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Homer1 Monoclonal antibody and application thereof

A monoclonal antibody, his-homer1b technology, applied in the field of biomedicine, can solve the problems of low sensitivity and specificity, and achieve the effect of strong specificity, good thermal stability and wide application space

Active Publication Date: 2018-04-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given the limitations of polyclonal antibodies, their sensitivity and specificity are not high

Method used

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  • Homer1 Monoclonal antibody and application thereof
  • Homer1 Monoclonal antibody and application thereof
  • Homer1 Monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] [Example 1] Preparation of anti-Homer1 monoclonal antibody

[0032] 1. Construction of His-Homer1b (Genebank: NM_004272.4), His-Homer1b (110-354aa) and His-Homer1b (130-180aa) recombinant expression vectors

[0033] RNA was extracted from human liver tissue, reverse-transcribed into cDNA, specific PCR primers were designed, and the DNA sequences corresponding to His-Homer1b, His-Homer1b (110-354aa) and His-Homer1b (130-180aa) were cloned. The amino acid sequence of the full-length Homer1b is shown in SEQ NO:1. His-Homer1b and His-Homer1b (110-354aa) were recombined into pET28a vector to construct pET28a-Homer1 and pET28a-Homer1b (110-354aa) prokaryotic expression vector; His-Homer1b (130-180aa) DNA sequence was recombined into pET28a-Sumo plasmid Among them, the pET28a-Sumo-Homer1b (130-180aa) prokaryotic expression vector was constructed. DNA sequencing verifies the accuracy of the sequence.

[0034] 2. Expression and purification of His-Homer1b, His-Homer1b (110-35...

Embodiment 2

[0055] [Example 2] Obtaining ascites and purifying monoclonal antibodies

[0056] 1. Take 8-week-old female Balb / C mice and inject 0.5 ml Freund's incomplete adjuvant intraperitoneally. After 7-12 days, suspend the expanded hybridoma cells in PBS and inject 1×10 per mouse intraperitoneally. 6 a hybridoma cell. Seven days after the injection of hybridoma cells, the abdomen of the mouse was obviously enlarged. At this time, the ascites was extracted with a sterile syringe, and centrifuged at 4000 rpm for 10 min at 4°C in a centrifuge tube. The supernatant was collected to obtain the monoclonal antibody ascites.

[0057] 2. Equilibrate the column packed with Protein G agar with 1% NaAc.

[0058] 3. Filter the ascites with a filter membrane with a pore size of 0.2 um, dilute the ascites with 1% NaAc, then add it to the equilibrated column, and control the flow rate with a valve.

[0059] 4. After the ascites is loaded on the column, wash it with 1% NaAc, and check the washing s...

Embodiment 3

[0062] [Example 3] Monoclonal antibody specificity and titer

[0063] 1. Western blot experiment

[0064] 1. The 293T cells transfected with Homer1a, Homer1c, Homer2, and Homer3, as well as the mouse cortex, cerebellum, and hippocampus tissues were lysed with RIPA lysate. Centrifuge at 12000 r / min at 4°C for 20 min. The supernatant was collected and the BCA kit was used to detect the protein concentration.

[0065] 2. Add the cell and tissue lysate to 12% PAGE, and electrophoresis at 120V for 1 hour. Then the PAGE gel was placed on the PVDF membrane, and transferred to the membrane at 40 mA for 90 min.

[0066] 3. Put the PVDF membrane into 1% casein to block at room temperature for 1 hour. Then, the Homer1B1 and Homer1B3 monoclonal antibodies of the present invention were used as primary antibodies at 1:2000 and incubated overnight at 4°C.

[0067] 4. Take out the PVDF membrane and rinse it with PBST for 3 times, 3 minutes each time. Place in 1:5000 diluted HRP-labeled ...

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Abstract

The invention discloses Homer1 monoclonal antibodies and application thereof. Two hybridoma cell strains capable of stably secreting monoclonal antibodies Homer1 1B1 and Homer1 1B3 capable of specifically recognizing Homer1 protein are provided herein; collection numbers of the two hybridoma cell strains include CCTCC NO: C2017191 and CCTCC NO: C2017190. An immunoassay tool for detecting Homer1 protein, an immunohistochemical kit containing Homer1 1B1 and Homer1 1B3 and a kit for preparing diagnosing Homer1 protein-related tumors are prepared with the monoclonal antibodies provided herein. Themonoclonal antibodies Homer1 1B1 and Homer1 1B3 may specifically combine with Homer1 protein and exhibit no cross reaction with other proteins in cells, and Homer1 protein immunoassay specificity andsensitivity are significantly improved.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the monoclonal antibody of Homer1, which produces the anti [0002] The hybridoma cell line of the body, and the immunological detection application of the antibody. Background technique [0003] Homer protein is a very important skeleton protein in the process of intracellular signal transduction. It participates in the activities of many nerve cells, including regulating the balance of calcium ions and the formation of the cytoskeleton of synaptic remodeling in the process of learning and memory, etc. . The Homer protein family includes three members, Homer1, Homer2 and Homer3, which form various isoforms through different splicing of exons. All subtypes contain an N-terminal EVH1 domain that can bind to mGluR, IP 3 R, TRPC and other proline-rich proteins are combined, and the long-form Homer protein has a CC region at the C-terminus, and homologous or heterologous dimers ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577G01N33/574
CPCC07K16/18G01N33/57415G01N33/577G01N2333/47
Inventor 涂建成张献伟沈鹤霄吴春燕柴红燕陈浩井宣王美钧保罗·沃利
Owner WUHAN UNIV
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