US-12617840-B2 - Hybridoma cell line for secreting anti-rabies virus M protein monoclonal antibody and application thereof

Abstract

The present invention is a hybridoma cell line for secreting monoclonal antibody against rabies virus M protein and its application, relates to the field of biotechnology. A classification of the hybridoma cell line is named as hybridoma cell line 4A1, and the hybridoma cell line was deposited on Apr. 1, 2019 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit number CCTCC NO: C201947. The monoclonal antibody prepared by the hybridoma cell line has high titer, good specificity and excellent biological characteristics. The present invention identifies the variant antigen epitope recognized by the RABV M protein, the hybridoma cell line can be used to distinguish Flury strain and other RABV strains, prepare kit for detecting rabies virus RABV, detect RABV infection and differential diagnosis vaccine Flury strain and other RABV strains.

Inventors

• Jiyong ZHOU
• Min Liao
• Yan Yan
• Boli HU
• Jinyan Gu
• Xiaojuan Zheng
• Weiren Dong
• Yulan Jin

Assignees

• ZHEJIANG UNIVERSITY

Dates

Publication Date

20260505

Application Date

20200330

Priority Date

20190401

Claims (3)

1. 1 . A hybridoma cell line for secreting a monoclonal antibody against rabies virus M protein, wherein the classification designation of the hybridoma cell line is Hybridoma cell line 4A1 with a deposit number of the Hybridoma cell line China Center for Type Culture Collection (CCTCC) NO: C201947.
2. 2 . A monoclonal antibody against rabies virus M protein, wherein the monoclonal antibody is prepared from the hybridoma cell line according to claim 1 .
3. 3 . A kit for detecting rabies virus, comprising the monoclonal antibody against rabies virus M protein according to claim 2 .

Description

This is a U.S. national stage application of PCT Application No. PCT/CN2020/081974 under 35 U.S.C. 371, filed Mar. 30, 2020 in Chinese, claiming priority of Chinese Application No. 201910258004.2, filed Apr. 1, 2019, all of which is hereby incorporated by reference. TECHNICAL FIELD The present invention relates to the field of biotechnology, in particular to hybridoma cell line for secreting anti-rabies virus M protein monoclonal antibody and use thereof. DESCRIPTION OF RELATED ART Rabies is a highly lethal zoonotic disease caused by the rabies virus (RABV). After the onset of RABV infection, the fatality rate is almost 100%, which is by far the acute infectious disease with the highest mortality rate in human history. There are still no treatments, and only vaccines can be used to prevent RABV infection. Rabies cases have been reported in more than 150 countries around the world. About 50,000 to 70,000 people die from rabies every year, 95% of rabies cases occur in Africa and Asia. The number of human rabies infections in China is second only to India and the second largest in Asia. Dogs are the most common virus reservoir of RABV, and 99% of human infections with rabies are mediated by canine transmission. RABV belongs to the Rhabdoviridae rabies virus genus (Lyssavirus) in virus taxonomy. It is a single-stranded non-segmented RNA virus. Its genome mainly encodes nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase protein (L). M protein is composed of 202 amino acids and about 23 kDa, and is the smallest and most abundant non-glycosylated protein of RABV virus particles. The M protein is located between the viral envelope and the nucleocapsid and serves to connect the G protein and the nucleus. The M protein has multiple functions: participating in the assembly and budding of virus particles, playing a decisive factor in the shape of virus particles, regulating the transcription and translation process of the virus, participating in determining the pathogenicity of the virus, inducing cell apoptosis, and inhibiting the expression of host-related genes, etc. The monoclonal antibodies against RABV proteins that have been prepared are mainly NP protein and G protein. Monoclonal antibody to NP protein can be used to distinguish different serotypes of rabies virus; Monoclonal antibody to G protein shows a higher neutralizing activity. There are few reports on the monoclonal antibody anti-RABV M protein, and the fine epitopes recognized by the monoclonal antibody to RABV M protein are still unclear, and lacking understanding of the biological characteristics of these monoclonal antibodies, so the application in virus diagnosis of anti-M protein monoclonal antibodies and research on the structure and antigenic properties of M protein are restricted. SUMMARY OF THE INVENTION The object of the present invention is to provide a hybridoma cell line for secreting a monoclonal antibody that specifically recognizes a RABV M protein, and the monoclonal antibody can specifically react with RABV field strain but not with the vaccine Flury strain. In order to achieve the above object, the present invention adopts the following technical solutions: A hybridoma cell line for secreting monoclonal antibody to rabies virus M protein, a classification of the hybridoma cell line is named as hybridoma cell line 4A1, and the hybridoma cell line was deposited on Apr. 1, 2019 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit number CCTCC NO: C201947. The present invention first obtains 12 strains of hybridoma cells stably secreting monoclonal antibody to RABV M protein, and finally screens out the hybridoma cell 4A1 strain, which has excellent biological characteristics. The titer of the prepared monoclonal antibody 4A1 in mouse ascites was 1×105 measured by indirect enzyme-linked immunosorbent assay. The present invention also provides a monoclonal antibody to rabies virus M protein prepared from the hybridoma cell 4A1 strain. The present invention identifies the variant antigen epitopes recognized by the monoclonal antibody for the first time, and its amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:2. These epitopes are relatively conserved on the M protein of different RABV strains, but there is an antigenic variation on the vaccine Flury strain (the amino acid sequence of the Flury strain is shown in SEQ ID NO: 3), which makes the monoclonal antibody unable to recognize the epitope. Therefore, the monoclonal antibody provided by the present invention can specifically react with the rabies virus strain, but cannot react with the vaccine Flury strain. The present invention provides an application of the hybridoma cell 4A1 strain in preparing a kit for detecting RABV. The hybridoma cell 4A1 strain can be applied to the methods such as ELISA, immunofluorescence, Western Blot, immunohistochemistry, etc., and can be used i