PD-1 nanoantibody as well as cloning and expression method and application thereof

A PD-1, nanobody technology, applied in the direction of antibodies, chemical instruments and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of low immunogenicity and short half-life, To achieve the effect of high relative activity, small molecular weight and low cost

Active Publication Date: 2018-07-20
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nanobodies have the characteristics of high affinity, high stability, high water solubility, high antigen binding, high expression, strong tissue permeability, low im

Method used

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  • PD-1 nanoantibody as well as cloning and expression method and application thereof
  • PD-1 nanoantibody as well as cloning and expression method and application thereof
  • PD-1 nanoantibody as well as cloning and expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Expression, purification and identification of human PD-1 extracellular segment protein in eukaryotic cells

[0070] According to the PD-1 extracellular domain (PD-1ECD) gene sequence (NCBI Gene ID: 5133) and referring to the pCMV (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) multi-cloning site information, design appropriate primers and add Nhe I, BamH I restriction site, and 6XHis tag, using pEZ-M02-PD-1 (purchased from Guangzhou Funeng Gene Co., Ltd.) as a template, using TaKaRa's PrimerSTAR HS DNA Polymerase for PCR reaction, PCR products were washed with 1% agarose Gel electrophoresis test, there is a clearly amplified band above 500bp, consistent with the expected size of 546bp. The pCMV vector and the amplified PD-1ECD gene were double digested with FastDigestNhe I and BamH I from Fermentas, and inserted into the pCMV plasmid to construct the eukaryotic expression vector pCMV-PD-1ECD encoding the PD-1ECD recombinant protein. Through trans...

Embodiment 2

[0071] Example 2 PD-1 Nanobody Screening

[0072] 1. VCSM13 helper phage preparation and titer determination

[0073] Different dilutions of VCSM13 (purchased from Agilent Technologies) were added to the newly amplified E.coli TG1 (purchased from China Plasmid Vector Strain Cell Gene Collection Center) bacterial solution, and incubated at room temperature for 15 minutes. Add 3mL of liquefied (below 50°C) LB top agar to each mixture, mix well and pour into LB plates, culture overnight at 37°C, count the number of plaques and obtain a single plaque. Pick a single phage plaque into 5mL of newly amplified E.coli TG1 bacterial liquid, shake and culture at 37°C for 3h, transfer the culture to 100mL 2YT liquid medium (containing kanamycin 70μg / mL), shake at 37°C Incubate overnight. The culture supernatant was collected by centrifugation, and the supernatant was collected by centrifugation after a water bath at 70°C, added with 7% DMSO, and stored at -20°C or -80°C.

[0074] 2. Lar...

Embodiment 3

[0086] Example 3 Expression and Purification of PD-1 Nanobody

[0087] 1. Competent preparation of E.coli WK6 electroporation

[0088] Take E.coli WK6 (purchased from China Plasmid Carrier Strain Cell Gene Conservation Center) seed solution stored at -80°C and inoculate it into 250mL LB medium at a ratio of 1% (v / v), and store at 37°C Shake culture to bacterial solution OD 600 =0.35~0.4, ice bath for 30min. Centrifuge at 4°C, add sterile ultrapure water to resuspend the pellet, repeat centrifugation once, add 20mL cold 10% sterile glycerol solution to resuspend the bacterial pellet. After centrifugation at 4°C, carefully aspirate the supernatant, resuspend the bacterial pellet in 1mL of cold 10% sterile glycerol solution, aliquot into pre-cooled sterilized 1.5mL EP tubes, and store at -80°C for later use.

[0089] 2. pMECS-Nb PD-1 electroconversion

[0090] The electroporation cup was pre-cooled on ice, and the WK6 competent cells prepared in step 1 were taken out and tha...

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Abstract

The invention discloses a PD-1 nanoantibody as well as a cloning and expression method and an application thereof. The PD-1 nanoantibody has small molecular weight, high specificity and high affinityand can inhibit the interaction between PD-L1 and PD-1 molecules, specifically recognize cell surface natural conformation PD-1 and block combination of PD-L1 and PD-1 to keep activity of T cells; thePD-1 nanoantibody has the combining capacity the same as a PD-1 monoclonal antibody (IgG complete antibody); the PD-1 nanoantibody can reverse and activate the PD-L1 inhibited state of T cells. Meanwhile, compared with the PD-1 monoclonal antibody, the nano antibody has higher relative activity and better thermal stability and has the potential of being developed into an immune checkpoint inhibitor after the same high-temperature treatment. Besides, the cloning and expression method of the nanoantibody has higher yield, the obtained protein purity can reach 93% or above, and the preparation method is simple and low in cost and has good application prospects in preparation of anti-tumor drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a PD-1 nanobody and its cloning and expression method and application. Background technique [0002] Tumor is one of the diseases with the highest fatality rate in the world, which seriously threatens human health. In addition to surgery and radiation therapy, tumor treatment methods also include: chemotherapy, hormone therapy, targeted drug therapy and tumor immunotherapy. Since "Science" magazine listed tumor immunotherapy as the most important scientific breakthrough of the year in 2013, tumor immunotherapy has attracted more and more attention in recent years. [0003] Targeted blockade of immune checkpoint tumor immunotherapy has gradually become a hot spot in cancer treatment. Since 2014, the FDA has approved Merck's Keytruda and Bristol-Myers Squibb's Opdivo for the treatment of melanoma, targeting programmed death molecule 1 (programmed death-1, PD-1, CD2...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/62C12N15/85A61K39/395A61P35/00
CPCA61K2039/505C07K16/2818C07K2317/565C07K2317/567C07K2317/569
Inventor 熊盛邓颖莎陈纯谢秋玲卢加李军洪岸
Owner JINAN UNIVERSITY
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