CN111662854A - Cell culture method and application of Chlamydia abortus - Google Patents
Cell culture method and application of Chlamydia abortus Download PDFInfo
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Abstract
The invention relates to a cell culture method of Chlamydia abortus and application thereof. The culture method comprises the following steps: washing the cells growing into a monolayer to obtain washed cells; inoculating the chlamydia abortus inoculation liquid, mixing the inoculation liquid with a DNA transfection reagent Lipofectamine2000, standing, inoculating the mixture to washed cells, incubating, removing the liquid after incubation, and culturing for 48-72 hours by using a culture solution. The culture method of the invention does not need centrifugal operation, can realize high-efficiency infection of the in vitro culture cells by the Chlamydia abortus, and has convenient operation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cell culture method of Chlamydia abortus and application thereof.
Background
Chlamydia (Chlamydia spp.) is a gram-negative, obligate intracellular parasitic prokaryotic microorganism with a unique biphasic developmental cycle, which can infect humans and various animals and cause disease, and is an important pathogenic microorganism. All chlamydia species have the same life cycle, including infection and 2 stages of intracellular proliferation. Chlamydia (EBs) are metabolically inactive but infectious. After the free EBs attach to the surface of the host cell, they enter the cell by endocytosis and form inclusion bodies (RBs), in which the EBs are transformed into metabolically vigorous networks (RBs), which proliferate by binary fission. After a certain period of proliferation, RBs are converted into EBs again and released outside cells to complete the development cycle.
Chlamydia abortus (Chlamydia abortus) is the main pathogen of sheep endemic abortion (OEA) and can cause the infected ewe to have abortion, stillbirth lambs or weak lambs which can not survive for more than 48 hours under term birth within the last 2-3 weeks of gestation. Late pregnancy abortions caused by chlamydial infections cause severe economic losses in many sheep-raising areas of the world, especially in areas with a high population of antenatal sheep. In addition to sheep, domestic animals such as goats, cattle, pigs, horses, etc. infected with Chlamydia abortus also cause abortion in the late gestation period. The abortion chlamydia can also infect people, cause atypical pneumonia, abortion of pregnant women and the like, and belongs to the pathogen of zoonosis. Timely immunization is the most effective measure for preventing and controlling the abortion chlamydia disease of animals.
Chlamydia abortus can also be propagated in large quantities in chicken embryos, so that the antigen is harvested for vaccine preparation, and the inactivated vaccine for the chlamydia disease of the dairy cows developed by Lanzhou veterinary research institute of Chinese agricultural academy of sciences is used for antigen propagation through the chicken embryos. The Chlamydia abortus can also be isolated and cultured in vitro by using various cell lines such as mouse fibroblast (McCoy, L929), green monkey kidney cell (BGM), small hamster kidney cell (BHK), human cervical cancer epithelial cell (HeLa) and the like. Compared with the separation by using chick embryos, the separation of the Chlamydia abortus strain from a clinical sample by using cultured cells is more convenient to operate and has higher sensitivity, thus being widely used at present. However, since various chlamydia pathogens, including Chlamydia abortus, have a weak ability to spontaneously infect in vitro cultured cells, isolation or proliferation of Chlamydia abortus using the in vitro cultured cells often requires appropriate treatment of the cells to enhance susceptibility to Chlamydia. At present, the method is commonly used, which is to pretreat the cells with DEAD-D of 30. mu.g/mL for 15min to neutralize the surface charge of the cells, then centrifuge the chlamydia inoculation liquid together with the cells for 1h at a centrifugal force of 1500g, and finally replace it with a culture liquid containing cycloheximide, thus the chlamydia can be effectively cultured. Although the culture method has good effect, the steps are complicated, and the method is not suitable for large-scale production of the antigen. Although the chick embryos can be used for producing antigens on a large scale, the production process is too complex, and the chick embryos are living organisms, so that certain ethical disputes exist, and the chick embryos do not accord with the development concept and trend of the modern vaccine production process. Therefore, the technical bottleneck that the chlamydia can effectively infect in-vitro cultured cells only by centrifugal assistance is broken through, and the method becomes a necessary way for realizing large-scale production of chlamydia antigens by utilizing cell culture.
Disclosure of Invention
The invention aims to provide a cell culture method of Chlamydia abortus and application thereof. The culture method of the invention does not need centrifugal operation, can realize high-efficiency infection of the in vitro culture cells by the Chlamydia abortus, and has convenient operation.
The invention provides a cell culture method of Chlamydia abortus, which comprises the following steps:
washing the cells to be infected which grow into a single layer to obtain washed cells;
mixing the abortion chlamydia inoculation liquid with a DNA transfection reagent Lipofectamine2000, standing, inoculating the mixture to the washed cells, incubating, and removing the incubated liquid to obtain infected cells;
and continuously culturing the infected cells for 48-72 hours by using a culture solution.
Preferably, the cells to be infected include mouse fibroblast McCoy, mouse fibroblast L929, mouse hamster kidney cell BHK-21 or human cervical cancer cell HeLa.
Preferably, the chlamydia abortus inoculation liquid is a serum-free culture liquid containing chlamydia abortus, and the serum-free culture liquid comprises RPMI-1640.
Preferably, the volume ratio of the DNA transfection reagent Lipofectamine2000 to the Chlamydia abortus inoculation liquid is 1: (250 to 4000).
Preferably, the time of standing is 10 min.
Preferably, the incubation condition is 37 ℃ and 30-60 min.
The invention also provides application of the DNA transfection reagent Lipofectamine2000 in enhancing the infection capacity of the cultured cells by the Chlamydia abortus.
The invention provides a cell culture method of Chlamydia abortus. The culture method disclosed by the invention does not need centrifugal operation, can realize efficient infection of the in-vitro culture cells by the Chlamydia abortus, is convenient to operate, can be applied to laboratory subculture of the Chlamydia abortus, preparation of vaccine antigens, screening of anti-Chlamydia drugs and the like, and can also be applied to other Chlamydia so as to improve the culture efficiency.
Drawings
FIG. 1 is a graph of the ability of Lipofectamine2000 provided by the present invention to enhance infection of cells by Chlamydia abortus;
FIG. 2 is a graph comparing the effect of Lipofectamine2000 provided by the present invention on enhancing Chlamydia abortus infection and proliferation in cells compared to conventional centrifugation methods.
Detailed Description
The invention provides a cell culture method of Chlamydia abortus, which comprises the following steps:
washing the cells to be infected which grow into a single layer to obtain washed cells;
mixing the abortion chlamydia inoculation liquid with a DNA transfection reagent Lipofectamine2000, standing, inoculating the mixture to the washed cells, incubating, and removing the incubated liquid to obtain infected cells;
and continuously culturing the infected cells for 48-72 hours by using a culture solution.
The present invention is not particularly limited to the aforementioned Chlamydia abortus, and in a specific example of the present invention, infection of cells is carried out using Chlamydia abortus GN6 Strain (ZHAOCAI Li, Jinchan Cai, Xiaoan Cao, Zhongzi Lou, Yilin Chao, Wei Kan, Juzhang Zhou, white-Genome Sequence of Chlamydia abortus Strain 6 Isolattedfrom Abed Yak Fetus. Genome Announc,2017,5(35): e00893-17) and Chlamydia abortus SX5 Strain (Chanqiu Qing, Zhou Chi Chun, Shucheng Gao, Xiao Cao, Gao, Giangyamo. development of inactivated vaccine for bovine protozoonosis Chlamydia, Chinese veterinary science 2006 343: (347)).
In the present invention, the cells to be infected preferably include mouse fibroblast McCoy, mouse fibroblast L929, mouse hamster kidney cell BHK-21, human cervical cancer cell HeLa and the like. The source of the cells is not particularly limited in the present invention, and the cells may be purchased from a cell bank known to those skilled in the art. In the present invention, the cells to be infected are preferably cultured in RPMI-1640 medium containing 5% Fetal Bovine Serum (FBS), 100U/mL kanamycin and 100. mu.g/mL streptomycin, preferably at 37 ℃ with 5% CO2In the present invention, the cells to be infected are preferably cultured by being seeded on a 6-well plate, more preferably at 1 × 10 per well6The density of individual cells was seeded in 6-well plates and cultured overnight to allow the cells to be infected to adhere and grow into a monolayer.
The invention washes the cells to be infected which grow into a single layer to obtain the washed cells. Before washing, the present invention preferably discards a culture solution used for culturing cells. The washing method of the present invention is not particularly limited, but washing with PBS (0.01mol/L, pH7.2) is preferably performed 2 times.
After the washed cells are obtained, the method preferably mixes the chlamydia abortus inoculation liquid with a DNA transfection reagent Lipofectamine2000, stands, inoculates the mixture on the washed cells, incubates, and sucks away the incubated liquid to obtain infected cells; and continuously culturing the infected cells for 48-72 hours by using a culture solution.
In the invention, the chlamydia abortus inoculation liquid is a serum-free culture liquid containing chlamydia abortus, and the serum-free culture liquid comprises RPMI-1640. The inoculation dose of the Chlamydia abortus is not particularly limited in the present invention.
In the present invention, the volume ratio of the DNA transfection reagent Lipofectamine2000 to the chlamydia abortus inoculation solution is preferably 1: (250-4000), more preferably 1: 500. The DNA transfection reagent Lipofectamine2000 can obviously enhance the infection capacity of the Chlamydia abortus on in vitro cells.
In the present invention, the time for the standing is preferably 10 min. In the invention, the incubation condition is preferably 37 ℃ and 30-60 min, and more preferably 60 min. The present invention preferably performs the incubation in an incubator. The standing of the invention can combine the Chlamydia abortus in the inoculation liquid with lipofectamine2000, promote the combination of the Chlamydia abortus and the cells to be infected during inoculation, and is beneficial to the Chlamydia entering the cells. The invention adds the inoculation liquid to the cells to be infected and places the cells at 37 ℃, and the process is incubation, and during the incubation process, chlamydia and cells are identified and enter the cells.
In the present invention, the culture solution is preferably RPMI-1640 culture solution containing 5% by mass of fetal bovine serum, 100U/mL kanamycin, 100. mu.g/mL streptomycin, and 1. mu.g/mL cycloheximide.
The method can obviously enhance the infection capacity of the Chlamydia abortus to cells, and does not need to perform complex operations such as centrifugation and the like.
The invention also provides application of the DNA transfection reagent Lipofectamine2000 in enhancing the infection capacity of the cultured cells by the Chlamydia abortus.
The cell culture method and application of Chlamydia abortus of the present invention will be described in further detail with reference to the following embodiments, but the technical solution of the present invention includes, but is not limited to, the following embodiments.
Example 1 culture of Chlamydia abortus based on McCoy cells
1 Material Process
1.1 strains and cell lines
The Chlamydia abortus SX5 strain is separated from a flowing dairy cow sample, is a vaccine-making strain of a dairy cow chlamydia disease inactivated vaccine, and has been approved by a national class 1 new veterinary medicine certificate. The Chlamydia abortus GN6 strain is a clinical isolate and is isolated from a yak aborted fetus. These two Chlamydia abortus strains were maintained at the Lanzhou veterinary institute of Chinese academy of agricultural sciences. The Chlamydia abortus strain is subcultured in McCoy cells, and the titer is determined after harvesting, and the strain is stored at-80 ℃ as seeds for later use. The titer of the Chlamydia abortus strain is expressed in inclusion body formation units per milliliter (IFU/mL), and the amount of inoculum on the cells is expressed in multiplicity of infection (MOI), which is the ratio of the IFU of Chlamydia abortus to the number of infected cells in the inoculum, i.e., 1 cell is infected per how many IFUs of Chlamydia abortus.
Mouse fibroblast McCoy, purchased from the Qingqi (Shanghai) Biotech development Co., Ltd. McCoy cells were cultured in RPMI-1640 medium containing 5% Fetal Bovine Serum (FBS), 100U/mL kanamycin, and 100. mu.g/mL streptomycin at 37 ℃ in 5% CO2Culturing in an incubator.
1.2 methods of the invention
McCoy cells were plated at 1 × 10 per well6The density of each cell was inoculated on a 6-well plate, cultured overnight to grow the cell as a monolayer, the medium was discarded, washed 2 times with PBS (0.01mol/L, pH7.2), and the Chlamydia abortus SX5 strain and GN6 strain were diluted to 8 × 10 in serum-free RPMI-1640 medium, respectively5IFU/mL of the inoculation liquid of the Chlamydia abortus, adding different doses of commercial DNA transfection reagents Lipofectamine2000 into the inoculation liquid, wherein the Lipofectamine2000 in each milliliter of the inoculation liquid is 0.25 mu L, 0.5 mu L, 1 mu L, 2 mu L and 4 mu L respectively, uniformly mixing, standing for 10min at room temperature, then inoculating the inoculation liquid of the Chlamydia abortus onto McCoy cells (MOI is 0.8) in 1mL of each well, incubating for 1h in a 37 ℃ cell incubator, finally sucking and removing the liquid after incubation in the wells, replacing the liquid with RPMI-1640 culture liquid containing 5% FBS, 100U/mL kanamycin, 100 mu g/mL streptomycin and 1 mu g/mL cycloheximide, and continuing culturing for 48-72 h.
1.3 conventional culture method
McCoy cells were plated at 1 × 10 per well6The cells were seeded at a density of 6-well plates and cultured overnight to grow a monolayer, the medium was aspirated off, washed 2 times with PBS (0.01mol/L, pH7.2), the cells were treated with 1mL of diethylaminoethyl dextran (DEAE-D) at a concentration of 30. mu.g/mL for 15min and then seeded, the Chlamydia abortus SX5 strain and the GN6 strain were diluted with serum-free RPMI-1640 medium to 8 × 10-containing cells, respectively5IFU/mL of Chlamydia abortus inoculum. The aborted chlamydia inocula were seeded onto McCoy cells at 1mL per well (MOI ═ 0.8), respectively. Placing the 6-well plate in a centrifuge at 33 deg.CCentrifuging at 1500g centrifugal force for 1 hr, removing liquid, culturing with RPMI-1640 culture solution containing 5% FBS, 100U/mL kanamycin, 100 μ g/mL streptomycin, and 1 μ g/mL cycloheximide at 37 deg.C under 5% CO2And (5) continuously culturing for 48-72 h in the incubator.
1.4 Indirect immunofluorescence staining
After 48h of inoculation and culture, the growth of Chlamydia abortus in McCoy cells was observed by indirect immunofluorescence staining. Cells were fixed with pure methanol for 10min, blocked with 1% BSA for 2h at room temperature, then incubated with commercial Chlamydia abortus Major Outer Membrane Protein (MOMP) -specific monoclonal antibody (sc-101594, 1:200) overnight at 4 ℃ and then FITC-labeled goat anti-mouse IgG for 1h at room temperature, and finally the nuclei were counterstained with 0.1mol/mL DAPI. The ability of chlamydia abortus to infect cells was expressed as the rate of inclusion body formation (inclusion body formation rate ═ number of inclusion bodies/number of cells × 100%).
2 results
2.1 the method of the invention can significantly enhance the ability of Chlamydia abortus to infect cells
The addition of different doses of Lipofectamine2000 to the Chlamydia abortus inoculation solution can increase the number of inclusion bodies formed by strains of Chlamydia abortus SX5 and GN6 in McCoy cells, so that the formation rate of the inclusion bodies is increased by 26-66 times and 41-84 times respectively (Table 1), and the obvious statistical difference is shown (A and B in figure 1. the effect of different concentrations of Lipofectamine2000 on the infection of McCoy cells by Chlamydia abortus is shown in the figure, and Lipo in the figure is the abbreviation of Lipofectamine 2000.
In FIG. 1, A shows that the enhancement effect of different concentrations of Lipofectamine2000 on the infection of McCoy cells by Chlamydia abortus strain SX5 is dose-dependent, the enhancement effect can reach the maximum when the concentration of Lipofectamine2000 in the inoculation liquid is 2 muL/mL, B shows that the enhancement effect of different concentrations of Lipofectamine2000 on the infection of cells by Chlamydia abortus strain GN6 can obviously enhance the infection capacity of GN6 strain on the cells when the concentration of Lipofectamine2000 in the inoculation liquid is 2 muL/mL, and the enhancement effect is dose-dependent, and the enhancement effect can reach the maximum when the concentration of Lipofectamine2000 in the inoculation liquid is 2 muL/mL). Lipofectamine2000 is dose-dependent in its ability to enhance the infection of Chlamydia abortus strains. The inclusion body formation rate of Chlamydia abortus in infected cells was maximized when 2. mu.L of Lipofectamine2000 was added per ml of inoculum (Table 1, FIG. 1).
TABLE 1 enhancing effect of Lipofectamine2000 on infection by Chlamydia abortus strains
2.2 the enhancement effect of the method of the present invention on the infection ability of Chlamydia abortus is equivalent to that of the conventional centrifugation method
The method of the present invention was compared with conventional centrifugation to enhance the ability of Chlamydia abortus to infect cells. The effect of Lipofectamine2000 treatment and conventional centrifugation on Chlamydia abortus infection and proliferation is shown in FIG. 2 (note: Lipo in the figure is the abbreviation of Lipofectamine 2000), wherein, A in FIG. 2 is the treatment of Lipofectamine2000 at 2. mu.L/mL (the method of the invention) and the conventional centrifugation method are respectively inoculated with Chlamydia abortus SX5 or GN6 strains with the same multiplicity of infection (MOI is 0.8) in McCoy cells, and the inclusion body formation rate of Chlamydia abortus in the cells has no significant difference; in FIG. 2, B is 2 μ L/mL Lipofectamine2000 treatment (the method of the present invention) and conventional centrifugation, which were inoculated with the same multiplicity of infection (MOI ═ 0.8) of Chlamydia abortus SX5 or GN6 strain respectively, in McCoy cells, and cultured for 72h, the titer of the harvested progeny was not significantly different.
The results show that the chlamydia abortus SX5 strain was inoculated with the same multiplicity of infection (MOI ═ 0.8) in McCoy cells using the method of the present invention or the conventional method, respectively, and the inclusion body formation rate was not significantly different (a in fig. 2). The filial generation of the Chlamydia abortus SX5 strain obtained by the method of the invention and the conventional method is collected, and the titer (expressed as IFU/mL) of the filial generation Chlamydia is determined by the method of subculture. The results show that there was no significant difference in titer between the progeny harvested by the method of the invention and conventional centrifugation (B in fig. 2). Similarly, the strain Chlamydia abortus GN6 cultured by the method of the present invention or the conventional method showed no significant difference in the rate of inclusion body formation and the titer of progeny in McCoy cells (A, B in FIG. 2). This demonstrates that the method of the invention is comparable to conventional centrifugation methods in its ability to enhance Chlamydia abortus infection.
The results show that the method can replace the conventional centrifugation method, and is more convenient and faster for routine laboratory culture, subculture proliferation and the like of the Chlamydia abortus.
Example 2 Mass propagation of Chlamydia abortus in spinner flasks based on BHK-21 cells
1 Material Process
1.1 strains and cell lines
The Chlamydia abortus SX5 strain is a vaccine-making strain of inactivated vaccine of cow chlamydia disease, and is preserved in Lanzhou veterinary research institute of Chinese academy of agricultural sciences. The Chlamydia abortus SX5 strain was subcultured in McCoy cells, and the titer was measured after harvesting and stored at-80 ℃ as seeds for future use. The titer of Chlamydia abortus is expressed in inclusion body formation units per milliliter (IFU/mL) and the amount inoculated on cells is expressed as multiplicity of infection (MOI), which is the ratio of the IFU of Chlamydia abortus to the number of infected cells in the inoculum, i.e., 1 cell is infected per how many IFUs of Chlamydia abortus.
Baby hamster Kidney cell BHK-21, a Chinese academy of sciences type culture Collection cell Bank was purchased. BHK-21 was cultured in RPMI-1640 medium containing 5% Fetal Bovine Serum (FBS), 100U/mL kanamycin and 100. mu.g/mL streptomycin at 37 ℃ in 5% CO2Culturing in an incubator.
1.2BHK-21 cells were cultured in spinner flasks
BHK-21 cell density was adjusted to 0.5 × 106150mL of the seed/mL are inoculated into 2000mL rotary bottles (surface area: 850 cm)2) Horizontally placed on a bottle rotating machine at the rotating speed of 6r/h and the temperature of 37 ℃ and the CO content of 5 percent2Culturing for 6-8 h in an incubator to allow the wall to grow into a monolayer.
1.3 the method of the invention
The culture medium in the spinner flask was discarded, BHK-21 monolayer cells were washed 2 times with PBS (0.01mol/L, pH7.2), and Chlamydia abortus SX5 strain was diluted to a titer of 5 × 10 in serum-free RPMI-1640 culture medium630mL of IFU/mL inoculum was added 60. mu.L of Lipofectamine2000 and gently mixed wellAnd standing for 10 min. Adding the inoculum into washed BHK-21 monolayer cells, horizontally placing on a rotary bottle machine, rotating at 6r/h at 37 deg.C and 5% CO2The culture box is incubated for 2h, so that the Chlamydia abortus can fully infect the BHK-21 cells. Then, the liquid in the spinner flask was discarded, and the culture was changed to 150mL of RPMI-1640 medium containing 5% FBS, 100U/mL kanamycin, 100. mu.g/mL streptomycin, and 1. mu.g/mL cycloheximide, and the culture was continued for 72 hours.
1.4 direct inoculation method
The culture medium in the spinner flask was discarded, BHK-21 monolayer cells were washed 2 times with PBS (0.01mol/L, pH7.2), and Chlamydia abortus SX5 strain was diluted to a titer of 5 × 10 in serum-free RPMI-1640 culture medium630mL of IFU/mL of the inoculation solution, adding the inoculation solution into a washed BHK-21 monolayer cell rotary bottle, horizontally placing the rotary bottle on a rotary bottle machine, rotating at the speed of 6r/h at 37 ℃ and 5% CO2The culture box is incubated for 1h, so that the Chlamydia abortus can fully infect the BHK-21 cells. Then, the liquid in the spinner flask was discarded, and the culture was changed to 150mL of RPMI-1640 medium containing 5% FBS, 100U/mL kanamycin, 100. mu.g/mL streptomycin, and 1. mu.g/mL cycloheximide, and the culture was continued for 72 hours.
1.5 progeny Chlamydia abortus Titer assay
Discarding the culture solution, collecting the cell culture in the spinner flask in 50mL serum-free RPMI-1640 culture solution, ultrasonically breaking the cells on ice, centrifuging at 4 ℃ for 3min at 3000r/min, and collecting the supernatant as the filial generation Chlamydia abortus. The titer of the progeny Chlamydia abortus was determined by inoculation into McCoy cells using conventional centrifugation methods.
2 results
Directly inoculating Chlamydia abortus vaccine strain to BHK-21 cells cultured in a spinner flask, and averagely proliferating each flask to obtain progeny Chlamydia abortus with titer of 9.03 × 105IFU/mL, whereas using the method of the invention, on average each bottle of BHK-21 cells can be propagated to obtain 2.05 × 1011IFU/mL progeny aborted Chlamydia (Table 2). The method can be used for the mass propagation of the vaccine strain of the Chlamydia abortus on the BHK-21 cells, and further can be used for the mass preparation of the Chlamydia abortus vaccine antigen.
TABLE 2 culture of Chlamydia abortus SX5 Strain in spinner flasks based on BHK-21 cells
In view of the above, the present invention provides a method for culturing Chlamydia abortus in cells which can replace the centrifugation process. The method can simplify the culture operation of the chlamydia, and can be conveniently used for the conventional passage and enrichment culture of the abortive chlamydia in a laboratory so as to improve the efficiency; can also be used for preparing vaccine antigens and challenge agents in large quantities with infectious chlamydia abortus.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A cell culture method of Chlamydia abortus comprises the following steps:
washing the cells to be infected which grow into a single layer to obtain washed cells;
mixing the abortion chlamydia inoculation liquid with a DNA transfection reagent Lipofectamine2000, standing, inoculating the mixture to the washed cells, incubating, and removing the incubated liquid to obtain infected cells;
and continuously culturing the infected cells for 48-72 hours by using a culture solution.
2. The culture method according to claim 1, wherein the cell to be infected comprises a mouse fibroblast McCoy, a mouse fibroblast L929, a small hamster kidney cell BHK-21, or a human cervical cancer cell HeLa.
3. The culture method according to claim 1, wherein the Chlamydia abortus inoculation liquid is a serum-free culture liquid containing Chlamydia abortus, and the serum-free culture liquid comprises RPMI-1640.
4. The culture method according to claim 1, wherein the volume ratio of the DNA transfection reagent Lipofectamine2000 to the Chlamydia abortus inoculum is 1: (250 to 4000).
5. The culture method according to claim 1, wherein the time for the standing is 10 min.
6. The culture method according to claim 1, wherein the incubation is carried out at 37 ℃ for 30 to 60 min.
Use of the DNA transfection reagent Lipofectamine2000 to enhance the ability of chlamydia abortus to infect cultured cells.
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