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CN-120574783-B - Construction of EBV-infected epidermal cells and B cell models

CN120574783BCN 120574783 BCN120574783 BCN 120574783BCN-120574783-B

Abstract

The invention provides EBV-susceptible 293T cells which simultaneously express an epidermal cell receptor EphA2 and a key protein RTA of lytic replication, and EBV-susceptible BJAB cells which simultaneously express a B cell receptor CD21 and a key protein ZTA of lytic replication, so that the infectivity of the cells to EBV is obviously improved. The titer of infection of EBV by the cell line of the invention is more than three times that of normal control under the same condition. The EBV susceptible cells have important significance for researching an EBV infection mechanism, screening antiviral drugs and researching and developing vaccines.

Inventors

  • LIANG XIAOZHEN
  • Wei Wendi
  • Yi Yannan
  • KUANG LINLIN

Assignees

  • 中国科学院上海免疫与感染研究所

Dates

Publication Date
20260505
Application Date
20250611

Claims (10)

  1. 1. An EBV-susceptible stable cell line, wherein the cell line overexpresses an EBV receptor and a transcriptional switch protein that is a viral gene-cleaving replication transcriptional activator; Wherein the cell line is an epidermic-like cell line or a B cell line; the ratio X1/X0 of the EBV infection rate X1 of the cell line to the EBV infection rate X0 of the normal control is more than or equal to 3; Wherein, the If the cell line is an epidermoid cell line, the EBV receptor is EphA2 and the transcriptional switch protein is a replication transcriptional activator RTA; if the cell line is a B cell line, the EBV receptor is the CD21 receptor and the transcriptional switch protein is the replication transcriptional activator ZTA; Wherein, the The Genbank accession number of EphA2 is NP-004422.2; the Genbank accession number of the CD21 receptor is NP-001006659.1.
  2. 2. The cell line of claim 1, wherein the normal control is a wild-type cell line that does not overexpress an EBV receptor and does not overexpress a transcriptional switch protein under the same culture conditions.
  3. 3. The cell line of claim 1, wherein the ratio Y1/Y0 of the EBV infection titre Y1 of the cell line to the EBV infection titre Y0 of the normal control is ≡3.
  4. 4. The cell line of claim 1, wherein the epidermoid cell line is a 293T cell line.
  5. 5. The cell line of claim 1, wherein the B cell line is a BJAB cell.
  6. 6. Use of the cell line of claim 1 for one or more of the following: (Z1) a mechanism for studying EBV-infected host cells; (Z2) for screening of EBV neutralizing antibodies; (Z3) for screening anti-EBV virus drugs; (Z4) for screening of EBV vaccines; (Z5) A method for preparing a living cell preparation for detecting an EBV neutralizing antibody in a sample.
  7. 7. A living cell preparation, characterized in that, the viable cell preparation comprises the cell line of claim 1.
  8. 8. The method of preparing a cell line according to claim 1, comprising the steps of: (A1) Providing pLVX-puro-RTA plasmid and pLVX-hygro-EphA2 plasmid, and (A2) Transfecting said pLVX-puro-RTA plasmid and said pLVX-hygro-EphA2 plasmid into an epidermoid cell line, thereby obtaining an EBV-susceptible stable epidermoid cell line; Or alternatively (B1) Providing pLVX-hygro-CD21 plasmid and pLVX-puro-ZTA plasmid; (B2) Electrotransferring said pLVX-hygro-CD21 plasmid into a B cell line to obtain BJAB-CD21 cells, and (B3) Packaging said pLVX-puro-ZTA into lentivirus for introducing into said BJAB-CD21 cells, thereby obtaining an EBV susceptible stable B cell line.
  9. 9. A method for evaluating EBV neutralization activity of a sample for non-diagnostic non-therapeutic purposes, comprising the steps of: (S1) providing a cell line according to any one of claims 1-5; (S2) incubating the sample with the cell line in step (S1) to provide EBV neutralization activity of the sample.
  10. 10. A platform for screening anti-EBV virus drugs and/or developing EBV vaccines, the platform comprising an EBV susceptible cell line that is: (i) An epidermoid cell line in which EphA2 and a replication transcriptional activator RTA are overexpressed, or (Ii) A B cell line in which CD21 receptor and replication transcriptional activator ZTA are overexpressed; Wherein, the The Genbank accession number of EphA2 is NP-004422.2; the Genbank accession number of the CD21 receptor is NP-001006659.1.

Description

Construction of EBV-infected epidermal cells and B cell models Technical Field The invention belongs to the field of bioengineering, and particularly relates to construction of an EBV infected epidermal cell and B cell model. Background Epstein-Barr virus (EBV) is a human herpesvirus widely spread among people and has a global infection rate of over 90%. EBV infection is closely associated with a variety of serious human diseases including infectious mononucleosis, nasopharyngeal carcinoma, gastric cancer, burkitt lymphoma, and other types of lymphoproliferative diseases. When EBV is primarily infected, it primarily infects epidermal cells for lytic replication, and subsequently infects B cells and establishes latent infection in the B cells, allowing the infected person to carry over. However, research into EBV and antiviral drug development face a significant challenge in that EBV is difficult to efficiently infect conventional laboratory cell lines, and this limitation severely hampers in-depth research into its infectious mechanisms, high-throughput screening of potent antiviral drugs, and vaccine evaluation. At present, raji cells are the most commonly used B cell infection model for analyzing EBV neutralizing antibodies in a laboratory, but exogenous virus infection can interfere with endogenous virus products because the cells naturally carry the EBV, so that the accuracy of experimental results is affected, and the infection efficiency is low. Furthermore, there is a lack of epidermal cells and B cell models that are highly efficient in EBV infection, possibly related to the replication characteristics of the receptor and EBV. Thus, to overcome the research bottleneck associated with EBV infection, there is an urgent need to provide EBV infection-sensitive cells in order to screen effective antiviral drugs and evaluate vaccine effects. Disclosure of Invention The invention provides an EBV-sensitive type epidermal cell line 293T which simultaneously overexpresses receptor EphA2 of an EBV-infected epidermal cell and a viral gene-lytic replication transcription activated switching protein RTA, and an EBV-sensitive type B cell line BJAB which simultaneously overexpresses B cell receptor CD21 and a viral gene-lytic replication transcription activated switching protein ZTA, and provides the type of epidermal cell line and application of the type of epidermal cell line. In a first aspect of the invention, an EBV susceptible cell line is provided that overexpresses an EBV receptor and a transcriptional switch protein. In another preferred embodiment, the cell line comprises an epidermoid cell line and/or a B cell line. In another preferred embodiment, the EBV receptor comprises the CD21 receptor, the EphA2 receptor, or a combination thereof. In another preferred embodiment, the transcriptional switch protein is a viral gene cleavage replication transcriptional activator. In another preferred embodiment, the transcriptional switch protein comprises RTA encoded by the BRLF1 gene, ZTA encoded by the BZLF1 gene, or a combination thereof. In another preferred embodiment, the cell line is an epidermoid cell line, the EBV receptor is adrenergic receptor A2, and the transcriptional switch protein is a replication transcriptional activator RTA. In another preferred embodiment, the epidermoid cell line is a 293T cell line. In another preferred embodiment, the cell line is a B cell line, the EBV receptor is the CD21 receptor, and the transcriptional switch protein is the replication transcriptional activator ZTA. In another preferred embodiment, the B cell line is a BJAB cell. In another preferred embodiment, the cell line has an EBV infection titer Y1 that is significantly higher than the EBV infection titer Y0 of the normal control. In another preferred embodiment, the normal control is a wild-type cell line that does not overexpress the EBV receptor and does not overexpress the transcriptional switch protein under the same culture conditions. In another preferred embodiment, the ratio Y1/Y0 of the EBV infection titer Y1 of the cell line to the EBV infection titer Y0 of the normal control is ≡3, preferably ≡5, most preferably ≡8. In another preferred embodiment, the EBV infection rate X1 of the cell line is significantly higher than the EBV infection rate X0 of the normal control. In another preferred embodiment, the ratio X1/X0 of the EBV infection rate X1 of the cell line to the EBV infection rate X0 of the normal control is ≡2, preferably ≡3. In a second aspect of the invention there is provided the use of a cell line according to the first aspect of the invention for one or more of the following uses: (Z1) a mechanism for studying EBV-infected host cells; (Z2) for screening of EBV neutralizing antibodies; (Z3) for screening anti-EBV virus drugs; (Z4) for screening of EBV vaccines; (Z5) is used to prepare a viable cell preparation for detecting EBV neutralizing antibodies in a sample. In another preferred embodim