identify dull and stereotyped the taking-up and since bacterium GZ excretory lipase hydrolysis identify and sweet oil emulsion on the flat board therefore around little agar block, produced transparent circle, be not oyster white by the zone of lipase effect.With scanner flat board is scanned, obtain image.
(6) the hydrolysis collar region on " Color Range " selection function selected digital image of use photoshop software uses " measurement facility " to measure hydrolysis circle size, reads dimension information on " information " hurdle.The dimension information of each transparent circle is imported Excel software, carry out statistical study, choose the purpose bacterial strain.
(7) inoculation on the little agar block that will choose is preserved to the beef-protein medium inclined-plane.
Embodiment 2
(1) will produce bacterium GZ through the extracellular lipase of ultraviolet radiation mutagenic treatment and be located away from the solid medium flat board (substratum is traditional beef-protein medium), 37 ℃ of constant temperature culture 48 hours.
(2) the sterilising medium C (%) of adding 200 μ L in each hole of disposable sterilized 96 porocyte culture plates: wheat bran 7, soybean cake powder 3, (NH
4)
2SO
40.25, pH7.0.If reuse 96 orifice plates, can use cobalt 60 sterilization, perhaps 96 orifice plates are cleaned up, use 75% alcohol-pickled 24 hours, place ultraviolet ray irradiation 1 hour down again.Add substratum and can use devices such as multichannel pipettor or continuous sample introduction device.
(3) use the sterilization toothpick to be inoculated on 96 orifice plates in the substratum each hole in, cover plate and cover, place on the constant temperature shaking table cultivating each bacterial colony that obtains in the step (1), with the rotating speed of 220rpm, 37 ℃ of constant temperature culture 48 hours.
(4) the evaluation flat board in the preparation example 1 (4), use culture plate reproducer is got the nutrient solution in the micro-step (3), and point is added on to be identified on the flat board, will identify that flat board is positioned over 48 ℃ and cultivated 24 hours.
(5) with example one (5) (6)
(6) bacterial strain of choosing is shifted from the hole be inoculated into the beef-protein medium inclined-plane and preserve.
Embodiment 3
(1) with example 2 (1) (2) (3)
(2) use equipment such as multichannel pipettor or continuous sample introduction device to add the sweet oil emulsion of 10 μ L in each hole of 96 orifice plates, mixing is cultivated adding brilliant green evaluation liquid (final concentration is 0.004%) after 24 hours for 48 ℃.The room temperature that is placed on mixing left standstill 5 minutes.
(3) 96 orifice plates are put into microplate reader, measure the absorbancy at 640nm place.
(4) the data importing Excel software that records is carried out statistical study and handle, select the purpose bacterial strain.
(5) bacterial strain of choosing is shifted from the hole be inoculated into the beef-protein medium inclined-plane and preserve.
Embodiment 4
(1) the plate lid with Tissue Culture Plate uses marking pen marking corresponding to the hole place, uses the Electric drill-bit punching, and the hole diameter of punching diameter and culture plate quite is advisable, eight layers of gauze on the ware exterior surface covers, sterilising treatment.Radioactive substance sterilization (as cobalt 60 methods) is preferably used in sterilization.
(2) with embodiment 2 or embodiment 3.
Claims (3)
1. one kind is passed through the little agar block of preparation aseptic culture medium, cultivates the solid fermentation method for the treatment of screening sample.It is characterized in that: preparation volume and the little agar block of the duplicate aseptic culture medium of shape, the sample inoculation is cultivated thereon, can guarantee the nutrition unanimity that each sample obtains.
2. method that the growth plate lid is transformed of cultivating that aerobic sample uses.It is characterized in that: use awl to cover punching, then at plate lid outer side covers last layer ventilation filtration sterilization layer at plate.
3. one kind comprises the screening method of cultivating and detecting, and its concrete steps comprise:
A. cultivation stage
(1) solid culture
Be applicable to the biology that is fit to solid culture.It is characterized in that: the suitable peptizer that in being suitable for waiting screening the substratum of material, adds proper concn.Substratum is poured under aseptic condition in the ware with horizontal bottom,, therefore, controls the volume of the substratum of pouring into, just can control the thickness of the solid medium flat board of formation because the bottom volume of ware can measure.Use the sterilization punch tool to prepare duplicate little agar block, it is transferred in the sterilization culture dish with the sterilization tweezers.Use the sterilization toothpick, the sample transfer in the library to be screened is inoculated on the little agar block cultivates, culture condition is decided on waiting the characteristic of screening material.
(2) liquid culture
Be applicable to the biology that is fit to liquid culture.It is characterized in that: use the porous cell culture plate, add with the nutrient solution of liquid-transfering device with prior degerming, add-on is decided on the cultivation situation.Use the sterilization toothpick, the sample transfer in the library to be screened is inoculated in the nutrient solution cultivates, culture condition is decided on waiting the characteristic of screening material.If sample needs aerated culture, then in advance the growth plate lid is improved by the described method of claim 2.
B. detecting stage
(1) identification plate detects
It is characterized in that: can be laid on transparent level with the solid-state form of gel with the material that active substance to be measured reacts and energy is detected and identify on the flat board.If test substance is cultivated acquisition in the mode of solid agar block, the little agar block after then will cultivating under aseptic condition is arranged on the identification plate; If test substance obtains in the mode of liquid culture, then use the culture plate reproducer to get the nutrient solution of trace, transfer on the identification plate.After test substance and the detection material reaction, generally can around test substance, form the zone that color change takes place for transparent circle, variable color circle or the like.Use scanner that identification plate is scanned into electronic pictures, the image processing software that uses a computer identification is also distinguished background colour and color change zone, thereby can measure the size and the color in color change zone, the numerical value input data statistics process software with obtaining carries out statistical study.
(2) microplate reader detects
It is characterized in that: for the material of liquid culture, can join in the nutrient solution, after the mixing reaction, use microplate reader to measure its absorbancy at a certain wavelength place with the detection material of test substance generation color reaction.
C. preserve the stage
It is characterized in that: selected purpose bacterial strain is shifted being inoculated into strain inclined plane or the frozen pipe of preparation bacterial classification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100098122A CN101307292A (en) | 2007-11-15 | 2007-11-15 | Easy and rapid screening method for bioactive substance |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100098122A CN101307292A (en) | 2007-11-15 | 2007-11-15 | Easy and rapid screening method for bioactive substance |
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| Publication Number | Publication Date |
|---|---|
| CN101307292A true CN101307292A (en) | 2008-11-19 |
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ID=40123980
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|---|---|---|---|
| CNA2007100098122A Pending CN101307292A (en) | 2007-11-15 | 2007-11-15 | Easy and rapid screening method for bioactive substance |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
| CN106011219A (en) * | 2016-05-12 | 2016-10-12 | 上海交通大学 | High-throughput screening method for phenazine-1-formamide high-yield strains |
-
2007
- 2007-11-15 CN CNA2007100098122A patent/CN101307292A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
| CN102268378B (en) * | 2011-07-14 | 2014-01-01 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
| CN106011219A (en) * | 2016-05-12 | 2016-10-12 | 上海交通大学 | High-throughput screening method for phenazine-1-formamide high-yield strains |
| CN106011219B (en) * | 2016-05-12 | 2019-09-03 | 上海交通大学 | The high-throughput screening method of azophenlyene -1- formamide superior strain |
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Open date: 20081119 |