CN113234665A - Primary endometrial cell line for PEDV replication and separation method thereof - Google Patents

Abstract

The invention discloses a primary endometrial cell line for PEDV replication and a separation method thereof, wherein the primary endometrial cell line is obtained by the following steps: (1) taking the uterus of a healthy adult sow, washing the uterus with a PBS (phosphate buffer solution) containing double antibodies, cutting the endometrium, and washing the uterus with a PBSS (phosphate buffer solution); (2) collecting porcine endometrium epithelium, and cutting to 1mm³After the small pieces are washed by PBS buffer solution, 0.1% collagenase (0.1 mg/ml) with the volume being two times that of the small pieces is added, and the small pieces are digested for 2 hours, and are shaken during the digestion to be uniformly digested; terminating digestion with an equal volume of cell culture medium containing 10% serum; (3) sieving with 100nm cell sieve, centrifuging at 500g for 5min, removing supernatant, and precipitating to obtain pig endometrium epithelial cells; (4) adding the precipitate into culture medium, performing heavy suspension culture to obtain porcine endometrium cells, and continuously passaging for 7-8 times, or freezing and storing in liquid nitrogen. Cells of the inventionThe system can realize better infection of PEDV on primary porcine endometrial cells.

Description

Primary endometrial cell line for PEDV replication and separation method thereof

Technical Field

The invention belongs to the technical field of biological products, and particularly relates to a primary endometrial cell line for PEDV replication and a separation method thereof.

Background

Porcine Epidemic Diarrhea Virus (PEDV) is an acute, highly contagious Porcine intestinal infectious disease that causes diarrhea, vomiting, dehydration and high mortality to suckling piglets. PEDV can infect pigs of various ages, but suckling piglets are more susceptible and their mortality and morbidity approaches 100%. In recent years, the popularity of PED is more and more extensive, new variant strains continuously appear, the antigenicity of commercial vaccines and epidemic strains is not matched, and the immune effect is poor, so that the healthy development of the pig industry in China is seriously influenced. In the prior art, the research on the PEDV on primary endometrial cells is not carried out, and the research needs to be accelerated in the later period. PEDV can effectively replicate on small intestinal mucosal epithelial cells (IPEC-J2), but other cell infection studies in pigs are few, so that a novel PEDV infected cell model is provided, which has important biological significance and lays a foundation for the subsequent studies on virus receptors, pathogenic mechanisms and the like.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a primary endometrial cell line for PEDV replication and a separation method thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for isolating a primary endometrial cell line for PEDV replication comprising the steps of:

(1) taking the endometrium epithelium of the pig: taking the uterus of healthy adult sows for useWashing with PBS buffer solution containing double antibody, cutting endometrium, washing with PBSS buffer solution, collecting pig endometrium epithelium, and cutting to 1mm³Small pieces of (2);

(2) digestion: washing the porcine endometrium epithelium with PBS buffer, adding 0.1% collagenase (0.1 mg/ml) with twice volume, digesting for 2 hours, and shaking to make it digest uniformly; terminating digestion with an equal volume of cell culture medium containing 10% serum;

(3) separation: sieving with 100nm cell sieve, centrifuging at 500g for 5min, removing supernatant, and precipitating to obtain pig endometrium epithelial cells;

(4) culturing: and adding the precipitate into a culture medium, and performing heavy suspension culture to obtain the porcine endometrial cells, wherein the porcine endometrial cells can be continuously passed for 7-8 times, or the porcine endometrial cells are frozen and stored in liquid nitrogen, and the cell state is not influenced after recovery.

Further, the PBS buffer solution containing the double antibody adopts a penicillin-streptomycin solution to prevent bacterial infection.

The invention also provides a primary endometrial cell line for PEDV replication, obtained by the isolation method of the invention.

Has the advantages that: the cell line disclosed by the invention can support the replication of PEDV, the PEDV can better infect primary porcine endometrial cells to cause cytopathy, and the difference between the PEDV and TCID50 of PEDV infected Vero cells is smaller, so that the cell line can be used for the passage and related research of the PEDV. Meanwhile, researches show that the vaccine has reference significance for later-stage production of the PEDV vaccine, and lays a foundation for researches on whether breeding disorder diseases are caused after the PEDV is infected with porcine endometrial cells and vertical spread of PEDV placenta.

Drawings

FIG. 1 is a graph of the lesion section changes induced by infection of primary porcine endometrial cells with PEDV in the examples;

FIG. 2 is an IFA immunofluorescence assay profile following infection of primary porcine endometrial cells by PEDV in the examples;

FIG. 3 is a line graph comparing TCID50 after infection of PEDV-infected porcine trophoblast cells with IPEC-J2, Vero cells in the examples.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

A primary endometrial cell line for PEDV replication, isolated by the steps of:

(1) taking the endometrium epithelium of the pig: collecting uterus of healthy adult sow, washing with PBS buffer solution containing double antibody, cutting endometrium, washing with PBSS buffer solution, collecting endometrium epithelium of pig, and cutting to 1mm³Small pieces of (2);

(2) digestion: washing the porcine endometrium epithelium with PBS buffer, adding 0.1% collagenase (0.1 mg/ml) with twice volume, digesting for 2 hours, and shaking to make it digest uniformly; terminating digestion with an equal volume of cell culture medium containing 10% serum;

(3) separation: sieving with 100nm cell sieve, centrifuging at 500g for 5min, removing supernatant, and precipitating to obtain pig endometrium epithelial cells;

(4) culturing: adding the precipitate into a culture medium, and performing heavy suspension culture to obtain porcine endometrial cells, wherein the porcine endometrial cells can be continuously passed for 7-8 times, or the porcine endometrial cells are frozen and stored in liquid nitrogen, and the cell state is not influenced after recovery; thereby obtaining a primary endometrial cell line.

To observe the lesion sectioning changes caused by PEDV infection of primary porcine endometrial cells, the primary endometrial cell lines obtained in this example were subjected to a PEDV infection control experiment, and divided into an infected group (MOI =1 group) and an uninfected virus control group (Mock group). The results are shown in FIG. 1, where the cell infection pattern is shown with lesions and the cell status is changed: endometrial epithelial cells were infected with PEDV-CV777 at MOI =1, and after 48 h of infection (48 hpi), a difference in status was observed compared to cells in the uninfected virus control group (Mock group), and by 72 h after infection, a more pronounced cytopathy was observed in the virus-infected cells.

To observe the IFA immunofluorescence profile after infection of primary porcine endometrial cells by PEDV, the primary endometrial cell lines obtained in this example were subjected to a PEDV infection control experiment, and divided into an infected group (MOI =1 group), a virus-uninfected control group (Mock group), an infected group to which pancreatin was added (MOI =1+ pancreatin group), and a control group to which pancreatin was added (Mock + pancreatin group). As a result, as shown in FIG. 2, it can be seen that the virus particles emit green light by IFA: endometrial epithelial cells were infected with PEDV-CV777 at a MOI =1, while 2 μ l of EDTA-free pancreatin per ml of control and infected media were added. After infection for 48 h, IFA detection was performed. PEDV was found to replicate efficiently 48 h after infection (48 hpi) compared to control uninfected cells, but no significant enhancement of the PEDV virus was found in the infected group with pancreatin.

For comparison of PEDV-infected porcine endometrial epithelial cells with IPEC-J2 and Vero cells in TCID50 after infection, the primary endometrial cell lines obtained in this example were subjected to a PEDV infection comparison experiment, and porcine endometrial epithelial cells, IPEC-J2 and Vero cells were infected with PEDV, respectively, and virus solutions were collected at different infection time points. The collected virus fluid was then assayed for TCID50 on Vero cells. As a result, shown in FIG. 3, it was found that PEDV on Vero cells had the highest TCID50, and that the TCID50 of porcine endometrial epithelial cells was significantly higher than the TCID50 on IPEC-J2.

Claims (3)

1. A method for isolating a primary endometrial cell line that is capable of being used for replication of PEDV, comprising the steps of:

(1) taking the endometrium epithelium of the pig: collecting uterus of healthy adult sow, washing with PBS buffer solution containing double antibody, cutting endometrium, washing with PBSS buffer solution, collecting endometrium epithelium of pig, and cutting to 1mm³Small pieces of (2);

(2) digestion: washing the porcine endometrium epithelium with PBS buffer, adding 0.1% collagenase (0.1 mg/ml) with twice volume, digesting for 2 hours, and shaking to make it digest uniformly; terminating digestion with an equal volume of cell culture medium containing 10% serum;

(3) separation: sieving with 100nm cell sieve, centrifuging at 500g for 5min, removing supernatant, and precipitating to obtain pig endometrium epithelial cells;

(4) culturing: and adding the precipitate into a culture medium, and performing heavy suspension culture to obtain the porcine endometrial cells, wherein the porcine endometrial cells can be continuously passed for 7-8 times, or the porcine endometrial cells are frozen and stored in liquid nitrogen, and the cell state is not influenced after recovery.

2. The method of claim 1, wherein the PBS buffer containing the diabody is a penicillin-streptomycin solution to prevent bacterial infection.

3. A primary endometrial cell line for PEDV replication, obtained by the isolation method of claim 1 or 2.

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