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Polypeptide composition for detecting serum marker of autoimmune disease patient and application of polypeptide composition

A technology of serum markers and polypeptide compositions, applied in the field of medicine, can solve the problems of complex pathogenesis and unclear specific mechanisms of autoimmune diseases

Active Publication Date: 2017-10-24
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenesis of autoimmune diseases is very complex, involving many factors including genetics, infection, immune dysfunction, etc., but the specific mechanism has not yet been clarified

Method used

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  • Polypeptide composition for detecting serum marker of autoimmune disease patient and application of polypeptide composition
  • Polypeptide composition for detecting serum marker of autoimmune disease patient and application of polypeptide composition
  • Polypeptide composition for detecting serum marker of autoimmune disease patient and application of polypeptide composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Serum coating and phage adsorption and elution

[0045] Select 30 cases of autoimmune disease patients and 30 normal human sera, mix them separately, and add them to a 96-well plate after 1:10 dilution, 150 microliters per well, incubate overnight at 4℃, 30r / min with slight shaking, then discard the wells Fill up the blocking buffer and incubate at 4°C for 2h. Pour out the blocking buffer in the well, wash it quickly with TBST (TBS+0.1v / v%Tween-20) for 6 times, and dilute 2×10 with 100μL of TBST buffer 11 The original library of pfu phage (including E.coli ER2738 host bacteria kit, produced by New England Biolabs, USA), the library plasmid map is as follows figure 1 , Add it to the wells that have been coated with normal human serum, and shake gently at room temperature for 60 minutes. Collect the phage supernatant that has not been combined with the normal human mixed serum in a sterile microcentrifuge tube. Dilute with 1 mL of TBST buffer, add to the well plat...

Embodiment 2

[0046] Example 2 Titer determination and clonal expansion of phage bound to patient mixed serum

[0047] Pick the recovered single colony of E.coli ER2738 in 10ml LB medium. When the culture reaches the logarithmic growth phase, divide it into 1.5mL centrifuge tubes, each tube 200μL, and add 10μL to each tube to combine with the patient's mixed serum Incubate the phage stock solution at room temperature for 5 min, add it to the pre-warmed top agar culture tube, and immediately pour it on the LB / IPTG / Xgal plate pre-warmed at 37°C. After the plate is cooled, it is placed upside down at 37°C and incubated overnight. Check the plate and count the number of plaques on the plate with image 3 . Pick blue plaques in 1 mL of 1:100 diluted ER2738 overnight culture medium. Incubate on a shaker at 37°C at 150r / min for 4.5h. The culture was transferred to a 1.5mL centrifuge tube and centrifuged for 30s, the supernatant was transferred to a new tube, and centrifuged for another 30s. Trans...

Embodiment 3

[0048] Example 3 Determination of the ssDNA sequence of the positive phage bound to the serum of patients with autoimmune diseases

[0049] Transfer 500 μl of phage-containing supernatant to a new centrifuge tube. Add 200μl PEG / NaCl, place at room temperature for 10min, centrifuge at 10,000r / min at 4℃ for 10min, and discard the supernatant. Centrifuge for another 2 minutes and carefully remove the remaining supernatant. The pellet was completely resuspended in 100 μL of iodide buffer and 250 μL of ethanol was added. Incubate at room temperature for 10 min. Centrifuge for 10 min and discard the supernatant. The precipitate was washed with 70% ethanol and dried briefly in vacuum. The pellet was resuspended in 30μL TE [10mM Tris-HCl (pH 8.0), 1mM EDTA], and the -96gIII sequencing primer 5'-CCC TCA TAG TTA GCG TAA CG-3', 100pmol / μL, 1pmol / μL was used for automatic sequencing. The read sequence corresponds to the antisense strand of the template, write out the complementary stran...

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Abstract

The invention discloses a polypeptide composition for detecting a serum marker of an autoimmune disease patient and an application of the polypeptide composition and belongs to the technical field of medicines. Polypeptide with bonding capability on the serum marker of an autoimmune disease is screened out from a phage random peptide library, and is applied to immunological detection of the serum marker through artificial synthesis. The polypeptide composition comprises all or partial sequences as shown in SEQID NO.1-19. In addition, the invention further discloses a chip comprising the polypeptide composition, an enzyme-linked immune response plate and a kit. The obtained polypeptide sequences have good bonding capability on the serum marker of the autoimmune disease patient, and can be applied to diagnosis and evaluation of the autoimmune disease.

Description

Technical field [0001] The invention relates to a set of polypeptides for detecting serum markers of patients with autoimmune diseases, and also relates to the application of the polypeptide combination in the preparation of polypeptide chips and enzyme-linked immunosorbent antibody detection kits, which can realize the detection of autoimmune diseases in plasma / serum. Detection of objects. The invention belongs to the field of medical technology. Background technique [0002] Autoimmune disease (AID) is a large group of diseases that are common clinically and difficult to treat. It is caused by the destruction of the tolerance mechanism of the body to recognize "self". The body's immune system responds excessively to self-antigens and destroys normal tissue structure. The occurrence of a series of lesions. The pathogenesis of autoimmune diseases is very complex, involving many factors including heredity, infection, and immune function abnormalities, but the specific mechanism ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
CPCG01N33/543G01N33/577G01N2800/065G01N2800/067G01N2800/10G01N2800/101G01N2800/102G01N2800/104G01N2800/24G01N2800/2892G01N2800/60
Inventor 张凤民王垚宋武琦李洋牟凤云黄鹤
Owner HARBIN MEDICAL UNIVERSITY
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