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Anti-GST tag protein nano antibody and application thereof

A technology for labeling proteins and nanobodies, applied in anti-enzyme immunoglobulins, applications, biochemical equipment and methods, etc., can solve the problems of short half-life and prolong half-life, and achieve the effect of good plasticity, high affinity and stable performance

Active Publication Date: 2018-01-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current nanobodies have the disadvantage of short half-life, and modification measures to extend the half-life are needed

Method used

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  • Anti-GST tag protein nano antibody and application thereof
  • Anti-GST tag protein nano antibody and application thereof
  • Anti-GST tag protein nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Panning and Identification of Anti-GST Tag Protein Single Domain Heavy Chain Antibody

[0030] Using the method of solid-phase panning, the GST-tagged protein was diluted to 30-100ug / ul with 1xPBS, coated on the ELISA plate, and 100ul was added to each blank, and coated overnight at 4°C. Wash three times with PBS, add 300ul 4% skimmed milk to each well, and block for 2 hours at 37°C. After washing three times with PBS, add the phage display library (approximately 1x10 12 CFU), incubated at 37°C for 1 hour. Aspirate unbound phage, add PBS to wash 5-10 times (increase the number of washes with each round), and then wash three times with PBST, add 100ul of glycine-hydrochloric acid solution with pH=2.2, and incubate at 37°C for 5 minutes. Gently blow the wells of the plate to wash off the adsorbed phages, then add 15ul of Tris-Hcl solution (PH=8.8), take 10ul to measure the titer, and the rest will be amplified and used for the next round of panning.

[0031]...

Embodiment 2

[0044] Example 2: Expression and purification of Nanobodies

[0045] The above-mentioned base sequence was sent to the biological company and inserted into the PET28a plasmid to construct an expression vector. Take 1 ul of the constructed PET28a plasmid and add it to 50 ul of BL21 competent, place it on ice for 30 minutes, then place it in a 42° water bath for heat shock for 90 seconds, place it on ice for 5 minutes, then add 600 ul of LB medium and put it in 37 ° Incubate for 1 hour in an incubator. Take 100ul of the above-mentioned culture solution and spread it on the ampicillin plate, put it into a 37° constant temperature incubator and cultivate it overnight. The next day, pick a single clone from the plate and inoculate it into 1L culture medium, culture at 37°, 220rmp / min, when the bacterial solution OD=0.5, add IPTG with a final concentration of 0.1mM, 16°, 180rmp / min, overnight induced. The next day, the bacterial liquid was collected by centrifugation and resuspen...

Embodiment 3

[0046] Example 3: Western blot verification

[0047] Western blot step: Take 500ng of anti-GST tagged protein, load the sample, electrophoresis with sodium dodecylsulfonate-polyacrylamide gel (SDS-PAGE), transfer to membrane, add specific primary antibody and corresponding secondary antibody successively for incubation , and then analyzed the effect of anti-GST tag protein nanobody by chemiluminescence colorimetry.

[0048] 1. Protein denaturation

[0049] Mix the protein sample whose concentration has been determined with 5× loading buffer solution at a ratio of 4:1, cook in boiling water for 10 minutes to denature the protein, and store it at -70°C after aliquoting for later use or directly use it for loading.

[0050] 2. Preparation of separating gel

[0051] 1) Prepare two matching 1.5mm glass plates, including a thin plate and a thick plate;

[0052] 2) Rinse the glass plate with tap water and dry it;

[0053] 3) Wash the comb with water and let it dry naturally;

[...

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Abstract

The invention relates to an anti-GST tag protein nano antibody and an application thereof. The anti-GST tag protein nano antibody contains a VHH chain composed of a framework region FR and a complementary determining region CDR, wherein the framework region FR comprises the following group of FR amino acid sequences: FR1 shown as SEQ ID NO:1, FR2 shown as SEQ ID NO:2, FR3 shown as SEQ ID NO:3, andFR4 shown as SEQ ID NO:4, the complementary determining region CDR comprises the following group of CDR amino acid sequences: CDR1 shown as SEQ ID NO:5, CDR2 shown as SEQ ID NO:6, and CDR3 shown as SEQ ID NO:7. The invention also comprises an application of the anti-GST tag protein nano antibody. The anti-GST tag protein nano antibody has the advantages of small molecules, easy expression, high affinity, good solubility, strong stability, and low immunogenicity.

Description

technical field [0001] The invention relates to the field of proteomics and molecular protein interaction, in particular to a nanobody against GST tag protein and its application. Background technique [0002] The hotspot in today's pharmaceutical industry, the development of therapeutic antibody drugs called "biomissiles" is extremely active, but their common characteristics, such as too large molecules, too complex structures, and too expensive prices limit its production and clinical application promotion. For example, each molecule of an ordinary antibody contains two sets of heavy chains and light chains, which overlap with sugar molecules, thus forming an antibody drug. The macromolecular characteristics restrict its wide use and curative effect, and many diseases cannot be treated with monoclonal antibody drugs. At the same time, monoclonal antibodies will decompose under the conditions of high temperature and strong acid and strong alkali, and must be stored at abso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13
Inventor 林坚房景刚周鹏陈鹏
Owner PEKING UNIV
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