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Anti-procalcitonin high-affinity nano antibody and application thereof

A nanobody and procalcitonin technology, applied in the field of peptides, can solve the problems of low molecular weight affinity of nanobodies, hindering the application of nanobodies, short serum half-life, etc., and achieve excellent detection results

Active Publication Date: 2020-03-17
深圳市国创纳米抗体技术有限公司
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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 and functional defects such as low affinity, easy aggregation, and short serum half-life hinder the further application of Nanobodies.
In the specific application of PCT immunoassay, if the antigenic determinant of PCT recognized by the anti-PCT antibody is too single or the sites are too close or overlapping, the specific antigen-antibody binding reaction will be affected, which will seriously affect the detection efficiency.

Method used

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  • Anti-procalcitonin high-affinity nano antibody and application thereof
  • Anti-procalcitonin high-affinity nano antibody and application thereof
  • Anti-procalcitonin high-affinity nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 anti-PCT nanobody

[0035] 1.1 Construction and screening of anti-PCT nanobody phage display library

[0036] 1.1.1 Immunization of alpaca: Select a healthy adult alpaca, mix human recombinant PCT antigen with Freund's adjuvant at a ratio of 1:1, and immunize with 6-7μg / Kg by subcutaneous multi-point injection on the back Alpacas were immunized four times with an interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0037] 1.1.2 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.

[0038] 1.1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the conce...

Embodiment 2

[0060] Example 2. Preparation of Anti-PCT Nanobody 6H6

[0061] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL 21 (DE 3 )

[0062] 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, transform 1 μl of the above plasmid into 100 μl competent cells, mix gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and cool in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and incubate at 37°C for 60 minutes. 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.

[0063] 2.2 Induced expression of nanobodies

[0064] Th...

Embodiment 3

[0067] Example 3. Affinity Activity Determination of Anti-PCT Nanobodies and Antigens

[0068] 3.1 Chip antigen coupling

[0069] The PCT 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, and the Biacore T100 protein interaction analysis system instrument was used 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 coup...

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Abstract

The invention discloses a anti-procalcitonin high-affinity nano antibody. The nano antibody has three unique complementary determining regions CDR1, CDR2 and CDR3. The invention further provides an expression vector containing the variable region coding sequence of the nano antibody, and a host cell containing the expression vector. The invention further provides a fusion protein of the nano antibody variable region and alkaline phosphatase, the application of the nano antibody in preparation of a procalcitonin detection kit, a method for carrying out procalcitonin immunodetection by using thenano antibody, and a corresponding detection kit. The anti-procalcitonin nano antibody provided by the invention has specific recognition and binding capacity on procalcitonin. The affinity of the nano antibody can reach 3.773 E to 9. The nano antibody has a unique antigenic determinant recognition site, and can acquire an excellent detection effect in procalcitonin detection, and especially in adouble-antibody sandwich method.

Description

technical field [0001] The invention discloses a nanobody and belongs to the technical field of polypeptides. Background technique [0002] Procalcitonin (PCT) is a calcitonin propeptide substance without hormonal activity, composed of calcitonin, calcitonin, and N-terminal residue fragments, with a relative molecular weight of 13KDa. Calcitonin is produced only when thyroid C cells are stimulated by hormones, whereas PCT is secreted by different cell types in many organs in response to proinflammatory stimuli, especially those caused by bacteria. [0003] PCT can be used as an important marker that can specifically distinguish bacterial infection from other causes of inflammatory response. When severe bacterial, fungal, parasitic infection, sepsis and multiple organ failure, its level in plasma raised. Clinical data show that when the PCT concentration is greater than 0.1ng / ml, it indicates that there is a clinically relevant bacterial infection, which needs to be treated...

Claims

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Application Information

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IPC IPC(8): C07K16/26C07K19/00C12N9/16C12N15/13C12N15/70C12N1/21G01N33/74
CPCC07K16/26C12N9/16G01N33/74C12Y301/03001C07K2317/22C07K2317/569C07K2317/92C07K2319/00G01N2333/585
Inventor 林景涛宋海鹏于建立刘原源
Owner 深圳市国创纳米抗体技术有限公司
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