CN117343910A - Method for improving PED virus expression - Google Patents
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 50
- 239000011780 sodium chloride Substances 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 22
- 238000011081 inoculation Methods 0.000 claims description 5
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Abstract
The present invention provides a method for increasing expression of PED virus. The method comprises the following steps: the PED virus is inoculated into a culture medium containing ST cells, and the culture is carried out by adjusting the osmotic pressure value of the culture medium, so that the improvement of the expression of the PED virus is realized. The cell strain used in the invention is an ST cell strain, the effect of improving the virus yield is achieved by adjusting and maintaining the osmotic pressure of a cell culture solution in a virus expression stage, and finally, a proper osmotic pressure control range value is selected, and the improvement of PED virus expression is achieved by adjusting the osmotic pressure value of a culture medium for culture, so that the virus titer of virus expression can be remarkably improved.
Description
Technical Field
The invention relates to a virus production process, in particular to a method for improving PED virus expression.
Background
Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute diarrhea disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) with diarrhea, vomiting and dehydration of piglets as main clinical symptoms, and finally, the electrolyte balance is destroyed to cause death of pathogenic pigs. At present, the scope of the spread of PED is wider, case reports are all available worldwide, meanwhile, the incidence rate of PED is high, the infection rate of young pigs is up to 100%, the infection mortality rate of piglets below 7 days old is 80% -100%, and the frequent occurrence of PED causes serious economic loss to the pig industry.
At present, many studies have been conducted to clinically isolate PED virus and to conduct cell infection experiments in vitro in an attempt to propagate the virus by in vitro culture for use in the development of PED virus vaccines. The ST cells are pig testicular cells (SFVs), which are widely used for the isolation, in vitro proliferation and production of various veterinary vaccines of Swine Fever Virus (SFV), pseudorabies virus (PRV), porcine parvovirus (SFV) and the like.
In the aspect of virus inoculation process, the traditional PED virus vaccine production process refers to that after ST cells grow to a certain density, liquid is changed and then PED virus is inoculated or PED virus is inoculated directly. However, the primary virus culture process is slow, the virus titer is not high enough, and the problem to be solved is that the vaccine is developed. In the prior art, a CD ST culture medium is used for culturing ST cells and inoculating PED virus for PED virus expression, and the effect of improving the PED virus titer is achieved by mainly adjusting parameters of cell culture and optimizing culture control parameters such as rotating speed, pH and temperature of a virus expression stage when the PED virus expression is improved, so that the effect of improving the PED virus titer is limited.
In view of this, the present invention has been made.
Disclosure of Invention
It is an object of the present invention to provide a method for increasing expression of PED virus. The method has the effects of improving the virus yield by adjusting and maintaining the osmotic pressure of the cell culture fluid in the virus expression stage, and finally screening out the proper osmotic pressure control range value.
It is a second object of the present invention to provide a PED virus expression product. By usingThe method in the technical proposal can adjust the osmotic pressure value of the cell culture solution in the virus expression stage to be controlled and maintained at 360+/-10 mOsm/Kg by adding sodium chloride reagent, and can control the virus titer of virus expression from 7.50Log (TCID) 50 Increase in/mL) by 8.50Log (TCID) 50 /mL), the virus titer of PED antigen or seed virus at harvest is increased.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
in a first aspect, the present invention provides a method of increasing expression of a PED virus, the method comprising the steps of:
the PED virus is inoculated into a culture medium containing ST cells, and the culture is carried out by adjusting the osmotic pressure value of the culture medium, so that the improvement of the expression of the PED virus is realized.
In the animal cell culture process, most culture mediums have osmotic pressure values of 280-320 mOsm/Kg, and the feed culture medium and other reagents added in the culture process have certain influence on the osmotic pressure; the cell strain used in the invention is an ST cell strain, the effect of improving the virus yield is achieved by adjusting and maintaining the osmotic pressure of a cell culture solution in a virus expression stage, and finally, a proper osmotic pressure control range value is selected, and the improvement of PED virus expression is achieved by adjusting the osmotic pressure value of a culture medium for culture, so that the virus titer of virus expression can be remarkably improved.
Preferably, the osmotic pressure value is regulated in the range of 320 to 410mOsm/Kg, for example, 320mOsm/Kg, 330mOsm/Kg, 340mOsm/Kg, 350mOsm/Kg, 360mOsm/Kg, 370mOsm/Kg, 380mOsm/Kg, 390mOsm/Kg, 400mOsm/Kg, 410mOsm/Kg, etc., preferably 350 to 370mOsm/Kg.
The invention adds sodium chloride reagent to adjust the osmotic pressure value of the cell culture liquid in the virus expression stage, when the osmotic pressure is controlled and maintained at 360+/-10 mOsm/Kg, the virus titer of virus expression can be controlled from 7.50Log (TCID) based on the original PED virus expression process 50 Per mL) to 8.50Log (TCID) 50 Per mL), the virus titer of PED antigen or seed virus at harvest is greatly improved.
Preferably, the osmotic pressure value of the culture medium is adjusted by adding sodium chloride to the culture medium.
Preferably, the concentration of sodium chloride in the medium is 0 to 10.0g/L, for example, 0.001g/L, 0.01g/L, 0.1g/L, 0.3g/L, 0.5g/L, 0.7g/L, 1.0g/L, 2.0g/L, 5.0g/L, 10.0g/L, etc., preferably 0.3 to 1.0g/L.
In the invention, the mass and the volume of the added sodium chloride have better linear relation, and the formula is shown in the following formula I:
m= 1.1302 ×v+0.4535 formula I
Wherein m represents the mass of sodium chloride and V represents the volume of sodium chloride.
Preferably, the ST-cell-containing medium is prepared by the steps of:
inoculating ST cells into a culture medium, and performing cell culture; and diluting the cultured ST cells by adopting a fresh culture medium, and adjusting the density of living cells in the culture medium.
Preferably, the medium is a serum-free CD ST medium, preferably CD ST 258 medium.
Preferably, the ST cells are seeded at a density of 0.3X10 6 ～4.0×10 6 The cell/mL may be, for example, 0.3X10 6 cells/mL、0.4×106cells/mL、0.5×10 6 cells/mL、0.6×10 6 cells/mL、0.7×10 6 cells/mL、0.8×10 6 cells/mL、0.9×10 6 cells/mL、1.0×10 6 cells/mL、2.0×10 6 cells/mL、3.0×10 6 cells/mL、4.0×10 6 cells/mL, etc., preferably 1.0X10 6 cells/mL。
Preferably, the time for culturing the cells is 60 to 84 hours, for example, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, etc., preferably 72 hours.
Preferably, the ST viable cell density in the diluted medium is 1.0X10 6 ～6.0×10 6 The cell/mL may be, for example, 1.0X10 6 cells/mL、2.0×10 6 cells/mL、3.0×10 6 cells/mL、4.0×10 6 cells/mL、5.0×10 6 cells/mL、6.0×10 6 cells/mL, etc., preferably 3.0X10 6 cells/mL。
Preferably, the virus receiving time TOI of the PED virus is 22-26 h, for example, 22h, 23h, 24h, 25h, 26h and the like, preferably 24h.
Preferably, the inoculation amount of the PED virus is 0.03-0.1 MOI, for example, 0.03MOI, 0.04MOI, 0.05MOI, 0.06MOI, 0.07MOI, 0.08MOI, 0.09MOI, 0.1MOI, etc., preferably 0.065MOI.
The incubation time after inoculation with PED virus is preferably 54 to 96 hours, and may be, for example, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, etc., and preferably 72 hours.
In the present invention, after the cell density was adjusted to a proper range by supplementing fresh medium, which is referred to herein as virus expression for 0h, and sampling was performed every 6h during the virus expression period, biochemical parameters of the cell culture broth were measured using NOVA, and the osmolality value of the culture broth was measured using a freezing point osmometer, preferably harvested when PED virus was expressed for 72h.
As a preferred technical scheme of the invention, the method for improving the expression of PED virus specifically comprises the following steps:
(1) ST cell culture: according to 0.3X10 6 ～4.0×10 6 cell/mL inoculation density inoculating cells in serum-free CD ST culture medium, cell culturing for 60-84 h, and counting cells every day;
(2) Regulation of ST cell density: according to the viable cell density at the ST cell culture stage, fresh serum-free CD ST medium is added for dilution, and the cell density is adjusted to 1.0X10 6 ～6.0×10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating the ST-containing cells into the culture medium according to TOI of 0.03-0.1 h and MOI of 0.03-0.1 MOI;
(4) Culturing: the culture is carried out by adjusting the osmotic pressure value of the culture medium in such a way that sodium chloride is added into the culture medium, and the osmotic pressure value is adjusted to be 350-370 mOsm/Kg.
Preferably, after expression of the PED virus is complete,PED virus titer was 7.5Log (TCID 50 Per mL) or more, for example, 7.5Log (TCID) 50 /mL)、7.6Log(TCID 50 /mL)、7.8Log(TCID 50 /mL)、8.0Log(TCID 50 /mL)、8.1Log(TCID 50 /mL)、8.2Log(TCID 50 /mL)、8.3Log(TCID 50 /mL)、8.4Log(TCID 50 /mL)、8.5Log(TCID 50 /mL)、8.6Log(TCID 50 /mL)、8.7Log(TCID 50 /mL)、8.8Log(TCID 50 /mL)、8.9Log(TCID 50 /mL)、9.0Log(TCID 50 /mL), etc., preferably 8.5Log (TCID) 50 /mL) of the above.
Compared with the prior art, the invention has the following beneficial effects:
the cell strain used in the invention is an ST cell strain, the effect of improving the virus yield is achieved by adjusting and maintaining the osmotic pressure of a cell culture solution in a virus expression stage, and finally, a proper osmotic pressure control range value is selected, and the improvement of PED virus expression is achieved by adjusting the osmotic pressure value of a culture medium for culture, so that the virus titer of virus expression can be remarkably improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a linear graph showing the effect of NaCl addition on the osmotic pressure of the CD ST 258 medium.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.
It is noted that in the following description, specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than those herein described, and those skilled in the art may readily devise numerous other arrangements that do not depart from the spirit of the invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.
Preparation examples 1 to 9
This preparation provides a medium for ST cells with different NaCl contents, the NaCl addition and the osmotic pressure for the CD ST 258 medium are shown in Table 1 below:
TABLE 1
Preparation examples 10 to 14
The 200g/L sodium chloride addition volume (volume conversion formula I below) and the osmotic pressure for the CD ST 258 medium are shown in Table 2 below:
m= 1.1302 ×v+0.4535 formula I
Wherein m represents the mass of sodium chloride and V represents the volume of sodium chloride.
TABLE 2
Note that: in practice, the osmotic pressure of the culture broth was adjusted using 200g/L NaCl solution according to the data in Table 1.
The NaCl solution with the concentration of 200g/L is selected as an osmotic pressure regulating reagent, the NaCl concentration in 100mL of culture solution can be increased by 1g/L only by adding 0.5mL at a time, and the osmotic pressure value of the CD ST 258 culture medium can be increased by about 30mOsm/Kg according to the graph by 1g/L of NaCl, so that the requirement on osmotic pressure regulation of the shake flask culture solution is met and the culture volume is not influenced.
Example 1
The present embodiment provides a method for increasing expression of PED virus, the method comprising the steps of:
(1) ST cell culture: according to 1.0X10 6 cells/mL were inoculated in CD ST 258 medium, cell cultured at 37℃for 72h, cell counts per day;
(2) Regulation of ST cell density: according to the living cell density from ST cell culture stage to 72h, fresh culture medium is added to adjust the cell density to 3.0X10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating virus (isolated virus XJ-HM strain, KM 386647.1) into the culture medium containing ST cells according to TOI 24h and 0.065MOI, and adjusting the culture temperature to 35 ℃;
(4) Culturing: sampling cell counts at each of 18h, 24h, 42h, 48h, 72h and 96h for virus expression, detecting biochemical parameters of the cell culture fluid using NOVA, detecting osmotic pressure value of the culture fluid using a freezing point osmometer, and harvesting PED virus expression for 72 h; wherein, the culture is carried out by adjusting the osmotic pressure value of the culture medium in such a way that sodium chloride is added into the culture medium, and the osmotic pressure value is adjusted within the range of 300mOsm/Kg.
Example 2
The present embodiment provides a method for increasing expression of PED virus, the method comprising the steps of:
(1) ST cell culture: according to 1.0X10 6 cells/mL were inoculated in CD ST 258 medium, cell cultured at 37℃for 72h, cell counts per day;
(2) Regulation of ST cell density: according to the living cell density from ST cell culture stage to 72h, fresh culture medium is added to adjust the cell density to 3.0X10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating viruses into the culture medium containing ST cells according to the inoculum size of TOI for 24h and 0.065MOI, and adjusting the culture temperature to 35 ℃;
(4) Culturing: sampling cell counts at each of 18h, 24h, 42h, 48h, 72h and 96h for virus expression, detecting biochemical parameters of the cell culture fluid using NOVA, detecting osmotic pressure value of the culture fluid using a freezing point osmometer, and harvesting PED virus expression for 72 h; wherein, the culture is carried out by adjusting the osmotic pressure value of the culture medium in such a way that sodium chloride is added into the culture medium, and the osmotic pressure value is adjusted to be 330mOsm/Kg.
Example 3
The present embodiment provides a method for increasing expression of PED virus, the method comprising the steps of:
(1) ST cell culture: according to 1.0X10 6 cells/mL were inoculated with cells in CD ST 258 medium, cultured at 37℃for 72h, and counted daily
(2) Regulation of ST cell density: according to the living cell density from ST cell culture stage to 72h, fresh culture medium is added to adjust the cell density to 3.0X10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating viruses into the culture medium containing ST cells according to the inoculum size of TOI for 24h and 0.065MOI, and adjusting the culture temperature to 35 ℃;
(4) Culturing: sampling cell counts at each of 18h, 24h, 42h, 48h, 72h and 96h for virus expression, detecting biochemical parameters of the cell culture fluid using NOVA, detecting osmotic pressure value of the culture fluid using a freezing point osmometer, and harvesting PED virus expression for 72 h; wherein, the culture is carried out by adjusting the osmotic pressure value of the culture medium in such a way that sodium chloride is added into the culture medium, and the adjusting range of the osmotic pressure value is 360mOsm/Kg.
Example 4
The present embodiment provides a method for increasing expression of PED virus, the method comprising the steps of:
(1) ST cell culture: according to 1.0X10 6 cells/mL were inoculated with cells in CD ST 258 medium, cultured at 37℃for 72h, and counted daily
(2) Regulation of ST cell density: according to the living cell density from ST cell culture stage to 72h, fresh culture medium is added to adjust the cell density to 3.0X10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating viruses into the culture medium containing ST cells according to the inoculum size of TOI for 24h and 0.065MOI, and adjusting the culture temperature to 35 ℃;
(4) Culturing: sampling cell counts at each of 18h, 24h, 42h, 48h, 72h and 96h for virus expression, detecting biochemical parameters of the cell culture fluid using NOVA, detecting osmotic pressure value of the culture fluid using a freezing point osmometer, and harvesting PED virus expression for 72 h; wherein, the culture is carried out by adjusting the osmotic pressure value of the culture medium in such a way that sodium chloride is added into the culture medium, and the osmotic pressure value is adjusted to be 400mOsm/Kg.
Comparative example 1
This comparative example improves a method of improving expression of PED virus, the method comprising the steps of:
(1) ST cells were cultured using CD ST 258 medium and inoculated with PED virus for PED virus expression. In the ST cell culture stage: according to 1.0X10 6 Inoculating cells in cells/mL, culturing the cells for 72 hours, and counting cells every day;
(2) Regulation of ST cell density: according to the living cell density from ST cell culture stage to 72h, fresh culture medium is added to adjust the cell density to 3.0X10 6 cells/mL, recorded as virus expression for 0h at this time;
(3) Virus inoculation: inoculating viruses into the culture medium containing ST cells according to the inoculum size of TOI for 24h and 0.065MOI, and adjusting the culture temperature to 35 ℃;
(4) Culturing: sampling cell counts at each of 18h, 24h, 42h, 48h, 72h and 96h for virus expression, detecting biochemical parameters of the cell culture fluid using NOVA, detecting osmotic pressure value of the culture fluid using a freezing point osmometer, and harvesting PED virus expression for 72 h;
wherein, the PED virus expression is improved by optimizing culture control parameters, taking a 10L reactor as an example: specifically, the rotation speed of the culture is 70-100 rpm, the pH of the culture medium is 7.0-7.2, and the temperature of the virus expression stage is 35 ℃.
Test example 1
Test sample: the methods for expressing the PED virus provided in examples 1 to 4, the method for expressing the PED virus provided in comparative example 1, and the samples of the positive control group during the expression for 54 to 96 hours;
test items: virus titer of the sample during 54-96 h expression;
the positive control group preparation method comprises the following steps: according to PCD 3.0X10 6 The cells/mL, TOI 0h and 0.04MOI were inoculated with the virus in the above ST-cell-containing medium, and the culture temperature was adjusted to 35℃and harvested for 72h. Reactor culture parameters: taking a 10L reactor as an example: specifically, the rotation speed of the culture is 70-100 rpm, the pH of the culture medium is 7.0-7.2, and the temperature of the virus expression stage is 35 ℃.
The details are shown in table 3 below:
TABLE 3 Table 3
As shown in Table 3, the present invention can achieve the effect of improving virus productivity by adjusting and maintaining the osmotic pressure of the cell culture solution during the virus expression stage.
Among them, in 54-96 hours of culture after inoculating PED virus, a more suitable osmotic pressure control range value of 360+ -10 mOsm/Kg was found, and the virus titer at each time point in the range was significantly improved over that of comparative example 1 (the process method for improving PED virus expression in the prior art) and the positive control group.
In particular, when the culture time after inoculating PED virus reaches 72 hours, and the osmotic pressure value of the cell culture solution in the virus expression stage is controlled and maintained within the preferred range of 360+/-10 mOsm/Kg by adding sodium chloride reagent, the virus titer of virus expression can be improved from 7.50Log (TCID 50/mL) to 8.80Log (TCID 50/mL) on the basis of the original PED virus expression process (comparative example 1), and the virus titer of PED antigen or seed virus at the time of harvest is remarkably improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A method of increasing expression of PED virus, said method comprising the steps of:
the PED virus is inoculated into a culture medium containing ST cells, and the culture is carried out by adjusting the osmotic pressure value of the culture medium, so that the improvement of the expression of the PED virus is realized.
2. A method of increasing expression of PED virus according to claim 1, wherein the osmolality value is adjusted in the range 320 to 410mOsm/Kg, preferably 350 to 370mOsm/Kg.
3. The method of increasing expression of PED virus according to claim 1 or 2, wherein the osmolality value of the culture medium is adjusted by adding sodium chloride to the culture medium.
4. A method of increasing expression of PED virus according to claim 3 wherein the concentration of sodium chloride in the culture medium is from 0 to 10.0g/L, preferably from 0.3 to 1.0g/L.
5. The method of claim 1, wherein the ST cell-containing medium is prepared by:
inoculating ST cells into a culture medium, and performing cell culture; and diluting the cultured ST cells by adopting a fresh culture medium, and adjusting the density of living cells in the culture medium.
6. The method of increasing the expression of PED virus according to any one of claims 1 to 5, wherein the medium is a serum-free CD ST medium, preferably a CD ST 258 medium.
7. The method of claim 5, wherein the ST cells are seeded at a density of 0.3 x 10 6 ～4.0×10 6 cells/mL, preferably 1.0X10 6 cells/mL。
Preferably, the time for the cell culture is 60 to 84 hours, preferably 72 hours.
8. The method of claim 5, wherein the density of ST viable cells in the diluted medium is 1.0x10 6 ～6.0×10 6 cells/mL, preferably 3.0X10 6 cells/mL。
9. The method of claim 1, wherein the PED virus has a virus pickup time TOI of 22 to 26 hours, preferably 24 hours;
preferably, the inoculation amount of the PED virus is 0.03-0.1 MOI, preferably 0.065MOI;
preferably, the incubation time after inoculation with PED virus is 54-96 hours, preferably 72 hours.
10. The method of increasing expression of PED virus according to claim 1, wherein said PED virusAfter expression was complete, the PED virus titer was 7.5Log (TCID 50 Per mL) or more, preferably 8.5Log (TCID) 50 /mL) of the above.
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