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Application of Glutamine Transporter 1 Fusion Protein Expression Vector

A transporter and glutamine technology, applied in the field of bioengineering, can solve problems such as expensive antibodies, difficult membrane proteins, and unsatisfactory results

Inactive Publication Date: 2014-10-29
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Due to the difficulty of preparing effective antibodies against membrane proteins, current antibodies against SNAT1 are very expensive and the effect is not ideal

Method used

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  • Application of Glutamine Transporter 1 Fusion Protein Expression Vector
  • Application of Glutamine Transporter 1 Fusion Protein Expression Vector
  • Application of Glutamine Transporter 1 Fusion Protein Expression Vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Mass acquisition of the original eukaryotic expression vector plasmid containing the SNAT1-HA gene and the vector plasmid containing the EGFP gene:

[0072] The original eukaryotic expression vector plasmid containing rat glutamine transporter 1 and HA tag protein fusion gene (SNAT1-HA) is pBK-CMV (△[1098-1300])-SNAT1-HA, containing enhanced green fluorescent protein ( EGFP) gene vector plasmid is pMD-19T-EGFP.

[0073] Add 1ng pBK-CMV (△[1098-1300])-SNAT1-HA or pMD-19T-EGFP plasmid to 100μl of DH5α competent cells (TIANGEN). Gently rotate the centrifuge tube to mix the contents. Let stand for 30 min in the bath. Place the centrifuge tube in an ice bath at 42°C for 60-90 seconds, then quickly transfer the tube to the ice bath to cool the cells for 2-3 minutes. Add 900μl of sterile LB medium (without antibiotics) to each centrifuge tube. After mixing, incubate with shaking at 37°C for 45min (150 revolutions / min), mix the contents, and gently coat the plate contain...

Embodiment 2

[0076] Example 2 Amplification of EGFP gene

[0077] Using the plasmid pMD-19T-EGFP obtained in Example 1 as a template, polymerase chain reaction was performed to amplify the EGFP gene; reaction conditions: 94℃, 2min, one cycle; 94℃, denaturation for 30s, 66.1℃ annealing for 30s, 72 Extend at ℃ for 60s, 33 cycles in total; extend at 72℃ for 20 min. The reaction product is purified and recovered for sequencing and identification and the construction of the fusion protein SNAT1-HA-EGFP vector;

[0078] The specific primers used for PCR amplification of EGFP gene are:

[0079] EGFP-P1: 5’-gaatctatta GCGGCCGC aatggtgagcaagggcgag-3’,

[0080] EGFP-P2: 5’-ctatatagat GCGGCCGC tcacttgtacagctcgtccatg-3’.

[0081] The underlined part indicates the sequence of the NotI restriction site;

Embodiment 3

[0082] Example 3 Construction of pBK-CMV△-SNAT1-HA-EGFP recombinant

[0083] (1) Take 5.0 μg of pBK-CMVΔ-SNAT1-HA vector plasmid obtained in Example 1, perform a Not I single digestion reaction, incubate at 37°C for 3 hours, and recover the large vector fragment;

[0084] Take the 4.0 μg PCR amplification product obtained in Example 2 and perform a Not I single enzyme digestion reaction, and incubate at 37°C for 10 hours to recover the EGFP gene fragment;

[0085] (2) Use T4DNA ligase to ligate the large vector fragment and the small EGFP fragment:

[0086] The large fragment of pBK-CMVΔ-SNAT1-HA vector and the small fragment of EGFP which have been purified by restriction digestion were ligated with a molar ratio of 4:1, and the ligation condition was 16°C for 5 hours. The large fragment of pBK-CMVΔ-SNAT1-HA vector and the small fragment of EGFP are connected by NotI restriction site.

[0087] The resulting vector pBK-CMV△-SNAT1-HA-EGFP recombinant plasmid as figure 1 As shown in B, ...

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Abstract

The invention relates to the field of biological engineering, provides fused protein vector construction and expression detection technology and discloses an expression vector of a fused protein of glutamine transporter 1. The expression vector is prepared by the following steps that: the gene segment of the glutamine transporter 1, a gene segment of an HA (hemagglutinin) tag protein and a gene segment of an enhanced green fluorescence protein are sequentially connected and constructed on an eukaryotic expression vector pBK-CMV delta; the C terminal of the glutamine transporter 1 is directly connected with the N terminal of the HA tag protein; the C terminal of the HA tag protein is connected with the N terminal of the enhanced green fluorescence protein; and a hinge formed by 4 amino acid is arranged between the HA tag protein and the first amino acid M of the enhanced green fluorescence protein, wherein the sequence is GAAA, and the base sequence is ggagcggccgca. The prepared vector can be used for transporting SNAT1 (sodium-coupled neutral amino acid transporters) gene into eukaryocyte, so that the SNAT1 gene is massively expressed in the eukaryocyte; and the vector is an effective method for detecting expression, location and quantification of SNAT1 on an eukaryocyte membrane, so that the method can be applied to research of structure and functions of SNAT1.

Description

Technical field [0001] The invention relates to the field of bioengineering. It is a fusion protein vector construction and expression detection technology, and discloses a glutamine transporter 1 (SNAT1), HA tag protein and enhanced green fluorescent protein (EGFP) fusion protein expression vector and its construction Methods and applications. Background technique [0002] Fusion protein vector construction technology is one of the most commonly used and important molecular biology methods. It is mainly a process in which two or more different protein genes are fused and constructed on the same expression vector through genetic engineering, and expressed in specific living cells to obtain a new fusion protein. [0003] The HA tag protein has only 9 amino acids, which generally does not affect the structure and function of the target protein. Moreover, the HA antibody has strong specificity and high sensitivity, and is a commonly used tag. [0004] Enhanced Green Fluorescent Protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66G01N33/68
Inventor 张舟王函孟雯董晓云李洋
Owner SHANGHAI NORMAL UNIVERSITY
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