Cell system for indicating bovine foamy virus infection and application thereof

Abstract

The invention discloses a cell line of indicating cow foam viral infection and the application, which expresses EGFP in the specific condition of BFV infection and indicates cow foam viral infection indirectly by detecting the expression of the protein. The construction process of cell line (the preservation number is CGMCC NO 1989) comprises the following steps: inserting full-length gene of EGFP in the response unit downstream of a specific response cow foam viral trans-activation factor and constructing a report plasmid answering cow foam viral infection firstly; infecting BHK-21cell with the plasmid by means of liposome secondly; proceeding 1:5 cell passage after the second day of infection; screening by adding G418; dying a good deal of cells after a week; growing cells in-line until 20 days; proceeding cell monoclonal operation with 1 / 2 finite dilution gauging after first-screening; getting positive clone(indicating cell line of BFV) without background expression and with a EGFP high expression quantity after virus attack.

Description

Technical field

[0001] The invention relates to a cell line for detecting bovine foamy virus (BFV) infection, a method for establishing the cell line and its specific application.

Background technique

[0002] Before the emergence of indicator cell lines, the identification methods of virus infection in cell culture mainly include observation of cytopathic method, plaque test, neutralization test, immunofluorescence, metabolic inhibition, erythrocyte adsorption, interference phenomenon, etc. Viruses multiply in sensitive cells, often causing changes in cell metabolism, cell pathology and release of virus into the medium. In this case, cytopathic, color change and detection of virus antigens in the medium can be used to determine the virus. presence and titer. However, the virus titer determined by this method depends on the naked eye observation of the experimenter, and different experimenters may obtain different results.

[0003] Foamyvirus belongs to the Retrovirida...

Examples

Embodiment 1

[0025] Production of indicator cell lines

[0026] Firstly, the full-length gene of EGFP protein was inserted into the downstream of a response element specifically responding to the bovine foamy virus transactivator isolated from the bovine foamy virus genome, and a reporter plasmid that could respond to bovine foamy virus infection was constructed. (Note: The EGFP gene comes from the commercialized plasmid pEGFP-N1, and the response element of the bovine foamy virus transactivator was isolated by our laboratory, and its sequence is shown in the attached table.)

[0027] Afterwards, the plasmid was transfected into BHK-21 cells by liposome method, and on the second day after transfection, the cells were subcultured at 1:5, and 600 μg / ml of G418 was added to the medium at the same time. A week later, the cells began to die in large numbers, and by the 14th day, all the cells in the control wells (non-transfected normal cells) died. By 20 days, the cells began to grow in clust...

Embodiment 2

[0032] Determination of indicator cell lines

[0033] The cell line was co-cultured with BFV-infected fetal bovine lung cells (fetal bovine lung, FBL). During the co-cultivation, the BFV-infected FBL was inserted into the BFV-infected FBL at a dilution ratio of 1:10, and the expression of EGFP was observed after 48 hours of culture. Express the situation.

[0034] If the cell line responds to the stimulation of the virus and the expression of EGFP is significantly increased, it is the bovine foamy virus infection indicator cell line in this patent.

Embodiment 3

[0036] Comparison of indicator cell lines and CPE methods

[0037] Traditional virus determination is based on cytopathic, one of the classic methods is TCID50 (Tissue Culture Infectious Dose 50%), which is the determination of 50% tissue cell infection. In the determination of foamy virus titers, this method was widely used until the emergence of indicator cell lines.

[0038] BFV3026 was co-cultured with FBL, and after the typical nucleosomes appeared in the cells, the cells were digested with 0.25% trypsin and divided into equal parts. Divide FBL (P10) and indicated cell lines into 1×10 4 The cells were plated in a 96-well plate, and were inserted into BFV3026-infected FBL at a dilution ratio of 1:10. From the next day, the expression of cytopathic (FBL) and green fluorescent protein was observed every day, and it was recorded until the virus maintained the highest titer. The maximum titers detected for the indicated cell lines occurred at 5 days post-infection, while th...

Technical field

[0001] The invention relates to a cell line for detecting bovine foamy virus (BFV) infection, a method for establishing the cell line and its specific application.

Background technique

[0002] Before the emergence of indicator cell lines, the identification methods of virus infection in cell culture mainly include observation of cytopathic method, plaque test, neutralization test, immunofluorescence, metabolic inhibition, erythrocyte adsorption, interference phenomenon, etc. Viruses multiply in sensitive cells, often causing changes in cell metabolism, cell pathology and release of virus into the medium. In this case, cytopathic, color change and detection of virus antigens in the medium can be used to determine the virus. presence and titer. However, the virus titer determined by this method depends on the naked eye observation of the experimenter, and different experimenters may obtain different results.

[0003] Foamyvirus belongs to the Retrovirida...

Examples

Embodiment 1

[0025] Production of indicator cell lines

[0026] Firstly, the full-length gene of EGFP protein was inserted into the downstream of a response element specifically responding to the bovine foamy virus transactivator isolated from the bovine foamy virus genome, and a reporter plasmid that could respond to bovine foamy virus infection was constructed. (Note: The EGFP gene comes from the commercialized plasmid pEGFP-N1, and the response element of the bovine foamy virus transactivator was isolated by our laboratory, and its sequence is shown in the attached table.)

[0027] Afterwards, the plasmid was transfected into BHK-21 cells by liposome method, and on the second day after transfection, the cells were subcultured at 1:5, and 600 μg / ml of G418 was added to the medium at the same time. A week later, the cells began to die in large numbers, and by the 14th day, all the cells in the control wells (non-transfected normal cells) died. By 20 days, the cells began to grow in clust...

Embodiment 2

[0032] Determination of indicator cell lines

[0033] The cell line was co-cultured with BFV-infected fetal bovine lung cells (fetal bovine lung, FBL). During the co-cultivation, the BFV-infected FBL was inserted into the BFV-infected FBL at a dilution ratio of 1:10, and the expression of EGFP was observed after 48 hours of culture. Express the situation.

[0034] If the cell line responds to the stimulation of the virus and the expression of EGFP is significantly increased, it is the bovine foamy virus infection indicator cell line in this patent.

Embodiment 3

[0036] Comparison of indicator cell lines and CPE methods

[0037] Traditional virus determination is based on cytopathic, one of the classic methods is TCID50 (Tissue Culture Infectious Dose 50%), which is the determination of 50% tissue cell infection. In the determination of foamy virus titers, this method was widely used until the emergence of indicator cell lines.

[0038] BFV3026 was co-cultured with FBL, and after the typical nucleosomes appeared in the cells, the cells were digested with 0.25% trypsin and divided into equal parts. Divide FBL (P10) and indicated cell lines into 1×10 4 The cells were plated in a 96-well plate, and were inserted into BFV3026-infected FBL at a dilution ratio of 1:10. From the next day, the expression of cytopathic (FBL) and green fluorescent protein was observed every day, and it was recorded until the virus maintained the highest titer. The maximum titers detected for the indicated cell lines occurred at 5 days post-infection, while th...