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High-affinity anti-carcinoembryonic antigen nanobody and application thereof

A nanobody and affinity technology, applied in the field of immunoglobulin, can solve the problems of affecting detection efficiency, easy aggregation, single antigenic determinant, etc., to improve detection efficiency and ensure specificity.

Active Publication Date: 2018-10-16
长春力太生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low molecular weight of Nanobodies, some structural defects such as low affinity, easy aggregation, and short serum half-life hinder the further application of Nanobodies.
In the specific application of CEA immunoassay, if the antigenic determinant of CEA recognized by the anti-CEA antibody is too single or the sites are too close or overlapped, the specific antigen-antibody binding reaction will be interfered, which will seriously affect the detection efficiency.

Method used

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  • High-affinity anti-carcinoembryonic antigen nanobody and application thereof
  • High-affinity anti-carcinoembryonic antigen nanobody and application thereof
  • High-affinity anti-carcinoembryonic antigen nanobody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and Screening of Anti-CEA Nanobody Phage Display Library

[0026] 1.1 Immunization of alpaca: Select a healthy adult alpaca, mix the recombinant protein CEA and Freund's adjuvant at a ratio of 1:1, and immunize the alpaca by subcutaneous multi-point injection on the back at 6-7μg / Kg , a total of four immunizations, immunization interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0027] 1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, every 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store the rest at -80°C.

[0028] 1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the concentration to 1 μg / μL with RNase-free water.

[002...

Embodiment 2

[0056] Example 2. Preparation of anti-CEA nanobody 2C5

[0057] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0058] Inoculate the original strain TG1 glycerolbacterium containing Nanobody nucleic acid in 5 mL of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, 1 μl of the above plasmid was transformed into 100 μl of competent cells, mixed gently, placed on ice for 30 minutes, heat-shocked in a water bath at 42°C for 90 seconds, and cooled in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and culture at 37°C for 60min. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37°C overnight.

[0059] 2.2 Induced expression of nanobodies

[0...

Embodiment 3

[0063] Example 3 Affinity Activity of Anti-CEA Nanobody and CEA Antigen

[0064] 3.1 Chip antigen coupling

[0065] The CEA antigen was prepared into a 20 μg / mL working solution with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0), and a 50 mM NaOH regeneration solution was prepared at the same time, using the Biacore T100 protein interaction analysis system instrument The template method in the analysis of the electrostatic binding between the antigen and the surface of the chip (GE company) under different pH conditions, with the signal increase amount reaching 5 times RL as the standard, select the most appropriate neutral pH system and The antigen concentration was adjusted as the conjugation conditions. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical coupling amount. The cou...

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Abstract

The invention discloses a high-affinity anti-carcinoembryonic antigen nanobody. The nanobody has three unique complementary determining regions CDR1, CDR2 and CDR3. The invention further provides an expression vector containing a nanobody variable region coding sequence and a host cell containing the expression vector, and further provides fusion protein of a nanobody variable region and alkalinephosphatase, the application of the nanobody to preparation of a carcinoembryonic antigen detection kit, a method for performing immunodetection on a carcinoembryonic antigen by applying the nanobody,and the corresponding detection kit. The anti-carcinoembryonic antigen (CEA) nanobody provided by the invention has a specific CEA recognizing and bonding ability, has the affinity which can reach 4.791E-10, and has unique antigen-determining cluster recognition sites; an excellent detection result can be obtained in CEA immunodetection, especially in a double-antibody sandwich method.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an immunoglobulin. Background technique [0002] Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domains. CEA, originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEA is observed in many types of cancers, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers, and thus, CEA has been identified as a tumor-associated antigen. CEA is easily cleaved from the cell surface and shed from the tumor into ...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N9/16G01N33/574
CPCC07K16/3007C07K2317/22C07K2317/33C07K2317/56C07K2317/565C07K2317/569C07K2317/92C07K2319/61C12N9/16C12Y301/03001G01N33/57473
Inventor 李文秋谢英林徐雪松王珂刘永红
Owner 长春力太生物技术有限公司
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