CN-118047859-B - Heavy chain and light chain variable region gene sequences of three LSDV ORF monoclonal antibodies, two expressed immunoglobulin complete gene sequences and application thereof

Abstract

The invention relates to the biomedical technical field, in particular to heavy chain and light chain variable region gene sequences of three LSDV ORF monoclonal antibodies, two expressed immunoglobulin complete gene sequences and application thereof, which extracts total RNA of hybridoma cells (1C 1-4D11, 3C4-2E9 and 1E1-2C 11) by utilizing hybridoma sequencing, and then, carrying out reverse transcription by taking mRNA in the total RNA as a template to obtain cDNA of a corresponding antibody gene, obtaining a heavy chain variable region (Variable region ofheavy chain, V H ) and a light chain variable region (Variable region oflight chain, V L ) of the corresponding antibody by using specific PCR, and carrying out cloning and sequencing analysis. Finally, the nucleotide sequences of the heavy chain and the light chain genes of three monoclonal antibodies (1C 1-4D11, 3C4-2E9 and 1E1-2C 11) of the targeted LSDV ORF protein are obtained, and the invention is realized. Sequence analysis of the heavy chain (V H ) and light chain antibodies (V L ) of the three monoclonal antibodies indicated that the gene subtypes 1C1-4D11, 3C4-2E9 and 1E1-2C11 were all of the IgG type, and the light chain was all of the kappa type.

Inventors

• Ren Shanhui
• SUN YUEFENG
• YIN XIANGPING
• CHEN HAOTAI
• GAO XIAOHONG
• WANG XIANGWEI
• GAO XIAOLONG

Assignees

• 中国农业科学院兰州兽医研究所

Dates

Publication Date

20260512

Application Date

20231113

Claims (2)

1. 1. Two LSDV ORF monoclonal antibodies, comprising a heavy chain and a light chain, are characterized in that: The nucleotide sequences for the 3 Complementarity Determining Regions (CDRs) of the light chain variable region were written as follows: CDR 1: CAGAATGTGGGTACAAAT of 1C1-4D11 CDR2: TCGGCATCC of 1C1-4D11 1C1-4D11 CDR3: CAACAATATAACAGCTTTCCGCTCACG CDR 1: CAGAATGTGGATACTAAT of 3C4-2E9 CDR2: TCGGCATCC of 3C4-2E9 CDR3: CAGCAATATAACAGCTATCCGCTCACG of 3C4-2E9 The nucleotide sequences for the 3 Complementarity Determining Regions (CDRs) of the heavy chain variable region were written as follows: CDR1: GGCTACACCTTCACAAGCTACTAT of 1C1-4D11 CDR2: ATTTATCCTGGAAGTGTTAATGCT of 1C1-4D11 1C1-4D11 CDR3: GCGAACTACGGTAGTAGTGACTTCGATGTC CDR1: GGCTACACCTTCACATCCTACTAT of 3C4-2E9 CDR2: ATTTATCCTGGAAATGTTAATACT of 3C4-2E9 CDR3: GCGAACTACGGTAGTAGGGACTTCGATATC of 3C4-2E 9.
2. 2. The two LSDV ORF monoclonal antibodies of claim 1, wherein the amino acid sequences of the light chain variable regions are: v L for 1C1-4D 11: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSRSGTDFTLTISNVQSEDLADYFCQQYNSFPLTFGAGTKLELK v L for 3C4-2E 9: DIVMTPSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVKPEDLAEYFCQQYNSYPLTFGAGTKLELK The amino acid sequences of the heavy chain variable regions are respectively as follows: V H for 1C1-4D 11: QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGSVNAKYNEKFKGKATLTADKSSSTAYIHLNSLTSEDSAVYFCANYGSSDFDVWGAGTTVTVSS V H for 3C4-2E 9: QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWLKQRPGQGLEWIGWIYPGNVNTKYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCANYGSRDFDIWGAGTTVTVSS.

Description

Heavy chain and light chain variable region gene sequences of three LSDV ORF monoclonal antibodies, two expressed immunoglobulin complete gene sequences and application thereof Technical Field The invention relates to the technical field of biomedicine, in particular to heavy chain and light chain variable region gene sequences of three LSDV ORF monoclonal antibodies, two expressed immunoglobulin complete gene sequences and application thereof. Background Niu Jiejie dermatological disorders (Lumpy SKIN DISEASE, LSD) are an acute, subacute contact infectious disease caused after infection with bovine nodular skin disease virus (Lumpy SKIN DISEASE virus, LSDV). LSDV cause fever, papules or skin nodules in cattle, such as head, neck, shoulders and breasts, and in severe cases present pustular lesions and necrotic crusting of tissue. Niu Jiejie skin diseases (LSD) are an important overseas input animal epidemic disease in recent years in China, cause great economic loss to cattle raising industry and seriously affect the healthy development of the cattle raising industry in China. LSD was first found in prandial republic in 1929, and then the disease spread rapidly to the middle east, eastern europe, and middle asia. However, LSD has been diagnosed in 14 provinces, municipalities and municipalities nationally since 2019 and has shown a gradual spread trend, increasingly severe prevention and control situations, due to the lack of effective treatments and specific vaccines against the epidemic. LSDV differ greatly from vaccinia virus (Cowpox virus, CPXV) and are similar to Sheep pox virus (SPPV) and goat pox virus (Goat pox virus, GTPV), belonging to the genus capripoxvirus (CaPV). Currently, our understanding of the poxvirus genome comes largely from related studies on vaccinia virus (Vaccinia Virus, VACV), and studies specific for LSDV viral genome functionality are extremely poor. At present, little research is done specifically for the biological function of LSDV-encoded proteins. The ORF29 protein is a LSDV important structural protein, and the specific function of the ORF29 protein has not been clearly reported, so that deep elucidation of the biological function of the LSDV ORF protein is helpful for enhancing understanding of LSDV etiology and pathogenesis. Currently, LSDV has been studied as a member of the poxviridae family, and LSDV ORF29 protein is a membrane protein structure functionally similar to the F15 protein of vaccinia virus and plays an important role in the packaging process of envelope proteins and virions. In the early research, aiming at LSDV ORF protein, LSDV ORF protein is obtained through prokaryotic expression, and purified and immunized with BALB/C mice, after mice positive for corresponding antibodies are detected, spleen cells are separated and fused with myeloma cells, and then the mice are screened in a HAT selective medium, and finally three hybridoma cell strains 1C1-4D11, 3C4-2E9 and 1E1-2C11 which can stably secrete LSDV ORF monoclonal antibodies are screened. Monoclonal antibodies with high purity can be prepared by utilizing the hybridoma technology, and large-scale production of monoclonal antibodies can be performed, however, hybridoma cells generated in the hybridoma process are likely to lose high specificity and high activity sequence risks, and the cell state is also likely to influence subsequent sustainable research. In this case, it is necessary to perform hybridoma sequencing. After the corresponding antibody sequence is obtained through hybridoma sequencing, the antibody can be stably expressed and obtained in a recombinant protein mode, so that a research and development or producer does not need to reserve the antibody by taking hybridoma cells as a carrier, but stores the antibody in a base sequence mode, and the risk of losing specific antibodies is avoided. Disclosure of Invention Aiming at the defects of the prior art, the invention provides three heavy chain and light chain variable region gene sequences of LSDV ORF monoclonal antibodies, two expressed immunoglobulin complete gene sequences and application thereof, and solves the problems in the background art. In order to achieve the above purpose, the present invention provides the following technical solutions: Three LSDV ORF monoclonal antibodies, including a heavy chain and a light chain, characterized in that: The 3 Complementarity Determining Region (CDR) sequences of the light chain variable region are: CDR1(1C1-4D11):CAGAATGTGGGTACAAAT CDR2(1C1-4D11):TCGGCATCC CDR3(1C1-4D11):CAACAATATAACAGCTTTCCGCTCACG CDR1(1E1-2C11):CAGAATGTGGGTACAAAT CDR2(1E1-2C11):TCGACATCC CDR3(1E1-2C11):CAACAATATAACAGCTTTCCGCTCACG CDR1(3C4-2E9):CAGAATGTGGATACTAAT CDR2(3C4-2E9):TCGGCATCC CDR3(3C4-2E9):CAGCAATATAACAGCTATCCGCTCACG the 3 Complementarity Determining Region (CDR) sequences of the heavy chain variable region are: CDR1(1C1-4D11):GGCTACACCTTCACAAGCTACTAT CDR2(1C1-4D11):ATTTATCCTGGAAGTGTTAATGCT CDR3(1C1-4D11):GCGAACT