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Method for rapidly acquiring nano-antibodies and application of method

A nanobody, fast technology, applied in the biological field, can solve problems such as complex technical process, high cost, difficult to be effectively expressed and displayed

Inactive Publication Date: 2017-01-04
康众(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] (3) Since the phage display system depends on the expression of genes in cells, some molecules that are toxic to cells, such as biotoxin molecules, are difficult to be effectively expressed and displayed
[0009] (4) Phage library surface display technology is complex and costly

Method used

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Embodiment Construction

[0074] A method for rapidly obtaining nanobodies, said method comprising the steps of:

[0075] Step 1, acquisition of negative control;

[0076] Step 2, isolation of alpaca peripheral blood mononuclear cells (PBMCs);

[0077] Step 3, flow cytometry sorting;

[0078] Step 4, extract positive cell RNA, reverse transcribe into cDNA;

[0079] Step 5, amplification of the VHH fragment;

[0080] Step 6, linking the VHH fragment into the expression vector;

[0081] Step 7, PCR identification of recombinant prokaryotic expression vector;

[0082] Step eight, expression of pet28a-CD19-VHH;

[0083] Step 9, ELISA activity identification of the expressed antibody.

[0084] The first step includes purchasing the alpaca, marking the alpaca, taking 30 mL of blood from the jugular vein before immunization, and putting it into a pre-prepared 50 mL centrifuge tube with anticoagulant, the amount of anticoagulant is 20-50 U per mL of blood , isolate peripheral blood mononuclear cells (PB...

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Abstract

The invention discloses a method for rapidly acquiring nano-antibodies. The method comprises the following steps: (1) acquiring a negative control; (2) separating peripheral blood mononuclear cells (PBMC) of an alpaca; (3) carrying out flow cytometry sorting; (4) extracting RNA of positive cells, and carrying out inverse transcription to form cDNA; (5) amplifying VHH segments; (6) linking the VHH segments into an expression vector; (7) carrying out PCR identification on a recombinant prokaryotic expression vector; (8) carrying out expression of pet28a-CD19-VHH; and (9) carrying out ELISA activity identification on the expression vector.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly obtaining nanobodies and an application thereof. Background technique [0002] Antibodies are immunoglobulins produced by plasma cells differentiated from B cells under the stimulation of antigenic substances, and can specifically bind and react with corresponding antigens. [0003] In 1993, Hamers Casterman of Belgium reported that in camel serum, there were not only IgG1 conventional antibodies composed of 2 H chains and 2 L chains, but also heavy-chain antibodies of IgG2 and IgG3 subtypes lacking light chains. , hcAb), with full antigen-binding ability. These heavy chain antibodies (HcAbs) account for about 50% of the total Ig in the serum of the genus Alpaca (Lama) and about 75% of the total Ig of the serum of the genus Camelus (Camelus). The heavy chain antibodies can bind some conventional antibodies that cannot be accessed antigenic epitopes....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C07K16/28
CPCC12N15/70C07K16/2803C07K2317/22C07K2317/569C12N15/66
Inventor 李福胜李洪利庄文超
Owner 康众(北京)生物科技有限公司
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