Method for separating and amplifying duck circovirus by using duck embryo primary hepatocytes

Abstract

The invention discloses a method for separating and amplifying duck circovirus by using duck embryo primary hepatocytes, and relates to the technical field of animal virology and cell culture. According to the method, a trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) and collagenase IV double-enzyme digestive system is creatively combined and the HGF is added, so that the yield and the activity of primary hepatocytes are remarkably improved, and the duck circovirus is quickly separated on the basis of the yield and the activity of the primary hepatocytes. According to the technical scheme, the duck embryo liver is adopted as a cell source, the cell survival rate is increased by 16.5% by shortening the digestion time and optimizing the enzyme ratio, an HGF stable cell membrane structure is innovatively introduced, a complete DuCV in-vitro proliferation system is established, and the virus titer reaches a peak value 60 hours after inoculation. According to the method, the duck circovirus is rapidly separated and amplified by using the duck embryo primary hepatocytes, and the living cell percentage of the primary cells is improved, so that the problems of low duck circovirus separation efficiency, low primary cell utilization rate, high cost and the like in the existing method are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal virology and cell culture technology, and in particular to a method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes. BACKGROUND

[0002] Duck embryo liver cell (DEL) is a core in vitro model in the study of avian virology, developmental biology and drug metabolism. The traditional isolation method generally relies on single collagenase digestion, and the subsequent adjustment of digestion time, centrifugation parameters and basic medium formula to obtain hepatocytes. However, this method has defects such as low cell yield, poor viability and short functional maintenance time, which cannot meet the needs of high-sensitivity virus isolation and long-term culture. As a unique primary cell source of waterfowl, DEL plays an irreplaceable role in analyzing pathogen replication mechanisms, vaccine research and drug screening. However, its in vitro culture is prone to dedifferentiation, and the metabolic function maintenance time is short, so it is necessary to optimize the culture system to prolong its active period.

[0003] Duck circovirus disease (DuCVD) is an immunosuppressive infectious disease caused by duck circovirus (DuCV), which can infect all age and breed of duck flocks, and is scattered or endemic throughout the year, especially in densely populated areas. The disease can be transmitted vertically and horizontally through contact, aerosol and other horizontal routes, often forming synergistic infection with duck parvovirus, duck Riemerella anatipestifer and other pathogens, increasing the clinical mortality rate and seriously restricting the healthy breeding development of China's waterfowl industry.

[0004] Currently, the in vitro culture of DuCV faces three major technical bottlenecks: (1) the existing cell lines support low efficiency of virus replication; (2) the survival time of primary hepatocytes is short; (3) the virus titer is difficult to meet the requirements of vaccine preparation. Therefore, it is of urgent practical significance to develop a DEL high-efficiency isolation and culture method that is simple to operate, reproducible and can be implemented under ordinary laboratory conditions, and to establish a DuCV rapid proliferation platform based on it for in-depth study of pathogeny, immunology and vaccinology. SUMMARY

[0005] To solve the above technical problems, the purpose of the present application is to provide a method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes, which can quickly isolate and amplify duck circovirus using duck embryo primary hepatocytes, and improve the percentage of viable cells of primary cells, in order to solve the problems of low isolation efficiency of duck circovirus, low utilization rate of primary cells and high cost of existing methods.

[0006] The technical scheme for solving the above technical problems is as follows: a method for separating and amplifying duck circovirus by using duck embryo primary hepatocytes is provided, and the method comprises the following steps:

[0007] After the liver tissue of the SPF duck embryo is washed, a digestive solution composed of trypsin-EDTA and collagenase IV is added for digestion treatment, and after filtering and sterilizing, a duck embryo primary hepatocyte suspension is obtained; hepatocyte growth factor HGF is added for continuous cell culture, duck circovirus is inoculated into the obtained monolayer cells, and after incubation, the virus liquid is cultured and harvested.

[0008] Further, after the 16-17 day old SPF duck embryo with good development is wiped with alcohol, it is washed in D-Hank's buffer solution, and the duck embryo primary hepatocytes are separated under sterile conditions.

[0009] Further, the duck embryo primary hepatocytes are added into 3-4 times the volume of the digestive solution for digestion treatment; the digestive solution is composed of 0.25% trypsin-EDTA and 0.4% collagenase IV mixed at a volume ratio of 3:1.

[0010] The simultaneous addition of trypsin-EDTA and collagenase IV in the digestive solution can preferentially degrade the collagen fibers in the liver tissue, reduce the excessive digestion of a single enzyme, and increase the percentage of viable primary cells, compared with single enzyme digestion, the percentage of viable primary cells is increased by 16.5% after 48 h of culture. During the culture of the primary cells, the addition of hepatocyte growth factor HGF stabilizes the cell membrane, reduces cell apoptosis caused by enzyme treatment, and improves the quality of duck embryo primary hepatocytes.

[0011] Further, the concentration of hepatocyte growth factor HGF is 5 ng / mL.

[0012] Further, the concentration of collagenase IV is 0.3-0.5 mg / mL.

[0013] Preferably, the concentration of collagenase IV is 0.4 mg / mL.

[0014] The above method for separating and amplifying duck circovirus by using duck embryo primary hepatocytes comprises the following steps:

[0015] S1, the 16-17 day old SPF duck embryo with good development is placed on the clean bench, the eggshell surface is wiped with 75 vt% alcohol, the eggshell air chamber is tapped with sterile forceps, the embryo body is taken out, D-Hank's buffer solution is added for immersion and washing 2-3 times to remove residual blood on the surface; then the liver is taken out using sterile forceps, the gallbladder is removed, and then transferred to a culture flask for washing to remove impurities, and the supernatant is aspirated;

[0016] S2, the cleaned liver tissue is added into 3-4 times of the digestion solution of 0.25% trypsin-EDTA and 0.4% collagenase IV, the volume ratio is 3:1, and the mixture is subjected to magnetic stirring digestion and decomposition at 37 DEG C for 8-10 min, then an equal volume of DMEM medium containing 10% fetal bovine serum FBS is added to terminate the digestion, the mixture is filtered with sterilized gauze, and the cells are collected after centrifugation and cultured to the adherent state;

[0017] S3, the mixture is transferred to a culture bottle, and hepatocyte growth factor HGF is added, and the mixture is continuously cultured at 37 DEG C and 5% carbon dioxide.

[0018] Further, in step S3, when the confluence is 85%, duck circovirus is inoculated by using a multi-point adsorption method, and the inoculation amount is 0.5 mL / 25 cm 2 of culture area, after adsorption for 1-2 h, the virus liquid is sucked off, DMEM medium containing 2% fetal bovine serum FBS is added, and the supernatant is collected.

[0019] Further, after continuous culture, cell suspensions are collected at different time points, 200 uL of the diluted cell suspension is taken, 200 uL of 0.4% trypan blue staining solution is added, the mixture is mixed, and then added dropwise to a cell counting plate for counting, so as to calculate the percentage of living cells of duck embryo primary hepatocytes.

[0020] Further, the obtained duck embryo primary hepatocytes are subjected to PAS staining function evaluation, and the glycogen storage capacity of the hepatocytes after being digested by the trypsin-EDTA and collagenase IV double enzyme digestion method is compared by staining.

[0021] Further, when the confluence is about 85%, duck circovirus is inoculated by using a multi-point adsorption method, and the inoculation amount is 0.5 mL / 25 cm 2 of culture area, after adsorption for 1-2 h, the virus liquid is sucked off, DMEM medium containing 2% fetal bovine serum FBS is added, and the supernatant is collected. 50 The TCID

[0022] The present application has the following beneficial effects:

[0023] 1. The existing duck embryo primary hepatocyte separation and culture method needs to use a complex device in the treatment process, and may face the challenge of external environmental pollution, and is strict in the requirements for technology and environment, while the method of the present application selects 16-17 day old SPF duck embryos, which can be easily realized on a clean bench, reduces the risk of pollution, and provides a safe guarantee for the in vitro culture research of duck embryo hepatocytes.

[0024] 2. Existing culture methods often result in over-digestion and cell damage during liver cell isolation due to the difficulty in controlling the digestion process, leading to a decrease in the survival rate of live cells. In the method of this invention, trypsin-EDTA and collagenase IV are added simultaneously, requiring only one digestion process, which is time-saving and yields a high live cell yield.

[0025] 3. Since the conventional cell sieving method yields a small number of primary hepatocytes and undoubtedly increases the separation cost and operational complexity, the method of this invention uses sterilized gauze to filter and resuspend the cells, which facilitates the subsequent acquisition of a large number of duck embryo hepatocytes.

[0026] 4. After primary hepatocytes were isolated, the addition of hepatocyte growth factor (HGF) to the culture medium stabilized the cell membrane and reduced cell apoptosis caused by enzyme treatment. After 48 h of culture, the percentage of viable cells increased by 16.5% compared with the control group.

[0027] 5. The isolated duck embryo primary hepatocytes were inoculated into cell flasks, and duck circovirus was inoculated into DEL cells. A high titer of duck circovirus was produced in just 60 hours, achieving DuCV in a short time.

[0028] 6. This invention innovatively combines a trypsin-EDTA and collagenase IV dual-enzyme digestion system with the addition of hepatocyte growth factor (HGF), significantly improving the yield and activity of primary hepatocytes, and enabling rapid isolation of duck circovirus. The technical solution of this invention uses the livers of 16-17 day old SPF duck embryos as the cell source. By shortening the digestion time (8-10 minutes) and optimizing the enzyme ratio, the cell viability is increased by 16.5%. The innovative introduction of 5 ng / mL HGF effectively stabilizes the cell membrane structure, establishing a complete in vitro DuCV proliferation system. The viral titer reaches its peak 60 hours after inoculation. This method is simple to operate, has good reproducibility, and provides an efficient technical platform for DuCV etiological research, vaccine development, and antiviral drug screening, with broad application prospects. Attached Figure Description

[0029] Figure 1 This is a diagram of primary hepatocytes from duck embryos cultured for different times in Example 1;

[0030] Figure 2 This is a diagram showing the number of viable cells determined by the trypan blue staining method in Example 1.

[0031] Figure 3 This is a diagram showing the identification of AFP and ALB genes in Example 2;

[0032] Figure 4 This is a glycogen staining image of primary hepatocytes from duck embryos in Example 2;

[0033] Figure 5 Growth curve of duck embryo primary hepatocytes for Example 3;

[0034] Figure 6 CPE of duck circovirus in duck embryo primary hepatocytes for Example 4;

[0035] Figure 7 Proliferation curve of duck circovirus in duck embryo primary hepatocytes for Example 4. DETAILED DESCRIPTION

[0036] The principles and features of the present application are described below, and the examples are used only to explain the present application and not to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0037] Example 1: Isolation and culture of SPF duck embryo primary hepatocytes

[0038] Test method: Take 16-17 day old well developed SPF duck embryos, place the air chamber upwards on the egg tray, place it on the clean bench, wipe the duck egg surface with 75 vt% alcohol, make a small hole at the air chamber end, cut off the eggshell with sterile tweezers and tear open the allantoic membrane, hold the duck embryo neck, take out the embryo and place it in a small culture dish, add D-Hank's buffer in advance, wash 2-3 times to remove the residual blood on the surface; use sterile tweezers to take out the liver, carefully remove the gallbladder, do not need to cut it, transfer it to the cell culture flask, wash repeatedly to remove blood cells and other impurities in the hepatocytes, and aspirate the supernatant.

[0039] The washed liver tissue obtained by sterile treatment is added with 3-4 times the volume of 0.25% trypsin-EDTA (control group) and 3-4 times the volume of 0.25% trypsin-EDTA + 0.4% collagenase IV (test group) respectively, and the primary hepatocytes are digested by single enzyme digestion and enzyme combination digestion respectively, and the digestion is carried out on a magnetic stirrer at 37 ℃ for 8-10 min. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum FBS is added to terminate the digestion, and the filtered and sterilized gauze is filtered, and the cells are collected by centrifugation; fresh primary DEL cells are inoculated into 6-hole cell culture plates, 6 holes per plate, 1 mL of cell suspension per hole, and the volume of medium in each hole is supplemented to 2 mL with DMEM growth medium. After dispersing the cells on the cross-shaking plate, they are placed in a CO2 cell culture incubator with a set culture condition of 5% CO2 and 37 ℃.

[0040] The test group cells obtained above were added with 5 ng / mL hepatocyte growth factor HGF and continued to be cultured in a 37℃ 5% carbon dioxide incubator, and the cell suspensions of the control group and the test group were collected at different time points, 200 μL of the diluted cell suspension was taken, 200 μL of 0.4% trypan blue staining solution was added, and then it was mixed and dropped into a cell counting plate for counting, so as to calculate the percentage of live duck embryo primary hepatocyte cells.

[0041] Test results: The duck embryo primary hepatocytes were separated and cultured by rapid digestion method, and were observed at 24 h, 48 h and 72 h respectively. Figure 1 The cells were epithelial cell-like, the nucleus was in the middle of the cell, and binucleate hepatocytes could be seen, which was consistent with the typical characteristics of hepatocyte morphology. The cell suspension and 0.4% trypan blue solution were mixed at a ratio of 9:1, and the results of duck embryo primary hepatocyte live cells showed that with the increase of time, the number of cells was the most at 48 h, compared with the control group, the percentage of primary cell live cells increased by 16.5% when cultured for 48 h. Figure 2

[0042] Example 2: Identification of duck embryo primary hepatocytes

[0043] I. AFP and ALB gene identification

[0044] Test method: The test group DEL cells were separated and cultured to a six-well cell plate, when the cell confluence reached 90%, the original cell culture solution was discarded, washed with PBS for 2 times, and the cell RNA was extracted by using a cell RNA extraction kit, and the albumin (Albumin, ALB) and alpha fetoprotein (Alpha fetoprotein, AFP) were amplified by reverse transcription PCR (Reverse Transcription Polymerase Chain Reaction, RT-PCR) using identification primers ALB-F / R, AFP-F / R, and the primer sequences are shown in Table 1.

[0045] Table 1 AFP and ALB gene identification primers

[0046]

[0047] Test results: ALB is secreted by hepatocytes, and AFP is mainly derived from embryonic hepatocytes. The results show that when the identification primers ALB-F / R, AFP-F / R are used to identify the DEL cells, the amplification product sizes are 1934 bp and 1829 bp respectively. Figure 3 ), the amplification band is correct, and the PCR successfully identifies the ALB and AFP genes.

[0048] II. Glycogen staining identification of duck embryo primary hepatocytes ​

[0049] Test method: DEL cells were inoculated in 48-well cell plates, divided into 2 groups, each group repeated 3 holes, after the cells grow monolayer, using PAS fixing fluid fixation 10-15 min, after fixation PBS wash the fixing fluid, dry naturally after adding oxidant, room temperature oxidation 15-20 min, PBS wash 2-3 times, each time 2 min, drop Schiff staining solution, room temperature avoid light staining 10-20 min; sodium sulfite solution drop wash 2 times, each time 2 min, PBS rinse, drop Mayer hematoxylin staining solution re-staining, washed with water, dry, placed under inverted microscope observation.

[0050] Test results: after PAS glycogen staining, the test group and the control group, the glycogen can be observed purple red cell distribution, compared with the control group, the test group hepatocyte cytoplasm contains rich glycogen (Fig. 2) Figure 4 ), homogeneous or granular distribution.

[0051] Example 3: determination of the growth characteristics of primary hepatocytes

[0052] Test method: DEL cells were inoculated in 96-well plates, 6.0×10 3 cells per well; each group was divided into 5 groups, each group 3 repeats. The inoculation time was recorded as 0 h, then 10 μL of CCK-8 was added 1 h before the continuous different time points, and the absorbance value at 450 nm was determined by enzyme marker; with time as abscissa, absorbance value as ordinate, draw duck embryo primary hepatocyte growth curve, as shown in Figure 5 .

[0053] Test results: as shown in Figure 5 , the DEL cell growth curve showed "S" type. Among them, the cell proliferation was slow before 24 h, the logarithmic growth period was from 24 h to 72 h, the cell number reached the maximum at 72 h, the cell growth entered the platform period from 72 h to 96 h, and the cell number decreased at 96 h. Compared with the control group, the duck embryo primary hepatocytes in the test group could survive and proliferate in the environment of in vitro culture, and the growth condition was good.

[0054] Example 4: duck circovirus infected duck embryo primary hepatocytes

[0055] Test method: according to the culture conditions of Example 3 to prepare duck embryo primary hepatocytes, when the growth confluence degree reaches about 85%, use D-Hank's buffer to wash 2-3 times, inoculate 0.5 mL duck circovirus liquid to each duck embryo primary hepatocyte, adsorb for 1-2 h, then discard the virus liquid, add DMEM culture medium containing 2% fetal bovine serum FBS, collect the virus liquid at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h after infection, draw the growth curve of duck circovirus by determining the median tissue culture infectious dose (TCID 50 ) of duck circovirus.

[0056] Test result: inoculate duck circovirus to duck embryo primary hepatocytes, the attack group produces obvious cytopathic effect (CPE) Figure 6 ), the cell intercellular space increases, part of the cell is stretched into a filamentous shape, a large number of cells are shed, gathered, and obvious CPE changes occur; the control group cells grow well without cytopathic effect. Collect the virus liquid at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h after infection, and determine the TCID 50 Results are shown in Figure 7 , the duck circovirus titer continuously increases with the increase of infection time, reaches the peak at about 60 h after infection, and then slowly decreases.

[0057] The above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for isolating and amplifying duck circovirus by using duck embryo primary hepatocytes, characterized in that, It comprises the following steps: The SPF duck embryo liver tissue is washed, then a digestive solution composed of trypsin-EDTA and collagenase IV is added for digestion treatment, and the duck embryo primary hepatocyte suspension is obtained after filtering and sterilization; hepatocyte growth factor HGF is added for cell culture, and duck circovirus is inoculated into the obtained single-layer cells, and the virus liquid is harvested after incubation and culture.

2. The method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes according to claim 1, wherein, The 16-17 day old SPF duck embryo is wiped with alcohol, then washed in D-Hank's buffer, and the duck embryo primary hepatocytes are isolated under sterile conditions.

3. The method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes according to claim 1, wherein, The duck embryo primary hepatocytes are added into 3-4 times the volume of digestive solution for digestion treatment; the digestive solution is composed of 0.25% trypsin-EDTA and 0.4% collagenase IV mixed at a volume ratio of 3:

1.

4. The method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes according to claim 1, wherein, The concentration of the hepatocyte growth factor HGF is 5 ng / mL.

5. The method for isolating and amplifying duck circovirus using duck embryo primary hepatocytes according to claim 1, wherein, The concentration of the collagenase IV is 0.3-0.5 mg / mL.

6. The method for isolating and amplifying duck circovirus by using duck embryo primary hepatocytes according to any one of claims 1-5, characterized in that, It comprises the following steps: S1, the 16-17 day old SPF duck embryo is placed on the clean bench, the eggshell surface is wiped with 75 vt% alcohol, the eggshell air chamber end is tapped with sterile forceps, the embryo body is taken out, added into D-Hank's buffer, immersed and washed 2-3 times to remove residual blood on the surface; then the liver is taken out using sterile forceps, the gallbladder is removed, and then transferred to a culture flask, washed to remove impurities, and the supernatant is aspirated; S2, the washed liver tissue is added into 3-4 times the volume of digestive solution composed of 0.25% trypsin-EDTA and 0.4% collagenase IV, the volume ratio is 3:1, and the digestion and decomposition are carried out at 37℃ under magnetic stirring for 8-10 min, then an equal volume of DMEM medium containing 10% fetal bovine serum FBS is added to terminate the digestion, filtered with sterile gauze, centrifuged to collect the cells, and cultured to the adherent state; S3, transfer to a culture flask, add hepatocyte growth factor HGF, continue to culture at 37℃ and 5% carbon dioxide, and inoculate duck circovirus when the confluence is 85% and culture.

7. The method for isolating and amplifying duck circovirus by using duck embryo primary hepatocytes according to claim 6, wherein, In step S3, when the confluence is 85%, duck circovirus is inoculated by multi-point adsorption method, the inoculation amount is 0.5 mL / 25 cm2 culture area, after adsorption for 1-2 h, the virus liquid is aspirated, DMEM medium containing 2% fetal bovine serum FBS is added, and the supernatant is collected.

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