Method for constructing membrane protein cDNA library and use thereof

A membrane protein and library technology, applied in the field of construction of membrane protein cDNA library, can solve the problems of membrane protein research interference, lack of high-throughput specific screening of membrane proteins, etc., and achieve the effect of improving screening efficiency and screening specificity

Active Publication Date: 2009-01-07
IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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Problems solved by technology

However, this approach has significant limitations due to the need to utilize known antibodies and ligands
In recent years, a signal peptide trap technology has been applied to the screening of membrane proteins (Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane prot...

Method used

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  • Method for constructing membrane protein cDNA library and use thereof
  • Method for constructing membrane protein cDNA library and use thereof
  • Method for constructing membrane protein cDNA library and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Construction of eukaryotic expression vector pSecTag-attR

[0030] Using the eukaryotic expression vector pcDNA-DEST47 as a template, PCR amplification was used to obtain the restriction endonuclease HindIII and Xho I restriction site, polyhistidine recognition site, attR recombination site and suicide gene ccdB Gene fragment, estimated length 1800bp, such as figure 1 As shown, two independent positive clones at positions 2 and 3. The parameters are: 94°C for 40 seconds, 58°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and the last extension is 7 minutes. The construction process of the pSecTag-attR recombinant vector is as follows figure 2 Shown: The fragments obtained by PCR are recovered after double digestion with restriction endonucleases HindIII and Xho I, and the fragments recovered after double digestion with restriction endonucleases HindIII and Xho I contain a signal peptide for guiding the protein to pass through the membrane The Igk secreti...

Embodiment 2

[0031] Example 2: Construction and screening of mouse liver lymphocyte membrane protein cDNA expression library

[0032] 2.1 Construction of total protein cDNA expression library of mouse liver lymphocytes

[0033] The mouse liver lymphocyte cDNA clone library was stored in the pDONR207 vector containing the attL recombination site, and the LR recombination reaction was carried out through the lambda phage-specific recombination site attL and attR, and the genes in the cloned library were recombined into the eukaryotic expression vector pSecTag-attR , to obtain the total protein cDNA expression library of lymphocytes, the library vector was transformed into Escherichia coli competent DH5 to amplify, and the library titer reached 10 9 pfu / ml.

[0034] 2.2 Expression of the expression library in COS-1 cells

[0035] The cDNA expression library was transfected into COS-1 cells by liposome-mediated method. First, COS-1 cells were cultured in DMEM medium containing 10% calf se...

Embodiment 3

[0038] Example 3: Identification of a mouse liver lymphocyte membrane protein cDNA expression library

[0039] 3.1 RT-PCR analysis of mouse liver lymphocyte membrane protein cDNA expression library

[0040] The COS-1 cells expressing the cDNA library of mouse liver lymphocyte membrane protein were amplified and cultured, the total RNA was extracted by Trizol method, and the cDNA after reverse transcription was identified by PCR with different detection primers. The results are as follows: Figure 5 As shown, all detected membrane proteins (TLR2, TLR3, TLR4, NKG2A, NKG2D, Bcl-2, Bcl-xl) were expressed in the library, proving that the library has high throughput, while the non-membrane protein-actin no expression. It was proved that the library has high specificity.

[0041] 3.2 Flow cytometry analysis of mouse liver lymphocyte membrane protein cDNA expression library

[0042] The COS-1 cells expressing the cDNA library of mouse liver lymphocyte membrane protein were amplifie...

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Abstract

The invention relates to a construction method of a Membrane protein cDNA library and an application thereof. The method constructs a eukaryotic expression vector used for the specificity selection of the membrane protein. The expression vector can perform clone recombination with any cDNA clone library provided with the recombination site attL of Lambada bacteriophage so as to acquire a cDNA expression library. After the transfection of COS-1 cell, expression membrane protein clone is further selected by immunofluorescence so as to acquire the membrane protein cDNA expression library. As the application of the method, the liver lymphocyte membrane protein cDNA library of a C57BL/6 rat is built up. The method is proved to be an effective proposal of the high flux specificity selection membrane protein through the identification and analysis of the library, and the method provides a powerful method to describe the map of the membrane protein of the cell surface of certain issue and to research the unknown membrane protein.

Description

technical field [0001] The invention relates to a method for constructing a membrane protein cDNA library, including the construction of a eukaryotic expression vector for screening membrane proteins, the expression of membrane proteins on the surface of mammalian cell COS-1 and fluorescence screening; and applying it to mice Specific high-throughput screening of liver lymphocyte membrane proteins. Background technique [0002] Membrane proteins are an important part of the plasma membrane and play a vital role in the biological functions of the plasma membrane. For example, transport proteins, connexins, and some enzymes play an important role in the material transport and exchange of cells, the formation and maintenance of cytoskeleton, and the generation of cellular energy and signal transduction. Among them, receptor protein, as a class of functionally diverse proteins, is essential in maintaining cell survival and development; and plays a decisive role in the process o...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/85C12N5/10G01N33/53G01N21/64G01N15/00
Inventor 田志刚葛葵葵孙汭魏海明
Owner IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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