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Method for screening multi-transmembrane region protein antibody and application of multi-transmembrane region protein antibody

A screening method and technology of transmembrane region, which are applied in the field of multi-transmembrane region protein antibody screening, can solve the problem that the antigen cannot be directly coated, and achieve the effect of improving transportation and high concentration

Inactive Publication Date: 2018-05-04
深圳市中瑞牛津生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve: 1) the antigen cannot be directly coated; 2) the choice of detergent for washing the plate; 3) the choice of antibodies only targeting the extracellular region

Method used

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  • Method for screening multi-transmembrane region protein antibody and application of multi-transmembrane region protein antibody
  • Method for screening multi-transmembrane region protein antibody and application of multi-transmembrane region protein antibody
  • Method for screening multi-transmembrane region protein antibody and application of multi-transmembrane region protein antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Expression strategy of copper ion transporter CTR1 for antibody screening

[0056] The human copper ion transporter spans the cell membrane three times, belongs to multi-transmembrane protein, and consists of 270 amino acids. The N-terminus of the protein contains a divalent cation binding site, which can be used for purification by metal chelation affinity chromatography. In addition, we fused a stretch of (DYKDDDDKG) epitope recognized by anti-FLAG antibody at its C-terminus. The entire DNA was synthesized by the American Invitrogen Company and cloned into the American InvitrogenBac-to-Bac expression system. The preparation of recombinant baculovirus was carried out in strict accordance with the operating procedures set by Invitrogen. In the sequence listing, SEQ ID NO: 1 is the amino acid sequence of hCTR1, SEQ ID NO: 2 is the DNA sequence of hctr1, SEQ ID NO: 3 and SEQ ID NO: 4 are the amino acids added at the C-terminus of the human copper ion transporte...

Embodiment 2

[0057] Example 2 Expression and purification of hCTR1 protein

[0058] The expression and purification of hCTR1 protein were divided into the following steps.

[0059] 1. Expression of hCTR1 protein and separation of cell membrane components

[0060] The expression system Sf9 obtained in Example 1 was cultured in a shake flask containing SF 900 II medium (Invitrogen), and the cell density was 2.0 × 10 6 cells / ml culture medium, inoculate the third-generation baculovirus with a multiplicity of infection of 0.5. Continue to culture the cells at 27°C at 100 rpm. Harvest cells when their viability has dropped to between 70% and 80% (usually 70 to 72 hours after infection). Cells were washed once with PBS, suspended in 50 mM HEPES (pH 7.5) buffer, and disrupted with EmulsiFlex-C5 at a pressure of 10,000 psi. Cell lysates were first centrifuged at 1000g for 10 min at low temperature (4°C) to remove unlysed cells and nuclei. The supernatant was centrifuged at 100,000g for 1 h a...

Embodiment 3

[0063] Example 3 Antigen Preparation and Animal Immunization

[0064] Glycerol monooleate was melted at 40°C and added to a Haminton syringe, and the hCtr1 protein solution (5 mg / ml) and 1mg / ml phospholipid A to form a mixed solution (the volume ratio of protein solution and phospholipid A is 4:1; the volume ratio of glycerol monooleate to the mixed solution is 3:2), and the two syringes are connected with a special connector Then mix the contents of the two syringes until a clear lipid cubic phase (LCP) forms. Suspend the prepared lipid cubic phase in PBS so that each milliliter of phosphate buffer contains a protein concentration of 2 mg per milliliter, and then use a small high-pressure cell breaker to disperse the lipid cubic phase into microparticles with 20Kpsi twice.

[0065] Mix and emulsify the lipid cubic phase prepared above with an equal volume of Freund's complete adjuvant, and immunize rabbits. The dose of 600 μg protein per rabbit is used for the first immuniz...

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Abstract

The invention provides a method for screening a multi-transmembrane region protein antibody and an application of the multi-transmembrane region protein antibody. The method comprises the following steps: by taking a multi-transmembrane region protein as an antigen, screening an antibody for complete proteins, and by taking a baculovirus with the multi-transmembrane region protein as an antigen, identifying the antibody for an extracellular region of the multi-transmembrane region protein. The invention further relates to an antibody prepared by using the method and an application of the antibody.

Description

technical field [0001] The present invention relates to a screening method and application of multi-transmembrane domain protein antibody, in particular to the screening method and application of multi-transmembrane domain protein extracellular region conformation antibody. Background technique [0002] Proteins that cross the biomembrane more than twice are collectively referred to as multi-transmembrane integral membrane proteins, including G protein-coupled receptors, ion channels, transmembrane transporters, etc. Integral membrane proteins account for about 30% of total cell proteins and are the targets of more than two-thirds of drugs. For example, the human copper ion transporter (hCtr1) belongs to the integral membrane protein with multiple transmembrane domains. It is one of the main transmembrane transporters of copper ions required by the human body. In addition, Cisplatin is also transported across the membrane through the hCtr1 protein. Cisplatin is a chemothe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395A61P35/00C07K14/705C07K16/06
CPCA61K39/0011A61K39/39516A61K39/39558A61K2039/55566C07K14/705C07K16/06
Inventor 夏小兵彭艳春李子剑陈智杰
Owner 深圳市中瑞牛津生命科学技术有限公司
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