CN107955803B - Method for improving culture titer of trypsin-dependent porcine epidemic diarrhea virus - Google Patents
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Abstract
The invention relates to a method for improving culture titer of trypsin-dependent porcine epidemic diarrhea viruses. The method solves the problem of proliferation of porcine epidemic diarrhea virus, and is suitable for improving culture titer of pancreatin-dependent porcine epidemic diarrhea virus. Compared with the common culture conditions, the culture titer of the trypsin-dependent porcine epidemic diarrhea virus can be improved by at least 20 times by the method.
Description
Technical Field
The invention belongs to the technical field of animal virus culture, and particularly relates to a method for improving culture titer of a pancreatin-dependent Porcine Epidemic Diarrhea Virus (PEDV).
Background
Porcine Epidemic Diarrhea (PED) is an acute and highly contagious disease of pigs, the etiology of which is Porcine Epidemic Diarrhea Virus (PEDV), and can cause symptoms of diarrhea, vomiting, dehydration, imbalance of electrolyte balance and the like of pigs, especially newborn piglets, with extremely high mortality rate. The virus infects epithelial cells of small intestine of pig to cause mucosa atrophy to cause malabsorption, and in recent years, the disease is widely spread in China, causing serious economic loss.
PEDV is difficult to culture, and researchers have tried primary and secondary cells of many pigs using various methods, all failing to complete the process. In 1988, Hoffman et al successfully isolated and cultured PEDV by adding a proper amount of pancreatin to a cell culture solution at one time for the first time, and using Vero to successfully isolate and culture the PEDV, which has great difficulty in adapting to cells, mainly because the pancreatin is needed for promoting the proliferation of viruses in the cells or activating S protein to enter the cells to form endosomes for membrane fusion, while most of the wild strains obtained by isolation are cultured by adding a certain amount of pancreatin at one time, but generally the successful culture is very difficult, and the proliferation titer is very low, which is also the main difficulty faced by the virus research at present, restricts the research and development of PEDV molecular biology to a certain extent, and seriously hinders the research and development of PED vaccines.
Therefore, for porcine epidemic diarrhea virus, which is a virus that is difficult to culture stably and efficiently, further research on culture methods and optimization of culture conditions are urgently needed in the art.
Disclosure of Invention
The invention aims to provide a method for improving culture titer of trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV).
In a first aspect of the present invention, there is provided a method of increasing the culture titer of a trypsin-dependent porcine epidemic diarrhea virus, the method comprising: inoculating the trypsin-dependent porcine epidemic diarrhea virus into Vero cells for 2 + -1 hr, preferably 2 + -0.5 hr, and culturing the Vero cells in an initial medium containing a certain amount of trypsin to propagate the trypsin-dependent porcine epidemic diarrhea virus; and in the culture process, 1.5-5 ug/ml of pancreatin is supplemented every 24 +/-4 hours.
In a preferred embodiment, the vero cells are cultured in an initial medium containing a certain amount of pancreatin, wherein the pancreatin concentration of the initial medium is 0.1-30 ug/ml; 2-20 ug/ml; more preferably 10-20 ug/ml.
In another preferred example, 2-4 ug/ml of pancreatin is added every 24 +/-2 hours in the culture process.
In another preferred embodiment, the virus is inoculated in vero cells cultured (preferably, with DMEM medium) for 2 to 5 days (preferably, for example, 3 to 4 days).
In another preferred embodiment, the virus is inoculated in Vero cells cultured for 3-4 days.
In another preferred embodiment, before inoculation of the virus, the method further comprises the following steps: vero cells were washed.
In another preferred embodiment, Vero cells are washed 2-4 times; preferably washed with PBS.
In another preferred embodiment, the vero cells are initially passaged at a concentration of 5X 105±5×104Each/ml.
In another preferred embodiment, the vero cells are initially passaged at a concentration of 2X 105±5×104Each/ml.
In another preferred example, the pancreatin-containing initial culture medium contains 15 +/-5 ug/ml of pancreatin; preferably 15 + -3 ug/ml; more preferably 15. + -.2 ug/ml.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Detailed Description
The present inventors have conducted extensive studies and conducted various analyses of conditions to provide a method for increasing the culture titer of trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV). The method solves the problem of the proliferation of the porcine epidemic diarrhea virus, and is suitable for improving the culture titer of the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV). Compared with the common culture conditions, the culture titer of the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV) can be improved by at least 20 times by the method.
In the process of researching the culture condition of the pancreatin-dependent porcine epidemic diarrhea virus, the inventor finds that the enzyme activity of the pancreatin is gradually reduced under the culture condition of 37 ℃, and particularly the enzyme activity of the pancreatin can be reduced by more than half after 1 day. Based on this finding, the present inventors have further studied and found that reduction in pancreatin enzyme activity has an effect on proliferation of PEDV in Vero cells. Therefore, the present inventors tried a method of promoting the proliferation of PEDV in culture by adding pancreatin during the culture, and conducted experiments in terms of the time of culture before cell inoculation, the amount of pancreatin added, the amount of inoculation, and the like, and verified that the titer of PEDV in Vero cells could be suitably increased by adding a fixed amount of pancreatin at regular time.
Based on the above-mentioned new findings of the present inventors, the present invention provides a method for increasing the culture titer of a trypsin-dependent porcine epidemic diarrhea virus, the method comprising: inoculating the trypsin-dependent porcine epidemic diarrhea virus into vero cells, and culturing the vero cells in an initial medium containing a fixed amount of trypsin to thereby propagate the trypsin-dependent porcine epidemic diarrhea virus; and, pancreatin was supplemented during the cell culture.
The pancreatin can be added in a timed and quantitative manner in the cell culture process. As a preferred mode of the invention, after the pancreatic enzyme-dependent porcine epidemic diarrhea virus is inoculated to African green monkey kidney cells for 2 plus or minus 1 hour, preferably 2 plus or minus 0.5 hour, the cells are cultured by an initial culture medium containing pancreatic enzyme, and 1.5-5 ug/ml of pancreatic enzyme is supplemented every 24 plus or minus 4 hours; preferably 2-4 ug/ml. More preferably, the step of supplementing pancreatin during the culture comprises the following steps: pancreatin was added 2 + -0.3 hours after the pancreatin-dependent porcine epidemic diarrhea virus was inoculated into vero cells. In a preferred embodiment of the present invention, pancreatin is added in an amount of 2.5. + -. 0.5ug/ml, and 2.5. + -. 0.5ug/ml of pancreatin is added every 24. + -.2 hours. In the preferred culture mode of the invention, proper amount of pancreatin with good activity is given to the virus, so that good conditions are created for the propagation of the virus in cells.
Media known to those skilled in the art to be useful for culturing vero cells may be used in the present invention, preferably as DMEM media. In the preparation of the initial medium containing pancreatin, pancreatin can be added to the medium to a final concentration of 15 + -5 ug/ml; preferably the final concentration is 15 plus or minus 3 ug/ml; more preferably the final concentration is 15 plus or minus 2 ug/ml; even more preferably the final concentration is 15. + -.1 ug/ml.
By observing the whole cell state of the kidney cells of the African green monkey in the culture process, the inventor finds that the cell culture for about 3 days is ideal for virus inoculation. Therefore, it is preferable to inoculate the virus after culturing the vero cells for 2 to 5 days, preferably 3 to 4 days, so as to maintain the overall cell status of the vero cells good.
In a preferred embodiment of the present invention, the method further comprises, before the inoculation of the virus: vero cells were washed. The number of washing may be determined according to actual operating conditions, for example, 2 to 4 times of washing. Preferably, the wash is performed with PBS.
As a preferred mode of the present invention, the initial cell passage concentration of Vero cells is adjusted to 2X 105±1×105Per ml; more preferably, the initial cell passage concentration of the Vero cells is adjusted to 2X 105±5×104One per ml. The initial passage concentration can ensure that the subsequent Vero cells maintain a more ideal growth state.
In the present invention, the viral Titer (TCID)50) The determination of (b) can be performed according to a method conventional in the art. For example, according to Korean Mr. King, Wang hong Wei, Wang jin Bao, porcine reproductive and respiratory syndrome Virus TCID50Determination of (D), Laiyang academy of agricultural sciences, proceedings 22 (2): 132-134, 2005; alternatively, the method set forth in the embodiments of the present invention may be followed.
In the specific embodiment of the present invention, the test strain PEDV-DR13S was obtained by comparing the time of culturing cells before inoculation, the amount of trypsin supplement, the amount of inoculation, and the likeKF650375FCS(pancreatic enzyme-dependent), the following optimal culture protocol is preferred, comprising: the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV) preferably adopts an optimal culture scheme: initial cell passage concentration of 2 x 105Culturing in a T25 cell bottle (10% FBS DMEM) for 3 days, washing, adding 1ml (30ul virus solution +15ug/ml Trypsin + DMEM) to incubate the virus for 2h, removing virus inoculation liquid, supplementing 5ml DMEM +75ug Trypsin for culturing, and supplementing 12.5ug Trypsin every 24 h.
The method effectively promotes the improvement of the culture titer of the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV), and compared with the common culture conditions, the culture titer of the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV) can be improved by at least 20 times.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
I. Experimental materials and methods
1. Experimental Material
Cell: vero cells, purchased from ATCC.
Strain: PEDV-DR13SKF650375FCS(pancreatin-dependent) was constructed as follows: the S gene in p-rPEDV vector is replaced by the S gene of a popular strain (KF650375), and the GTA of the S gene is replaced by2671-2673ntMutation to CGC2671-2673ntArtificially introducing a furin site to obtain p-PEDV-DR13SKF650375FCSvector is subjected to a padv reverse genetic manipulation system to obtain a recombinant virus PEDV-DR13SKF650375FCS(pancreatic enzyme-dependent), detailed methods are described in this document, Li C, Li Z, Zou Y, et al].Plos One,2013,8(8)::e69997.。
The reagent comprises: DMEM, FBS (Total bone Serum), PBS, 0.25% Trypsin and 0.25% Trypsin-EDTA, all available from Gibco.
2. Preliminary test for the reduction of the activity of pancreatin over time
(1) Every 24h, taking an EP tube filled with 1ml of 0.25% Trypsin, putting the EP tube into an incubator at 37 ℃, and marking the EP tube with the number shown in the table 1; for 5 consecutive days, 5 flasks (T25 flasks) of Vero cells were prepared on day 2 last;
(2) on day 5, ultraviolet irradiating on superclean bench for more than 30min, taking out the packaged PBS from 4 deg.C refrigerator, placing in water bath at 37 deg.C, and water bathing for 5-10 min;
(3) observing 5 bottles of Vero cells (fully filled) prepared before under a light mirror, then placing the Vero cells into a super clean workbench, sucking 2ml of PBS, gently shaking the cell bottles, sucking off the PBS after the bottoms of the cell bottles are all contacted with the PBS, and repeating for three times;
(4) taking out 5 0.25% Trypsin EP tubes in a 37 deg.C incubator, and separatingRespectively taking 1ml of 0.25% Trypsin from each tube, adding into a cell culture flask, shaking, and placing in CO at 37 deg.C2And (5) timing incubation in the incubator until the cells shrink round and fall off along with beating, and timing is finished.
TABLE 1 incubation time of pancreatin at 37 ℃
3. Passage of Vero cells
(1) Performing ultraviolet irradiation on an ultra-clean bench for more than 30min, simultaneously taking out the PBS, 0.25% Trypsin-EDTA and 10% FBS DMEM which are subpackaged from a refrigerator at 4 ℃, and putting the DMEM into a water bath kettle for 5-10min in water bath at 37 ℃;
(2) when the cells were observed to be completely confluent under light (typically 2-3 days since the last passage), the cells could be passaged.
(3) Sucking 2ml of PBS, gently shaking the cell bottle to ensure that the bottom of the cell bottle is contacted with the PBS, sucking off the PBS, and repeating for 3 times;
(4) adding 1ml of 0.25% Trypsin-EDTA, shaking, and placing in CO at 37 deg.C2Incubating in incubator for 1-2min (time varies with seasons, generally in warm days in spring and summer, only 3 min is needed, and in cold days in autumn and winter, 10min or more is needed); until the cells are round and contracted, and fall off with beating;
(5) adding 2ml 10% FBS DMEM to stop digestion, blowing uniformly, counting cells with a counter, and preparing 2X 105Cell sap per ml 5ml of culture medium was added to T25 flasks (9 flasks in two batches), mixed well and returned to the incubator. The specific incubation time for each tube of pancreatin is shown in table 1.
4. Proliferation of PEDV Virus
(1) Preparing DMEM with the pancreatin concentration of 15ug/ml by using 0.25% Trypsin;
(2) taking out Vero cells cultured in the previous step, slowly and gently washing the cell monolayer with PBS for 3 times, removing PBS, and adding PEDV-DR13SKF650375FCS(pancreatin-dependent) was mixed homogeneously with 1ml of pancreatin-containing DMEM (final pancreatin concentration 15ug/ml), inoculated into a Vero cell monolayer, shaken up and labeled with the sample number as shown in Table 1.
(3) Putting the cells added with the virus back to the corresponding position of the incubator at 37 ℃, incubating for 2h, then discarding the inoculation liquid, adding 5ml of warm DMEM with the pancreatin concentration of 15ug/ml, and continuing to put the cells in the incubator for culture;
(4) observing the cytopathic condition of each bottle every 24h, adding pancreatin with corresponding amount in a superclean workbench, observing the cell state and the cytopathic condition, and moving the cell bottle to a low-temperature refrigerator at minus 80 ℃ for temporary storage when most cell samples fall off more than 80%.
5. Detoxification of PEDV virus
(1) Repeatedly freezing and thawing the virus cell culture solution temporarily stored in the low-temperature refrigerator at-80 ℃ for 3 times at-80 ℃ and 37 ℃;
(2) transferring to a 10ml centrifuge tube during the last melting, and centrifuging at 4 ℃ and 3000rpm for 10 min;
(3) taking out the supernatant, and subpackaging into 2ml freezing tubes, wherein each tube contains 1.5 ml;
(4) the mark is made and stored at-80 ℃.
6. Titration of PEDV Virus
(1) Plating 2X 10 into flat bottom 96-well plates5Each/ml of Vero cells, each 100 mu L, after marking, putting the Vero cells in an incubator for culturing for 48 h;
(2) after the cells grow into a monolayer, washing the cells twice by PBS (100 ul per well), adding DMEM (100 ul per well), and putting the cells into an incubator for 24 hours;
(3) after the cells grew into monolayers, the PEDV was diluted 10-fold in a round bottom 96-well plate, and 6 replicates were performed for each dilution for each generation;
(4) sucking and removing cell culture supernatant, inoculating diluted PEDV to a cell monolayer, shaking uniformly, marking the number and date of a sample, and culturing at 37 ℃;
(5) observing day by day until no new lesion hole appears;
(6) calculation of TCID 50:
calculated according to the formula log TCID50 ═ Xa-D (SP-0.5);
xa is the log of the dilution at which the last row all appeared to be diseased;
d is the log of the dilution factor (e.g., 10-fold dilution, log10 is 1);
SP is the number of positive wells between the last row with all CPE and the first row without all CPE/number of parallel wells made per dilution;
the total number of the test samples was 18, and details of the time of cell culture before inoculation, the amount of trypsin supplementation, and the amount of inoculation of each sample are shown in Table 2.
TABLE 2
Example 1 preliminary test for the reduction of the pancreatic enzyme Activity with time
In this example, it was examined whether or not the activity of pancreatin decreases with time at 37 ℃. The enzymatic activity of pancreatin was indirectly demonstrated by the time the cells were digested with pancreatin. The procedure of the method is as described above "2 preliminary test for the reduction of the activity of pancreatin over time".
The time for incubation of the cells at 37 ℃ for different periods of time for trypsinization is given in Table 3.
TABLE 3
As is clear from Table 3, the longer the pancreatin was cultured at 37 ℃ the longer the time required for the cells to be digested. After 1 day of culture, the time required for the pancreatin to digest the cells is prolonged by 4 times, which indirectly suggests that the enzymatic activity of the pancreatin is reduced by about 4 times. This result also suggests that since PEDV virus (pancreatin-dependent) requires pancreatin to promote its proliferation in Vero cells, this decrease in pancreatin activity has a great influence on the proliferation of PEDV in Vero cells.
Example 2 incubation time of cells before sample inoculation
Cell cultures were performed and the time of culture of the cells before inoculation of the samples was examined as described in the method steps of "Experimental materials and methods 4-6" above.
The details of the respective sample states are shown in Table 4.
As can be seen from Table 4, the whole cell state and titer of samples Nos. 1 to 9 were better than those of samples Nos. 10 to 18, so that the inoculation was more desirable after 3 days of cell culture, and the initial cell passage concentration was 2X 105And each cell is cultured for 3 days or more than 3 days, otherwise, the Vero cells have poor state at the pancreatin concentration of 15ug/ml after inoculation.
TABLE 4
In the table, the titer after harvest, i.e., the titer 2 days after the addition of pancreatin, was determined by the "titer determination".
Example 3 differentiation of samples not supplemented with pancreatin from supplemented with pancreatin
Cell culture was performed as described in the method steps described in "test materials & methods 4-6" above, and the samples were examined for differences between not supplemented pancreatin and supplemented pancreatin.
The results of the titer determination for each sample are shown in Table 5.
TABLE 5
As can be seen from Table 5, the titer of the pancreatin-supplemented sample (supplemented with a certain amount of pancreatin every 24 h) was significantly higher than that of the sample No. 10 (not supplemented with pancreatin), indicating that the regular supplementation of pancreatin to the medium can promote the proliferation of the virus.
Example 4 sample supplementation with Trypsin amount
Cell culture was performed and the samples were examined for the preferred range of trypsin supplementation as described in "titration of PEDV Virus", 6, supra.
The mean titers of samples supplemented with different amounts of pancreatin are given in Table 6.
TABLE 6
As can be seen from Table 6, when the amount of pancreatin added every 24h is controlled to be 2.5ug/ml, the titer of the virus is about 20 times different from that of the virus without adding pancreatin, and the titer is not increased linearly when the pancreatin is added in large dose, so that the analysis is probably related to the fact that the pancreatin concentration is too high, which causes bad cell state, even death in advance, and the virus cannot proliferate.
According to the above embodiments, the present inventors consider that: the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV) preferably adopts an optimal culture scheme (T25 cell culture flask cells): initial cell passage concentration of 2X 105Culturing cells in a 96-well plate (10% FBS DMEM) for 3 days, washing, adding 1ml (30ul virus solution and DMEM containing pancreatin) to incubate the virus for 2h, removing virus inoculation liquid, supplementing 5ml DMEM, and culturing at a final pancreatin concentration of 15ug/ml in a culture system; pancreatin (concentration 2.5ug/ml) was added every 24 h. When the trypsin-dependent Porcine Epidemic Diarrhea Virus (PEDV) is cultured by the above method, the virus titer can be increased by at least 20 times compared with the conventional culture conditions.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (8)
1. A method of increasing culture titer of a trypsin-dependent porcine epidemic diarrhea virus, comprising: culturing Vero cells for 3-4 days; inoculating the porcine epidemic diarrhea virus with the porcine epidemic diarrhea virus, inoculating the porcine epidemic diarrhea virus into the Vero cells for 2 +/-1 hours, and culturing the Vero cells with an initial culture medium containing 15 +/-2 ug/ml of pancreatic enzyme so as to propagate the porcine epidemic diarrhea virus; and 2.5ug/ml pancreatin is supplemented every 24 plus or minus 2 hours in the culture process; wherein the pancreatin-dependent porcine epidemic diarrhea virus is PEDV-DR13SKF650375 FCSIt is constructed as follows: in p-rPEDV vectorThe S gene of (1) is replaced with the S gene of the epidemic strain KF650375, and the GTA of the S gene is substituted with the GTA of the S gene2671-2673ntMutation to CGC2671-2673 ntArtificially introducing a furin site to obtain p-PEDV-DR13SKF650375FCSvector is subjected to a padv reverse genetic manipulation system to obtain a recombinant virus PEDV-DR13SKF650375FCS。
2. The method of claim 1, wherein vero cells are cultured in a starter medium containing a quantitative amount of pancreatin, the starter medium having a pancreatin concentration in an amount of 15 ug/ml.
3. The method of claim 2, wherein pancreatin is added at 2.5ug/ml every 24 hours.
4. The method of claim 1, further comprising, prior to inoculation with the virus: vero cells were washed.
5. The method of claim 4, wherein vero cells are washed 2-4 times.
6. The method of claim 5, wherein vero cells are washed with PBS.
7. The method of claim 1, wherein vero cells are initially passaged at a concentration of 5 x 105±5×104One per ml.
8. The method of claim 7, wherein vero cells are initially passaged at a concentration of 2 x 105±5×104One per ml.
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