CN-121975744-A - Method for preparing cell-binding virus from specific MHC haplotype

CN121975744ACN 121975744 ACN121975744 ACN 121975744ACN-121975744-A

Abstract

The invention relates to the fields of molecular biology and virology, and provides a method for preparing cell-binding viruses derived from specific MHC haplotypes. In particular to a method for rapidly obtaining chicken Marek's Disease Virus (MDV) with specific MHC haplotypes, which comprises the steps of firstly obtaining chicken embryos with different haplotypes, separating CEF cells, transiently expressing exogenous fluorescent proteins through electrotransfection, then carrying out virus inoculation, obtaining a cell population carrying MDV with positive fluorescence through flow separation, and obtaining purified MDV virus with specific haplotype sources through in vitro propagation. The purified virus can provide test materials for researching haplotypes on virus infection, and can be widely applied to in-vitro purification of different cell-binding viruses and related research of presenting virus peptides by specific MHC molecules. The invention is favorable for developing related researches aiming at host immune response of the virus, analyzes the genetic basis of the differential resistance of haplotypes to MDV, and is expected to be applied to the preparation of vaccine viruses.

Inventors

LIAN LING
ZHENG GANG
YANG YUQIN
Du Yushuang
WEI YUNJIE

Assignees

中国农业大学

Dates

Publication Date: 20260505
Application Date: 20260106

Claims (9)

1. A method for preparing a cell-associated virus of particular MHC haplotype origin comprising the steps of: (1) Providing a host cell derived from a specific MHC haplotype individual; (2) Transfecting the host cell to express a reporter gene; (3) Infecting the transfected host cell with the cell-binding virus; (4) Isolating host cells expressing the reporter gene and carrying the virus by a sorting technique; (5) Expanding the virus-carrying host cells isolated in step (4) to obtain the cell-associated virus of specific MHC haplotype origin.
2. The method of claim 1, wherein the cell-binding virus is chicken Marek's Disease Virus (MDV) and the host cell is Chicken Embryo Fibroblast (CEF).
3. The method of claim 1, wherein the reporter gene is a fluorescent protein gene.
4. The method of claim 1, wherein the transfection in step (4) is transient transfection.
5. The method of claim 1, wherein the expanding in step (5) comprises co-culturing the virus-carrying host cells isolated in step (4) with uninfected host cells derived from the same specific MHC haplotype individual.
6. The method of claim 1, wherein the sorting technique in step (4) employs flow cytometry.
7. The method of claim 6, wherein the flow cytometry sorting is performed when the virus plaque density in the host cell reaches 40% -60%.
8. The method of claim 2, wherein step (1) is preceded by the step of isolating primary chick embryo fibroblasts from the specific MHC haplotype chick embryos incubated for 5-7 days.
9. The method of any one of claims 2-8, wherein the specific MHC haplotype is selected from at least one of B15, B19 or B21 haplotypes.

Description

Method for preparing cell-binding virus from specific MHC haplotype Technical Field The invention relates to the fields of molecular biology and virology, in particular to a method for preparing cell-binding viruses derived from specific MHC haplotypes. Background The chicken major histocompatibility complex (Major histocompatibility complex, MHC) plays a central role in the immune system, whose central functions include antigen presentation by presenting intracellular or extracellular antigen fragments (peptides) to T lymphocytes, thereby initiating specific immune responses, self/non-self recognition, i.e., the basis by which the immune system distinguishes itself from foreign pathogens (e.g., viruses, bacteria), immune rejection, MHC incompatibility among individuals, which can lead to rejection of transplanted organs or tissues. Individuals of different MHC haplotypes often exhibit differences in resistance to different diseases, a classical example of which is Marek's Disease (MDV), a highly infectious lymphomatosis caused by infection with the herpes Virus family member Marek's Disease Virus (MDV), which has a significant economic impact on the poultry industry worldwide. The disease is extremely contagious and almost all commercial chicken flocks are exposed to the virus. After viral activation, lymphocytic tumors can develop in internal organs, nerves, skin, etc. Viruses can further destroy immune organs, resulting in impaired immune systems in chickens and more susceptible to other diseases. Currently, the disease cannot be treated, and prevention is the only and most cost-effective control means. Prevention relies on a comprehensive strategy of (1) on-time, effective vaccination, (2) stringent biosafety measures, and (3) breeding of genetic disease-resistant lines. Despite the great success of vaccine applications, the continual evolution of viruses continues to challenge existing control systems. This also motivates the development of resistant lines and the study of the genetic basis for resistance/susceptibility. The B21 haplotype was found to be associated with very high resistance to marek's disease. Chickens carrying the B21 haplotype, even if infected with virulent MDV, have a much lower probability of tumor occurrence and death than other haplotypes. B19 haplotype is associated with a high susceptibility to Marek's disease, and the incidence and mortality of chickens carrying this haplotype are high after infection. Other haplotypes, such as B2, B5, B13, etc., exhibit moderate disease resistance. By mechanism, MHC is able to present antigenic peptides of MDV to cytotoxic T cells (cd8+ T cells) to recognize and clear virus-infected cells, preventing tumor formation. While the possible differences in antigen presentation efficiency of resistant/susceptible haplotype MHC molecules lead to a tremendous difference in resistance among individuals of different haplotypes. MDV is a strict cell-binding virus, production, preparation and propagation of which are strictly dependent on chicken embryo fibroblasts, and conventional propagation processes do not distinguish haplotypes, which also results in that MDV sources are mixed and have complex genetic background information. This presents difficulties for related studies, such as the inability to distinguish the background of the virus-loaded cells in vitro studies of antigenic peptides. Thus, there is a need to establish a method for preparing/propagating MDV virus with clear context for MHC haplotypes. Disclosure of Invention It is an object of the present invention to provide a method for preparing cell-binding viruses of specific MHC haplotype origin. To achieve the object of the present invention, the present invention provides a method for preparing a cell-binding virus derived from a specific MHC haplotype, comprising the steps of: (1) Providing a host cell derived from a specific MHC haplotype individual; (2) Transfecting the host cell to express a reporter gene; (3) Infecting the transfected host cell with the cell-binding virus; (4) Isolating host cells expressing the reporter gene and carrying the virus by a sorting technique; (5) Expanding the virus-carrying host cells isolated in step (4) to obtain the cell-associated virus of specific MHC haplotype origin. Further, the cell-binding virus is chicken Marek's Disease Virus (MDV) and the host cell is Chicken Embryo Fibroblast (CEF). Further, the reporter gene is a fluorescent protein gene, such as GFP, RFP or other fluorescent proteins. Further, the transfection in step (4) is transient transfection. Further, the expanding in step (5) comprises co-culturing the virus-carrying host cells isolated in step (4) with uninfected host cells derived from the same specific MHC haplotype individual. Further, the sorting technique in step (4) employs flow cytometry. Preferably, flow cytometry sorting is performed when the virus plaque density in the host cell reaches 40% -60%. Further, the step (1)