WO2019051642A1 - Kit for transfecting intracellular parasites and use thereof - Google Patents

Abstract

A kit for transfecting intracellular parasites and the use thereof, the kit comprising polyethyleneimine, the kit further comprising saponin and/or polyethylene glycol octylphenyl ether. The kit has rapid transfection, high transfection efficiency and high transfection success rate; the positive insect strain after transfection takes a short time and does not require long-term drug screening; only a very small amount of chemical transfection reagent is needed, and it is not expensive. Transfection equipment; low cost of raw materials, low cost of single-transfection reagents; potential for transfection of multiple gene expression vectors at one time.

Description

Kit for transfecting intracellular parasites and application thereof Technical field

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.

Background technique

AIDS, tuberculosis and malaria are the three most deadly infectious diseases in the world. According to the 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.

In fact, there are still about 50% of the functions of the P. falciparum gene 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 have been applied to the study of the function of the P. falciparum gene in the erythrocytic stage, but the progress is slow (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, Mesén-Ramírez P, Jonscher E, Bergmann B, Spielmann TA genetic system to study Plasmodium falciparum protein function. Nat Methods.2017,14( 4): 450-456.). The unsuccessful genetic manipulation of specific genes in Plasmodium is often due to the transfection efficiency of Plasmodium The rate is too low and the survival rate of the malaria parasite is too low after transfection. Although researchers have developed a variety of alternative methods for the transfection of Plasmodium, red blood cell electroporation-based DNA spontaneous absorption is still the preferred method for introducing foreign DNA into P. falciparum (Deitsch K, 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-Adams TS, Lawrie PM, Hawthorne PL, Gardiner DL, Trenholme KR .Comparison of Plasmodium falciparum transfection methods. Malar J. 2003, 2:19.). However, this electroporation transfection method requires long-term culture and multiple rounds of drug screening to obtain positive transgenic strains (Birnbaum J, Flemming S, Reichard N, Soares AB, Mesén-Ramírez P, Jonscher E, Bergmann B). , Spielmann TA genetic system to study Plasmodium falciparum protein function. Nat Methods. 2017, 14(4): 450-456.).

"Lu et al., 2016; Birnbaum et al., 2017" reported that it takes several dozen days to obtain a positive worm for simple green fluorescent protein (GFP) gene knock-in at a non-essential locus of the P. falciparum genome. Plant (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. Parasit Vectors.2016, 9: 198. Birnbaum J, Flemming S, Reichard N, Soares AB, Mesén-Ramírez P, Jonscher E, Bergmann B, Spielmann TA genetic system to study Plasmodium falciparum protein function. Nat Methods. 2017, 14(4): 450-456. ). Moreover, this method requires an expensive electroporation transfection device and a matching cytomix to carry out, and the amount of DNA required is also large, and it takes hundreds of micrograms of DNA molecules to load DNA for one transfection. The red blood cells and the malaria parasite used for transfection need to be separately prepared, and frequent fluid changes are required at the initial stage of transfection, which is relatively time consuming, labor intensive, and costly.

Unlike other species, 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. 1991, 37(1): 34-40.), probably because the dense erythrocyte membrane skeleton increases the strength of the membrane lipid bilayer (Lux SE 4th.Anatomy of the red cell membrane skeleton:unanswered questions.Blood.2016,127 (2): 187-99.) In turn, internalization of membrane vesicles is prevented. This makes it difficult to achieve transfection of Plasmodium erythropolis by chemical transfection methods such as the conventional cationic liposome method and cationic polymer method. Despite this, P. falciparum itself still has spontaneous endocytosis during the development of the red inner phase (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 KA, Hoppe HC. Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum. Cell Microbiol. 2008, 10(2): 452-64.) Therefore, how to pass the transfection complex containing the gene sequence through the host cell membrane is the key to the success of chemical transfection.

Polyethylenimine (PEI) 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.). Coincidentally, another cationic polymer, polyamidoamine dendrimer, has been reported to be useful for transfection of 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 studies have found that the method is used to transfect P. falciparum and add drugs Multiple replicates of screening to obtain stable transfected strains were not successful (Skinner-Adams TS, Lawrie PM, Hawthorne PL, Gardiner DL, Trenholme KR. Comparison of Plasmodium falciparum transfection methods. Malar J. 2003, 2:19 .).

Historically, researchers have developed a series of transfection methods for P. falciparum. Among them, the most widely used and most commonly used is electroporation transfection of red blood cell-loaded DNA, which relies on expensive electroporation transfection instruments and transfection reagents. The cost of the instrument alone is as high as several hundred thousand RMB, a single transfection. The reagent cost is also a few hundred yuan, and the fresh human red blood cells used in a single transfection and the P. falciparum (a few hundred microliters of insect blood with a infection rate of more than 10%) in the schizont stage need to be prepared separately. DNA It is also used in a large amount (tens to hundreds of micrograms). It takes 2-3 hours to complete a single electrical transfection, and it takes at least 30 days of in vitro culture and drug screening to obtain a positive strain. Transfection Both efficiency and success rate are low (approximately one in a million transfection rates). There are also a few researchers who have tried to use chemical transfection. However, the highest transfection efficiency reported is less than 10%, and the experimental success rate is very low (nearly impossible to repeat), and the reagents used are also expensive.

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.

Summary of the invention

In view of the existing problems, 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.

To this end, the present invention employs the following technical solutions:

In one aspect, 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.

In the present invention, 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). The whole process takes only about 30 minutes, the transfection efficiency can reach more than 50%, and the highest can reach 100%.

According to the invention, the kit comprises PEI and saponin.

In the present invention, 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.

According to the invention, 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.

In a second aspect, the present invention provides a method of transfecting an intracellular parasite using the kit of the first aspect, comprising the steps of:

(1) treating the cells infected with the parasite to be transfected with saponin and/or polyethylene glycol octylphenyl ether in the kit;

(2) Adding a transfected gene sequence to the cell to be transfected with the parasite-infected cell after the step (1), and transfecting the complex with polyethyleneimine.

According to the present invention, 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%.

In the present invention, 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. The 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.

According to the invention, 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.

In the present invention, 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.

In the present invention, the gene sequence is a gene capable of transfecting a parasite, and 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.

According to the present invention, 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.

In a specific embodiment, 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 plasmid is exemplified by a molar concentration of phosphate (P) = weight of plasmid DNA (1 μg) / (base pair of double-stranded plasmid DNA (6669 bp) × average molecular weight of DNA base pair (sodium salt) (650 g/mol/bp) )) 0.23 pmol, calculated as a ratio of nitrogen to phosphorus (N/P) of 30, the amine nitrogen (N) of PEI requires 6.9 pmol, and the PEI used in the present invention contains 11% of N-propionylated amine Base, while PEI has an average molecular weight of about 25,000 g/mol, so the amount of PEI required is (6.9 pmol/0.11) × 25,000 g/mol ≈ 1.6 μg, that is, 1.6 μl of PEI working solution (1 μg/) is required. Ll).

According to the invention, the molecular weight of the polyethyleneimine is directly related to the amount of subsequent PEI addition, PEI The average molecular weight is about 25000, and 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.

According to the present invention, 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.

Preferably, the step of culturing is further included after step (2).

According to the present invention, the step of culturing specifically comprises: removing the medium containing the transfection reagent, and adding the fresh medium for culturing.

Preferably, 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.

Preferably, 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.

Preferably, the culture uses a three-gas incubator.

In the present invention, since 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.

In the present invention, the drug screening in the stable transfection is blasticidin S. blasticidin.

According to the invention, the method comprises the steps of:

(1) in vitro culture and synchronization of parasites;

(2) treating the parasitic infected cells to be transfected with saponin and/or polyethylene glycol octyl phenyl ether in a kit, the saponin and/or polyethylene glycol octyl phenyl ether The mass to volume ratio is 0.001-0.05%;

(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;

(4) 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.

As a preferred technical solution, the method includes the following steps:

(1) in vitro culture and synchronization of Plasmodium;

(2) treating the cells infected with the Plasmodium to be transfected with the saponin in the kit, the mass to volume ratio of the saponin being 0.001-0.01%;

(3) adding a transfected gene sequence to a cell to be transfected with the step of (2), and transfecting the transfected gene sequence with a polyethyleneimine, the amino nitrogen and the The molar ratio of the phosphate group in the gene sequence is 5-100, and the molecular weight of the polyethyleneimine is 10000-30000;

(4) 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.

Compared with the prior art, the present invention has the following beneficial effects:

(1) 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;

(2) 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. The reagent is low in cost and can transfect multiple gene expression vectors at one time, and the efficiency is high;

(3) The method of the invention is simple, convenient to operate, and convenient for popularization and application.

DRAWINGS

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, and Figure 2 (B) is based on CRISPR- for stable transfection of P. falciparum strains. Cas9 gene editing pPfInt-GFP & NanoLuc Donor vector map;

Figure 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, and 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, and Figure 6 (B) is a Hoechst. After the 33258 dye treatment, the result of photographing in the UV channel, Figure 6 (C) is red The photographing result of the color fluorescence detecting channel, 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, and Figure 8 (B) is a PCR identification result of transient transfection of P. falciparum 3D7 strain of pPfEps-mCherry free expression vector, wherein M represents DNA molecular weight standard, S1, S2 PCR identification product of PEI transfected strain treated with 0.001% (wt/v) saponin, S3, S4 was 0.01% (wt/v) saponin-treated PCR product of PEI transfected strain WT is the PCR product of wild-type P. falciparum 3D7 strain as a negative control group, and PC is purified pPfEps-mCherry plasmid DNA as a positive control group;

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, and Figure 9 (B) shows the stable transfection of the CRISPR-Cas9 gene editing expression vector. PCR identification results of P. falciparum 3D7 strain, wherein M represents the DNA molecular weight standard, WT is the PCR product of the wild type P. falciparum 3D7 strain as a negative control group, and S1 is a stable transfected strain of P. falciparum 3D7. PCR product;

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. In the test group, 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, and 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, and Fig. 13 (D) is Fig. 13 (A), Fig. 13 (B), and Fig. 13 (C). Overlay of ).

Detailed ways

The technical solutions of the present invention will be further described with reference to the accompanying drawings and the embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

Example 1: In vitro culture and synchronization of human P. falciparum strain P. falciparum 3D7

In vitro culture and synchronization of P. falciparum 3D7 of human P. falciparum strains using standardized conditions (Radfar A, Méndez D, Moneriz C, Linares M, Marín-García P, Puyet A, Diez A, Bautista JM. Synchronous culture Of Plasmodium falciparum at high parasitemia levels. Nat Protoc. 2009, 4(12): 1899-915.): Includes complete medium: 10.4 g/L RPMI 1640, 25 mM HEPES, 0.5% (wt/vol) AlbuMAX I, 1.77 mM sodium bicarbonate, 100 μM hypoxanthine, 12.5 μg/ml gentamicin sulfate, adjusted to pH 7.2, 1% hematocrit, 37 ° C three-gas incubator (1% O ₂ , 3% CO ₂ , And 96% N ₂ ). The medium was changed once a day, and the infection rate was counted by Giemsa staining. The infection rate of the protozoa reached 10%, and the sorbitol cleavage method was used to synchronize the malaria parasites.

Example 2: P. falciparum transfected plasmid vector and related gene sequences

To test the feasibility of the chemical transfection method, 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) gene was inserted into the end, and a linker peptide consisting of serine (S) and alanine (A) was fused at the C-terminus of mCherry protein. Strep tag protein tag; a 5'-end promoter of the translation elongation factor from P. falciparum (5'-PfEf1α, 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 DH5α) , screening of positive plasmids in XL-10, Stbl3 and NEB Stable plasmid DNA clones) and maintaining the stability of the plasmid in E. coli; and the basic skeleton of plasmid DNA and the origin of plasmid DNA replication in E. coli (ColE1 origin) sequence and the like.

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). GFP&NanoLuc Donor 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. Parasit Vectors .2016, 9:198.); and a recombinant donor plasmid flanked by the GFP and NanoLuc fusion protein expression cassettes with the 5'-end and 3'-end homologous arm sequences of the Pf47 site, with pARM- The construction process of the GFP/RUCki 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. Parasit Vectors.2016, 9:198.), the only difference is that the renilla luciferase in the pARM-GFP/RUCki vector is replaced by the NanoLuc fluorescing with higher detection sensitivity. The enzyme gene (Hall MP, 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 GGPSG short peptide DNA sequence Linked to the green fluorescent protein GFPm3 gene to form a new fusion protein expression cassette.

Example 3: P. falciparum transfection experiment

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:

The Knob-associated histidine-rich protein (KAHRP) 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 μl of iRBCs. Add 1 ml of pre-warmed washing medium at 37 ° C: 10.4 g / L RPMI 1640, 25 mM HEPES, 100 μM hypoxanthine, 12.5 μg / ml gentamicin sulfate, 350 × g, centrifuge at room temperature for 5 minutes to remove the supernatant . Add 500 μl of different concentrations of saponin (Sigma-Aldrich, Cat. #S4521) solution prepared in washing medium at concentrations ranging from 0.001% (wt/v) to 0.1% (wt/v), and incubate for 3 minutes at room temperature. Thereafter, the supernatant was removed by centrifugation at 1900 x g for 3 min at room temperature, and then washed once with 1 ml of P. falciparum complete medium preheated at 37 °C. The perforated iRBCs were smeared and microscopically examined to determine the optimal saponin concentration for cell permeation. The results are shown in Figure 5(A)-Fig. 5(E), saponin concentration (wt/v, saponin). A mass/solution total volume greater than 0.03% will result in 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 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.

Transfection

First, prepare 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 μg/μl with endotoxin-free sterile deionized water, sterilized by filtration through a 0.22 μm filter, and stored at -20 ° C until use.

The ratio of the number of moles of the amine nitrogen (N) to the plasmid DNA phosphate (P) of PEI is determined by calculation, for example, the free expression plasmid pPfEps-mCherry for transient transfection, and its phosphate molar concentration (P ) = plasmid DNA weight (1 μg) / (base pair of double-stranded plasmid DNA (6669 bp) × average molecular weight of DNA base pair (sodium salt) (650 g / mol / bp)) ≈ 0.23 pmol, according to the ratio of nitrogen to phosphorus ( The N/P) is calculated as 30, and the amine nitrogen (N) of PEI requires 6.9 pmol, while the PEI (Polysciences, Cat. #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 μg, that is, 1.6 μl of PEI working solution (1 μg/μl) 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.

According to the N/P ratio of 30, 1 μg of DNA and the corresponding amount of PEI were sequentially added to 200 μl of serum-free DMEM minimal medium (C11995500BT, Gibco), thoroughly mixed, and incubated at room temperature for 20 min. During this time, iRBCs that pass through a suitable concentration of saponin solution (0.001% to 0.05%) are prepared. The PEI/DNA complex was gently mixed with the prepared iRBCs and transferred to a T25 flask (Cat. #169900, Thermo Scientific) pre-filled with 7 ml of P. falciparum complete medium preheated at 37 ° C. The fresh red blood cells of the human body washed by the washing medium maintain the hematocrit at about 1%. The culture flask was placed in a three-gas incubator at 37 ° C for 8 hours, then changed and added. The fresh complete medium preheated at 37 °C was continued to be cultured in a three-gas incubator at 37 °C.

Transiently transfected strains were screened without any drug during the culture. The stably transfected strains were screened 48 hours after transfection, and 5.0 μg/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 μg/mL.

Example 4: Transfection of other parasites

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.

Example 5: Real-time detection by fluorescence microscope

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 μg/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).

It has been identified that the transient transfection of positive insect strains can be obtained within 72 hours by chemical transfection combined with saponin and PEI. The results of fluorescence microscopy are shown in Figure 6(A)-Fig. 6(D). The statistical results of efficiency are shown in Table 1;

Within 2-3 weeks, after 5 rounds of drug screening (screening 5.0 μg/mL blasticidin S from the two days after transfection, changing daily until a positive strain was obtained), stable transfected positive strains were obtained. Figure 7 (A) - Figure 7 (C).

Table 1. Statistics of in vitro transient transfection efficiency of P.falciparum 3D7

It can be seen from Table 1 that the transfection success rate can reach 100% and the transfection efficiency is above 50%. However, we only found any positive insect strains by transfection with PEI alone.

From 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%.

Example 6: PCR identification

72 hours after transfection, 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.

Table 2 PCR detection target and primer list

It can be seen from Fig. 8 that 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

Using the above method for isolating the malaria parasite from iRBCs, 20 μl of the isolate isolated after transfection was collected, and 100 μl of 2×SDS-PAGE protein loading buffer (Takara, Cat. #9173) was added, resuspended and transferred to a 1.5. Ml EP tube (Eppendorf tube). Heat at 100 ° C for 5 minutes. After cooling, 13,000 The supernatant was collected by centrifugation at room temperature for 1 minute to obtain a sample of Plasmodium total protein SDS-PAGE electrophoresis. Take appropriate samples directly, analyze them by 10% SDS-PAGE gel or store at -20 °C for use.

Before Western blot analysis, 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. #ab125247), incubated for 1 hour at room temperature. Wash the membrane 3 times (6 minutes each) with ice-cold TBST buffer and add horseradish peroxidation The enzyme (HRP)-labeled goat anti-mouse IgG H&L secondary antibody was diluted 1:5000 with TBST buffer, abcam, Cat. #ab205719), and incubated for 1 hour at room temperature. The membrane was washed 3 times with ice-cold TBST buffer (6 minutes each time), then ECL reagent (Millipore, Cat.#WBKLS0500) was added and incubated for 5 minutes at room temperature in the dark, using a chemiluminescence detector (Tanon, China) The results of photographing Western blots at different exposure times are shown in FIG.

As can be seen from Figure 10, 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

Take a black 96-well plate (ThermoFisher Scientific, Cat. #07-200-565), add 100 μl of sterile deionized water to the test wells, and pipet 3 μl of P. falciparum 3D7 stably transfected strains of infected red blood cells ( Infection rate is about 10%) added to the test well containing sterile deionized water, fully mixed uniform. The reaction substrate of NanoLuc luciferase was prepared according to the NanoLuc Luciferase Assay Kit (Promega, Cat. #N1110), ie, the substrate and the enzyme reaction buffer were diluted 1:50 and mixed uniformly. 100 μl of the prepared reaction substrate of NanoLuc luciferase was added to the sample wells containing P. falciparum 3D7-infected erythrocytes, and the reaction was read at room temperature for 3 minutes, and the chemiluminescence signal (λmax) was read with a microplate reader (BioTek Synergy H1). = 460 nm), the experimental results were recorded and counted as shown in FIG.

As can be seen from Fig. 11, the fusion protein of GFP and NanoLuc luciferase of P. falciparum 3D7 stably transfected strain was normal.

Example 9: Triton X-100 and PEI combined with transfection of P. falciparum 3D7

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%.

First, as in Example 3, the perforated iRBCs were subjected to smear and microscopic examination to determine the optimal Triton X-100 concentration for cell permeation.

The results are shown in Figure 12. 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.

As in Example 3, 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.

Comparative example 1

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.

As a result, it was confirmed that the gene sequence could not be transferred into intracellular malaria parasites.

Comparative example 2

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.

As a result, it was confirmed that the gene sequence could not be transferred into intracellular malaria parasites.

In summary, 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. .

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims (9)

A kit for transfecting intracellular parasites, characterized in that the kit comprises polyethyleneimine; The kit also includes saponin and/or polyethylene glycol octyl phenyl ether. The kit according to claim 1, wherein the kit comprises polyethyleneimine and saponin; Preferably, the parasite is any one or a combination of at least two of Plasmodium, Babesia, human Colpodella-like parasite, Leishmania or Trypanosoma, preferably Plasmodium, further preferably intracellular Plasmodium. A method for transfecting intracellular parasites using the kit of claim 1 or 2, comprising the steps of: (1) treating the cells infected with the parasite to be transfected with saponin and/or polyethylene glycol octylphenyl ether in the kit; (2) Adding a transfected gene sequence to the cell to be transfected with the parasite-infected cell after the step (1), and transfecting the complex with polyethyleneimine. The method according to claim 3, wherein the mass to volume ratio of the saponin and/or polyethylene glycol octylphenyl ether is 0.001 to 0.05%, preferably 0.001 to 0.03%, respectively, further preferably 0.001-0.01%. The method according to claim 3 or 4, wherein the gene sequence is any one or a combination of at least two of a plasmid DNA vector, a DNA expression frame sequence or an RNA expression frame sequence. The method according to any one of claims 3 to 5, wherein the molar ratio of the amino nitrogen in the polyethyleneimine to the phosphate group in the gene sequence is from 1 to 6,000, preferably from 1 to 1. 2500, further preferably 5-100; Preferably, the polyethyleneimine has a molecular weight of from 1,000 to 750,000, preferably from 10,000 to 30,000. The method according to any one of claims 3-6, further comprising the step of in vitro culture and synchronization of the parasite prior to step (1); Preferably, the step of culturing is further included after step (2). The method according to any one of claims 3-7, wherein the step of culturing comprises: removing the medium containing the transfection reagent, and adding the fresh medium for culturing; Preferably, the temperature of the culture is 30-40 ° C, preferably 35-38 ° C; Preferably, the culture time is 48h or more, preferably 48-96h; Preferably, the culture uses a three-gas incubator. A method according to any one of claims 3-7, comprising the steps of: (1) in vitro culture and synchronization of parasites; (2) treating the parasitic infected cells to be transfected with saponin and/or polyethylene glycol octyl phenyl ether in a kit, the saponin and/or polyethylene glycol octyl phenyl ether The mass to volume ratio is 0.001-0.05%; (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; (4) 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.

Images