Nano-antibody aiming at procalcitonin and application thereof

A nanobody and procalcitonin technology, which is applied in the fields of biomedicine and clinical diagnosis, can solve the problems of no nanobody, no suitable clinical diagnosis nanobody and its detection kit products, etc., and achieve a good development and application prospect. , Overcome the effect of long development cycle, good specificity and stability

Inactive Publication Date: 2018-08-10
北京科卫临床诊断试剂有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are no relevant reports on the use of nanobody technology for the detection of procalcitonin, and there are no nanobodies and detection kits for procalcitonin that are suitable for clinical diagnosis.

Method used

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  • Nano-antibody aiming at procalcitonin and application thereof
  • Nano-antibody aiming at procalcitonin and application thereof
  • Nano-antibody aiming at procalcitonin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0026] Example: Preparation of Nanobodies against Procalcitonin

[0027] (1) Construction of PCT nanobody library:

[0028] (1) Mix 2 mg / mL PCT antigen with an equal volume of Freund's adjuvant, immunize a Bactrian camel, once every two weeks, and immunize 3 times in total to stimulate the camel's B lymphocytes to secrete PCT-specific nanobodies;

[0029] (2) extract 100mL camel peripheral blood, and extract the total RNA of lymphocyte;

[0030] (3) synthesize cDNA and utilize nested PCR to amplify the VHH gene fragment;

[0031](4) Digest 10 μg of the VHH gene fragment and 20 μg of the phage display vector pComb3 with restriction endonucleases Pst I and Not I;

[0032] (5) Ligate the above digested products and transform them into competent cells TG1 to construct a PCT nanobody library, and measure the size of the library to be 1.0×10 9 .

[0033] (2) Screening nanobodies against PCT:

[0034] (1) Dissolve 20 μg PCT antigen into 100 mM NaHCO 3 (PH9.6), and then added to...

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Abstract

The invention relates to the technical field of biomedicines and clinical diagnosis and discloses a novel nano-antibody aiming at procalcitonin (PCT), a coding sequence of the novel nano-antibody andapplication of the novel nano-antibody to diagnosis. According to the novel nano-antibody provided by the invention, camels are immunized by utilizing the procalcitonin to obtain a high-quality nano-antibody gene library; then an enzyme-coupled plate is coated with the PCT to display a correct space structure of protein; the PCT is used as an antigen for screening a camel heavy-chain antibody bacteriophage display gene library, so that a nano-antibody gene aiming at the PCT is obtained; then the gene is transformed into escherichia coli and a nano-antibody strain which can be efficiently expressed in the escherichia coli is established. According to the novel nano-antibody provided by the invention, two different antigen epitopes, which can be combined with the nano-antibody, in the PCT are finally determined, and the nano-antibody has good specificity and stability; furthermore, the obtained nano-antibody is coated and labeled and a detection method of the PCT is established; a firm foundation is provided for researches and development of a PCT clinical diagnosis kit.

Description

technical field [0001] The invention relates to the technical fields of biomedicine and clinical diagnosis, in particular to a nanobody directed against procalcitonin, its coding sequence and its application in diagnosis. Background technique [0002] Procalcitonin (PCT for short) is a protein in human blood. Its level in plasma increases in severe bacterial, fungal, and parasitic infections, as well as sepsis and multiple organ failure. Autoimmunity, allergies, and viral infections do not increase PCT, and local limited bacterial infections, minor infections, and chronic inflammation do not cause it to increase. PCT reflects the active degree of systemic inflammatory response. Factors affecting PCT level include the size and type of infected organ, the type of bacteria, the degree of inflammation and the status of immune response. [0003] The detection of PCT is of great significance for guiding clinical treatment and evaluating the effect of treatment. Traditional detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26G01N33/74C12N15/13C12N15/70
CPCC07K16/26C07K2317/22C07K2317/565C07K2317/567C07K2317/569C12N15/70G01N33/74G01N2333/585
Inventor 王保君万亚坤冯长访张希忠汤双双史素娟刘耀飞郑德森李良红聂章林
Owner 北京科卫临床诊断试剂有限公司
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