Expression vector of fusion protein of neutral amino acid transporter 2, SNAT2 and enhanced green fluorescent protein and construction method and application thereof

A green fluorescent protein and transport protein technology, applied in the field of bioengineering, can solve problems such as unsatisfactory results, expensive antibodies, and difficult membrane proteins

Inactive Publication Date: 2012-01-18
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Due to the difficulty in preparing effective antibodies against membrane proteins, current antibodies against SNAT2 are expensive and not ideal

Method used

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  • Expression vector of fusion protein of neutral amino acid transporter 2, SNAT2 and enhanced green fluorescent protein and construction method and application thereof
  • Expression vector of fusion protein of neutral amino acid transporter 2, SNAT2 and enhanced green fluorescent protein and construction method and application thereof
  • Expression vector of fusion protein of neutral amino acid transporter 2, SNAT2 and enhanced green fluorescent protein and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A large number of original eukaryotic expression vector plasmids containing SNAT2 gene and vector plasmids containing EGFP gene:

[0058] The original eukaryotic expression vector plasmid containing rat glutamine transporter 2 (SNAT2) gene is pBK-CMV(Δ[1098-1300])-SNAT2-myc, and the vector plasmid containing enhanced green fluorescent protein (EGFP) gene is pMD -19T-EGFP.

[0059] Add 1 ng of pBK-CMV(Δ[1098-1300])-SNAT2 or pMD-19T-EGFP plasmid to 100 μl DH5α competent cells (TIANGEN Company) respectively, gently rotate the centrifuge tube to mix the contents, and place in an ice bath Let stand for 30min. Place the centrifuge tube in a 42-degree ice bath for 60-90 seconds, then quickly transfer the tube to the ice bath to cool the cells for 2-3 minutes. Add 900 μl of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, shake and culture at 37 degrees for 45 minutes (150 rpm), mix the contents, and gently smear the plate containing Kana Mycin (30m...

Embodiment 2

[0062] The amplification of embodiment 2SNAT2 gene and EGFP gene:

[0063] Using the plasmid pBK-CMVΔ-SNAT2-myc obtained in Example 1 as a template, the SNAT2 gene was amplified by polymerase chain reaction. Reaction conditions: 94°C, 2min, one cycle; denaturation at 94°C for 30s, annealing at 67.5°C for 30s, extension at 72°C for 90s, a total of 31 cycles; extension at 72°C, 20min; the reaction product was purified and recovered for sequencing identification and next fusion Construction of protein SNAT2-EGFP vector.

[0064] Using the plasmid pMD-19T-EGFP obtained in Example 1 as a template, perform polymerase chain reaction to amplify the EGFP gene; reaction conditions: 94°C, 2min, one cycle; 94°C, denaturation for 30s, 66.1°C for 30s, 72°C Extend at ℃ for 60s, a total of 33 cycles; extend at 72°C for 20min. The reaction product was purified and recovered for sequencing identification and the construction of the fusion protein SNAT2-EGFP vector in the next step;

[0065] ...

Embodiment 3

[0072] Example 3 Construction of pBK-CMVΔ-SNAT2-EGFP recombinant:

[0073] (1) pBK-CMVΔ-SNAT2 vector plasmid, digestion and recovery of SNAT2 and EGFP gene fragments:

[0074] Take 5.8 μg of the pBK-CMVΔ-SNAT2-myc vector plasmid obtained in Example 1, perform double enzyme digestion reaction with XbaI and NotI, incubate at 37°C for 4 hours, and recover the large pBK-CMVΔ fragment;

[0075] Take the 6.6ug PCR amplified and purified recovered SNAT2 gene fragment obtained in Example 2, perform double enzyme digestion reaction with XbaI and BlpI, and recover the SNAT2 gene fragment after incubating at 37°C for 6 hours;

[0076] Take 3.35ug PCR amplified and purified recovered EGFP gene fragment, carry out BlpI and NotI double enzyme digestion reaction, and recover EGFP fragment after incubation at 37°C for 10 hours;

[0077] (2) Use T4 DNA ligase to connect SNAT2, EGFP and pBK-CMVΔ-SNAT2 vector large fragment:

[0078] The digested and purified SNAT2, EGFP and the large fragment...

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Abstract

The invention relates to the field of biological engineering, and discloses a fusion protein expression vector. The C end of neutral amino acid transporter 2, SNAT2 and the N end of enhanced green fluorescent protein (EGFP) are connected and are constructed on an eukaryotic expression vector pBK-CMV (delta[1098-1300]), and the expression vector of the fusion protein of neutral amino acid transporter 2,SNAT2 and enhanced green fluorescent protein is obtained. Through the vector, the expression, the positioning and the quantity of SANT2 on a mammalian cell (e.g. an HEK293T cell) membrane can be detected through a Western blot technology with a laser confocal electronic microscope and a green fluorescent protein (GFP) antibody.

Description

technical field [0001] The invention relates to the field of bioengineering, and discloses a fusion protein expression vector of glutamine transporter 2 (SNAT2) and enhanced green fluorescent protein (EGFP) as well as its construction method and application for fusion protein carrier construction and expression detection technology. Background technique [0002] Fusion protein carrier construction technology is one of the important molecular biology methods commonly used. It is mainly through the method of genetic engineering that two different protein genes are fused and constructed on the same expression vector, and expressed in specific living cells to obtain a new fusion protein. [0003] Enhanced green fluorescent protein (EGFP) is often used as an autofluorescent fusion protein tag to express fusion with the target protein, and is used for the localization and movement of the target protein in living cells. [0004] Small aliphatic neutral amino acids such as alanine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/79G01N33/68G01N21/64
Inventor 张舟孟雯王函董晓云李洋
Owner SHANGHAI NORMAL UNIVERSITY
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