CN110168093A - It is a kind of transfect cytozoon kit and its application - Google Patents

It is a kind of transfect cytozoon kit and its application Download PDF

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CN110168093A
CN110168093A CN201780001027.4A CN201780001027A CN110168093A CN 110168093 A CN110168093 A CN 110168093A CN 201780001027 A CN201780001027 A CN 201780001027A CN 110168093 A CN110168093 A CN 110168093A
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transfection
kit
culture
saponin
falciparum
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CN110168093B (en
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胡文
单雪凤
姚永超
秦莉
陈小平
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Zhongke Lanhua Guangzhou Biomedical Technology Co ltd
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Guangzhou Cas Lamvac Biotech Co Ltd
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Abstract

It include polyethyleneimine the present invention relates to a kind of kit for transfecting cytozoon and its application, the kit, the kit further includes saponin and/or Triton X-100.Quickly, transfection efficiency is high for kit transfection of the present invention, and transfection success rate is high；Positive worm strain obtains time-consuming short after transfection, does not need long-time drug screening；Minimal amount of chemical transfection reagent is only needed, expensive transfection apparatus is not needed；Cost of material is low, and the reagent of single transfection expends lower；With the potentiality for once transfecting multiple expression vectors.

Description

It is a kind of transfect cytozoon kit and its application Technical field
The present invention relates to bioengineering field more particularly to a kind of kit for transfecting cytozoon and its application, the method for the specially a kind of kit for transfecting cytozoon and transfection cytozoon.
Background technique
AIDS, tuberculosis and malaria are the big most lethal infections in the world three.According to the statistics of the World Health Organization, worldwide, by the end of 2015, still 43.8 ten thousand people die of this disease of malaria, wherein 90% death rate concentrates on African Territories, 7% occurs to occur in south east asia, 2% at east Mediterranean Region (World Health Organization.World Malaria Report 2015.).And the generation of clinical malaria is mainly due to caused by Plasmodium falciparum (Plasmodium falciparum, P.falciparum) this protozoan parasite infection in erythrocyte stage.Up to the present, since the basic biological study to P.falciparum still has many tera incognitas, to limit the development of anti-malaria medicaments treatment and malaria vaccine.
In fact, still the function of having about 50% P. falciparum genes is unknown (Webster WA, McFadden GI.From the genome to the phenome:tools to understand the basic biology of Plasmodium falciparum.J Eukaryot Microbiol.2014,61 (6): 655-71.).A variety of genetic tools are applied to the research of erythrocytic stage P. falciparum genes function, but make slow progress (de Koning-Ward TF, Gilson PR, Crabb BS.Advances in molecular genetic systems in malaria.Nat Rev Microbiol.2015,13 (6): 373-87.Birnbaum J, Flemming S, Reichard N, Soares AB, Mes é n-Ram í rez P, Jonscher E, Bergmann B, Spielmann T.A gene Tic system to study Plasmodium falciparum protein function.Nat Methods.2017,14 (4): 450-456.).And unsuccessful transfect often caused by plasmodium of the genetic manipulation of specific gene is imitated in plasmodium body Rate it is too low and transfection after plasmodium survival rate it is too low caused by.Although researchers develop transfection of a variety of alternatives for plasmodium, DNA spontaneous absorption method based on red blood cell electroporation is still by preferred manner (the Deitsch K of Exogenous DNA transfered erythrocytic stage P.falciparum, Driskill C, Wellems T.Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes.Nucleic Acids Res.2001,29 (3): 850-3.Skinner-A Dams TS, Lawrie PM, Hawthorne PL, Gardiner DL, Trenholme KR.Comparison of Plasmodium falciparum transfection methods.Malar J.2003,2:19.).But this electroporation transfection method needs to be possible to by long-term culture and more wheel drug screenings to obtain positive transgenosis worm strain (Birnbaum J, Flemming S, Reichard N, Soares AB, Mes é n-Ram í rez P, Jonscher E, Bergmann B, Spielmann T.A genetic system to study Plasmodium falciparum protein function.Nat Methods.2017,14 (4): 450-456.).
" Lu et al., 2016；Birnbaum et al., 2017 " report and carry out knocking in for simple green fluorescent protein (GFP) gene in the dispensable gene site of P.falciparum genome and be also required to could obtain for tens days positive worm plant (Lu J; Tong Y; Pan J; Yang Y; Liu Q; Tan X, Zhao S, Qin L, Chen X.A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.Parasit Vectors.2016,9:198. Birnbaum J, Flemming S, Reichard N, Soares AB, Mes é n-Ram í rez P, Jonscher E, Bergmann B, Spielmann T.A genetic system to study Plasmodium falciparum protein function.Nat Methods.2017,14 (4): 450-456.).And this method needs expensive electroporation transfection device and the electricity to match to turn liquid (cytomix) and just can be carried out, the amount of the DNA needed is also larger, primary transfection will expend the DNA molecular of several hectogammas, plasmodium for loading the red blood cell of DNA and for transfection needs to prepare respectively, and it needs frequently to change liquid at transfection initial stage, relatively time-consuming, effort, expensive.
Different from other species, the P.falciparum persistently cultivated in vitro must be colonized in the red blood cell of people Can survive (Trager W, Jensen JB.Human malaria parasites in continuous culture.Science.1976,193 (4254): 673-5.), this means that exogenous DNA needs in the nucleus that human erythrocyte membrane, plasmodium cell membrane and plasmodium nuclear membrane trilamellar membrane system can just enter plasmodium.And, very low (the Colin FC of spontaneous endocytosis efficiency of people's mature erythrocyte, Schrier SL.Spontaneous endocytosis in human neonatal and adult red blood cells:comparison to drug-induced endocytosis and to receptor-mediated endocytosis.Am J Hematol.1991,37 (1): 34-40.), this is likely due to intensity (the Lux SE 4th. that fine and close membrane skeleton increases membrane lipid bilayer Anatomy of the red cell membrane skeleton:unanswered questions.Blood.2016,127 (2): 187-99.) and then prevent the internalization of membrane vesicle.This makes the transfection difficult to realize to erythrocytic stage plasmodium of the chemical transfections methods such as traditional cationic-liposome method, cationic polymer method.However, during erythrocytic stage development, P.falciparum itself still remains spontaneous endocytosis (Hoppe HC, van Schalkwyk DA, Wiehart UI, Meredith SA, Egan J, Weber BW.Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum.Antimicrob Agents Chemother.2004,48 (7): 2370-8.Smythe WA, Joiner K A, Hoppe HC.Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum.Cell Microbiol.2008,10 (2): 452-64.), therefore, how to allow the transfection composite comprising gene order to penetrate the cell membrane of host is that chemical transfection method is successfully crucial.
Polyethyleneimine (Polyethylenimine, it PEI) is a kind of stable cationic polymer, it is widely used in mammalian cell transfection (Longo PA, Kavran JM, Kim MS, Leahy DJ.Transient mammalian cell transfection with polyethylenimine (PEI) .Methods Enzymol.2013,529:227-40.), also it once be used to belong to gene transfection (Salehi N, the Peng C of the toxoplasma Toxoplasma gondii of protozoon parasite with P.falciparum A.Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes.J Microbiol Methods.2012,91 (1): 133-7.).PEI  DNA can be compressed into positively charged particle to the anion binding with cell surface, later, PEI/DNA compound is discharged into cytoplasm (Sonawane ND by intracellular annexation, Szoka FC Jr, Verkman AS.Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes.J Biol Chem.2003,278 (45): 44826-31.Huth S, Lausier J, Gersting SW, Rudo Lph C, Plank C, Welsch U, Rosenecker J.Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer.J Gene Med.2004,6 (8): 923-36.).Coincidentally, another cationic polymer, polyamide-amine type branch-shape polymer (polyamidoamine dendrimer), which was once reported, can be used for transfecting erythrocytic stage P.falciparum (Mamoun CB, Truong R, Gluzman I, Akopyants NS, Oksman A, Goldberg DE.Transfer of genes into Plasmodium falciparum by polyamidoamine dendrimers.Mol Biochem Parasitol.1999,103 (1): 117-21. ), but subsequent research discovery, P.falciparum, which is transfected, using this method and adds drug screening tests but none success (Skinner-Adams TS to obtain the multiplicating of stable transfection worm strain, Lawrie PM, Hawthorne PL, Gardiner DL, Trenholme KR.Comparison of Plasmodium falciparum transfection methods.Malar J. 2003,2:19.).
In history, researchers develop the transfection method of erythrocytic stage P.falciparum a series of.Wherein most extensively also the most commonly used is the electroporation transfection methods of red blood cell load DNA for use scope, it is dependent on expensive electroporation transfection instrument and transfection reagent, only instrument cost is as high as hundreds of thousands yuan, the reagent consuming of single transfection also wants several hundred yuans, and fresh human red blood cells used in single transfection need separately to prepare with the P.falciparum (a few hectolambda infection rates are more than 10% worm blood) for being in the schizont phase, DNA dosage is also more (tens arrive several hectogammas), electrotransfection, which needs to expend 2-3 hours, to be completed, and nearly 30 days in vitro cultures and drug screening is at least needed to be possible to obtain positive worm strain, transfection efficiency and success rate are all very low (about millionth transfection efficiency).Also a few studies person attempts using chemical transfection method, But the highest transfection efficiency having been reported that is less than 10%, and experiment success rate is very low (can not almost repeat), agents useful for same is also costly.
Other than plasmodium, other kinds of cytozoon is also faced with the problem of chemical transfection.In order to solve the problems, such as Plasmodium falciparum chemical transfection, essentially consist in can develop a kind of method allow the human erythrocyte membrane of falciparum infection to perforate but and do not damage the plasmodium of its endoparasitism, recycle such as polyethyleneimine/DNA complex particle then to penetrate the human erythrocyte membrane of falciparum infection and realize that plasmodium transfects.
Summary of the invention
Presently, there are aiming at the problem that, the present invention provide it is a kind of transfect cytozoon kit and its application, carrying out transfection cytozoon using kit can solve the problem that helminth transfection efficiency is low, success rate is low, realize the system of multiple Plasmid DNA cotransfections.
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of kit for transfecting cytozoon, and the kit includes polyethyleneimine；
The kit further includes saponin and/or Triton X-100.
In the present invention, pass through polyethyleneimine (PEI) and saponin and/or the synergistic effect of Triton X-100 (Triton X-100), the high-efficiency transfection of gene pairs cytozoon may be implemented, whole process only needs about 30min, transfection efficiency reaches as high as 100% up to 50% or more.
According to the present invention, the kit includes PEI and saponin.
In the present invention, inventor's discovery can generate certain toxicity using Triton X-100 and PEI cooperation transfection helminth to helminth, cause the death rate of helminth high, and saponin and PEI is used to be used cooperatively, it has no toxic side effect, helminth can survive, and gene can also stablize expression in helminth.
According to the present invention, the helminth is plasmodium, Babesia, mankind's Colpodella sample helminth (kidney Shape mesh helminth), any one or any two kinds of combination in Leishmania or trypanosome, preferably plasmodium, further preferably intracellular plasmodium.
Second aspect, the present invention provide a kind of method using kit transfection cytozoon as described in relation to the first aspect, include the following steps:
(1) saponin and/or Triton X-100 in kit is used to handle the cell of parasitic infection to be transfected；
(2) incubation transfection is carried out to the gene order of cell addition transfection of step (1) treated parasitic infection to be transfected and the compound of polyethyleneimine.
According to the present invention, the mass volume ratio of the saponin and/or Triton X-100 is respectively 0.001-0.05%, such as it can be 0.001%, 0.002%, 0.003%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.012%, 0.013%, 0.015%, 0.016%, 0.018%, 0.02%, 0.022%, 0.025%, 0.026%, 0.028%, 0.03%, 0.032%, 0.035%, 0.038%, 0.04%, 0.042%, 0.045%, 0.048% or 0.05%, preferably 0.001-0.03%, into one Step is preferably 0.001-0.01%.
In the present invention, inventor's discovery, when saponin concentration is controlled in 0.03% (wt/v, quality/overall solution volume) below, the cell of parasitic infection is relatively complete, the case where seldom cell cracks, especially concentration of the saponin concentration between 0.001% (wt/v) -0.01% (wt/v) is best, in this concentration range, the integrality of the cell of parasitic infection can be kept to greatest extent, also it is just able to maintain the activity of cytozoon, while being also applied for subsequent PEI transfection.
According to the present invention, the gene order be plasmid DNA vectors, DNA expression cassette sequence or rna expression frame sequence in any one or at least two combination.
In the present invention, the transfection can transfect multiple expression vectors simultaneously, and the transfection includes transient transfection and stable transfection, and described transiently transfect is the free DNA expressed of transfection, and the stable transfection is to transfect energy The gene editing system of genome conformity enough occurs.
In the present invention, the gene order is that can be transfected into the gene of helminth be all feasible, resistant gene, reporter gene or the functional gene for specifically including that the worm strain of drug screening Positive transfections, wherein the reporter gene for example can be green fluorescent protein (GFP) and/or red fluorescent protein (RFP) and luciferase etc.；The functional gene includes the functional gene for knocking out plasmodium itself, or introduces the functional gene for needing to overexpress, and functional gene can come from any helminth, can be from other species.
According to the present invention, inventor has found that the molar ratio of the ammonia nitrogen in polyethyleneimine and the phosphate in the gene order is extremely important, the efficiency of transfection can be had a huge impact, the molar ratio of ammonia nitrogen in heretofore described polyethyleneimine and the phosphate in the gene order is 1-16000, such as it can be 1, 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1500, 1800,2000,2300,2400,2500,2800,3000,4000,5000,6000,7000,8000,9000,10000,110000,120000,130000,140000,150000 or 160000, preferably 1-2500, further preferably 5-100.
In a specific embodiment, the specific calculating that the ratio of the molal quantity of the amido nitrogen (N) and Plasmid DNA phosphate (P) according to PEI calculates the additive amount of PEI is as follows: for the free expression plasmid of transient transfection, its phosphate molarities (P)=Plasmid DNA weight (1 μ g)/(average molecular weight (650g/mol/bp) of base-pair (the 6669bp) × DNA base of Double stranded plasmids DNA to (sodium salt)) ≈ 0.23pmol, it is calculated by N/P ratio (N/P) is 30, the amido nitrogen (N) of PEI needs 6.9pmol, and PEI used in the present invention contains the 11% propionating amido of N-, and the average molecular weight of PEI is about 25, 000g/mol, so the PE needed The additive amount of I is 1.6 μ g of (6.9pmol/0.11) × 25,000g/mol ≈, that is, needs to be added the PEI working solution (1 μ g/ μ l) of 1.6 μ l.
According to the present invention, the additive amount of the molecular weight of the polyethyleneimine and subsequent PEI have direct relation, PEI Average molecular weight be about 25000, the molecular weight of PEI of the present invention is 1000-750000, for example, can be 1000,1100,1200,1300,1500,1600,1800,2000,2200,2300,2500,2800,3000,3200,3500,3800,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10000,12000,13000,15000,18000,20000,22000,25000,28000,30000,35000,40000, 45000,50000,55000,60000,70000,80000,90000,100000,130000,150000,200000,250000,300000,350000,400000,450000,500000,550000,600000,650000,700000 or 750000, preferably 10000-30000.
According to the present invention, it further include the in vitro culture and synchronized step of helminth before step (1), heretofore described in vitro culture with synchronize turn to the ordinary skill in the art, those skilled in the art can select according to the difference of the helminth of culture, be not particularly limited herein.
Preferably, culture is further included the steps that after step (2).
According to the present invention, the step of culture specifically includes: culture medium of the removal containing transfection reagent is added fresh culture medium and is cultivated.
Preferably, the temperature of the culture is 30-40 DEG C, such as can be 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 35-38 DEG C.
Preferably, the time of the culture is 48h or more, such as can be 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 58h, 60h, 62h, 63h, 65h, 68h, 70h, 72h, 75h, 78h, 80h, 82h, 85h, 88h, 90h, 92h, 95h, 96h, 98h or 100h, preferably 48-96h.
Preferably, the culture uses three gas incubators.
In the present invention, drug screening is needed not move through due to transfection efficiency height or only can obtain Positive transfections worm strain by short time screening, it is described due to transiently transfecting the difference with rotaring redyeing gene in stable transfection in the present invention It transiently transfects without being screened, the stable transfection needs drug screening in 2-3 weeks.
In the present invention, drug screening is using blasticidin S blasticidin-S in the stable transfection.
According to the present invention, described method includes following steps:
(1) in vitro culture and synchronization of helminth；
(2) using in kit saponin and/or Triton X-100 handle the cell of parasitic infection to be transfected, the mass volume ratio of the saponin and/or Triton X-100 is respectively 0.001-0.05%；
(3) incubation transfection is carried out to the gene order of cell addition transfection of step (2) treated parasitic infection to be transfected and the compound of polyethyleneimine, the molar ratio of ammonia nitrogen in the polyethyleneimine and the phosphate in the gene order is 1-16000, and the molecular weight of the polyethyleneimine is 1000-750000；
(4) culture medium containing transfection reagent is removed, fresh culture medium 30-40 DEG C of culture 48h or more of temperature in three gas incubators is added, obtains the worm strain for stablizing expression.
As optimal technical scheme, described method includes following steps:
(1) in vitro culture and synchronization of plasmodium；
(2) cell of Infected With Plasmodium to be transfected is handled using the saponin in kit, the mass volume ratio of the saponin is respectively 0.001-0.01%；
(3) incubation transfection is carried out to the gene order of cell addition transfection of step (2) treated Infected With Plasmodium to be transfected and the compound of polyethyleneimine, the molar ratio of ammonia nitrogen in the polyethyleneimine and the phosphate in the gene order is 5-100, and the molecular weight of the polyethyleneimine is 10000-30000；
(4) culture medium containing transfection reagent is removed, fresh culture medium 35-38 DEG C of culture 48-96h or more of temperature in three gas incubators is added, obtains the worm strain for stablizing expression.
Compared with prior art, the invention has the following beneficial effects:
(1) high-efficiency transfection of gene pairs helminth, entire mistake may be implemented by polyethyleneimine (PEI) and saponin and/or the synergistic effect of Triton X-100 (Triton X-100) in kit of the present invention Journey only needs about 30min, and transfection efficiency reaches as high as 100% up to 50% or more, and the worm strain after transfection at most only needs to screen within 2-3 weeks to obtain positive worm strain without screening for a long time；
(2) transfection reagent box of the present invention only needs a small amount of polyethyleneimine (PEI) and saponin and/or Triton X-100 (Triton X-100), cost of material is low, the reagent of single transfection expends lower, and multiple expression vectors can be once transfected, it is high-efficient；
(3) the method for the present invention is simple, easy to operate, easy to promote and utilize.
Detailed description of the invention
Fig. 1 is the free expression vector map for transiently transfecting P.falciparum worm strain；
Fig. 2 (A) is for stable transfection P.falciparum worm strain based on CRISPR-Cas9 gene editing pPfInt-Pf47Site Cleavage Vector map, and Fig. 2 (B) is for stable transfection P.falciparum worm strain based on CRISPR-Cas9 gene editing pPfInt-GFP&NanoLuc Donor Vector map；
Fig. 3 is the basic procedure of cytozoon chemical transfection；
Fig. 4 (A) is the result that the P.falciparum worm strain before gelatin enrichment is dyed through Giemsa；Fig. 4 (B) is the result that the P.falciparum worm strain after gelatin enrichment is dyed through Giemsa；
Fig. 5 (A) is that the saponin that mass volume ratio is 0.001% handles influence to P.falciparum 3D7 parasitized erythrocyte, Fig. 5 (B) is that the saponin that mass volume ratio is 0.01% handles influence to P.falciparum 3D7 parasitized erythrocyte, Fig. 5 (C) is that the saponin that mass volume ratio is 0.03% handles influence to P.falciparum 3D7 parasitized erythrocyte, Fig. 5 (D) is that the saponin that mass volume ratio is 0.05% handles influence to P.falciparum 3D7 parasitized erythrocyte, Fig. 5 (E) is that the saponin that mass volume ratio is 0.1% handles influence to P.falciparum 3D7 parasitized erythrocyte；
Fig. 6 is the fluorescence microscope testing result that pPfEps-mCherry free expression vector transiently transfects the strain of P.falciparum 3D7 worm using saponin and PEI cooperation, wherein, Fig. 6 (A) takes pictures for light field, Fig. 6 (B) is after the processing of 33258 dyestuff of Hoechst, the channel UV take pictures as a result, Fig. 6 (C) be it is red Color fluorescence detection channel is taken pictures as a result, Fig. 6 (D) is the stacking chart of Fig. 6 (A), Fig. 6 (B) and Fig. 6 (C)；
Fig. 7 is the fluorescence microscope testing result of CRISPR-Cas9 gene editing expression vector stable transfection P.falciparum 3D7 worm strain, wherein, Fig. 7 (A) takes pictures for light field, Fig. 7 (B) is that green fluorescence sense channel is taken pictures as a result, Fig. 7 (C) is the stacking chart of Fig. 7 (A) and Fig. 7 (B)；
Fig. 8 (A) is pPfEps-mCherry Plasmid DNA schematic diagram, Fig. 8 (B) is the PCR qualification result that pPfEps-mCherry free expression vector transiently transfects the strain of P.falciparum 3D7 worm, wherein, M indicates DNA molecular amount standard, S1, S2 is the PCR identification product of the worm strain through PEI transfection after the processing of 0.001% (wt/v) saponin, S3, S4 is the PCR identification product of the worm strain through PEI transfection after the processing of 0.01% (wt/v) saponin, WT is the PCR product of wild type P.falciparum 3D7 worm strain as negative control group, PC is the pPfEps-mCherry Plasmid DNA of purifying as positive controls；
Fig. 9 (A) is the double exchange reorganization schematic diagram of Pf47 gene loci on pPfInt-GFP&NanoLuc Donor genetic donor Plasmid DNA and No. 13 chromosomes of P.falciparum 3D7 genome, Fig. 9 (B) is the PCR qualification result of CRISPR-Cas9 gene editing expression vector stable transfection P.falciparum 3D7 worm strain, wherein, M indicates DNA molecular amount standard, WT is the PCR product of wild type P.falciparum 3D7 worm strain as negative control group, and S1 is the PCR product of P.falciparum 3D7 stable transfection worm strain；
Figure 10 is (protein imprinted) identification of Western blot that pPfEps-mCherry free expression vector transiently transfects the strain of P.falciparum 3D7 worm, wherein, M indicates DNA molecular amount standard, S1, S2 is the cell pyrolysis liquid of the P.falciparum 3D7 worm strain through PEI transfection after the processing of 0.001% (wt/v) saponin, S3, S4 is the cell pyrolysis liquid of the P.falciparum 3D7 worm strain through PEI transfection after the processing of 0.01% (wt/v) saponin, and WT is the cell pyrolysis liquid of wild type P.falciparum 3D7 worm strain；
Figure 11 is the NanoLuc luciferase assays of P.falciparum 3D7 stable transfection worm strain, wherein ordinate indicates the relative fluorescence signal strength of NanoLuc luciferase assays, and abscissa is real Group is tested, S1-S4 is 4 plants of stable transfections and the plasmodium worm strain for expressing GFP&NanoLuc Luciferase fusion protein, and number is GFP&NanoLuc respectively, and WT is that the strain of P.falciparum 3D7 wild type worm is used as negative control group；
Figure 12 is that mass volume ratio is respectively that 0.001%, 0.01%, 0.025% and 0.1% Triton X-100 handles the influence to P.falciparum 3D7 parasitized erythrocyte；
Figure 13 is the fluorescence microscope testing result that pPfEps-mCherry free expression vector transiently transfects the strain of P.falciparum 3D7 worm using Triton X-100 and PEI cooperation, wherein, Figure 13 (A) takes pictures for light field, Figure 13 (B) is after the processing of 33258 dyestuff of Hoechst, in the result that the channel UV is taken pictures, Figure 13 (C) is that red fluorescence sense channel is taken pictures as a result, Figure 13 (D) is the stacking chart of Figure 13 (A), Figure 13 (B) and Figure 13 (C).
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific embodiment to further illustrate the technical scheme of the present invention, but the present invention is not limited in scope of embodiments.
Embodiment 1: the in vitro culture and synchronization of Plasmodium falciparum worm strain P.falciparum 3D7
The in vitro culture of Plasmodium falciparum worm strain P.falciparum 3D7 is carried out using normalization condition and synchronizes operation (Radfar A, M é ndez D, Moneriz C, Linares M, Mar í n-Garc í a P, Puyet A, Diez A, Bautista JM.Synchronous culture of Plasmodium falciparum at high parasitemia levels.Nat Protoc.2009,4 (12): 1899-915.): including using complete medium: 10.4g/L RPMI 164 0,25mM HEPES, 0.5% (wt/vol) AlbuMAX I, 1.77mM sodium bicarbonate, 100 μM of hypoxanthine, 12.5 μ g/ml gentamicin sulphates, adjusting pH value is 7.2,1% hematocrit, 37 DEG C of three gas incubator (1%O2, 3%CO2And 96%N2) in culture.A subculture is changed daily, and infection rate is counted using Giemsa staining method (Giemsa staining), is reached to protozoan infection rate 10%, plasmodium synchronization is carried out using cracking sorbierite method.
The plasmid vector of embodiment 2:P.falciparum transfection and relevant gene order
In order to test the feasibility of chemical transfection method, used two sets of plasmids respectively: one is the free expression plasmid i.e. pPfEps-mCherry for transient transfection, as shown in Figure 1, its main element includes: constitutive promoter 5 '-CAM (calmodulin by P.falciparum, calmodulin, cam, Gene ID:PF3D7_1434200) promoter sequence of gene and 3 '-PbDT of terminator sequence (the difunctional dihyrofolate reductase thymidylate synthase of mouse P. berghei Plasmodium berghei, bifunctional dihydrofolate reductase-thymidylate synth Ase, DHFR-TS, Gene ID:PBANKA_0719300) the amalgamation and expression albumen frame of red fluorescent protein mCherry that is controlled of 3 '-end non-coding sequences, the membrane encoding transport signals peptide of a KAHRP (knob-associated histidine-rich protein) gene is inserted at the end N- of mCherry albumen, and has merged the Strep tag protein tag connected with serine (S) with the link peptide that alanine (A) forms at the end C- of mCherry albumen；5 '-end promoter (5 '-PfEf1 α of one translation elongation factor by P.falciparum, elongation factor 1-alpha, Gene ID:PF3D7_1357000) P.falciparum HRPII gene 3 '-end noncoding region (3 '-HRP2, histidine-rich protein IIHRPII, Gene ID:PF3D7_0831800) blasticidin S resistant gene (the Blasticidin-S deaminase that is controlled, BSD, EC number:3.5.4.23) expression cassette, for transgenosis plasmodium Resistance screening；With a kalamycin resistance gene (kanamycin phosphotransferase, kanamycin resistance gene, KmR expression cassette), for in Escherichia coli (such as DH5 α, the plasmid DNA clones such as XL-10, Stbl3 and NEB Stable bacterial strain) in carry out positive plasmid screening and maintain stability of the plasmid in Escherichia coli；And the basic framework and Plasmid DNA replication origin (ColE1 origin) sequence in Escherichia coli etc. of Plasmid DNA.
Another kind is the plasmid expression system for stable transfection, as shown in Fig. 2 (A)-Fig. 2 (B), the pPfInt-GFP&NanoLuc Donor of pPfInt-Pf47 Site Cleavage and Fig. 2 (B) including Fig. 2 (A) Two plasmids, the plasmid transfection system is constructed based on CRISPR-Cas9 gene editing technology, main includes the carrier of the expression cassette of the expression cassette of the sgRNA comprising the targeting site Pf47 and a Cas9 gene, the building of its building process and pCBS-Pf47 carrier constitutes consistent (Lu J, Tong Y, Pan J, Yang Y, Liu Q, Tan X, Zhao S, Qin L, Chen X.A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.Parasit Vectors.2016,9:198.)；And a flank in GFP and NanoLuc expressing fusion protein frame added the 5 '-ends in the site Pf47 and the recombination donor plasmid of 3 '-end homology arm sequences, (Lu J consistent with the building process of pARM-GFP/RUCki carrier, Tong Y, Pan J, Yang Y, Liu Q, Tan X, Zhao S, Qin L, Chen X.A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.Parasit Vectors.2016,9:198.) Renilla luciferase gene (renilla luciferase) in pARM-GFP/RUCki carrier higher NanoLuc luciferase gene (the Hall MP of detection sensitivity has been substituted for unlike unique, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F Encell LP, Wood KV.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chem Biol.2012,7 (11): 1848-57.), and a new expressing fusion protein frame is joined together to form with a GGPSG small peptide DNA sequence dna and green fluorescent protein GFPm3 gene.
Embodiment 3:P.falciparum transfection experiment
The process of transfection is as shown in figure 3, first by vitro culture and erythrocyte stage P.falciparum worm strain by gelatin enrichment carries out cell-permeant processing, then is incubated for transfection, specific as follows:
Since the relevant histidine rich protein (KAHRP) of Knob structure being located on No. bis- chromosomes of P.falciparum transports most important, signal peptide energy to the membrane of Erythrocyte Membrane Proteins 1 (PfEMP1) Its fusion protein is enough helped to be secreted into erythrocytic stage P.falciparum external.Therefore, when protozoan infection rate reaches 10%, complete P.falciparum worm strain (the Waterkeyn JG of No. 2 chromosomes is enriched with using the method for gelatin enrichment first, Cowman AF, Cooke BM.Plasmodium falciparum:gelatin enrichment selects for parasites with full-length chromosome 2.Implications for cytoadhesion assays.Exp Parasitol.2001,97 (2): 115-8.), result is as schemed Shown in 4 (A)-Fig. 4 (B), it is seen then that the worm strain many places after enrichment are in big trophozoite phase or schizont phase, conducive to the transfection of PEI/DNA composite particles.
Penetrating electroporation experiments are carried out to the red blood cell (iRBCs) of P.falciparum infection in accordance with the following steps: first, the worm blood that 5ml hematocrit is 1% is transferred in a sterile 15ml centrifuge tube, in 350 × g, room temperature is centrifuged 5 minutes, and collection obtains about 50 μ l iRBCs.1ml is added through 37 DEG C of preheated washing culture mediums: 10.4g/L RPMI 1640,25mM HEPES, 100 μM of hypoxanthine, 12.5 μ g/ml gentamicin sulphates, 350 × g, room temperature are centrifuged 5 minutes and remove supernatant.Saponin (the Sigma-Aldrich of the various concentration of 500 μ l washing culture mediums preparation is added, Cat.#S4521) solution, concentration range is 0.001% (wt/v)~0.1% (wt/v) respectively, after incubation at room temperature 3 minutes, 1900 × g, room temperature is centrifuged 3min and removes supernatant, then washed once with the P.falciparum complete medium that 1ml is preheated through 37 DEG C.The iRBCs of perforated processing determines the best saponin concentration of cell-permeant through smear and sediments microscope inspection, as a result as shown in Fig. 5 (A)-Fig. 5 (E), saponin concentration (wt/v, saponin quality/overall solution volume) it is greater than 0.03% cracking that will lead to iRBCs, be not suitable for subsequent PEI/DNA composite particles transfection.
Transfection experiment is completed with best saponin concentration penetrating iRBCs cell, saponin concentration control 0.03% (wt/v) below, iRBCs cell is relatively complete, the case where seldom cell cracks, especially concentration of the saponin concentration between 0.001% (wt/v)~0.01% (wt/v) is best, in this concentration range, the integrality of iRBCs can be kept to greatest extent, also it is just able to maintain the activity of plasmodium, while being also applied for subsequent PEI transfection.
Transfection
25kD L-PEI (Polyethylenimine is prepared first, PEI, Polysciences, Cat.#23966) working solution: the aseptic deionized water of the load weighted PEI endotoxin-free for being heated to 80 DEG C is dissolved, it is cooled to room temperature, with hydrochloric acid tune pH value to 7.0, the aseptic deionized water of solution endotoxin-free is settled to required 1 μ g/ μ l of concentration, with dispensed after 0.22 μm of membrane filtration degerming and be stored in -20 DEG C it is spare.
Need to determine the ratio of the amido nitrogen (N) of PEI and the molal quantity of Plasmid DNA phosphate (P) by calculating, by taking the free expression plasmid pPfEps-mCherry for transient transfection as an example, its phosphate molarities (P)=Plasmid DNA weight (1 μ g)/(average molecular weight (650g/mol/bp) of base-pair (the 6669bp) × DNA base of Double stranded plasmids DNA to (sodium salt)) ≈ 0.23pmol, it is calculated by N/P ratio (N/P) is 30, the amido nitrogen (N) of PEI needs 6.9pmol, and PEI (Polysciences used in the present invention, Cat.#23966) the amido propionating containing 11% N-, and the average molecular weight of PEI is about 2 5,000g/mol, so the additive amount of the PEI needed is 1.6 μ g of (6.9pmol/0.11) × 25,000g/mol ≈, that is, need to be added the PEI working solution (1 μ g/ μ l) of 1.6 μ l.Effect of the molar ratio (N/P) of ammonia nitrogen in inventor's verifying polyethyleneimine and the phosphate in the gene order in 1-16000, it is found that can all realize transfection when N/P is 1-16000, effect is best when N/P ratio is in 5-100.
It is 30 according to N/P ratio, 200 μ l successively is added without containing in the DMEM minimal medium (C11995500BT, Gibco) of serum in the PEI of 1 μ g DNA and corresponding amount, are incubated at room temperature 20min after mixing well.During this period, the iRBCs of saponin solution (0.001%~0.05%) penetrating mistake by suitable concentration is got out.It is transferred to after PEI/DNA compound and ready iRBCs are softly mixed in advance added with the T25 culture bottle (Cat.#169900 of the 7ml P.falciparum complete medium preheated through 37 DEG C, Thermo Scientific) in, suitable washed people's fresh red blood cell with culture medium rinse is added, maintains hematocrit 1% or so.Culture bottle is placed in 37 DEG C of three gas incubators after cultivating 8 hours and changes liquid, is added The fresh complete medium of 37 DEG C of preheatings, continuation are cultivated in 37 DEG C of three gas incubators.
The worm strain of transient transfection is during the cultivation process without any drug screening.And 5.0 μ g/mL blasticidin S (ThermoFisher Scientific is added in the worm strain of stable transfection in the medium after 48 hours of transfection, Cat.#R21001 it) is screened, it was changed the liquid once every 24 hours, the concentration of blasticidin S is reduced to 2.5 μ g/mL after fluorescence microscope to apparent positive worm strain.
Embodiment 4: the transfection of other helminths
According to mode in vitro culture similar to Example 1 and synchronize Babesia, mankind's Colpodella sample helminth (Colpodida helminth), Leishmania and trypanosome, plasmid vector similar to Example 2 is transfected by way of embodiment 3, inventor has found that the effect for transfecting other helminths is suitable with plasmodium, subsequent experiment inventor is illustrated using the result of plasmodium transfection, does not make extra repeat herein.
Embodiment 5: fluorescence microscope real-time detection
33258 (Invitrogen of Hoechst will be added in 1: 1000 ratio in the iRBCs culture solution of P.falciparum transfection worm strain, Cat.#H3569) to final concentration of 10 μ g/ml, 37 DEG C are incubated for 5 minutes, then are washed twice with PBS buffer solution (pH7.4).Washed iRBCs is applied to microscopy on the microscope slide of a low Poison background again to observe.What our photographing device was selected is the PLYMPUS IX73 type microscope and 100 times of oil mirror camera lens (UPLFLN 100X (Oil Immersion) objective lens) of Olympus company.
It is identified, using saponin and the united chemical transfection method of PEI, the positive worm strain of transient transfection can be obtained within 72 hours, shown in fluorescence microscope testing result such as Fig. 6 (A)-Fig. 6 (D), the statistical result of transfection efficiency is as shown in table 1；
Within 2-3 weeks, drug screening, which is taken turns, by 5 (5.0 μ g/mL blasticidin S screening is initially added within two days from after transfecting, liquid is changed daily until obtaining positive worm strain), the positive worm strain of stable transfection can be obtained, as a result such as Fig. 7 (A)-Fig. 7 (C).
The external transient transfection efficiency statistics of table 1, P.falciparum 3D7
Transfection success rate can achieve 100% as can be seen from Table 1, and transfection efficiency is 50% or more, still, individually only transfects us with PEI and fails to find that the worm strain of any positive occurs.
The expressing fusion protein that can be seen that reporter gene mCherry from Fig. 6 (A)-Fig. 6 (D) is normal, the plasmodium strain that the red fluorescence of reporter gene is obtained actually from chemical transfection；From Fig. 7 (A)-Fig. 7 (C) as can be seen that the expression of reporter gene GFP is normal, the plasmodium strain that the green fluorescence of reporter gene is obtained actually from chemical transfection, transfection success rate can achieve 100%.
Embodiment 6:PCR identification
72 hours after transfection, the infected erythrocytes of transient transfection are collected by centrifugation, and are washed twice with PBS buffer solution (pH7.4) spare.The saponin solution that 5 times of volumetric concentrations are 0.1% is added and cracks infected erythrocytes.Gentle inversion mixes incubation at room temperature 2 minutes.4 DEG C, 1900 × g is centrifuged 5 minutes collection infected erythrocytes The plasmodium discharged after cracking and relevant cell membrane fragments.It is spare after removing supernatant and being washed three times with the PBS buffer solution (pH7.4) of 3 times of volumes.The P.falciparum of wild type P.falciparum and stable transfection are collected using same method.Plasmodium cell is directly used to carry out PCR amplification identification as template.Experimental program is drafted according to the standard scheme of PCR kit specification (Takara, Cat.#R050Q).PCR detects target and primer is as shown in table 2, and PCR testing result is as shown in Figure 8 and Figure 9.
2 PCR of table detects target and list of primers
From figure 8, it is seen that using the DNA stripe size of primer pair P1/P2 examining report gene mCherry for 619bp, use the DNA stripe size of primer pair P3/P4 detection bsd gene for 399bp；From fig. 9, it can be seen that the PCR product of wild type P.falciparum 3D7 worm strain is as negative control group, amplified fragments size is 1390bp；The PCR product of S1, P.falciparum 3D7 stable transfection worm strain, amplified fragments size are 4293bp, it is seen then that either transient transfection (Fig. 8) or stable transfection (Fig. 9) all obtain positive transfection worm strain.
Embodiment 7:SDS-PAGE proteins gel electrophoresis and Western blot detection
Using the method for separating plasmodium from iRBCs above, collect the worm strain separated after 20 μ l transfection, 2 × SDS-PAGE albumen sample-loading buffer (Takara, Cat.#9173) of 100 μ l is added, is transferred to after resuspension in the EP pipe (Eppendorftube) of a 1.5ml.100 DEG C are heated 5 minutes.After cooling, 13,000 × g is centrifuged 1 minute collection supernatant at room temperature to get plasmodium total protein SDS-PAGE electrophoresis Sample.Directly take appropriate amount of sample, using 10%SDS-PAGE gel electrophoresis analysis or be stored in -20 DEG C it is spare.
Before Western blot analysis, first using the plasmodium total protein sample prepared using 10%SDS-PAGE gel electrophoresis separation, using wet transfer printing system (Tanon, China the protein band that PAGE gel electrophoresis is completed) is transferred to an opening and closing to fit on the pvdf membrane (BioRad, Cat.#1620177) of size.The pvdf membrane transferred 5%BSA (using TBST buffer) room temperature is closed 2 hours.The TBST buffer being pre-chilled through ice washes film 3 times (every time 6 minutes), source of mouse primary antibody is added, and (Anti-mCherry antibody is (with TBST buffer through 1: 1000 dilution, Biovision, Cat.#5993) or Anti-GAPDH antibody is (with TBST buffer through 1: 2000 dilution, abcam, Cat.#ab125247), it is incubated at room temperature 1 hour.Ice-cold TBST buffer washes film 3 times, and the mountain sheep anti-mouse igg H&L secondary antibody TBST buffer of horseradish peroxidase (HRP) label is added through 1: 5000 dilution in (every time 6 minutes) afterwards, abcam, Cat.#ab205719), it is incubated at room temperature 1 hour.Ice-cold TBST buffer washes film 3 times, and ECL reagent (Millipore is added in (every time 6 minutes) afterwards, Cat.#WBKLS0500), it is incubated at room temperature 5 minutes under the conditions of being protected from light, using chemiluminescence detector (Tanon, China), the results are shown in Figure 10 by photographic analysis Western blot under different exposure time.
As can be seen from Figure 10, it does not include KAHRP signal peptide sequence, the prediction average molecular weight size of mCherry- ' SA ' Linker-Strep tag fusion protein is 27.9kDa, and the prediction average molecular weight size of housekeeping gene GADPH albumen is 36kDa, as it can be seen that successfully being detected the expression of reporter gene in the worm strain of chemical transfection.
The NanoLuc luciferase assays of embodiment 8:P.falciparum 3D7 stable transfection worm strain
Take 96 orifice plates (the ThermoFisher Scientific an of black, Cat.#07-200-565), the sterile deionized water of 100 μ l is added in detection hole, the red blood cell (infection rate is about 10%) for drawing 3 μ l P.falciparum 3D7 stable transfection worm strain infection respectively is added in the detection hole containing aseptic deionized water, sufficiently mixed It is even.Prepare the reaction substrate of NanoLuc luciferase according to NanoLuc Luciferase Assay Reagent box (Promega, Cat.#N1110) specification, i.e., by substrate and enzyme reaction buffer solution by 1: 50 dilution proportion and be uniformly mixed.The reaction substrate of the prepared NanoLuc luciferase of 100 μ l is separately added into the sample well of the red blood cell infected containing P.falciparum 3D7, chemiluminescence signal (λ max=460nm) is read with microplate reader (BioTek Synergy H1) after reacting 3 minutes at room temperature, it records and statistical experiment is as a result, as shown in figure 11.
It can be seen from figure 11 that the expressing fusion protein of the GFP and NanoLuc luciferase of P.falciparum 3D7 stable transfection worm strain is normal.
The experiment of embodiment 9:Triton X-100 and PEI cooperation transfection P.falciparum 3D7
Compared with Example 3, in addition to using Triton X-100, and the mass volume ratio of Triton X-100 is except 0.001%-0.01%, and other conditions are same as Example 3 with method.
Firstly, in the same manner as in Example 3, the iRBCs of perforated processing determines the best Triton X-100 concentration of cell-permeant through smear and sediments microscope inspection.
As a result as shown in figure 12, Triton X-10 concentration (wt/v, saponin quality/overall solution volume) is greater than or equal to 0.01% cracking that will lead to iRBCs, is not suitable for subsequent PEI/DNA composite particles transfection.
Transfection experiment is completed with best Triton X-100 concentration penetrating iRBCs cell, Triton X-10 concentration control 0.01% (wt/v) below, iRBCs cell is relatively complete, the case where seldom cell cracks, especially concentration of the saponin concentration between 0.001% (wt/v) -0.005% (wt/v) is best, in this concentration range, the integrality of iRBCs can be kept to greatest extent, also it is just able to maintain the activity of plasmodium, while being also applied for subsequent PEI transfection.
In the same manner as in Example 3, it is transiently transfected using free expression plasmid pPfEps-mCherry, as shown in Figure 13 (A)-Figure 13 (D), although Triton X-100 and PEI cooperation can also transfect P.falciparum 3D7, and positive worm strain can be also observed in 72 hours after transfection, but subsequent observation finds Triton X-100 and PEI cooperation transfection helminth can generate certain toxicity to helminth, cause the death rate of helminth high, about transfect within latter week, all dead through the processed worm strain of Triton X-100.
Comparative example 1
Compared with Example 3, in addition to using saponin to carry out cell-permeant except the step of the step of only includes transfection, other conditions are same as Example 3 with method.
The results show that gene order can not be transferred to intracellular plasmodium.
Comparative example 2
Compared with Example 3, in addition to using PEI to be transfected, except being incubated for after saponin processing, other conditions are same as Example 3 with method.
The results show that gene order can not be transferred to intracellular plasmodium.
In summary, kit of the present invention passes through polyethyleneimine (PEI) and saponin and/or the synergistic effect of Triton X-100 (Triton X-100), the high-efficiency transfection of gene pairs helminth may be implemented, whole process only needs about 30min, transfection efficiency is up to 50% or more, 100% is reached as high as, the worm strain after transfection at most only needs to screen within 2-3 weeks to obtain positive worm strain without screening for a long time.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the invention is not limited to above-mentioned method detaileds, that is, do not mean that the invention must rely on the above detailed methods to implement.It should be clear to those skilled in the art, any improvement in the present invention, and addition, selection of concrete mode of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., all of which fall within the scope of protection and disclosure of the present invention.

Claims (9)

  1. A kind of kit transfecting cytozoon, which is characterized in that the kit includes polyethyleneimine；
    The kit further includes saponin and/or Triton X-100.
  2. Kit according to claim 1, which is characterized in that the kit includes polyethyleneimine and saponin；
    Preferably, the helminth be in plasmodium, Babesia, mankind's Colpodella sample helminth, Leishmania or trypanosome any one or at least two combination, preferably plasmodium, further preferably intracellular plasmodium.
  3. A method of cytozoon is transfected using kit as claimed in claim 1 or 2, which comprises the steps of:
    (1) saponin and/or Triton X-100 in kit is used to handle the cell of parasitic infection to be transfected；
    (2) incubation transfection is carried out to the gene order of cell addition transfection of step (1) treated parasitic infection to be transfected and the compound of polyethyleneimine.
  4. According to the method described in claim 3, it is characterized in that, the mass volume ratio of the saponin and/or Triton X-100 is respectively 0.001-0.05%, preferably 0.001-0.03%, further preferably 0.001-0.01%.
  5. The method according to claim 3 or 4, which is characterized in that the gene order is the combination of any one or at least two in plasmid DNA vectors, DNA expression cassette sequence or rna expression frame sequence.
  6. The method according to any one of claim 3-5, which is characterized in that the molar ratio of the ammonia nitrogen in the polyethyleneimine and the phosphate in the gene order is 1-16000, preferably 1-2500, further preferably 5-100；
    Preferably, the molecular weight of the polyethyleneimine is 1000-750000, preferably 10000-30000.
  7. The method according to any one of claim 3-6, which is characterized in that further include the in vitro culture and synchronized step of helminth before step (1)；
    Preferably, culture is further included the steps that after step (2).
  8. The method according to any one of claim 3-7, which is characterized in that the step of culture specifically includes: culture medium of the removal containing transfection reagent is added fresh culture medium and is cultivated；
    Preferably, the temperature of the culture is 30-40 DEG C, preferably 35-38 DEG C；
    Preferably, the time of the culture is 48h or more, preferably 48-96h；
    Preferably, the culture uses three gas incubators.
  9. The method according to any one of claim 3-7, which comprises the steps of:
    (1) in vitro culture and synchronization of helminth；
    (2) using in kit saponin and/or Triton X-100 handle the cell of parasitic infection to be transfected, the mass volume ratio of the saponin and/or Triton X-100 is respectively 0.001-0.05%；
    (3) incubation transfection is carried out to the gene order of cell addition transfection of step (2) treated parasitic infection to be transfected and the compound of polyethyleneimine, the molar ratio of ammonia nitrogen in the polyethyleneimine and the phosphate in the gene order is 1-16000, and the molecular weight of the polyethyleneimine is 1000-750000；
    (4) culture medium containing transfection reagent is removed, fresh culture medium 30-40 DEG C of culture 48h or more of temperature in three gas incubators is added, obtains the worm strain for stablizing expression.
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