CN109652384B - Method for culturing hepatitis E virus in vitro - Google Patents
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Abstract
The invention discloses a method for culturing hepatitis E virus in vitro, which adopts MDCK cells to culture the hepatitis E virus in vitro, and utilizes a method of RT-nPCR, PT-qPCR and immune confocal to confirm that the hepatitis E virus can be massively replicated in the MDCK cells and can be stably passed for more than 10 generations.
Description
Technical Field
The invention belongs to the field of biotechnology, and relates to a new method for successfully culturing and continuously replicating hepatitis E virus in vitro.
Background
Hepatitis E Virus (HEV), a viral Hepatitis pathogen transmitted through the intestinal tract, can infect humans and a variety of animals across species. HEV is a single-stranded positive-strand RNA virus, and is mainly found to have 8 genotypes (HEV-1- - -HEV-8) through current research, and a genotype 4 HEV strain (HEV-4) is mainly prevalent in China. HEV transmission has been demonstrated primarily as the fecal-oral route, but also by other routes such as blood transmission, contact transmission, vertical transmission, and organ transplantation.
HEV infection is generally acute self-limiting infection, and patients can recover after self-detoxification in 4-6 weeks. However, it is very likely to develop chronic hepatitis, and liver cirrhosis and liver cancer in patients with immunodeficiency and the elderly. According to the statistics of the world health organization, about 2000 million people infect HEV worldwide each year, and about 7 million people die. In addition, the mortality rate of pregnant women infected with HEV can reach 25%, and symptoms such as premature delivery, abortion, dead fetus and postpartum acute liver necrosis of pregnant women are frequently caused. The prevention and control situation of hepatitis E is urgent, and the development of an effective hepatitis E vaccine becomes an urgent and urgent problem related to public health, and virus culture is very important in the process.
Virus culture is the basis for the study of virus biological properties, pathogenesis and vaccine development. In vitro culture of HEV is a hotspot and difficulty of research, but because HEV cannot be efficiently replicated in vitro, research on a virus replication mechanism is seriously hindered, and further development of HEV vaccine is retarded.
Although the HEV cell culture system is successfully established by utilizing the A549 cell line and the PLC/PRF/5 cell line at present. There are many problems. On one hand, cell lines used in the currently established cell culture system are cancer cell lines, which cannot truly reflect relevant symptoms of normal organisms infected with HEV, and cannot adopt the cancer cell lines to develop and produce HEV vaccines. On the other hand, higher virus titers have to be inoculated when using the existing cell culture systems (10)8copy/mL) could be maintained in vitro, and in addition the virus titer in the progeny cell suspension decreased and stable serial subculture was not possible. Based on the defects of the HEV cell culture system, an efficient and stable HEV in-vitro culture system must be established.
Disclosure of Invention
The invention aims to provide a method for culturing HEV (Hepatitis E Virus) in vitro with high efficiency and stability, which adopts MDCK cells to culture HEV strains in vitro, finds that the Virus can be massively replicated in the MDCK cells and can be continuously passed for more than 10 times, and provides an effective in vitro experimental model for deeply researching HEV pathogenicity and researching and developing vaccines.
The hepatitis E virus in-vitro culture method of the invention adopts the following steps:
(1) filtering HEV virus suspension with 0.45 μm and 0.22 μm filter membrane for sterilization, adding double-resistant solution with a volume of 2-5% of the virus suspension, treating at 4-8 deg.C for 2-3 h, preserving in liquid nitrogen, and subjecting to Real-tThe virus copy number determined by ime qPCR was 2.4X 104～3.3×105Copy number/mL, wherein the double antibody solution is a solution containing 400U/mL penicillin and 1000U/mL streptomycin;
(2) MDCK cells were cultured at 1.8X 105～2×105Inoculation to 10 cm/mL3The cell culture flask of (1) is cultured in DMEM medium containing 10 vol% fetal bovine serum at 37 ℃ and 5% CO2Statically culturing the cells in an incubator until the cells grow into a monolayer;
(3) inoculating the HEV virus suspension in the step (1) into the monolayer cells in the step (2) according to the inoculation amount of 0.8-1 mL per bottle, incubating for 2-3 hours at 35-37 ℃, and slightly shaking up once every 15-30 min to enable the virus to be fully adsorbed to the cells; adding a DMEM culture medium containing 2-5% by volume of fetal bovine serum after 2-3 hours, and adding MgCl with the final concentration of 0.01mmol/L2Protecting the integrity of virus particles and enhancing the adsorption of virus to cells, placing at 37 deg.C and 5% CO2Continuously culturing in an incubator;
(4) observing whether the MDCK cells have pathological changes every day after inoculating the HEV virus, repeatedly freezing and thawing after 80% -85% of the MDCK cells have pathological changes, collecting the pathological changes and culture solution, and storing at-80 ℃ for later use.
(5) The MDCK cells can support in-vitro culture of the hepatitis E virus and can be continuously passed for more than 10 generations by adopting the culture method, and the virus replication efficiency is increased to 8.0 multiplied by 105Copy number/mL.
Although the establishment of HEV cell culture systems is reported in the prior publications, the disclosed cell systems all adopt cancer cell lines, and the HEV in vitro culture by adopting the prior cell culture systems needs to be inoculated with viruses with higher titer (10)8copy/mL) can be maintained in vitro, and moreover, the virus titer is reduced in the progeny cell suspension, and stable continuous subculture cannot be realized; in addition, cancer cell lines cannot be used for development and production of HEV vaccines.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the MDCK cells are adopted, so that continuous subculture in vitro can be realized;
(2) after the MDCK cells are adopted for HEV in-vitro culture, a large amount of HEV antigens can be detected, and the MDCK cells are proved to be capable of supporting HEV in-vitro replication;
(3) after the MDCK cells are adopted for HEV in-vitro culture, HEV RNA can be detected in continuous cell suspension of more than 10 generations, and the HEV RNA has higher virus copy;
(4) after the MDCK cell is adopted to carry out HEV in-vitro culture, virus copies higher than the inoculation amount are detected in a progeny cell suspension, and the fact that the MDCK cell can support the reproduction of HEV is proved;
(5) the MDCK cell is adopted to culture the HEV in vitro, and the HEV vaccine can be used for research and production of the HEV vaccine in the later period.
Drawings
FIG. 1 is a schematic diagram of RT-nPCR detection of HEV RNA in culture supernatant according to the present invention; wherein 1 is an uninfected MDCK cell; 2 is HEV infected MDCK cells;
FIG. 2 is a schematic diagram of RT-qPCR detection of HEV virus copy in culture supernatant after 10 serial culture in the present invention;
fig. 3 is a schematic view of the confocal immunization of MDCK cells used in the present invention to infect HEV; the upper panel of the graph is normal MDCK cells, and the lower panel of the graph is MDCK cells 24 hours after HEV infection.
Detailed Description
The invention is explained in more detail below with reference to the drawings and exemplary embodiments, without limiting the scope of the invention; in the examples, conventional cell biology methods are mainly used, and these methods are well known to those skilled in the art. The present invention is successfully practiced in accordance with the following examples with minor modifications and alterations as are within the scope of the claims of this application.
Example 1: in vitro culture of hepatitis E virus by MDCK cells
1. MDCK cells (purchased from ATCC in USA) were taken out from liquid nitrogen and immediately placed in warm water at 37 ℃ to rapidly thaw the cell suspension; 1500 turn off centerAfter 5 minutes, transfer to 10cm3Into the cell culture flask, DMEM culture solution (purchased from GIBCO Invitrogen Corporation) containing 10% by volume of newborn bovine serum was added, cultured at 37 ℃, after growing a dense monolayer, the cells were washed with PBS (purchased from GIBCO Invitrogen Corporation), digested with pancreatin-EDTA (purchased from GIBCO Invitrogen Corporation), added to the culture solution, placed at 37 ℃ with 5% CO2 Continuously culturing in an incubator;
2. preparing 10% PBS (pH7.4) feces suspension from collected HEV positive feces for HEV separation, vigorously shaking, emulsifying feces, centrifuging at 4 deg.C and 12000g for 10min, collecting supernatant, sequentially filtering with 0.45 μm and 0.22 μm filter membrane for sterilization, adding 2% double-antibody solution (400U/mL penicillin and 1000U/mL streptomycin) of virus solution volume, treating at 4 deg.C for 3 hr, storing in liquid nitrogen, and determining virus copy number to be 2.4 × 10 by Real-time qPCR4Copy number/mL, is separated from HEV RNA positive pig manure sample of Kunming city, Yunnan, China, and the genotype is type 4;
3. the cells of example 1 (MDCK cells) were cultured at 2X 105Inoculation to 10 cm/mL3In a cell culture flask, DMEM medium containing 10% by volume fetal bovine serum is used at 37 ℃ and 5% CO2Statically culturing cells in an incubator, when the cells grow to 70-80% of a monolayer, removing a supernatant culture medium, adding 1mL HEV virus suspension into each cell culture bottle, incubating at 37 ℃ for 2h, slightly shaking every 30 minutes to enable the viruses to be fully adsorbed onto the cells, adding a DMEM culture medium containing 2% fetal calf serum by volume percentage after 2h, and simultaneously adding MgCl with the final concentration of 0.01mmol/L2Protecting the integrity of virus particles and enhancing the adsorption of virus to cells, placing at 37 deg.C and 5% CO2Continuously culturing in an incubator; when the cytopathic effect reaches 85%, repeatedly freezing and thawing to completely drop the cells, and harvesting the viruses; detecting HEV RNA in culture supernatant by RT-PCR (reverse transcription-polymerase chain reaction) on the cell suspension obtained by the above culture method (figure 1); the results demonstrate that MDCK cells can culture HEV in vitro.
Example 2: continuous in vitro culture of hepatitis E virus by MDCK cells
1. MDCK cells (purchased from ATCC in USA) were taken out from liquid nitrogen and immediately placed in warm water at 37 ℃ to rapidly thaw the cell suspension; after centrifugation at 1500 rpm for 5 minutes, the cells were transferred to 10cm3Into the cell culture flask, DMEM (purchased from GIBCO Invitrogen Corporation) culture solution containing 10% by volume of newborn bovine serum was added, incubated at 37 ℃ until a dense monolayer had grown, and then the cells were washed with PBS (purchased from GIBCO Invitrogen Corporation), digested with pancreatin-EDTA (purchased from GIBCO Invitrogen Corporation), added with the above culture solution, incubated at 37 ℃ with 5% CO2 Continuously culturing in an incubator;
2. preparing 15% PBS (pH7.4) feces suspension from collected HEV positive feces for HEV separation, vigorously shaking, emulsifying feces, centrifuging at 4 deg.C and 12000g for 10min, collecting supernatant, sequentially filtering with 0.45 μm and 0.22 μm filter membrane for sterilization, adding 5% double-antibody solution (400U/mL penicillin and 1000U/mL streptomycin) of virus solution volume, treating at 6 deg.C for 2h, storing in liquid nitrogen, and determining virus copy number to be 3.1 × 10 by Real-time qPCR4Copy number/mL;
3. the cells of example 1 (MDCK cells) were cultured at 1.8X 105Inoculation to 10 cm/mL3In a cell culture bottle, DMEM medium containing 10% by mass of fetal calf serum is used at 37 ℃ and 5% CO2Statically culturing cells in an incubator, when the cells grow to 70-80% of a monolayer, removing a supernatant culture medium, adding 0.9mL HEV virus suspension into each cell culture bottle, incubating at 36 ℃ for 2.5h, slightly shaking every 15 minutes to enable the virus to be fully adsorbed onto the cells, adding a DMEM culture medium containing 5% fetal calf serum by mass percentage after 3 hours, and simultaneously adding MgCl with the final concentration of 0.01mmol/L2Protecting the integrity of the virus particles by exposure to 5% CO at 37 deg.C2Continuously culturing in an incubator; when the cytopathic effect reaches 83%, the cells are completely shed by repeated freeze thawing, and the viruses are harvested;
4. inoculating a new MDCK cell with the culture supernatant of the HEV MDCK cell obtained in the step 3, performing the same operation as the step 3, repeatedly freezing and thawing when the cytopathic effect reaches 85% to enable the cell to completely fall off, harvesting the virus, and continuously carrying out passage for 10 generations;
5. HEV virus copies were detected by RT-qPCR on HEV in vitro cell culture supernatants of different generations (10 generations) obtained by applying the above culture method (FIG. 2). The results demonstrate that MDCK cells can culture HEV in vitro and can be efficiently passaged for over 10 passages.
Example 3: fluorescent confocal microscope observation of HEV infected MDCK cells
1. MDCK cells (purchased from ATCC in USA) were taken out from liquid nitrogen and immediately placed in warm water at 37 ℃ to rapidly thaw the cell suspension; after centrifugation at 1500 rpm for 5 minutes, the cells were transferred to 10cm3Into the cell culture flask, DMEM (purchased from GIBCO Invitrogen Corporation) containing 10% by volume of newborn bovine serum was added and cultured at 37 ℃ to grow a dense monolayer, then the cells were washed with PBS (purchased from GIBCO Invitrogen Corporation), digested with pancreatin-EDTA (purchased from GIBCO Invitrogen Corporation), added to the culture solution and placed at 37 ℃ in a 5% CO atmosphere2 Continuously culturing in an incubator;
2. preparing 15% PBS (pH7.4) feces suspension from collected HEV positive feces for HEV separation, vigorously shaking, emulsifying feces, centrifuging at 4 deg.C and 12000g for 10min, collecting supernatant, sequentially filtering with 0.45 μm and 0.22 μm filter membrane for sterilization, adding 5% double-antibody solution (400U/mL penicillin and 1000U/mL streptomycin) of virus solution volume, treating at 6 deg.C for 2h, storing in liquid nitrogen, and determining virus copy number to be 2.8 × 10 by Real-time qPCR4Copy number/mL;
3. the cells of example 2 (MDCK cells) were cultured at 1.9X 105The cells were inoculated in 6-well plates and plated in DMEM medium containing 10% fetal bovine serum by mass at 37 ℃ with 5% CO2Statically culturing cells in an incubator, when the cells grow to 70-80% of a monolayer, removing a supernatant culture medium, adding 0.8mL HEV virus suspension into each hole, incubating at 35 ℃ for 3h, slightly shaking every 20 minutes to enable the viruses to be fully adsorbed onto the cells, adding a DMEM culture medium containing fetal calf serum with the mass percentage of 3% after 2.5 hours, and simultaneously adding MgCl with the final concentration of 0.01mmol/L2Protection of the integrity of viral particlesSex, standing at 37 deg.C and 5% CO2Continuously culturing in an incubator;
4. after 24 hours of incubation, the slides were removed and the cells were washed 3 times with PBS (purchased from GIBCO Invitrogen Corporation), 4% paraformaldehyde, fixed for 15 minutes at room temperature and washed three times with PBS. HEV ORF2 monoclonal antibody (MAB 8003, purchased from Millipore, USA, diluted 1:200 with PBS) was added and treated at 37 ℃ for 1 h; PBS was washed three times, and a Fluorescein (FITC) -conjugated Affinipure Goat Anti-Mouse IgG fluorescent secondary antibody (purchased from Abcam, USA, diluted with PBS 1: 1000) was added and left at room temperature for 45 minutes; washing with PBS for three times, adding 4', 6-diamidino-2-phenylindole (DAPI, 1 microgram/mL), and standing at room temperature for 15 minutes; washing with PBS for three times, and observing HEV ORF2 capsid protein expression by fluorescence confocal microscope (red fluorescence, see FIG. 3); the results confirmed that: the MDCK cells can successfully culture the HEV in vitro.
Claims (1)
1. A method for culturing genotype 4 hepatitis E virus in vitro, which is characterized in that: MDCK cells are adopted to carry out in-vitro culture on the E genotype 4 hepatitis E virus;
the method comprises the following specific steps:
(1) sequentially filtering genotype 4 hepatitis E virus suspension through 0.45 mu m and 0.22 mu m filter membranes for sterilization, adding a double-resistant solution with the volume of 2-5% of the virus suspension, treating at 4-8 ℃ for 2-3 h, preserving in liquid nitrogen for later use, and determining the virus copy number to be 2.4 multiplied by 10 by Real-time qPCR4～3.3×105Copy number/mL, wherein the double antibody solution is a solution containing 400U/mL penicillin and 1000U/mL streptomycin;
(2) MDCK cells were cultured at 1.8X 105～2×105Inoculation to 10 cm/mL3The cell culture flask of (1) is cultured in DMEM medium containing 10 vol% fetal bovine serum at 37 ℃ and 5% CO2Statically culturing the cells in an incubator until the cells grow into a monolayer;
(3) inoculating the genotype 4 hepatitis E virus suspension in the step (1) into the monolayer cells in the step (2) according to the inoculation amount of 0.8-1 mL per bottle, and incubating for 2-3 hours at 35-37 ℃ for every 1 hourSlightly shaking up once for 5-30 min to make the virus fully adsorbed on the cells; adding a DMEM culture medium containing 2-5% by volume of fetal bovine serum after 2-3 hours, and adding MgCl with the final concentration of 0.01mmol/L2Protecting the integrity of virus particles and enhancing the adsorption of virus to cells, placing at 37 deg.C and 5% CO2Continuously culturing in an incubator;
(4) and when 80% -85% of pathological changes appear in the MDCK cells, repeatedly freezing and thawing, collecting the pathological cells and culture solution, and storing at-80 ℃ for later use.
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