CN102206614A - Novel method for preparing mixed glue solution for purifying virus plaques and cloning viruses - Google Patents
Novel method for preparing mixed glue solution for purifying virus plaques and cloning viruses Download PDFInfo
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- CN102206614A CN102206614A CN2011101148002A CN201110114800A CN102206614A CN 102206614 A CN102206614 A CN 102206614A CN 2011101148002 A CN2011101148002 A CN 2011101148002A CN 201110114800 A CN201110114800 A CN 201110114800A CN 102206614 A CN102206614 A CN 102206614A
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Abstract
The invention provides a novel method for preparing a mixed glue solution for purifying virus plaques and cloning viruses, comprising the following steps of: preparing a 1*cell medium: uniformly mixing 4-6 grams of MEM (Minimum Essential Medium) dehydrated mediums, 0.02-0.04 grams of L-glutamine, 0.4-0.6 grams of lactoalbumin hydrolysate, 94-98 ml of water for injection and 3-5 ml of fetal calf serums; and regulating a pH value to be 7.2-7.3 for later use; preparing low-melting point glues: pouring 0.6 grams of low-melting point glue powder into a triangular flask; adding 40 ml of deionized water; fastening a bottleneck by using parchment paper and brown paper; and boiling on a microwave oven for 4 minutes for later use when in a semisolid state; preparing the mixed glue solution containing 1 percent of the low-melting point glues and spreading glues: completely sucking the medium contained in a cell plate by using a pipettor; then extracting a prepared glue solution; slowly spreading the low-melting point glues on the surfaces of cells along the wall of a culture plate, and then standing at room temperature for 10 minutes so that the low-melting point glues are sufficiently solidified; then placing into a CO2 incubator with a temperature of 37 DEG C and a relative humidity of 5 percent for culture; observing cytopathy for 3-5 days; and choosing single virus for cloning. According to the novel method, the conventional cell number is increased by 1.5 times, and the virus titer of the single cloned virus transferred for 3 generations is enhanced by 8.6 times.
Description
Technical field
The present invention relates to the preparation of viral plaque purification and clone's mixed glue solution, the novel method of employing is compared with ordinary method, has improved the quantity of the cell quantity and the mono-clonal purified virus particle of unit surface, has reduced experimental cost, improves the experiment effect.
Background technology
After conventional viral plaque, purifying and (cell) cloning process is to use (2～10) * substratum and low melting point glue mixes by corresponding proportion, be covered in the monolayer cell that connects behind the poison, but be to use (2～10) * substratum to be used for viral plaque, purifying and (cell) clone, need in advance (2～10) * substratum to be prepared, low melting point glue shifts to an earlier date behind the autoclaving standby; This method program is loaded down with trivial details, and substratum mix with glue the back solution pH value, osmotic pressure will change, influenced the growth conditions of cell, and then influenced the effect of virus clone.
The preparation of the virus clone purifying SOP:2% glue that literature search discloses; Take by weighing 2 gram glue, add 100 ml deionized waters, 0.112MPa autoclaving 30 minutes places 4 ℃ of preservations, and fully fusing places 40-45 ℃ of water-bath standby before using; The configuration of 2 * substratum: the HEPES of 10gMEM dehydrated medium, 45ml water for injection, foetal calf serum 20ml, 0.238 gram, the L-glutaminase of 0.06 gram, 2ml, the 0.5% toluylene red 1.2ml of 1 lactoalbumin hydrolysate that restrains, 3% hydrogen-carbonate are formulated, and the pH value transfers to 7.3; The sol solution that autoclaving is good in advance need be mixed with 2 * substratum 1:1 during use, sol solution to be mixed is reduced to about 37 ℃ and is covered cell.
The present invention's design mixes 1 * substratum (culturing cell conventional liq), (2～10) * substratum respectively with low melting point glue, cover low melting point glue culturing cell at cell surface under the situation that initial cell quantity equates; Cover low melting point glue with the virus back of inoculating the equivalent dilution under the situation about equating simultaneously and cultivate virus in initial cell quantity, carrying out cell counting and malicious valency measures, the result shows that the former can effectively improve the quantity of cell quantity and the mono-clonal and the purified virus particle of unit surface, simple and practical, reached design requirements.
Summary of the invention
The objective of the invention is to: the novel method that is used for viral plaque purification and the preparation of clone's epoxy glue solution that provides substitutes (2～10) * substratum and is used for viral plaque, purifying and (cell) clone ordinary method.
The present invention is achieved in that a kind of novel method that is used for viral plaque purification and the preparation of clone's epoxy glue solution, comprises the preparation of 1 * cell culture medium, the preparation of low melting point glue, the preparation and the shop glue of epoxy glue solution;
The wherein preparation of 1 * cell culture medium: by 4-6 gram MEM dehydrated medium, the L-glutaminase of 0.02-0.04 gram, the lactoalbumin hydrolysate of 0.4-0.6 gram, 94-98ml water for injection and foetal calf serum 3-5ml mix, and it is standby that the pH value transfers to 7.2-7.3;
The wherein preparation of low melting point glue: get the low melting point rubber powder of 0.6 gram, pour in the triangular flask, add the 40ml deionized water, bottleneck is tightened with template and kraft paper, on microwave oven, boil 4min, standby when being semi-solid;
Wherein contain the preparation and shop glue of the epoxy glue solution of 1% low melting point glue:
With the 1 * cell culture medium in last step, heat 38 ℃, the full dose low melting point glue of getting the preparation of 60ml and last step mixes, and blows and beats gently with aseptic suction pipe, glue is fully dissolved there is no the bubble generation; Take out the cultivation for preparing in advance and become individual layer and inoculation to produce cytopathic virus, be the cell plate of primary cell and passage cell; With pipettor the substratum in the cell plate is exhausted, with the sol solution sucking-off of preparation, glue slowly is layered on cell surface then, at room temperature placement 10min it is fully solidified after spread, put into 37 ℃, 5% CO then along cultivating wooden partition
2Cultivate observation of cell pathology 3-5 days, picking single virus clone in the incubator.
Described method can make conventional cell quantity improve 1.5 times, makes the malicious valency of mono-clonal virus biography after 3 generations improve 8.6 times (are the truth of a matter with 10).
Novel method of the present invention, using the every porocyte quantity of 1 * culture method culturing cell is 8.5 * 10
5Be that (cell count is 5.6 * 10 to ordinary method
5) 1.5 times, the viral TCID that clones with 1 * culture method
50Mean value be 10
7.2And the viral TCID that clones with 2 * culture method
50Mean value be 10
6.55, the former is 3.55 times of the latter; This method has not only improved the quantity of cell, has improved the malicious valency of mono-clonal virus simultaneously, has simplified program, the shortening time, shows technical progress.
Embodiment
Below in conjunction with embodiment invention is described further.
Adopt 1 * substratum to mix clone and the purifying that carries out virus with low melting point glue;
The wherein preparation of cell (is example with the Marc-145 cell):
The Marc-145 monolayer cell that growth is fine and close is washed 2 times with D～Hanks, and adding concentration is EDTA～trypsin digestion cell of 0.25%, after digestion finishes, adds an amount of substratum and blows and beats gently with suction pipe, makes cell be dispersed into individual cells; Get wherein 100 μ l, add the Hanks liquid of 900 μ l, drip behind the mixing on blood cell counting plate, by the white blood cell count(WBC) method, the cell in 4 big grids of four jiaos of the following countings of low power lens; The counting finish after with cell dilution to 2.0 * 10
4/ ml, the shop is gone in the 6 porocyte culture plates, and making every porocyte number average is 4.0 * 10
4Individual, place 37 ℃, 5% CO then
2Cultivate in the incubator, cultivate standby after 24 hours;
[being example with porcine reproductive and respiratory syndrome (PRRSV) living vaccine] used in wherein Bing Du inoculation, the preparation of different methods low melting point glue:
Extract 100 μ lPRRSV living vaccines, make 10 times gradient dilution, get 10 with cell culture medium
～6～10
～7The titre sample, add respectively in the above-mentioned fine and close individual layer, every hole 500 μ l establish the every hole of contrast simultaneously and add the cell substratum, put into 37 ℃, 5% CO
2Left standstill in the incubator 1 hour;
1 * substratum mixes with low melting point glue
Abandon or adopt cell culture medium after 1 hour, after at first the preheating of 1 * substratum being reached 38 ℃, take by weighing 0.4 gram low melting point rubber powder, rubber powder is poured in the triangular flask, add the 40ml deionized water, with template and kraft paper the triangle bottleneck is tightened, after boiling 4min with the high temperature shelves on the microwave oven, this moment, glue was the aseptic semi-solid (water evaporates) that is, getting the 1 * substratum 60ml of preheating and the full dose low melting point glue of preparation of last step mixes, blow and beat gently with aseptic suction pipe, make glue fully dissolve the generation of avoiding bubble simultaneously; With pipettor the liquid in the cell plate is exhausted then, with suction pipe just sol solution draw fast, along cultivating wooden partition glue slowly is layered on cell surface, at room temperature placement 10min it is fully solidified after spread, put into 37 ℃, 5% CO then
2Cultivate in the incubator, every day observation of cell state and viral pathology situation, observation of cell pathology 5 days, picking single virus clone with 3 inoculations of mono-clonal virus freeze thawing Marc-145 monolayer cell of picking, passed for 3 generations to survey TCID50 respectively;
2 * substratum mixes with low melting point glue
It is the same to connect malicious method; The preparation of 2% glue: take by weighing 2 gram rubber powders, add 100 ml deionized waters, 0.112MPa autoclaving 30 minutes places 4 ℃ of preservations, and fully fusing places 45 ℃ of water-baths standby before using; The HEPES of the configuration 10gMEM dehydrated medium of 2 * substratum, 45ml water for injection, foetal calf serum 20ml, 0.238 gram, the L～glutaminase of 0.06 gram, 2ml, the 0.5% toluylene red 1.2ml of 1 lactoalbumin hydrolysate that restrains, 3% hydrogen-carbonate are formulated, the pH value transfers to 7.3, during use the low melting point sol solution that autoclaving is good is in advance mixed than 1:1 with 2 * culture volume.
The used cell culture fluid of present method is by the 5gMEM dehydrated medium, 90ml water for injection, and foetal calf serum 10ml is formulated, and the pH value transfers to 7.3.
The used cell culture medium of present method is by the 5gMEM dehydrated medium, 98ml water for injection, and foetal calf serum 2ml is formulated, and the pH value transfers to 7.3.
Used 1 * the substratum of present method is by the 5gMEM dehydrated medium, the L～glutaminase of 0.03 gram, and the lactoalbumin hydrolysate of 0.5 gram, 96ml water for injection, foetal calf serum 4ml is formulated, and the pH value transfers to 7.3.
The Marc-145 cell that experiment is selected for use is available from China Veterinery Drug Inspection Office; The MEM substratum is produced article No.: SH30024.01B for Hyclone company; Foetal calf serum is produced article No.: SV30087.02 for Hyclone company; EDTA～trysinization liquid is produced for Lanzhou Braun Aktiengesellschaft; The low melting point rubber powder is produced article No.: Cat.no.16520-050 for invitrogen company.
High-pathogenicity porcine reproductive that experiment is selected for use and respiration syndrome (HPRRSV) living vaccine are the products that Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd produces, and the products production authentication code that the Ministry of Agriculture authorizes is: No. [2010] 24, agricultural medical informal letter.
Above-mentioned interpretation sees Table 1,2,3.
Annotate: " * " pH value is the cell required optimum range of growing 7.2～7.4; " # " osmotic pressure is the cell required optimum range of growing 260～320.
Comprehensive above list data result shows: after the present invention and the preparation of low melting point glue, do not change cell growth pH value, osmotic pressure, do not need to add the HEPES(pair cell certain toxicity arranged) etc. advantage, for the cell growth provides optimal environment, be that ordinary method is unrivaled.
Claims (3)
1. a novel method that is used for viral plaque purification and the preparation of clone's epoxy glue solution is characterized in that: comprise the preparation of 1 * cell culture medium, the preparation of low melting point glue, the preparation and the shop glue of epoxy glue solution;
The wherein preparation of 1 * cell culture medium: by 4-6 gram MEM dehydrated medium, the L-glutaminase of 0.02-0.04 gram, the lactoalbumin hydrolysate of 0.4-0.6 gram, 94-98ml water for injection and foetal calf serum 3-5ml mix, and it is standby that the pH value transfers to 7.2-7.3;
The wherein preparation of low melting point glue: get the low melting point rubber powder of 0.6 gram, pour in the triangular flask, add the 40ml deionized water, bottleneck is tightened with template and kraft paper, on microwave oven, boil 4min, standby when being semi-solid;
Wherein contain the preparation and shop glue of the epoxy glue solution of 1% low melting point glue:
With the 1 * cell culture medium in last step, heat 38 ℃, the full dose low melting point glue of getting the preparation of 60ml and last step mixes, and blows and beats gently with aseptic suction pipe, glue is fully dissolved there is no the bubble generation; Take out the cultivation for preparing in advance and become individual layer and inoculation to produce cytopathic virus, be the cell plate of primary cell and passage cell; With pipettor the substratum in the cell plate is exhausted, with the sol solution sucking-off of preparation, glue slowly is layered on cell surface then, at room temperature placement 10min it is fully solidified after spread, put into 37 ℃, 5% CO then along cultivating wooden partition
2Cultivate observation of cell pathology 3-5 days, picking single virus clone in the incubator.
2. in accordance with the method for claim 1, it is characterized in that: this method makes conventional cell quantity improve 1.5 times, makes the malicious valency of mono-clonal virus biography after 3 generations improve 8.6 times.
3. in accordance with the method for claim 1, it is characterized in that: the MEM dehydrated medium is produced article No.: SH30024.01B for Hyclone company; Foetal calf serum is produced article No.: SV30087.02 for Hyclone company; The low melting point rubber powder is produced article No.: Cat.no.16520-050 for invitrogen company.
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| CN 201110114800 CN102206614B (en) | 2011-05-05 | 2011-05-05 | Novel method for preparing mixed glue solution for purifying virus plaques and cloning viruses |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607082A (en) * | 2008-06-18 | 2009-12-23 | 洛阳普莱柯生物工程有限公司 | A kind of preparation method of Porcine reproductive and respiratory syndrome inactivated vaccine |
| CN101920010A (en) * | 2010-06-29 | 2010-12-22 | 西北农林科技大学 | Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof |
| CN102002481A (en) * | 2010-12-08 | 2011-04-06 | 成都天邦生物制品有限公司 | Production method of porcine reproductive and respiratory syndrome virus |
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607082A (en) * | 2008-06-18 | 2009-12-23 | 洛阳普莱柯生物工程有限公司 | A kind of preparation method of Porcine reproductive and respiratory syndrome inactivated vaccine |
| CN101920010A (en) * | 2010-06-29 | 2010-12-22 | 西北农林科技大学 | Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof |
| CN102002481A (en) * | 2010-12-08 | 2011-04-06 | 成都天邦生物制品有限公司 | Production method of porcine reproductive and respiratory syndrome virus |
Non-Patent Citations (3)
| Title |
|---|
| 《中国农学通报》 20091105 胡慧等 利用荧光定量PCR方法研究猪繁殖与呼吸综合征病毒在Marc-145细胞中的增殖规律 , 第21期 * |
| 《中国畜牧兽医》 20100331 李春梅, 邓雨修, 王东东, 苏润环, 招丽婵, 宋延华 不同猪繁殖与呼吸综合征病毒分离株在 全文 1－3 , * |
| 《畜牧与兽医》 20091231 周玉双等 Marc-145细胞接种PRRSV后多次收毒的探索 , 第12期 * |
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