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Application of single domain antibody specific for V5 tag protein

A single-domain antibody, tag protein technology, applied in the field of biotechnology or biomedicine, can solve the problems of restricting the research, purification and detection of the fine structure and function of cells, which take a lot of time, and the steric hindrance of antibodies is large. And the effect of high purification efficiency, low steric hindrance and stable quality between batches

Active Publication Date: 2019-08-20
REGENECORE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because super-resolution imaging requires very high sample quality, it often encounters some difficulties in practical application. For example, fluorescence imaging relies on the specific fluorescent labeling of molecules, but conventional immunofluorescence labeling depends on the quality of antibodies, and each batch The purification and detection after antibody production takes a lot of time. In addition, the steric hindrance of conventional antibodies is relatively large, with a diameter of 10nm, which has exceeded the size of many protein molecules and the effective resolution of super-resolution, so that the actual observation results are different. In addition, because the photon yield of fluorescent protein itself is much lower than that of fluorescent dyes, the accuracy of super-resolution imaging with fluorescent protein is not enough (Evgenia Platonova et al., 2015), or due to the Properties such as non-uniform labeling and spontaneous aggregation affect imaging results (Deschout et al., 2014)
These technical limitations limit the study of the structure and function of cellular haptics

Method used

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  • Application of single domain antibody specific for V5 tag protein
  • Application of single domain antibody specific for V5 tag protein
  • Application of single domain antibody specific for V5 tag protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of a single-domain antibody library against V5-tagged proteins:

[0058](1) Mix equal volume of 1 mg V5-KLH antigen with Freund's adjuvant to immunize a Xinjiang Bactrian camel once a week for a total of 7 consecutive immunizations. During the immunization process, B cells are stimulated to express specific nanobodies; ( 2) After immunization, 100ml of camel peripheral blood lymphocytes were extracted and total RNA was extracted; (3) cDNA was synthesized and VHH was amplified by nested PCR; (4) 20ug pMECS was digested with restriction enzymes PstI and NotI for phage display vector and 10ug VHH and ligated the two fragments; (5) Transform the ligated product into the electrotransformation competent cell TG1, construct the V5 tag protein phage display library and measure the library capacity, the size of the library capacity is about 2 × 10 8 ; At the same time, the correct insertion rate of the target fragment in the library was detected by colony...

Embodiment 2

[0059] Example 2: Screening process for V5-tagged protein single-domain antibodies:

[0060] (1) Take 200uL of recombinant TG1 cells and culture in 2×TY medium, add 40uL of helper phage VCSM13 to infect TG1 cells, and culture overnight to amplify the phages. The next day, the phages are precipitated with PEG / NaCl, and the amplified phages are collected by centrifugation. ; (2) NaHCO diluted in 100mM pH 8.3 3 500ug of neutravidin protein was coupled to the ELISA plate, placed at 4°C overnight, and a negative control well was set up; (3) 100uL of biotin-labeled V5-tagged protein (V5-Biotin) was added the next day, room temperature Incubate for 2 hours, add 100uL PBS to the negative control well; (4) After 2 hours, add 100ul of 3% BSA, and block at room temperature for 2h; (5) After blocking, add 100ul of amplified phage library (about 2×10 11 (6) After 1 hour of exposure, wash with PBS+0.05% Tween-20 for 5 times to remove unbound phages; (7) Use trypsin with a final concentrati...

Embodiment 3

[0061] Example 3: Screening of specific positive clones with phage-enzyme-linked immunosorbent assay (ELISA):

[0062] (1) Perform 3 rounds of screening on the V5-tagged protein according to the above single-domain antibody screening method. After the screening, the phage enrichment factor for the V5-tagged protein reaches more than 10, and select 100 single colonies from the positive clones obtained by screening. In a 96 deep-well plate containing 100ug / mL ampicillin in TB medium, and set a blank control, after culturing to log phase at 37 °C, add IPTG with a final concentration of 1 mM, and cultivate overnight at 28 °C; (2) Using permeation The crude antibody was obtained by the burst method; the neutravidin protein was diluted to 100 mM NaHCO, pH 8.3 3 Neutralize and coat 100ug of neutravidin protein in the ELISA plate overnight at 4°C, and add 100ug of V5-Biotin protein to the ELISA plate the next day; (3) Transfer 100uL of the crude antibody extract obtained in the above ...

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Abstract

The invention discloses application of a specific single domain antibody for a V5 tag protein. The single domain antibody has amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57 and SEQ ID NO:65-72; by adopting gene sequences and host cells of the single domain antibody disclosed by the invention, the singledomain antibody can be efficiently expressed and applied to stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM) to trace and detect target proteinsin a biological sample.

Description

technical field [0001] The invention relates to the field of biotechnology or biomedicine, in particular to the application of a single domain antibody specific for V5 tag protein. Background technique [0002] Antibodies are Y-type proteins secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. They are found in vertebrate body fluids and on the membrane surface of B cells. Antibodies are the most important component of biotechnology drugs and one of the most commonly used reagents in medical diagnosis and biological research. Therefore, antibody-related biological products have extremely high application prospects and commercial value. By immunizing animals to obtain antibodies specific for a certain protein (antigen), it can be widely used in academic research and industrial production. Antibodies bind to target proteins through their variable regions. Traditional antibodies contain light and he...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40G01N21/64
Inventor 苏志鹏谢维毕国强
Owner REGENECORE BIOTECH CO LTD
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