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Protein in situ expression chip and preparation method and application thereof

A protein and in situ technology, applied in the fields of biotechnology and tissue engineering, can solve the problems of single detection method and non-reusable use

Inactive Publication Date: 2014-01-29
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the object of the present invention is to provide a protein in situ expression chip and its preparation method and application in view of the shortcomings of the protein in situ expression chip detection method in the prior art that is single and cannot be reused. The chip can be reused, and Not only can it be used for traditional fluorescence detection, but more importantly, it can also be used for biosensors to monitor the kinetic process of real-time interaction

Method used

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  • Protein in situ expression chip and preparation method and application thereof
  • Protein in situ expression chip and preparation method and application thereof
  • Protein in situ expression chip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Construction of Plasmids

[0075]HG-GST plasmids, HG-P53 plasmids, pBH-GST plasmids and pBH-Fos plasmids were all constructed using Novagen's pET-Dut-1 as a template and modified it. The specific construction method is as follows:

[0076] 1. Using pET-Duet-1 (Novagen) as the template backbone, insert the GST tag gene (SEQ ID NO: 1) into the first group of multiple cloning sites (MCS1) to construct the expression vector HG-GST.

[0077] Using pANT-P53-GST as a template, using an upstream primer (5'-GCGGGATCCGCCTATACTAGGTTATTGG-3'(BamH I) (SEQ ID NO:2) and a downstream primer 5'-CGCGAATTCCTAACGCGGAACCAGATCCGATTTTGG-3'(EcoR I) (SEQ ID NO: 3) The GST tag fusion sequence product was amplified by PCR. After the PCR product was directly recovered by the Promega DNA recovery kit, it was subjected to restriction double digestion with BamH I and EcoR I, and pET-Duet after double digestion with the same enzyme. -1 (Novagen Company) for connection, and then transform...

Embodiment 2

[0088] Example 2 Construction of protein in situ expression chip and surface using microfluidic pipeline Ion resonance imaging (SPRi) detection

[0089] Proceed as follows:

[0090] 1. After cleaning the gold flakes with a plasma cleaner (PLasma), immobilize 500 μg / mL anti-GST antibody (anti-GST) (as a capture carrier) on the surface of the gold flakes, leave it at room temperature for 1 hour, wash with 1×PBS (phosphate buffer solution) ) after cleaning the gold slices, then seal the gold slices with 500mg / mL BSA (bovine serum albumin) for 1 hour, after cleaning, dry them with compressed air for later use.

[0091] 2. Will be as Figure 3a The microfluidic template (microfluidic template made of PDMS (polydimethylsiloxane) material) with microfluidic channels shown in , was attached to the surface of the gold sheet, and 50 μg / mL GST protein was injected into the reference channel As a reference signal, the microfluidic template is preset according to the required size an...

Embodiment 3

[0098] Example 3 The construction and expression of protein in situ expression chips using dotted microfluidic templates Surface plasmon resonance imager (SPRi) detection

[0099] Proceed as follows:

[0100] 1. After cleaning the gold flakes with a plasma cleaner (PLasma), immobilize 500 μg / mL anti-GST antibody (as a capture carrier) on the surface of the gold flakes, leave it at room temperature for 1 hour, wash the gold flakes with 1×PBS, and then wash them with 500 mg / mL The gold slices were blocked with mL BSA for 1 hour, and after cleaning, they were blown dry with compressed air for later use.

[0101] 2. Will be as Figure 3c The dot-like microfluidic template (microfluidic template made of PDMS (polydimethylsiloxane) material) was attached to the surface of the gold sheet, and 0.5 μL of plasmid DNA was added (the blank (Blank) was ddH 2 O, pBH-GST is a plasmid DNA that can express GST protein, and pBH-Fos is a plasmid DNA that can express Fos and GST fusion prot...

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Abstract

The invention provides a protein in situ expression chip and a preparation method and an application thereof. The chip comprises a substrate fixed with a capture carrier and a microfluidic template arranged on the substrate, wherein at least one protein combined with the capture carrier is also distributed on the substrate, and the protein is obtained by in situ expression on the chip; a flow cell inlet and a flow cell outlet are respectively formed in the two ends of a flow cell. According to the invention, the protein in situ expression chip is constructed to be not only applied in traditional fluorescence detection, but more importantly applied on a biological sensor for monitoring the interaction of a kinetic process in real time; and moreover, the protein in situ expression chip can be regenerated and recycled through certain regeneration buffer solution.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and tissue engineering. The invention relates to a protein chip, in particular to a protein in situ expression chip and a preparation method thereof. Background technique [0002] Protein is not only an important part of life, but also its function and its interaction with biomolecules is a key link in the formation of life. Protein array chip is an important method and means to study proteins (including the interaction between protein molecules, protein molecules and nucleic acids and other small molecules), which has high-throughput characteristics. At present, the development of protein chips is in the bottleneck period of development, and the reasons are analyzed as follows. [0003] The main preparation method of the traditional protein chip is to prepare the purified target protein by immobilizing it on a solid phase substrate in a specific way. This method is currently the most im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N21/553G01N33/68G01N2035/00158
Inventor 朱劲松郭碧红宋炉胜程志强李少鹏
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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