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WO2019051642A1 - Kit pour la transfection de parasites intracellulaires et son utilisation - Google Patents

Kit pour la transfection de parasites intracellulaires et son utilisation Download PDF

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Publication number
WO2019051642A1
WO2019051642A1 PCT/CN2017/101404 CN2017101404W WO2019051642A1 WO 2019051642 A1 WO2019051642 A1 WO 2019051642A1 CN 2017101404 W CN2017101404 W CN 2017101404W WO 2019051642 A1 WO2019051642 A1 WO 2019051642A1
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transfection
kit
saponin
polyethyleneimine
falciparum
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Chinese (zh)
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胡文
单雪凤
姚永超
秦莉
陈小平
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Guangzhou Cas Lamvac Biotech Co Ltd
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Guangzhou Cas Lamvac Biotech Co Ltd
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Priority to PCT/CN2017/101404 priority Critical patent/WO2019051642A1/fr
Priority to CN201780001027.4A priority patent/CN110168093B/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of bioengineering, in particular to a kit for transfecting intracellular parasites and an application thereof, in particular to a kit for transfecting intracellular parasites and a method for transfecting intracellular parasites.
  • AIDS, tuberculosis and malaria are the three most deadly infectious diseases in the world.
  • World Health Organization around the world, as of 2015, there are still 438,000 people who die of malaria, 90% of which are concentrated in Africa, 7% in Southeast Asia, and 2%. In the Eastern Mediterranean Region (World Health Organization. World Malaria Report 2015.).
  • the occurrence of clinical malaria is mainly caused by the infection of protozoan parasites of Plasmodium falciparum (P. falciparum) in the red blood cell stage. So far, the basic biological research on P. falciparum has many unknown areas, which limits the development of anti-malarial drugs and malaria vaccines.
  • P. falciparum which is continuously cultured in vitro, must be parasitic in human red blood cells. Survival (Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976, 193 (4254): 673-5.), which means that foreign DNA needs to cross human erythrocyte membrane, Plasmodium cell membrane and malaria The protozoal nuclear membrane trilayer membrane system can enter the nucleus of Plasmodium. Moreover, the spontaneous endocytosis of human mature red blood cells is very inefficient (Colin FC, Schrier SL. Toronto endocytosis in human neonatal and adult red blood cells: comparison to drug-induced endocytosis and to receptor-mediated endocytosis. Am J Hematol.
  • Polyethylenimine is a stable cationic polymer widely used in mammalian cell transfection (Longo PA, Kavran JM, Kim MS, Leahy DJ. Transient mammalian cell transfection with polyethylenimine (PEI). Enzymol. 2013, 529: 227-40.), has also been used for gene transfection of Toxoplasma gondii with the protozoan parasite of P. falciparum (Salehi N, Peng CA. Gene transfection of Toxoplasma gondii using PEI/ DNA polyplexes. J Microbiol Methods. 2012, 91(1): 133-7.).
  • PEI The DNA can be compressed into positively charged particles to bind to anion on the cell surface, after which the PEI/DNA complex is endocytosed and released into the cytoplasm (Sonawane ND, 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, Rudolph 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.).
  • Plasmodium falciparum In addition to Plasmodium, other types of intracellular parasites face the problem of chemical transfection. In order to solve the problem of chemical transfection of Plasmodium falciparum, it is mainly to develop a method for perforating human erythrocyte membrane infected by Plasmodium falciparum without damaging the parasitic Plasmodium, and then using such as polyethylene. The amine/DNA complex particles pass through the human erythrocyte membrane infected with Plasmodium falciparum and achieve transfection of the malaria parasite.
  • the present invention provides a kit for transfecting intracellular parasites and an application thereof, and the use of a kit for transfecting intracellular parasites can solve the problem of low parasitic transfection efficiency and low success rate, and realize A system in which a plurality of plasmid DNAs are co-transfected.
  • the invention provides a kit for transfecting intracellular parasites, the kit comprising polyethyleneimine;
  • the kit also includes saponin and/or polyethylene glycol octyl phenyl ether.
  • efficient transfection of intracellular parasites by genes can be achieved by the synergistic action of polyethyleneimine (PEI) and saponin and/or polyethylene glycol octylphenyl ether (Triton X-100).
  • PEI polyethyleneimine
  • Triton X-100 polyethylene glycol octylphenyl ether
  • the kit comprises PEI and saponin.
  • the inventors have found that the use of Triton X-100 and PEI in combination with transfection of parasites can cause certain toxicity to parasites, resulting in a high proportion of parasite deaths, and the use of saponin and PEI in combination, no toxic side effects, parasitic The worm can continue to survive, and the gene can be stably expressed in the parasite.
  • the parasite is Plasmodium, Babesia, human Colpodella-like parasite (kidney Any one or a combination of any two of Leishmania or Trypanosoma, preferably Plasmodium, is more preferably an intracellular Plasmodium.
  • the present invention provides a method of transfecting an intracellular parasite using the kit of the first aspect, comprising the steps of:
  • the mass to volume ratio of the saponin and/or polyethylene glycol octylphenyl ether is 0.001 to 0.05%, respectively, and may be, for example, 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%, further preferably 0.001-0.01%.
  • the inventors have found that when the concentration of saponin is controlled to be less than 0.03% (wt/v, mass/solution total volume), the cells infected by the parasite are relatively intact, and few cells are lysed, especially the concentration of saponin.
  • concentration between 0.001% (wt/v) and 0.01% (wt/v) is the best, in this concentration range, the integrity of the cells infected by the parasite can be maximized, and the intracellular parasitism can be maintained.
  • the activity of the worm is also applicable to subsequent PEI transfection.
  • the gene sequence is any one or a combination of at least two of a plasmid DNA vector, a DNA expression cassette sequence or an RNA expression cassette sequence.
  • the transfection can simultaneously transfect a plurality of gene expression vectors, the transfection comprising transient transfection and stable transfection, the transient transfection is transfection of freely expressed DNA, and the stable transfection is Transfection energy A gene editing system that is capable of genome integration.
  • the gene sequence is a gene capable of transfecting a parasite
  • the method mainly comprises: a drug resistance screening gene, a reporter gene or a functional gene for screening a positive transfectant strain, wherein the reporter gene is, for example, It may be green fluorescent protein (GFP) and/or red fluorescent protein (RFP), luciferase, etc.; the functional gene includes a functional gene that knocks out the Plasmodium itself, or a functional gene that needs to be overexpressed, and the functional gene may come from Any parasite can also come from other species.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the inventors have found that the molar ratio of the amino nitrogen in the polyethyleneimine to the phosphate group in the gene sequence is very important, and has a great influence on the efficiency of transfection.
  • the molar ratio of the amino nitrogen in the amine to the phosphate group in the gene sequence is 1-16000, and may be, for example, 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, 120,000, 130000, 140000 150000 or 160000, preferably 1 to 2500, further preferably 5 to 100.
  • the specific calculation of the amount of PEI added based on the ratio of the number of moles of the amine nitrogen (N) and the plasmid DNA phosphate (P) of PEI is as follows: free expression for transient transfection
  • the molecular weight of the polyethyleneimine is directly related to the amount of subsequent PEI addition, PEI
  • the average molecular weight is about 25000
  • the molecular weight of the PEI of the present invention is 1000-750000, for example, 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, 7000, 80000, 90000, 100000, 130000, 150,000, 200000, 250,000, 300000, 350000, 400000, 450000, 500000, 550,000, 600000, 650,000, 70000 or 750,000, preferably 10000- 30000.
  • the step of in vitro culture and synchronization of the parasite is further included before the step (1), and the in vitro culture and synchronization described in the present invention are conventional techniques in the art, and those skilled in the art can according to the cultured parasite. The choice is made differently and is not specifically limited herein.
  • step of culturing is further included after step (2).
  • the step of culturing specifically comprises: removing the medium containing the transfection reagent, and adding the fresh medium for culturing.
  • the culture temperature is 30-40 ° C, for example, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C or 40 ° C, It is preferably 35-38 °C.
  • the culture time is 48 h or more, for example, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 58 h, 60 h, 62 h, 63 h, 65 h, 68 h, 70 h, 72 h, 75 h. , 78h, 80h, 82h, 85h, 88h, 90h, 92h, 95h, 96h, 98h or 100h, preferably 48-96h.
  • the culture uses a three-gas incubator.
  • the transfection efficiency is high, it is not necessary to perform drug screening or only a short time screening to obtain a positive transfectant strain, which is different due to the difference in transfection genes in transient transfection and stable transfection in the present invention. Screening is not required for transient transfection, which requires 2-3 weeks of drug screening.
  • the drug screening in the stable transfection is blasticidin S. blasticidin.
  • the method comprises the steps of:
  • step (3) adding a transfected gene sequence to a cell to be transfected with the parasite-infected cell after step (2), and transfecting with a complex of polyethyleneimine, the amino nitrogen in the polyethyleneimine.
  • the molar ratio of the phosphate groups in the gene sequence is 1-16000, and the molecular weight of the polyethyleneimine is 1000-750000;
  • the medium containing the transfection reagent was removed, and the fresh medium was added and cultured in a three-gas incubator at a temperature of 30-40 ° C for 48 hours or more to obtain a stably expressed strain.
  • the method includes the following steps:
  • the medium containing the transfection reagent was removed, and the culture medium was cultured at a temperature of 35-38 ° C for 48-96 h or more in a fresh gas culture medium to obtain a stably expressed strain.
  • the present invention has the following beneficial effects:
  • the kit of the present invention can achieve efficient transfection of genes against parasites by synergistic action of polyethyleneimine (PEI) and saponin and/or polyethylene glycol octylphenyl ether (Triton X-100). Throughout The process takes only about 30 minutes, the transfection efficiency can reach more than 50%, and the highest can reach 100%.
  • the transfected insect strain does not need long-term screening, and the positive insect strain can be screened in at most 2-3 weeks;
  • the transfection kit of the present invention requires only a small amount of polyethyleneimine (PEI) and saponin and/or polyethylene glycol octylphenyl ether (Triton X-100), which has low raw material cost and single transfection.
  • PEI polyethyleneimine
  • Triton X-100 polyethylene glycol octylphenyl ether
  • Figure 1 is a map of a free expression vector for transient transfection of P. falciparum strains
  • Figure 2 (A) is a CRISPR-Cas9-based pPfInt-Pf47Site Cleavage vector map for stable transfection of P. falciparum strains
  • Figure 2 (B) is based on CRISPR- for stable transfection of P. falciparum strains. Cas9 gene editing pPfInt-GFP & NanoLuc Donor vector map;
  • FIG. 3 is the basic flow of chemical transfection of intracellular parasites
  • Figure 4 (A) shows the results of Giemsa staining of P. falciparum strain before gelatin enrichment
  • Figure 4 (B) shows the results of Giemsa staining of P. falciparum strain after gelatin enrichment
  • Fig. 5(A) shows the effect of saponin treatment with 0.001% by mass to volume ratio on P. falciparum 3D7 infected red blood cells
  • Fig. 5(B) shows the treatment of erythrocyte with P. falciparum 3D7 by saponin at a mass to volume ratio of 0.01%
  • Effect Figure 5 (C) is the effect of saponin treatment with P/faliparum 3D7 on erythrocytes in a volume-to-volume ratio of 0.03%
  • Figure 5 (D) is a saponin treatment with a mass-to-volume ratio of 0.05% on P. falciparum 3D7 infection.
  • the effect of red blood cells Figure 5 (E) is the effect of saponin treatment with mass to volume ratio of 0.1% on P. falciparum 3D7 infected red blood cells;
  • Figure 6 shows the results of fluorescence microscopy of pPfEps-mCherry free expression vector using saponin and PEI in transient transfection of P. falciparum 3D7 strain.
  • Figure 6 (A) is a bright field photograph
  • Figure 6 (B) is a Hoechst.
  • Figure 6 (C) is red
  • FIG. 6(D) is an overlay of FIG. 6(A), FIG. 6(B) and FIG. 6(C);
  • Figure 7 shows the results of fluorescence microscopy of the CRISPR-Cas9 gene editing expression vector stably transfected with P. falciparum 3D7 strain, wherein Fig. 7(A) is a bright field photograph, and Fig. 7(B) is a photograph of a green fluorescence detection channel.
  • Figure 7 (C) is an overlay of Figure 7 (A) and Figure 7 (B);
  • Figure 8 (A) is a schematic diagram of pPfEps-mCherry plasmid DNA
  • Figure 9(A) is a schematic diagram showing the double-crossover recombination of the pffInt-GFP&NanoLuc Donor gene donor plasmid DNA and the Pf47 gene locus on chromosome 13 of the P. falciparum 3D7 genome
  • Figure 9 (B) shows the stable transfection of the CRISPR-Cas9 gene editing expression vector.
  • Figure 10 is a Western blot (Western blot) identification of p. falciparum 3D7 strain transiently transfected with pPfEps-mCherry free expression vector, wherein M represents DNA molecular weight standard, S1, S2 is 0.001% (wt/v) after saponin treatment Cell lysate of P. falciparum 3D7 strain transfected with PEI, S3, S4 was 0.01% (wt/v) saponin-treated cell lysate of P. falciparum 3D7 transfected with PEI, WT was wild Cell lysate of the P. falciparum 3D7 strain;
  • Figure 11 shows the detection of NanoLuc luciferase activity of P. falciparum 3D7 stable transfected strains, wherein the ordinate indicates the relative fluorescence signal intensity detected by NanoLuc luciferase activity, and the abscissa is true.
  • S1-S4 were 4 strains of Plasmodium falciparum strains stably transfected and expressing GFP&NanoLuc Luciferase fusion protein, respectively GFP&NanoLuc, and WT was P.falciparum 3D7 wild type strain as negative control group;
  • Figure 12 shows the effect of Triton X-100 treatment on P. falciparum 3D7-infected erythrocytes in mass-to-volume ratios of 0.001%, 0.01%, 0.025%, and 0.1%, respectively;
  • Figure 13 shows the results of fluorescence microscopy of pPfEps-mCherry free expression vector using Triton X-100 in combination with PEI for transient transfection of P. falciparum 3D7 strain.
  • Figure 13 (A) is a bright field photograph
  • Figure 13 (B) is a photograph.
  • Figure 13 (C) shows the results of photographing the red fluorescence detection channel after treatment with Hoechst 33258 dye
  • Fig. 13 (D) is Fig. 13 (A), Fig. 13 (B), and Fig. 13 (C). Overlay of ).
  • Example 1 In vitro culture and synchronization of human P. falciparum strain P. falciparum 3D7
  • plasmids Two sets of plasmids were used: one is the free expression plasmid for transient transfection, pPfEps-mCherry, as shown in Figure 1, and its main components include: one by P.falciparum Promoter sequence of the constitutive promoter 5'-CAM (calmodulin, calmodulin, cam, Gene ID: PF3D7_1434200) and the terminator sequence of Plasmodium berghei 3'-PbDT (bifunctional dihydrofolate)
  • the fusion protein library of the red fluorescent protein mCherry which is controlled by the 3'-end non-coding sequence of the reductase thymidine synthase, bifunctional dihydrofolate reductase-thymidylate synthase, DHFR-TS, Gene ID: PBANKA_0719300), in the N-mCherry protein A membrane transport signal peptide of KAHRP (knob-associated histidine-rich protein
  • Strep tag protein tag a 5'-end promoter of the translation elongation factor from P. falciparum (5'-PfEf1 ⁇ , elongation factor 1-alpha, Gene ID: PF3D7_1357000) P.falciparu
  • the blasticidin S resistance gene (Blasticidin-S deaminase, BSD, EC number) controlled by the 3'-end non-coding region of the m HRPII gene (3'-HRP2, histidine-rich protein IIHRPII, Gene ID: PF3D7_0831800) :3.5.4.23) expression cassette for resistance screening of transgenic Plasmodium; and an expression cassette for kanamycin phosphotransferase (kanamycin resistance gene, KmR) for use in Escherichia coli (eg DH5 ⁇ ) , screening of positive plasmids in XL-10, Stbl3 and NEB Stable plasmid DNA clones) and maintaining the stability of the plasmi
  • the other is a plasmid expression system for stable transfection, as shown in Fig. 2(A) - Fig. 2(B), including pPfInt-Pf47 Site Cleavage of Fig. 2(A) and pPfInt- of Fig. 2(B).
  • Two plasmids which are constructed based on the CRISPR-Cas9 gene editing technology, mainly include a vector containing an expression cassette of the sgRNA targeting the Pf47 site and a expression cassette of the Cas9 gene, the construction process and pCBS-
  • the construction of the Pf47 vector is consistent (Lu J, Tong Y, Pan J, Yang Y, Liu Q, Tan X, Zhao S, Qin L, Chen XA redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.
  • the transfection process is shown in Figure 3.
  • the P. falciparum strain in vitro cultured in gelatin-enriched red blood cells is subjected to cell permeabilization treatment and then transfected, as follows:
  • KAHRP Knob-associated histidine-rich protein located on chromosome 2 of P. falciparum is critical for the membrane transport of human erythrocyte membrane protein 1 (PfEMP1), and its signal peptide energy Enough to help its fusion protein secrete into the red inner phase of P. falciparum in vitro. Therefore, when the protozoal infection rate reaches 10%, the gelatin enrichment method is first used to enrich the complete P. falciparum strain of chromosome 2 (Waterkeyn JG, 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.), the results are shown in Figure 4 (A) - Figure 4 (B), visible, after enrichment The strains are mostly in the trophozoite or schizont stage, which facilitates the transfection of PEI/DNA complex particles.
  • Permeabilization experiments were performed on P. falciparum-infected red blood cells (iRBCs) as follows: First, transfer 5 ml of 1% of the blood to a sterile 15 ml centrifuge tube and centrifuge at 350 ⁇ g at room temperature. Minutes were collected to obtain approximately 50 ⁇ l of iRBCs. Add 1 ml of pre-warmed washing medium at 37 ° C: 10.4 g / L RPMI 1640, 25 mM HEPES, 100 ⁇ M hypoxanthine, 12.5 ⁇ g / ml gentamicin sulfate, 350 ⁇ g, centrifuge at room temperature for 5 minutes to remove the supernatant .
  • Transfection experiments were performed with iRBCs cells permeable to the optimal saponin concentration.
  • the saponin concentration was controlled below 0.03% (wt/v).
  • the iRBCs cells were relatively intact, and few cells were lysed, especially the saponin concentration.
  • the concentration between 0.001% (wt/v) and 0.01% (wt/v) is the best. In this concentration range, the integrity of iRBCs can be maintained to the maximum, and the activity of Plasmodium can be maintained. Subsequent PEI transfection.
  • 25kD linear polyethyleneimine (Polyethylenimine, PEI, Polysciences, Cat. #23966) working solution dissolve the weighed PEI with endotoxin-free sterile deionized water heated to 80 ° C, and cool to room temperature. The pH was adjusted to 7.0 with hydrochloric acid, and the solution was made up to a desired concentration of 1 ⁇ g/ ⁇ l with endotoxin-free sterile deionized water, sterilized by filtration through a 0.22 ⁇ m filter, and stored at -20 ° C until use.
  • PEI Polyethylenimine
  • #23966 used in the present invention contains 11% of the N-propionylated amine group, and
  • the average molecular weight of PEI is about 25,000 g/mol, so the amount of PEI required is (6.9 pmol/0.11) x 25,000 g/mol ⁇ 1.6 ⁇ g, that is, 1.6 ⁇ l of PEI working solution (1 ⁇ g/ ⁇ l) needs to be added.
  • the inventors verified the effect of the molar ratio (N/P) of the amino nitrogen in the polyethyleneimine to the phosphate group in the gene sequence of 1-16000, and found that the conversion can be achieved when the N/P is 1-16000. Dyeing works best when the N/P ratio is 5-100.
  • Transiently transfected strains were screened without any drug during the culture.
  • the stably transfected strains were screened 48 hours after transfection, and 5.0 ⁇ g/mL blasticidin S (ThermoFisher Scientific, Cat. #R21001) was added to the culture medium for screening every 24 hours until the fluorescence microscope observed obvious.
  • the positive strain of the strain reduced the concentration of blasticidin S to 2.5 ⁇ g/mL.
  • a plasmid vector similar to that of Example 2 was passed through an in vitro culture and synchronization of Babesia, human Colpodella-like parasites (renal parasites), Leishmania, and trypanosomes in a manner similar to Example 1.
  • the method of transfection was carried out in the manner of 3, and the inventors found that the effect of transfecting other parasites was comparable to that of Plasmodium, and the subsequent experimental inventors used the results of transfection of Plasmodium to explain, and no redundant description is made here.
  • the IRBCs culture medium of the P. falciparum transfected strain was added to Hoechst 33258 (Invitrogen, Cat. #H3569) at a ratio of 1:1000 to a final concentration of 10 ⁇ g/ml, incubated at 37 ° C for 5 minutes, and then PBS buffer ( Wash at pH 7.4) twice.
  • the washed iRBCs were then coated onto a microscopic slide with a low fluorescence background for microscopic examination.
  • Our camera equipment uses Olympus' PLYMPUS IX73 microscope and 100x oil lens (UPLFLN 100X (Oil Immersion) objective lens).
  • Fig. 6(A)-Fig. 6(D) it can be seen that the fusion protein of the reporter gene mCherry is normal, and the red fluorescence of the reporter gene is indeed derived from the Plasmodium strain obtained by chemical transfection; from Fig. 7(A)-Fig. (C) It can be seen that the expression of the reporter gene GFP is normal, and the green fluorescence of the reporter gene is indeed derived from the Plasmodium strain obtained by chemical transfection, and the transfection success rate can reach 100%.
  • transiently transfected infected red blood cells were collected by centrifugation and washed twice with PBS buffer solution (pH 7.4) for use.
  • the infected red blood cells were lysed by adding a 5-fold volume concentration of 0.1% saponin solution. Gently invert and mix for 2 minutes at room temperature. Collecting infected red blood cells at 1900 ⁇ g for 5 minutes at 4 ° C Plasmodium and related cell membrane fragments released after lysis. The supernatant was removed and washed three times with 3 volumes of PBS buffer solution (pH 7.4) and used. Wild-type P. falciparum and stably transfected P. falciparum were collected in the same manner.
  • PCR amplification was performed directly using Plasmodium cells as a template.
  • the protocol was prepared according to the standard protocol of the PCR kit instructions (Takara, Cat. #R050Q).
  • the PCR detection target and primers are shown in Table 2, and the PCR detection results are shown in Figs. 8 and 9.
  • the DNA band size of the reporter gene pair P1/P2 detection gene mCherry is 619 bp, and the DNA band size of the bsd gene detected by the primer pair P3/P4 is 399 bp; as can be seen from Fig. 9, the wild The PCR product of P. falciparum 3D7 strain was used as a negative control group, and the amplified fragment size was 1390 bp; the PCR product of S1, P. falciparum 3D7 stably transfected strain, the amplified fragment size was 4293 bp, visible, whether Transient transfection (Figure 8) was also stable transfection (Figure 9), and positive transfected strains were obtained.
  • Example 7 SDS-PAGE protein gel electrophoresis and Western blot detection
  • the total protein sample of Plasmodium was prepared by 10% SDS-PAGE gel electrophoresis, and the protein band of SDS-PAGE gel electrophoresis was performed by wet transfer system (Tanon, China). Transfer to a suitable size PVDF membrane (BioRad, Cat. #1620177). The transferred PVDF membrane was blocked with 5% BSA (prepared with TBST buffer) for 2 hours at room temperature. The membrane was washed three times with ice-cold TBST buffer (6 minutes each), and the primary antibody (Anti-mCherry antibody (diluted 1:1000 with TBST buffer, Biovision, Cat. #5993) or Anti- was added. GAPDH antibody (diluted 1:2000 with TBST buffer, abcam, Cat.
  • HRP horseradish peroxidation
  • the predicted average molecular weight of the mCherry-'SA'Linker-Strep tag fusion protein is 27.9 kDa excluding the KAHRP signal peptide sequence, while the predicted average molecular weight of the housekeeping gene GADPH protein is 36 kDa.
  • the expression of the reporter gene was successfully detected in the chemically transfected strain.
  • Example 8 Detection of NanoLuc luciferase activity of P. falciparum 3D7 stably transfected strain
  • Example 3 Compared with Example 3, other conditions and methods were the same as those in Example 3 except that Triton X-100 was used, and the mass-to-volume ratio of Triton X-100 was 0.001% to 0.01%.
  • Example 3 the perforated iRBCs were subjected to smear and microscopic examination to determine the optimal Triton X-100 concentration for cell permeation.
  • Triton X-10 concentration (wt/v, saponin mass/total solution volume) greater than or equal to 0.01% leads to cleavage of iRBCs and is not suitable for subsequent PEI/DNA complex particle transfection. .
  • Transfection experiments were performed with iRBCs cells permeable to the optimal Triton X-100 concentration.
  • the concentration of Triton X-10 was controlled below 0.01% (wt/v).
  • the iRBCs cells were relatively intact, and few cells were lysed, especially
  • the concentration of saponin is preferably between 0.001% (wt/v) and 0.005% (wt/v). In this concentration range, the integrity of iRBCs can be maintained to the maximum, and the activity of Plasmodium can be maintained. It is also suitable for subsequent PEI transfection.
  • transient transfection was performed using the free expression plasmid pPfEps-mCherry, as shown in Figure 13(A)-Fig. 13(D), although Triton X-100 and PEI were able to transfect P. falciparum 3D7, Positive strains were also observed within 72 hours after transfection, but subsequent observations revealed Triton X-100 and PEI combined with transgenic parasites will cause certain toxicity to parasites, resulting in a high proportion of parasite deaths. All of the Triton X-100 treated strains died within about one week after transfection.
  • Example 3 The other conditions and methods were the same as in Example 3 except that the step of cell permeable without saponin was used, and only the step of transfection was included.
  • Example 3 In the same manner as in Example 3, except that the transfection was carried out without using PEI, and the incubation was carried out only after the treatment with saponin, the other conditions and methods were the same as in Example 3.
  • the kit of the present invention can achieve high efficiency of genes to parasites by synergistic action of polyethyleneimine (PEI) and saponin and/or polyethylene glycol octylphenyl ether (Triton X-100). Transfection, the whole process takes only about 30min, the transfection efficiency can reach more than 50%, and the highest can reach 100%. The transfected insect strain does not need long-time screening, and the positive insect strain can be screened in at most 2-3 weeks. .
  • PEI polyethyleneimine
  • Triton X-100 polyethylene glycol octylphenyl ether

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Abstract

L'invention concerne un kit pour la transfection de parasites intracellulaires et son utilisation. Le kit comprend de la polyéthylèneimine et comprend en outre de la saponine et/ou de l'éther octylphénylique de polyéthylèneglycol. Le kit permet une transfection rapide, il a une efficacité de transfection élevée et un taux de réussite de transfection élevé, il prend peu de temps pour acquérir des souches positives après transfection et ne nécessite pas de criblage de médicament à long terme, il ne nécessite qu'une très petite quantité de réactifs de transfection chimiques et ne nécessite pas d'instruments de transfection coûteux, il utilise des matières premières de faible coût, il a une consommation relativement faible des réactifs pour une seule transfection et il permet de transfecter de multiples vecteurs d'expression génique en une seule fois.
PCT/CN2017/101404 2017-09-12 2017-09-12 Kit pour la transfection de parasites intracellulaires et son utilisation Ceased WO2019051642A1 (fr)

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