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Human genetic engineering antibody TRD 109 as well as preparation method and application thereof

A genetically engineered antibody, antibody technology, applied in the direction of antibodies, anti-inflammatory agents, recombinant DNA technology, etc., can solve the problems of heavy economic burden, high treatment costs, low immunogenicity, etc. Simple structure and the effect of relieving symptoms of allergic rhinitis

Inactive Publication Date: 2010-04-21
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first two have obvious side effects due to their high immunogenicity. Abbott Company applied phage display technology to develop a human TNF-α antibody Adalimumab. Although its clinical effect has been widely recognized, it has no toxic side effects. Fewer and less immunogenic, but its high cost of treatment imposes a heavy financial burden on most patients

Method used

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  • Human genetic engineering antibody TRD 109 as well as preparation method and application thereof
  • Human genetic engineering antibody TRD 109 as well as preparation method and application thereof
  • Human genetic engineering antibody TRD 109 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Construction of ribosome-displayed single-chain antibody (VH / K) gene library

[0057] 1-1 Extraction of total cellular RNA and synthesis of cDNA

[0058] Extract 40ml of peripheral blood from patients clinically diagnosed as rheumatoid arthritis, add 5U heparin to each milliliter of blood; ) in a centrifuge tube, put it in a low-speed desktop centrifuge, 2500r / min, and centrifuge for 20min; gently absorb the lymphocyte layer with a capillary pipette, and place it in another 15ml centrifuge tube; dilute it with normal saline, 1000r / min, Centrifuge for 10 min, carefully remove the supernatant, and repeat this process twice; resuspend the pellet according to the ratio of adding 1 ml of normal saline to every 10 ml of blood, and distribute it in EP tubes; then use the total RNA isolation kit (Promega Company) to extract the total RNA of the cells .

[0059] Synthesis of cDNA (RNA reverse transcription kit purchased from TaKaRa Company) using total cell RNA as template, Hu...

Embodiment 2

[0084] In vitro transcription and translation

[0085] The in vitro transcription and translation were carried out by expresswayTM Plus ExpressionSystem of Invitrogen Company according to the operation manual. 20μL of IVPS Plus E.coli Extract, 20μL of 2.5×IVPS Plus E.coli Reaction Buffer, 1μL of 75mmol / L methionine, 1μL of 200μmol / L Anti-ssrA oligonucleotide, 1μL of PCR product, and 6μL of DNase-free water Add it into a 2.0mL EP tube; keep the reaction solution at 37°C for 30min, and use 4 times the volume of ice-precooled WBTH (50mmol / L Tris-acetate Ph 7.5, 150mmol / L NaCl, 50mmol / L magnesium acetate, 0.1% Tween-20, 2.5g / Lheparin, Sigma) After terminating the reaction, add pre-cooled 1 / 5 volume of WBT containing 10% (w / v) BSA to the supernatant; the reaction mixture was centrifuged at 1400g for 5min at 4°C Afterwards, transfer the supernatant to a new ice-cooled EP tube.

Embodiment 3

[0087] Affinity screening of ribosomal complexes

[0088] In order to screen the specific VH / K antibody gene against TNF-α, the 96-well plate was coated with recombinant TNF-α purified protein (PEPROTECH, USA), 40 μg / well, BSA-S.cerevisiae RNA-PBS blocking; the translation mixture Transfer to the coated wells, shake the microplate in a cold room for 1 hour, pour off the translation reaction solution, wash the microplate with WBT for 5 times; Shake for 5 minutes to elute the mRNA; the eluted mRNA was purified with RNeasy kit; the purified mRNA was dissolved in RNase-free water and treated with RNase-free DNase to remove the DNA molecules in the reaction solution; the purified mRNA was used as a template, RT-PCR was carried out, and the PCR product was used as the next cycle, namely: transcription-translation-screening, and a total of 3 rounds of screening were performed.

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Abstract

The invention provides a human genetic engineering antibody TRD 109 which is screened by utilizing a molecular biological technology-ribosome display technology, aims to TNF-alpha and realizes high-efficiency expression of the antibody to a prokaryotic system. A biological characteristic research demonstrates that the TRD 109 is a human genetic engineering antibody which has high affinity, better stability, simple structure and easy construction and can specifically neutralize the TNF-alpha, inhibit the generation of multiple kinds of cell factors, block the generation and development of AR in the source and is a human anti TNF-alpha antibody which can reduce the infiltration of eosinophils in a mouse nasal mucous membrane and relieve mouse allergic rhinitis symptom; and a gene and a gene product of the human anti TNF-alpha antibody can be used for preparing a medicaments for diagnosing and treating diseases caused by overexpression of the TNF-alpha, such as preparing a medicament for treating diseases including allergic rhinitis and rheumatoid arthritis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the human genetic engineering antibody TRD109 and its preparation method and application. Background technique [0002] Allergic rhinitis (AR) is a nasal inflammatory disease triggered by allergens and mediated by IgE. It is the most common disease in otolaryngology and one of the most common chronic diseases in the world. The main symptoms are runny nose, nasal congestion, nasal itching and sneezing. With the rapid changes in modern human life style and ecological environment, the incidence of AR is increasing globally, and the incidence in cities is much higher than that in rural areas. Although AR is not a major disease, it has a great impact on people's work, study and life. Serious impact, has gradually become a global health problem. The principle of AR treatment is mainly to avoid exposure to allergens, drug control and immune regulation. At present, there is no specific tre...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/63C12P21/08A61K39/395A61P11/02A61P37/08A61P29/00A61P19/02A61P19/04G01N33/577
Inventor 赵小玲杨志华陈伟强田露芳
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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