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V5-tag protein specifically targeted single-domain antibody and derived proteins thereof

A single-domain antibody and tag protein technology, which is applied in the field of biotechnology or biomedicine, can solve the problems that restrict the research, purification and detection of cell haptic fine structure and function, which take a lot of time, and the accuracy of the results is not enough, so as to achieve yield and High purification efficiency, low steric hindrance, and stable quality between batches

Active Publication Date: 2019-03-08
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • V5-tag protein specifically targeted single-domain antibody and derived proteins thereof
  • V5-tag protein specifically targeted single-domain antibody and derived proteins thereof
  • V5-tag protein specifically targeted single-domain antibody and derived proteins thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction of a single domain antibody library against V5 tagged protein:

[0055] (1) Mix 1 mg of V5-KLH antigen with Freund's adjuvant in equal volumes, immunize a Xinjiang Bactrian camel, once a week, and immunize continuously for 7 times in total, and stimulate B cells to express specific nanobodies during the immunization process; ( 2) After immunization, extract 100ml of camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Use restriction enzymes PstⅠ and NotⅠ to digest 20ug pMECS phage display Vector and 10ug VHH and connect the two fragments; (5) Transform the ligated product into electroporation competent cell TG1, construct the V5 tagged protein phage display library and measure the storage capacity, the size of the library storage capacity is about 2×10 8 ; At the same time, the correct insertion rate of the target fragment in the built library was detected by colony PCR, figure 1 Colon...

Embodiment 2

[0056] Example 2: Screening process for V5 tag protein single domain antibody:

[0057] (1) Take 200uL of recombinant TG1 cells and culture them in 2×TY medium, add 40uL of helper phage VCSM13 to infect TG1 cells during the period, and culture overnight to amplify the phages, use PEG / NaCl to precipitate the phages the next day, and centrifuge to collect the amplified phages ; (2) NaHCO diluted in 100mM pH 8.33 500ug of neutravidin protein in the medium was coupled to the microtiter plate, placed overnight at 4°C, and a negative control well was set up at the same time; (3) the next day, add 100uL of biotin-labeled V5-labeled protein (V5-Biotin), at 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 phage particles) at room temperature for 1 hour; (6) after 1 hour of reaction, wash 5 times with PBS+...

Embodiment 3

[0058] Example 3: Screening specific positive clones with phage enzyme-linked immunosorbent assay (ELISA):

[0059] (1) Perform 3 rounds of screening for V5-tagged proteins according to the above-mentioned single-domain antibody screening method. After the screening, the phage enrichment factor for V5-tagged proteins reaches more than 10, and select 100 single colonies from the positive clones obtained by screening to inoculate them respectively. In a 96 deep-well plate containing 100ug / mL ampicillin in TB medium, and set a blank control, after culturing at 37°C to the logarithmic phase, add IPTG with a final concentration of 1mM, and cultivate overnight at 28°C; (2) use osmotic The crude antibody was obtained by the expansion method; the neutravidin protein was diluted to 100mM NaHCO pH 8.3 3 Neutralize and coat 100ug neutravidin protein in the microtiter plate overnight at 4°C, and add 100ug V5-Biotin protein to the microtiter plate the next day; (3) transfer 100uL of the an...

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Abstract

The invention discloses a V5-tag protein specifically targeted single-domain antibody and derived proteins thereof. The V5-tag protein specifically targeted single-domain antibody has the amino acid sequences shown as 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. Through the gene sequences and the host cells of the V5-tag protein specifically targeted single-domain antibody, the V5-tag protein specifically targeted single-domain antibody can be expressed efficiently and can be applied to stochastic optical reconstruction microscopy (STORM) and Photo-activated localization microscopy (PLAM) for tracing and detecting target proteins in biological samples.

Description

technical field [0001] The invention relates to the field of biotechnology or biomedicine, in particular to a single-domain antibody specific for V5 tag protein and its derivative protein. Background technique [0002] Antibody is a Y-shaped protein secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. It is found in the body fluids of vertebrates and the membrane surface of B cells. Antibodies are the most important components of biotech 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. Obtaining antibodies specific to a certain protein (antigen) by immunizing animals can be widely used in academic research and industrial production. Antibodies bind to target proteins through their variable regions. Traditional antibodies include light chains...

Claims

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Application Information

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IPC IPC(8): C07K16/40G01N21/64
CPCC07K16/40C07K2317/565C07K2317/567C07K2317/569G01N21/6458
Inventor 苏志鹏谢维毕国强
Owner REGENECORE BIOTECH CO LTD
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