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CN-121991206-A - Specific monoclonal antibody of anti-pseudorabies virus IE180 protein, and preparation method and application thereof

CN121991206ACN 121991206 ACN121991206 ACN 121991206ACN-121991206-A

Abstract

The invention provides a specific monoclonal antibody for resisting pseudorabies virus IE180 protein, and a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a hybridoma cell strain pIE180 mAb for producing a monoclonal antibody specific to pseudorabies virus IE180 protein, and the monoclonal antibody aiming at PRV IE180 protein is successfully prepared. Through the overlapping peptide library technology, the monoclonal antibody can recognize a new B cell epitope, is highly conserved in various classical and mutant isolates, can be used for the specific detection of PRV infection, the development of diagnostic reagents, and provides an important molecular basis for the design of subunit vaccines, and has important clinical application and market prospects.

Inventors

  • GU JINYAN
  • CHEN HENG
  • ZHOU JIYONG
  • XU LINGYUN
  • Peng Xiran

Assignees

  • 浙江大学

Dates

Publication Date
20260508
Application Date
20260309

Claims (10)

  1. 1. The hybridoma cell strain pIE180 mAb for producing the anti-pseudorabies virus IE180 protein specific monoclonal antibody is characterized in that the hybridoma cell strain pIE180 mAb has been preserved, and the preservation number is CCTCC NO: C202638.
  2. 2. An anti-pseudorabies virus IE180 protein specific monoclonal antibody produced using the hybridoma cell line pIE180 mAb of claim 1.
  3. 3. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.3 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4.
  4. 4. A monoclonal antibody according to claim 2 or 3, wherein the nucleotide sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.1 and the nucleotide sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 2.
  5. 5. The method for preparing the monoclonal antibody according to any one of claims 2-4, which is characterized by comprising the steps of amplifying an IE180-S2 gene by taking genomic DNA of pseudorabies virus as a template, and performing biological expression and purification by using the amplified IE180-S2 gene to obtain recombinant IE180-S2 protein; Immunizing animals by utilizing the recombinant IE180-S2 protein, separating spleen cells and myeloma cells, performing cell fusion, and screening to obtain a hybridoma cell strain pIE180 mAb in claim 1; inoculating the hybridoma cell strain pIE180 mAb into the abdominal cavity of an animal, and separating and purifying the animal ascites fluid to obtain the monoclonal antibody.
  6. 6. The method of claim 5, wherein the primer pair for amplifying the IE180-S2 gene comprises an upstream primer having a nucleotide sequence shown in SEQ ID No.5 and a downstream primer shown in SEQ ID No. 6.
  7. 7. The method of claim 5, wherein the biological expression comprises expression using a prokaryotic bacterium.
  8. 8. Use of the monoclonal antibody according to any one of claims 2 to 4 or the monoclonal antibody prepared by the preparation method according to any one of claims 5 to 7 in the preparation of anti-pseudorabies virus drugs.
  9. 9. Use of the monoclonal antibody according to any one of claims 2 to 4 or the monoclonal antibody prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a reagent for detecting pseudorabies virus antigen.
  10. 10. The use according to claim 9, wherein the reagent for detecting pseudorabies virus antigen comprises an immunoassay reagent.

Description

Specific monoclonal antibody of anti-pseudorabies virus IE180 protein, and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a specific monoclonal antibody of anti-pseudorabies virus IE180 protein, and a preparation method and application thereof. Background Pseudorabies virus (PRV), a member of the subfamily Herpesviridae (ALPHAERPESVIRINAE), is a linear double stranded DNA virus. The PRV genome is about 150 kb in length and has a GC content of up to 74%. According to the strict time sequence of DNA replication and transcription after virus infection of cells, PRV coding genes can be divided into early genes, early genes and late genes, and the three genes are regulated in a cascade mode. The genome consists of a unique long region and a unique short region and internal and two-terminal repeats, has at least 70 ORFs, and can encode 70-100 proteins, whereas the mature virion contains only 50 more proteins. Studies have shown that nearly 50% of mature virions are composed of four parts, the genome, nucleocapsids, envelope and envelope. PRV immediate early gene 180 (IE 180) is its sole immediate early protein, activating transcription of downstream early and late genes, critical to viral replication. When the viral genome enters the nucleus, the gene immediately starts to transcribe, with a transcription size of 4.4 kb and a translation size of 1462 aa. The IE180 gene is the first gene transcribed and translated after virus infection, and IE180 protein can activate transcription of downstream genes and is a trans-acting factor expressed by the whole genes. Studies have shown that viruses lacking IE180 cannot replicate efficiently, and that after PRV infection, downstream genes (early and late genes) can begin transcription under their transcriptional activation only when the IE180 gene begins transcription and successfully translates to the IE180 protein, otherwise viral replication is blocked. That is, transcription and translation of the IE180 gene are a prerequisite for PRV replication, and expression of the IE180 protein is considered as a marker of early infection and latency-reactivation of PRV, and is also a key target protein in screening of antiviral drug targets. However, there is no commercial PRV IE180 monoclonal antibody, nor is there any report on the PRV IE180 monoclonal antibody. Disclosure of Invention The invention provides a specific monoclonal antibody of anti-pseudorabies virus IE180 protein, a preparation method and application thereof, wherein the monoclonal antibody can be specifically combined with PRV IE180 protein, can be used for establishing a ELISA, IFA, WB, IHC detection method for detecting PRV early infection and latent-reactivation state, and provides an important research tool for researching PRV early infection and latent-reactivation. The invention provides a hybridoma cell strain pIE180 mAb for producing a monoclonal antibody specific to pseudorabies virus IE180 protein, wherein the hybridoma cell strain pIE180 mAb has been preserved, and the preservation number is CCTCC NO: C202638. The invention also provides an anti-pseudorabies virus IE180 protein specific monoclonal antibody produced by using the hybridoma cell strain pIE180 mAb. In a specific embodiment of the invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4. In one specific embodiment of the invention, the nucleotide sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown as SEQ ID No.1, and the nucleotide sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 2. The invention also provides a preparation method of the monoclonal antibody, which comprises the following steps of amplifying IE180-S2 genes by taking genomic DNA of pseudorabies virus as a template, and performing biological expression and purification by utilizing the amplified IE180-S2 genes to obtain recombinant IE180-S2 protein; Immunizing animals by utilizing the recombinant IE180-S2 protein, separating spleen cells and myeloma cells, performing cell fusion, and screening to obtain the hybridoma cell strain pIE180 mAb; inoculating the hybridoma cell strain pIE180 mAb into the abdominal cavity of an animal, and separating and purifying the animal ascites fluid to obtain the monoclonal antibody. In one specific embodiment of the invention, the primer pair for amplifying the IE180-S2 gene comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.5 and a downstream primer shown as SEQ ID No. 6. In one embodiment of the invention, the biological expression comprises expression using a prokaryotic bacterium. The invention also provides application of the monoclonal antibody or th