Monoclonal antibody against human neurofilament light polypeptide (NEFL) and application thereof

A monoclonal antibody, microbial strain technology, applied in the biological field, can solve the problem of no detection reagent registration and other problems

Active Publication Date: 2019-12-27
无锡傲锐东源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Monoclonal antibody against human neurofilament light polypeptide (NEFL) and application thereof
  • Monoclonal antibody against human neurofilament light polypeptide (NEFL) and application thereof
  • Monoclonal antibody against human neurofilament light polypeptide (NEFL) and application thereof

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Experimental program
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Embodiment 1

[0022] Embodiment 1: the preparation of anti-NEFL monoclonal antibody

[0023] 1. Production of NEFL recombinant protein

[0024] The NEFL gene NM_006158 was selected from Genebank. The gene encodes 543 amino acids (aa) in the full length of NEFL, located at 355-1986nt. The immunogen of the antibody of the present invention is NEFL1-360aa, located at 355-1432nt. Known technical methods clone the gene into pET23a expression plasmid with his tag at the C-terminal, express in BL21 Escherichia coli, purify with nickel column, and identify the purity by SDS-PAGE electrophoresis. After electrophoresis, the target protein band with a molecular weight of about 43kDa was observed on the gel, and the purity was above 90%, which met the purity requirement for the preparation of monoclonal antibody.

[0025] 2. Animal immunity

[0026] The above-mentioned purified NEFL recombinant protein was emulsified with complete Freund's adjuvant, and immunized 6-8 week-old BALB / c mice by subcutane...

Embodiment 2

[0029] Embodiment 2: the identification of anti-NEFL monoclonal antibody

[0030] Through Example 1, 4 monoclonal antibody strains were obtained, namely OTI3G2, OTI11F6, OTI2F5, and OTI11F8, and were identified as follows.

[0031] 1. Specific identification of monoclonal antibodies

[0032] It was detected by indirect ELISA method. Coat the microtiter plate with NEFL, NEFM, NEFH, GFAP, UCH-L1, BSA, PET23a empty plasmid-transferred E. coli lysate, PCMV6 plasmid-transferred 293 cell lysate, etc., at a concentration of 5 μg / ml, overnight at 4°C. Block the plate with PBST containing 1% BSA, add 10 4 Two-fold diluted purified antibody, reacted at 37°C for 50min, washed the plate three times with PBST, and added HRP-labeled goat anti-mouse secondary antibody.

[0033]The results showed that the lysates of NEFM, NEFH, GFAP, UCH-L1, BSA, and PET23a empty plasmids transferred to E. coli, and the lysates of PCMV6 plasmids transferred to 293 cells were all negative for the 4 strains ...

Embodiment 3

[0043] Embodiment 3: gene sequencing and analysis of the variable region of monoclonal antibody

[0044] Procured from Takara Bio USA RACE 5' / 3' kit, using 5' RACE (Rapid Amplification of cDNA Ends, rapid amplification of cDNA ends) technology to clone the variable region sequence of functional antibody from the hybridoma cell line OTI11F6 secreting anti-NEFL monoclonal antibody . The specific experimental process is as follows, see Takara Bio USA company RACE 5’ / 3’Kit User Manual.

[0045] According to the IgG1 subtype of the antibody OTI11F6, specific gene primers pRace-H-GSP and pRace-K-GSP were designed for the 3' ends of its Ig and Kapa constant regions, and the primer sequences were as follows:

[0046] pRace-H-GSP:CATCDGTCTATCCACTGGCCCCTG

[0047] pRace-K-GSP:CTTCCCACCATCCAGTGAGCAGTT

[0048] The DNA fragments of the light and heavy chains of the antibody were amplified by RACE PCR, and the results are shown in figure 1 ,from figure 1 It can be seen that the...

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Abstract

The invention relates to the field of biotechnology, and discloses a monoclonal antibody against a neurofilament light polypeptide (NEFL). The antibody is secreted by a hybridoma cell line OTI11F6 with a preservation number of CGMCC No.18189. The immunogen of the antibody is a NEFL 1-360aa polypeptide expressed by prokaryotic cells. An amino acid sequence of a light polypeptide (VL) of the antibody is as shown in SEQ ID No.1, and an amino acid sequence of a heavy polypeptide (VH) of the antibody is as shown in SEQ ID No.2. The VL of the antibody comprises three antigenic determinants: CDR1, CDR2, and CDR3, amino acid sequences of the three antigenic determinants are respectively as shown in SEQ ID No.3-5, the VH region of the antibody comprises three antigenic determinants: CDR1, CDR2, andCDR3, and amino acid sequences of the three antigenic determinants are respectively as shown in SEQ ID No.6-8. The antibody OTI11F6 can be applied to preparation of various immunoassay kits of NEFL,such as preparation of double-antibody sandwich enzyme-linked immunosorbent assay kits or chemiluminescence assay kits, and assistant diagnoses for brain injury and chronic brain lesions are provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-human NEFL monoclonal antibody and its application in immune detection. Background technique [0002] Nerve intermediate filaments or neurofilaments include three subunits: neurofilament light chain (neurofilament lightpolypeptide, NEFL), neurofilament medium chain (NEFM) and neurofilament heavy chain (NEFH), with relative molecular masses of 68,000, 160,000 and 212,000. NEFL is the only subunit that can self-assemble. NEFL can self-assemble into homopolymeric 10nm fibers in vitro. When the NEFL subunit is absent, the neurofilament medium chain polypeptide and neurofilament heavy chain polypeptide subunits cannot assemble into functional Neurofilament protein, so NEFL is considered to be the most important neurofilament. [0003] NEFL is a key component of the nerve cytoskeleton and is interconnected with multiple protein targets that are critical to nerve cell shaping, playi...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/574C12R1/91
CPCC07K16/18C07K2317/35C07K2317/56C07K2317/565G01N33/57407G01N33/57415G01N33/57423G01N33/57484G01N33/6893G01N33/6896G01N2333/47G01N2800/28G01N2800/2821G01N2800/2835
Inventor 唐雨德付伟扈晓敏任琪钮倩王成林沈腾腾何为无
Owner 无锡傲锐东源生物科技有限公司
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