1. A method for screening a Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content, which comprises the following steps:

and (3) detection: detecting the percentage of the cells with CD163 receptors on the cell surface in the Marc-145 cell population in the Marc-145 monoclonal cell population formed by the propagation of the candidate Marc-145 monoclonal cell strain by using an indirect immunofluorescence method;

screening: taking the candidate Marc-145 monoclonal cell strain corresponding to the percentage of more than or equal to 50 percent as a Marc-145 cell strain which can be used for culturing PRRSV to obtain virus liquid with high virus content;

wherein the high virus content virus liquid is virus content more than or equal to 10^7.0TCID₅₀Viral fluid per ml.

2. The screening method according to claim 1, wherein the screening criteria are preferably: the percentage is more than or equal to 80 percent, and the virus content in the virus liquid with high virus content is more than or equal to 10 percent^8.0TCID₅₀/ml。

3. The screening method according to claim 1 or 2, wherein the cell having a CD163 receptor on the cell surface is a cell having specific fluorescence on the cell surface detected by indirect immunofluorescence.

4. The screening method according to any one of claims 1 to 3, wherein the candidate Marc-145 monoclonal cell strain is a candidate Marc-145 monoclonal adherent cell strain or a candidate Marc-145 monoclonal suspension cell strain, the cell population propagated from the candidate Marc-145 monoclonal adherent cell strain is a Marc-145 monoclonal adherent cell population, and the cell population propagated from the candidate Marc-145 monoclonal suspension cell strain is a Marc-145 monoclonal suspension cell population.

5. The screening method of claim 4, wherein the propagation of a population of Marc-145 monoclonal adherent cells from the candidate Marc-145 monoclonal adherent cell strain is performed by:

inoculating the candidate Marc-145 monoclonal adherent cell strain according to the ratio of 1 cell/holeInoculating to cell culture plate, and placing at 37 + -1 deg.C with 5% CO₂Culturing for 7-14 days by using a DMEM medium containing 10% newborn calf serum to obtain a Marc-145 monoclonal adherent cell population.

6. The screening method according to claim 4, wherein the propagation of the Marc-145 monoclonal suspension cell strain candidate to form a Marc-145 monoclonal suspension cell population is carried out by:

inoculating the candidate Marc-145 monoclonal suspension cell strain into a semi-solid agarose plate according to 1 cell/plate, and placing the semi-solid agarose plate at 37 +/-1 ℃ and 5% CO₂Culturing for 7-14 days by using a CD293 culture medium under the condition to obtain a Marc-145 monoclonal suspension cell population.

7. Screening method according to any one of claims 1 to 6, wherein the primary antibody used in the indirect immunofluorescence is an anti-CD 163 monoclonal antibody and the secondary antibody used is a FITC-labeled goat anti-mouse IgG secondary antibody.

8. A method for predicting the virus content in virus liquid obtained by culturing PRRSV by using a Marc-145 cell population comprises the steps of detecting the percentage of cells with CD163 receptors on the cell surfaces in the Marc-145 cell population by using an indirect immunofluorescence method, and predicting the virus content in the virus liquid according to the percentage value;

the prediction judgment standard is as follows:

if the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 50%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.0TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.0TCID₅₀/ml；

If the proportion of the cells with CD163 receptors on the cell surfaces in the Marc-145 cell population is less than 40 percent, culturing the Marc-145 cell populationThe virus content of PRRSV obtained virus liquid is less than 10^7.0TCID₅₀/ml。

9. The prediction method according to claim 8, wherein the prediction judgment criterion is:

if the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 90%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.44TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.11TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 70%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.61TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 50%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.22TCID₅₀/ml。

10. The prediction method according to claim 8 or 9, wherein the Marc-145 cell population is any one of a Marc-145 polyclonal adherent cell population, a Marc-145 polyclonal suspension cell population, a Marc-145 monoclonal adherent cell population, and a Marc-145 monoclonal suspension cell population; wherein the Marc-145 polyclonal adherent cell population is formed by propagation of a plurality of different Marc-145 adherent cell strains, and the Marc-145 polyclonal suspension cell population is formed by propagation of a plurality of different Marc-145 suspension cell strains.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as porcine reproductive and respiratory syndrome virus), is a contagious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which primarily affects the respiratory and reproductive systems of pigs. After infection with the virus, pigs replicate first in locally susceptible macrophages and then rapidly spread to systemic lymphoid tissues and lungs. About one week, the sick pigs have symptoms of high fever, diarrhea and the like, and the pregnant sows are easy to abortion, premature birth, stillbirth and the like after being infected with diseases. At present, the PRRSV vaccine is mainly used for preventing the porcine reproductive and respiratory syndrome in a pig farm, wherein mammalian cells are mainly used as a matrix for PRRSV proliferation in the production of the PRRSV vaccine, and the PRRSV vaccine has the advantages of high repeatability, stable production, closer antigen specificity to natural strains, less allergic reaction and the like.

The Marc-145 cell belongs to a rhesus monkey kidney cell line, is the most main host cell for in vitro proliferation of PRRSV at present, and is one of the mammalian cells most suitable for producing PRRSV vaccine. At present, in the production of PRRSV vaccines at home and abroad, virus PRRSV is usually propagated by adopting Marc-145 cells cultured by adherence or Marc-145 cells cultured by suspension, and the virus content in the obtained virus liquid is a key factor for determining the quality and the efficacy of the vaccine, so that the screening of the Marc-145 cell strain which can be used for culturing the PRRSV to obtain the virus liquid with high virus content has important significance for the research and the production of the PRRSV vaccine.

In the prior art, a screening method of a Marc-145 cell strain for culturing PRRSV to obtain a virus solution with high virus content is generally a virus infection method (for example, patent document CN103966157A, hereinafter referred to as document 1), which utilizes different Marc-145 cell strains to inoculate PRRSV after a monolayer of cells (referred to as a Marc-145 monoclonal cell group) are grown in a 96-well culture plate, observes the pathological changes of cells after the cells are cultured for 48 hours, and selects a cell strain with obvious pathological changes of cells for in vitro culture and proliferation of PRRSV to obtain a virus solution with high virus content. The cytopathic condition after virus infection directly reflects the proliferation capacity and the virulence of the virus in a culture system of the cell, so that a Marc-145 cell strain which can be used for culturing PRRSV to obtain virus liquid with high virus content can be accurately and objectively screened. However, the virus infection method disclosed in the above document 1 requires inoculation of virus into a cell line to be screened in screening a cell line capable of obtaining a high virus content, and then requires co-culture for about 48 hours to observe the cytopathic condition, so that there are problems that the operation is complicated, the time is long, and there is a possibility that there is a risk of infection during inoculation of virus.

Disclosure of Invention

In view of one or more problems in the prior art, one aspect of the present invention provides a method for screening a Marc-145 cell strain capable of being used for culturing PRRSV to obtain a virus solution with high virus content, which comprises the following steps:

and (3) detection: detecting the percentage of the cells with CD163 receptors on the cell surface in the Marc-145 cell population in the Marc-145 monoclonal cell population formed by the propagation of the candidate Marc-145 monoclonal cell strain by using an indirect immunofluorescence method;

screening: taking the candidate Marc-145 monoclonal cell strain corresponding to the percentage of more than or equal to 50 percent as a Marc-145 cell strain which can be used for culturing PRRSV to obtain virus liquid with high virus content;

wherein the high virus content virus liquid is virus content more than or equal to 10^7.0TCID₅₀Viral fluid per ml.

In the screening method, the screening standard is preferably as follows: the percentage is more than or equal to 80 percent, and the virus content in the virus liquid with high virus content is more than or equal to 10 percent^8.0TCID₅₀/ml。

In the screening method, the cell having a CD163 receptor on the cell surface is a cell in which specific fluorescence is present on the cell surface, which is detected by an indirect immunofluorescence assay.

In the screening method, the candidate Marc-145 monoclonal cell strain is a candidate Marc-145 monoclonal adherent cell strain or a candidate Marc-145 monoclonal suspension cell strain, a cell population formed by breeding the candidate Marc-145 monoclonal adherent cell strain is a Marc-145 monoclonal adherent cell population, and a cell population formed by breeding the candidate Marc-145 monoclonal suspension cell strain is a Marc-145 monoclonal suspension cell population.

In the screening method, the specific operation of breeding the candidate Marc-145 monoclonal adherent cell strain to form the Marc-145 monoclonal adherent cell population is as follows: inoculating the candidate Marc-145 monoclonal adherent cell strain on a cell culture plate according to 1 cell/hole, and placing at 37 +/-1 ℃ and 5% CO₂Culturing for 7-14 days by using a DMEM medium containing 10% newborn calf serum to obtain a Marc-145 monoclonal adherent cell population.

In the screening method, the concrete operation of breeding the candidate Marc-145 monoclonal suspension cell strain to form the Marc-145 monoclonal suspension cell population is as follows: inoculating the candidate Marc-145 monoclonal suspension cell strain into a semi-solid agarose plate according to 1 cell/plate, and placing the semi-solid agarose plate at 37 +/-1 ℃ and 5% CO₂Culturing for 7-14 days by using a CD293 culture medium under the condition to obtain a Marc-145 monoclonal suspension cell population.

In the screening method, the primary antibody used in the indirect immunofluorescence method is an anti-CD 163 monoclonal antibody, and the secondary antibody used in the indirect immunofluorescence method is a FITC-labeled goat anti-mouse IgG secondary antibody.

The invention provides a method for predicting the virus content in virus liquid obtained by culturing PRRSV by using a Marc-145 cell population, which comprises the steps of detecting the percentage of cells with CD163 receptors on the cell surfaces in the Marc-145 cell population by using an indirect immunofluorescence method, and predicting the virus content in the virus liquid according to the percentage value;

the prediction judgment standard is as follows:

if the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 50%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.0TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.0TCID₅₀/ml；

If CD163 exists on the cell surface in the Marc-145 cell populationThe cell ratio of the receptor is lower than 40%, and the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is lower than 10^7.0TCID₅₀/ml。

In the above prediction method, the prediction criterion is:

if the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 90%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.44TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.11TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 70%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.61TCID₅₀/ml；

If the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 50%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.22TCID₅₀/ml。

In the prediction method, the Marc-145 cell population is any one of a Marc-145 polyclonal adherent cell population, a Marc-145 polyclonal suspension cell population, a Marc-145 monoclonal adherent cell population and a Marc-145 monoclonal suspension cell population; wherein the Marc-145 polyclonal adherent cell population is formed by propagation of a plurality of different Marc-145 adherent cell strains, and the Marc-145 polyclonal suspension cell population is formed by propagation of a plurality of different Marc-145 suspension cell strains.

The screening method provided based on the technical scheme can detect the percentage of the cells (namely the cells with CD163 receptors on the cell surface) with specific fluorescence on the cell surface in the Marc-145 monoclonal cell population by using an indirect immunofluorescence method, and can quickly (namely the cells with CD163 receptors on the cell surface are detected by using the indirect immunofluorescence methodAbout 3 hours), can be conveniently, accurately and objectively screened to obtain the PRRSV with high virus content (not less than 10)^7.0TCID₅₀Per ml, even not less than 10^8.0TCID₅₀/ml) virus solution, is more efficient than the method (not less than 48h) for virus infection of the above document 1, and can avoid the potential infection risk caused by the need to inoculate virus to Marc-145 monoclonal cell groups in the virus infection method disclosed in the above document 1.

In another aspect, the present invention provides methods for predicting the amount of PRRSV virus in a fluid produced from a population of Marc-145 cells, wherein the average amount of PRRSV virus in a fluid obtained by culturing PRRSV using a population of Marc-145 cells is not less than 10 when the proportion of cells having CD163 receptors present on the cell surface of the cells in the population of Marc-145 cells (including the population of Marc-145 monoclonal cells and the population of Marc-145 polyclonal cells (propagated from a plurality of different Marc-145 cell strains)) reaches 50 percent, as shown in the following examples^7.0TCID₅₀Per ml; if the cell proportion of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.0TCID₅₀If the ratio of the cells with CD163 receptors on the cell surfaces in the Marc-145 cell population is less than 40 percent, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is less than 10^7.0TCID₅₀And/ml. Based on the method, in the production process of the PRRSV vaccine, before inoculating PRRSV seed viruses into the Marc-145 cell population for producing the PRRSV virus liquid, the PRRSV content in the virus liquid to be produced by the Marc-145 cell population can be estimated in advance by detecting the percentage of the cells with CD163 receptors on the cell surfaces in the Marc-145 cell population in the cell population, so that the virus content in the produced virus liquid can be estimated in advance without inoculating the seed viruses, for example, if the virus content in the virus liquid estimated according to the estimation standard is not less than 10^7.0TCID₅₀The virus solution can be directly produced if the estimated virus content in the virus solution is less than 10%^7.0TCID₅₀And/ml, the Marc-145 cell population which can be replaced by virus liquid with higher virus content is selected, so that the quality of the vaccine can be ensured, the production efficiency of the vaccine can be improved, and the production of the vaccine can be guided.

Drawings

FIG. 1 is an indirect immunofluorescence photograph of a Marc-145 polyclonal adherent cell population;

FIG. 2 is a photograph of an indirect immunofluorescence of a monoclonal adherent E7P cell population;

FIG. 3 is a photograph of an indirect immunofluorescence of a population of F9P monoclonal adherent cells;

FIG. 4 is a photograph of indirect immunofluorescence of a population of C8P monoclonal adherent cells;

FIG. 5 is a photograph of an indirect immunofluorescence of a population of D9P monoclonal adherent cells.

Detailed Description

Aiming at the defects that in the prior art, the safety problem and the time consumption are possibly caused by inoculating virus to a Marc-145 monoclonal cell population (a cell population formed by propagating a single Marc-145 cell strain) in a Marc-145 cell population screened by a virus infection method for culturing PRRSV to obtain a virus solution with high virus content, the invention aims to detect the percentage of cells (namely the cells with CD163 receptors on the cell surface) with specific fluorescence on the cell surface in the Marc-145 monoclonal cell population in the cell population by using an indirect immunofluorescence method, so that the PRRSV can be rapidly, conveniently, accurately and objectively screened to obtain the PRRSV with high virus content (not less than 10)^7.0TCID₅₀Per ml, even not less than 10^8.0TCID₅₀/ml) virus solution, can avoid the problems of potential infection risk and long time consumption caused by inoculating the virus to the Marc-145 monoclonal cell group in the existing virus infection method.

PRRSV invasion of target cells is known to be closely related to cell surface receptors, and a variety of cellular receptors and key molecules have been found to play a role in PRRSV infection, including CD163 receptors, sialic acid adhesins (Sn or CD169), heparan sulfate (HepS), Vimentin (Vimentin), CD151 receptors, and MYH9, CD209, and Siglecs (e.g., Siglec-10) molecules, among others. Wherein CD163 receptor, vimentin, CD151 receptor, MYH9 and Siglecs are receptors and key molecules existing on the surface of Marc-145 cells and mainly involved in PRRSV infection and invasion of Marc-145 cells, and all play important roles in PRRSV binding infection of Marc-145 cells and virus internalization processes, for example, vimentin can be involved in PRRSV binding with other cytoskeletal filaments and mediating PRRSV intracellular transport, MYH9 and CD163 receptor can play a role together in PRRSV infection cells to promote virus internalization, CD163 receptor can bind with different Siglecs to participate in virus invasion processes, and the like (see snowleaf et al, porcine reproductive and respiratory syndrome virus receptor and its role research progress in virus infection, animal medicine progress, 2020, 41 (12): 102-107, hereinafter referred to as reference 2). Obviously, the virus content in the virus liquid obtained by in vitro culture and propagation of PRRSV by using the natural Marc-145 cells is closely related to the total content of all receptors and key molecules which are present on the surface of the Marc-145 cells and participate in PRRSV infection and invasion into the Marc-145 cells, and is not dependent on the content of one of the receptors or key molecules, the higher the total content of these receptors and key molecules, the greater the number of viruses that infect and internalize the cell, the more likely it is to obtain a viral fluid with a high viral content, and the prior art (for example, patent document CN103525773A) has constructed Marc-145 cells capable of expressing one or more of these receptors and molecules (for example, heparan sulfate, sialoadhesin, vimentin, CD163, CD151, etc.) by gene recombination method to increase the content of receptors and molecules on the surface of Marc-145 cells, thereby increasing PRRSV infectious titer.

However, the inventors surprisingly found during PRRSV vaccine production that not all of the Marc-145 cell surfaces commonly recognized in the art for naturally occurring Marc-145 cell populations (including polyclonal cell populations propagated from different Marc-145 cell strains and monoclonal cell populations propagated from individual Marc-145 cells) have CD163 receptors on the surface (see, e.g., above-mentioned document 2, page 106, last paragraph on the left), and that cells having CD163 receptors on the surface of cells in the Marc-145 cell population (cells showing specific bright green fluorescence on the surface as measured by indirect immunofluorescence) account for the percentage of the cell population and the use of Marc-1Virus content (TCID) in virus liquid obtained by culturing and proliferating PRRSV in the outside of 45 cell population₅₀) Has a direct positive correlation without considering the total content of all receptors and key molecules on the surface of Marc-145 cells involved in PRRSV infection and invasion of Marc-145 cells. Therefore, the inventors considered that the virus content of the virus liquid obtained by culturing and proliferating PRRSV outside the cell population could be characterized by detecting the percentage of cells exhibiting specific fluorescence on the cell surface (i.e., cells having CD163 receptors on the cell surface) in the Marc-145 cell population by the indirect immunofluorescence method, and further that the percentage of cells exhibiting CD163 receptors on the cell surface in the monoclonal cell population formed by the proliferation of the individual Marc-145 cells could be detected by the indirect immunofluorescence method to screen PRRSV capable of being cultured and proliferated to obtain a high virus content (10)^7.0TCID₅₀More than or equal to 10/ml^8.0TCID₅₀More than ml) virus solution. Proved by verification, when the cell ratio of CD163 receptor on the cell surface in the Marc-145 monoclonal cell population reaches 50 percent, the virus content in the virus solution obtained by culturing and proliferating PRRSV in vitro by using the Marc-145 monoclonal cell population is not lower than 10^7.0TCID₅₀Per ml; when the cell ratio of CD163 receptor on the cell surface in the Marc-145 monoclonal cell population reaches 80 percent, the virus content in the virus solution obtained by culturing and proliferating PRRSV in vitro by using the Marc-145 monoclonal cell population is not less than 10^8.0TCID₅₀And/ml, so that Marc-145 cell strains can be screened, which can be used for culturing and propagating PRRSV to obtain virus liquid with high virus content. Based on this, the inventors also considered that the content of virus in a virus solution obtained by culturing and propagating PRRSV outside of a cell population can be predicted by detecting the percentage of cells having CD163 receptors on the cell surface in the Marc-145 cell population (including the Marc-145 polyclonal cell population (adherent or suspended) and the Marc-145 monoclonal cell population (adherent or suspended)) in the cell population by using an indirect immunofluorescence method, and thus can be used for guiding the production of PRRSV vaccines.

The present invention will be described in detail with reference to the following specific embodiments.

The methods used in the following examples are conventional methods unless otherwise specified. The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.

Example 1: detection of Marc-145 adherent cell CD163 receptor by indirect immunofluorescence method

In this embodiment, an indirect immunofluorescence method is used to detect CD163 receptors on the cell surface in a Marc-145 polyclonal adherent cell population (a polyclonal adherent cell population propagated from different Marc-145 adherent cell strains), and PRRSV is cultured and propagated outside the polyclonal adherent cell population to determine the virus content in the obtained virus solution.

1.1, culturing Marc-145 adherent cells: marc-145 adherent cells (stored in national engineering laboratories for veterinary vaccines of Jinyu Baoling biopharmaceutical GmbH) are cultured in a cell culture bottle, growth medium is DMEM medium (commercially available) containing 10% (volume percentage) newborn calf serum, and 5% CO is added at 37 DEG C₂Culturing in a constant-temperature incubator, carrying out passage for 1 time for 2-3 days, and growing monolayer cells. Digesting with pancreatin, adding growth liquid to make cell suspension at 3 × 10⁵The cells were seeded at a density of 100. mu.L/well in 96-well plates at 37 ℃ in 5% CO₂Culturing under the condition of (1), and after 48 hours, the cells are attached to the wall and spread to the bottom of the hole.

1.2 detection of cell surface CD163 receptors in Marc-145 adherent cell populations by indirect immunofluorescence

(1) Washing: discarding the growth solution in the wells of the 96-well plate in the step 1.1, and washing with PBS for 3 times, 5min each time;

(2) fixing: adding 80% acetone solution at-20 deg.C into the cell hole, fixing in refrigerator at 2-8 deg.C for 30min, washing with PBS for 3 times, each time for 5 min;

(3) primary antibody incubation: diluting anti-CD 163 monoclonal antibody (purchased from abcam) with PBS at a dilution rate of 200 times, adding into the cell well, incubating at 37 deg.C for 60min in a wet box, and washing with PBS for 5min 3 times;

(4) and (3) secondary antibody incubation: diluting FITC-labeled goat anti-mouse IgG secondary antibody (purchased from siama) with PBS (dilution factor of 300), adding into the cell well after primary antibody incubation, incubating at 37 ℃ for 60min in a wet box, and washing with PBS for 3 times (5 min each time);

(5) fluorescence observation and result judgment: the 96-well plate was placed on a fluorescent inverted microscope for observation, and the observation was performed in a dark room.

As shown in FIG. 1 (10-fold magnification), it can be seen that only a part of the cell membranes of the cells in the polyclonal adherent cell population propagated by different Marc-145 cell strains have specific fluorescence, and the proportion of the cells in the cell population is about 50%, which proves that the CD163 receptor is not present on the surface of all Marc-145 cells in the Marc-145 polyclonal adherent cell population.

1.3, culturing and proliferating PRRSV in vitro by utilizing polyclonal adherent cell population and measuring the virus content in the harvested virus liquid

Inoculating PRRSV seed virus (stored in national engineering laboratory of veterinary vaccine of Jinyubao biopharmaceutical GmbH) into the culture bottle full of the monolayer Marc-145 adherent cells in the step 1.1 according to the virus inoculation amount of 1 percent, adding DMEM maintenance solution (commercially available) containing 2 percent newborn bovine serum, culturing at 37 ℃ for 36-48 h to allow more than 80 percent of cells to have cytopathic effect, harvesting virus solution (repeating for three times), and calculating TCID according to Reed-Muench method₅₀Values are given as virus content and the results are given in table 1 below. Therefore, the virus content in the virus solution obtained by culturing and proliferating the PRRSV in the polyclonal cell population is basically kept at 10^7.33TCID₅₀About/ml.

Example 2: screening of Marc-145 monoclonal adherent cell strains that can be used for culturing PRRSV to obtain viral fluids with high viral content

In this embodiment, an indirect immunofluorescence method is used to detect CD163 receptors on the cell surface in a Marc-145 adherent cell population (a monoclonal adherent cell population propagated from a single Marc-145 adherent cell strain), and PRRSV is cultured and propagated outside the monoclonal adherent cell population to determine the virus content in the obtained virus solution.

2.1, obtaining a Marc-145 monoclonal adherent cell population: marc-145 adherent cells which are full of monolayer and have good state and no exogenous virus pollution in the step 1.1 in the example 1 are trypsinized into single scattered cells, the cells are diluted by a DMEM medium containing 10% newborn calf serum, the cells are inoculated on a 96-well cell culture plate according to 1-2 cells/well, and the 96-well cell culture plate is placed at 37 ℃ and 5% CO₂The wells containing only 1 cell were labeled, and these cell lines were named: d7, E7, G10, F9, F2, H5, C8 and D9, after the number of cells in each well is increased, carrying out expanded culture (about 7-14 days, DMEM culture medium of 10% newborn bovine serum) to obtain a Marc-145 monoclonal adherent cell population formed by propagation of a single Marc-145 monoclonal adherent cell strain, wherein the Marc-145 monoclonal adherent cell population is respectively named as follows corresponding to the monoclonal adherent cell strains: D7P, E7P, G10P, F9P, F2P, H5P, C8P, D9P.

2.2 detection of CD163 receptors on the cell surface in each Marc-145 monoclonal adherent cell population separately according to the indirect immunofluorescence detection method of step 1.2 in example 1.

The results are shown in fig. 2-5 (10 x magnification for each), which only exemplarily show fluorescence photographs of E7P, F9P, C8P and D9P monoclonal adherent cell populations, wherein fig. 2 is a fluorescence photograph of E7P monoclonal adherent cell population, fig. 3 is a fluorescence photograph of F9P monoclonal adherent cell population, fig. 4 is a fluorescence photograph of C8P monoclonal adherent cell population, and fig. 5 is a fluorescence photograph of D9P monoclonal adherent cell population. It is obvious that only part of the cell membranes of different monoclonal adherent cell populations have specific fluorescence, and the percentage of the cells with specific fluorescence on the cell membranes in different monoclonal adherent cell populations is different, i.e. the percentage of the cells with CD163 receptors on the cell surface in different monoclonal adherent cell populations is different, wherein the percentage of the cells in each monoclonal adherent cell population is shown in table 1 below. It is evident that cells with specific fluorescence of such membranes (i.e., cells with CD163 receptors present on the cell surface) account for the highest percentage of the E7P monoclonal adherent cell population, up to about 90%, while the lowest percentage of the F9P monoclonal adherent cell population is only about 20%.

2.3 propagation of PRRSV in vitro by culturing according to the method of step 1.3 of example 1 using each Marc-145 monoclonal adherent cell population obtained in step 2.1 and determining the virus content of the resulting virus fluid, the results of which are shown in Table 1 below. It can be seen that the virus content in the virus solution obtained by culturing and proliferating PRRSV outside the population of Marc-145 monoclonal adherent cells in which the percentage of the cells having CD163 receptors on the cell surface is different, and the virus content in the virus solution obtained by using the population of monoclonal adherent cells is also increased with the increase of the percentage of the cells having CD163 receptors on the cell surface in the population of monoclonal adherent cells, and the two are in a positive correlation.

From the data presented in Table 1, it is evident that when the percentage of cells having CD163 receptor present on the cell surface in the population of monoclonal adherent cells reaches about 50%, the virus content in the virus solution obtained by the multiple replicates of PRRSV propagated in culture outside the population of monoclonal adherent cells is no less than 10^7.0TCID₅₀A mean value of about 10/ml^7.22-10^7.39TCID₅₀Per ml; when the proportion of the cells with CD163 receptors on the cell surfaces in the monoclonal adherent cell population reaches about 70 percent, the virus content in the virus liquid obtained by using a plurality of repeated experiments of culturing and proliferating PRRSV outside the monoclonal adherent cell population is not less than 10^7.5TCID₅₀A mean value of about 10/ml^7.67TCID₅₀Per ml; when the proportion of the cells with CD163 receptors on the cell surfaces in the monoclonal adherent cell population reaches about 80 percent, the virus content in the virus liquid obtained by culturing and proliferating PRRSV outside the monoclonal adherent cell population for multiple times of repeated experiments is not less than 10^8.0TCID₅₀A mean value of about 10/ml^8.11TCID₅₀Per ml; when the proportion of the cells with CD163 receptors on the cell surfaces in the monoclonal adherent cell population reaches about 90 percent, multiple repeated tests for culturing and proliferating PRRSV in vitro by using the monoclonal adherent cell populationThe virus content in the virus liquid obtained by the test is not less than 10^8.33TCID₅₀A mean value of about 10/ml^8.44TCID₅₀Per ml; when the ratio of the cells with CD163 receptor on the cell surface in the monoclonal adherent cell population is less than about 50%, the virus content in the virus liquid obtained by culturing and proliferating PRRSV outside the monoclonal adherent cell population for multiple times is less than 10^7.0TCID₅₀And/ml. That is, when the proportion of the cells with CD163 receptor on the cell surface in the monoclonal adherent cell population reaches 50%, the virus content in the virus solution obtained by culturing and proliferating PRRSV outside the monoclonal adherent cell population can reach 10^7.0TCID₅₀Per ml; when the proportion of the cells with CD163 receptors on the cell surfaces in the monoclonal adherent cell population reaches 80 percent, the virus content in the virus solution obtained by culturing and proliferating PRRSV in vitro by using the monoclonal adherent cell population can reach 10^8.0TCID₅₀And/ml. Therefore, the proportion of the cells with CD163 receptor on the cell surface in the monoclonal adherent cell population can be directly utilized to screen out the cells which can be used for culturing and propagating PRRSV to obtain high virus content (not less than 10)^7.0TCID₅₀Per ml, even not less than 10^8.0TCID₅₀/ml) virus fluid, and thus among the various Marc-145 monoclonal cell strains listed in Table 1, the E7 monoclonal adherent cell strain was able to be used for the culture propagation of PRRSV to obtain the highest virus content (up to 10 on average)^8.44TCID₅₀Approximately/ml) virus fluid, and in addition, the monoclonal adherent cell strains of D7, F2, C8 and D9 can be used for culturing and propagating PRRSV to obtain the virus content of not less than 10^7.0TCID₅₀Marc-145 adherent cell strain in virus solution/ml.

Table 1: the cell ratio of CD163 receptor on the cell surface in each monoclonal cell population and the virus content in the virus solution obtained by culturing and proliferating PRRSV by using the monoclonal cell population

Example 3: detection of Marc-145 suspension cell CD163 receptor by indirect immunofluorescence method

This example uses an indirect immunofluorescence method to detect CD163 receptors on the cell surface of a Marc-145 polyclonal suspension cell population (a polyclonal suspension cell population propagated from different Marc-145 suspension cell strains), and uses this polyclonal suspension cell population for in vitro culture propagation of PRRSV to determine the viral content of the resulting viral fluid, and specifically includes the following operations.

3.1, culture of Marc-145 suspension cell population: marc-145 suspension cells (stored in national engineering laboratory of veterinary vaccine of Jinyubaoling biopharmaceutical Co., Ltd.) were cultured in a cell culture flask in a growth medium of CD293 (commercially available) at 37 deg.C with 5% CO₂The cells are cultured in a constant-temperature shaking incubator for 2 to 3 days until the cell density reaches 2 multiplied by 10⁶When the cell density is more than one/ml, the Marc-145 suspension cells are diluted to 0.5-0.8 multiplied by 10 by using a CD293 culture medium⁶And (4) carrying out passage on each cell/ml, taking the cell suspension when the cells grow to the logarithmic phase, dripping the cell suspension onto a glass slide, and placing the glass slide in an ultra-clean bench for air drying.

3.2 detection of cell surface CD163 receptors in Marc-145 suspension cell populations by Indirect immunofluorescence assay

(1) Fixing: dripping 80% acetone solution at-20 deg.C into the cell suspension on the glass slide, fixing in a refrigerator at 2-8 deg.C for 30min, and washing with PBS for 3 times, each time for 5 min;

(2) primary antibody incubation: diluting anti-CD 163 monoclonal antibody (purchased from abcam) with PBS (200 times dilution), dripping onto glass slide, incubating at 37 deg.C for 60min in a wet box, and washing with PBS for 5min for 3 times;

(3) and (3) secondary antibody incubation: diluting FITC-labeled goat anti-mouse IgG secondary antibody (purchased from sigma) with PBS (phosphate buffer solution) by 300 times, dripping onto a glass slide after primary antibody incubation, incubating at 37 ℃ for 60min in a wet box, and washing with PBS for 3 times (5 min each time);

(4) fluorescence observation and result judgment: the slide was observed on a fluorescent inverted microscope and the observation was performed in a dark room.

3.3 cell density in the flask of step 3.1 reached 2X 10⁶Inoculating PRRSV seed virus into Marc-145 suspension cell fluid per ml according to the virus inoculation amount of 1 percent, adding DMEM maintenance fluid containing 2 percent newborn bovine serum, culturing at 37 ℃ for 36-48 h to allow more than 80 percent of cells to have cytopathic effect, harvesting virus fluid (repeating for three times), and calculating TCID according to Reed-Muench method₅₀The value was taken as the virus content.

The above fluorescence observation results and virus content measurement results show that, in accordance with the results of example 1, it can also be seen that only a part of the cell membranes of the cells in the polyclonal suspension cell population propagated from different Marc-145 suspension cell strains have specific fluorescence, demonstrating that CD163 receptors are not present on the surface of all Marc-145 cells in the Marc-145 polyclonal suspension cell population. And the relation between the proportion of the cells with CD163 receptors on the cell surfaces detected by the indirect immunofluorescence method in the Marc-145 polyclonal suspension cell population and the virus content in the virus solution obtained by culturing and proliferating PRRSV in vitro by using the Marc-145 polyclonal suspension cell population conforms to the rule shown in the table 1.

Example 4: screening of Marc-145 monoclonal suspension cell strains that can be used for culturing PRRSV to obtain high virus content viral fluid

This example uses an indirect immunofluorescence method to detect CD163 receptors on the cell surface in a Marc-145 suspension cell population (a monoclonal suspension cell population propagated from a single Marc-145 suspension cell strain), and uses this monoclonal suspension cell population to culture and propagate PRRSV in vitro to determine the viral content of the resulting viral fluid, and specifically includes the following operations.

4.1, obtaining a Marc-145 monoclonal suspension cell population: culturing Marc-145 suspension cells in a triangular flask with a growth medium of CD293 at 37 deg.C with 5% CO₂The cells are cultured in a constant-temperature shaking incubator for 2 to 3 days until the cell density reaches 2 multiplied by 10⁶When the cell density is more than one/ml, the Marc-145 suspension cells are diluted to 0.5-0.8 multiplied by 10 by using a CD293 culture medium⁶Passage is carried out on each ml, and Marc-145 in logarithmic growth phase is obtainedDiluting the suspension cells into single scattered suspension cells by using a CD293 culture medium, culturing a plurality of single scattered suspension cells in a semi-solid agarose plate, and performing amplification culture (about 7-14 days, the CD293 culture medium) after the single suspension cells proliferate into cell clusters to obtain a corresponding series of Marc-145 monoclonal suspension cell populations formed by the propagation of the Marc-145 monoclonal cell strains.

4.2, when the cells in each suspension cell population in the step 4.1 grow to the logarithmic phase, dropwise adding the cell suspension onto a glass slide, placing the glass slide in a super clean bench for air drying, and detecting the CD163 receptor on the cell surface in each Marc-145 suspension cell population according to the indirect immunofluorescence method in the step 3.2 in the example 3.

4.3 propagation of PRRSV in vitro by culture using each Marc-145 monoclonal suspension cell population obtained in step 4.1, and determination of the virus content of the virus fluid obtained, according to the method of step 3.3 of example 3.

The results are consistent with those obtained in example 2, and the virus content in the virus solution obtained by culturing and propagating PRRSV in vitro using the population of Marc-145 monoclonal suspension cells in which the percentage of cells having CD163 receptors present on the cell surface is different, and the virus content in the virus solution obtained using the population of monoclonal suspension cells is increased as the percentage of cells having CD163 receptors present on the cell surface is increased, showing a clear positive correlation as described in example 2. That is, when the proportion of the cells with CD163 receptor on the cell surface in the monoclonal suspension cell population reaches 50%, the virus content in the virus solution obtained by culturing and proliferating PRRSV in vitro by using the monoclonal suspension cell population can reach 10^7.0TCID₅₀Per ml; when the proportion of the cells with CD163 receptors on the cell surfaces in the monoclonal suspension cell population reaches 80 percent, the virus content in virus liquid obtained by culturing and proliferating PRRSV in vitro by using the monoclonal suspension cell population can reach 10^8.0TCID₅₀And/ml. Therefore, the proportion of the cells with CD163 receptor on the cell surface in the monoclonal suspension cell population can be directly utilized to screen out PRRSV which can be cultured and proliferated to obtain high virus content (not less than 10)^7.0TCID₅₀/ml，Even not less than 10^8.0TCID₅₀/ml) Marc-145 monoclonal suspension cell strain of the virus fluid.

In conclusion, screening for PRRSV that can be used for in vitro culture propagation to achieve high virus content (e.g., not less than 10) can be performed by detecting the percentage of cells with CD163 receptors present on the cell surface in the Marc-145 cell population (Marc-145 monoclonal cell population (adherent or suspended)) by indirect immunofluorescence assay^7.0TCID₅₀Per ml, even not less than 10^8.0TCID₅₀/ml) virus fluid (including Marc-145 adherent cell strain and Marc-145 suspension cell strain). Based on the method, an indirect immunofluorescence method can be used for detecting the percentage of cells with CD163 receptors on the cell surfaces in the Marc-145 cell population (including the Marc-145 polyclonal cell population (attached or suspended) and the Marc-145 monoclonal cell population (attached or suspended)) to the cell population, so that the content of viruses in a virus solution obtained by culturing and propagating PRRSV outside the cell population can be predicted, the method can be further used for guiding the production of PRRSV vaccines, and the prediction judgment standard is as follows: if the cell ratio of CD163 receptor on the cell surface in the Marc-145 cell population reaches 50%, the average value of the virus content in virus liquid obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^7.0TCID₅₀Per ml; if the cell ratio of CD163 receptor on the cell surface in the Marc-145 cell population reaches 80%, the average value of the virus content in the virus solution obtained by culturing PRRSV by using the Marc-145 cell population is not less than 10^8.0TCID₅₀/ml。

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.