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Single-domain antibody HCV-E2 of hepatitis C virus E2 protein and application of single-domain antibody HCV-E2

A technology of HCV-E2 and hepatitis C virus, which is applied in the field of biomedicine or biotechnology, can solve the problem of lack of single-domain antibodies to hepatitis C virus, etc., and achieve the effect of less cross-reaction, broad application prospects and high sensitivity

Active Publication Date: 2022-07-29
BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is currently a lack of satisfactory single domain antibodies against hepatitis C virus in the art

Method used

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  • Single-domain antibody HCV-E2 of hepatitis C virus E2 protein and application of single-domain antibody HCV-E2
  • Single-domain antibody HCV-E2 of hepatitis C virus E2 protein and application of single-domain antibody HCV-E2
  • Single-domain antibody HCV-E2 of hepatitis C virus E2 protein and application of single-domain antibody HCV-E2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Immunopanning process of native single domain antibody against E2 protein

[0037] (1) Amplify the established natural single-domain antibody phage library: add 100µL of glycerol bacterial library to 2×YT medium, when OD600=0.5, add 20 MOI helper phage, let stand for 30min, and centrifuge the pellet with 2×YT medium. Resuspend in YT medium, culture for 1 hour, add antibiotics for 16 hours and then centrifuge, the supernatant is precipitated by pre-cooled PEG-NaCl (1 / 4 volume), and resuspended in 1 mL of PBS is the amplified single domain antibody library;

[0038] (2) Immune tube panning: E2 protein ( figure 1, biotin-labeled and purified) 50µg / tube coat the immunotube overnight, remove the coating solution, wash 3 times, block with 2mL BSA (1%) for 2h, wash 3 times with PBST, add 100µL (1) step amplification The single-domain antibody library was used as the primary antibody at 37°C for 2 h, washed with PBST for 3 times, eluted with Glycine-HCI (PH2.2), and...

Embodiment 2

[0041] Example 2: ELISA identification of single clones

[0042] (1) Panning a single positive clone: ​​Inoculate the natural single domain antibody library of the third round of panning in 2×YT medium, OD 600nm = 0.5, add 20 MOI helper phage, let stand for 30 min, resuspend the pellet in 2×YT medium after centrifugation, culture for another 1 h, then spread on 2×YT plate containing antibiotics, culture overnight, select 40 A single colony was inoculated into 2×YT medium. When OD600=0.5, 20 MOI of helper phage was added and left for 30 min. After centrifugation, the pellet was resuspended in 2×YT medium, re-cultured, and IPTG was added to induce expression for 8 h. .

[0043] (2) ELISA identification: 100µL / well of E2 protein (with biotin tag and purified, concentration 1ng / µL) was coated on ELISA plate overnight, washed 3 times after removing the coating solution, and blocked with 200µL / well BSA (3%). 2h, washed 3 times with PBST, added 100µL / well of the single domain antib...

Embodiment 2

[0045] Example 2 Specific identification of HCV-E2

[0046] The above sequence was sent to BGI (Beijing) to purify and synthesize the E2 protein single-domain antibody HCV-E2 for Western blot identification: A. Protein electrophoresis: Dilute the purified E protein with 6 x SDS protein electrophoresis loading buffer To 60μg, boil at 100 ℃ for 5 min, pre-stain Marker 2 μL, carry out protein electrophoresis on concentrated gel at 80 v and separation gel at 120 v. B. Transfer membrane: Put the gel on the nitrocellulose membrane (NC membrane), put 3 Whatman 3 mm filter papers on the top and bottom, put the above items in the transfer membrane electrophoresis buffer and soak for 15 minutes to drive out the residue on the filter membrane. on the bubbles. Install the electrotransfer device in sequence, place 3 pieces of filter paper, gel, NC membrane, and 3 pieces of filter paper in sequence on the negative electrode plate to ensure the precise alignment of each layer (from bottom t...

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Abstract

The invention provides a single-domain antibody HCV-E2 of hepatitis C virus E2 protein and application thereof, the single-domain antibody HCV-E2 of hepatitis C virus E2 protein can be used for a rapid detection test strip of hepatitis C virus, and the rapid detection test strip comprises a bottom plate, water absorption packing paper, an NC membrane, a gold pad and a sample chromatography pad. A contrast test proves that the single-domain antibody HCV-E2 is more sensitive than a commercial monoclonal antibody in HCV detection, the nano antibody HCV-E2 is combined with a colloidal gold labeling technology, the sensitivity is high, and the detection limit of HCV detection can reach 10ng / mL.

Description

technical field [0001] The invention relates to the application field of biomedicine or biotechnology, in particular to a single domain antibody HCV-E2 of hepatitis C virus E2 protein and its application. Background technique [0002] Hepatitis C is a group of systemic infectious diseases caused by hepatitis C virus (HCV). It is estimated that about 170 million people in the world are infected with HCV, and about 70% of them are transformed into chronic diseases, of which about 20% to 30% develop liver cirrhosis in 10 to 30 years, and 1% to 5% of them can lead to hepatocellular carcinoma. Hepatitis C infection has become a serious global public health problem. There is currently no effective hepatitis C vaccine available. [0003] At present, the most commonly used method for the detection of hepatitis C virus is immunoassay, which is based on the specific recognition of antigen-antibody, including the traditional enzyme-linked immunosorbent assay (ELISA) and the recently ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/58G01N33/577G01N33/576G01N33/558G01N33/543G01N33/52
CPCC07K16/109C07K16/005G01N33/587G01N33/577G01N33/5767G01N33/558G01N33/54346G01N33/52C07K2317/569G01N2333/186G01N2469/10
Inventor 陈丽娟刘秀颖庞星火邱倩孟璐璐付茵曹若湘安宁王慧孙美平
Owner BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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