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Expression vector of fused protein of glutamine transporter 1 as well as construction method and application thereof

A technology of transporter and glutamine, applied in the field of bioengineering, can solve problems such as unsatisfactory effect, difficult membrane protein, and expensive antibody

Inactive Publication Date: 2012-09-05
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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  • Expression vector of fused protein of glutamine transporter 1 as well as construction method and application thereof
  • Expression vector of fused protein of glutamine transporter 1 as well as construction method and application thereof
  • Expression vector of fused protein of glutamine transporter 1 as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 A large amount of 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(Δ[1098-1300])-SNAT1-HA, containing enhanced green fluorescent protein ( EGFP) gene carrier plasmid is pMD-19T-EGFP.

[0073] Add lng of pBK-CMV(Δ[1098-1300])-SNAT1-HA or pMD-19T-EGFP plasmid to 100μl DH5α competent cells (TIANGEN company), rotate the centrifuge tube gently to mix the contents, and store in ice Stand in the bath 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...

Embodiment 2

[0076] The amplification of embodiment 2EGFP gene

[0077] 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 SNAT1-HA-EGFP vector in the next step;

[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 represents the NotI restriction site sequence;

Embodiment 3

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

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

[0084] 4.0 μg of the PCR amplification product obtained in Example 2 was subjected to a Not I single enzyme digestion reaction, incubated at 37° C. for 10 hours, and the EGFP gene fragment was recovered;

[0085] (2) Ligate the vector large fragment and the EGFP small fragment with T4DNA ligase:

[0086] The digested and purified pBK-CMVΔ-SNAT1-HA carrier large fragment and EGFP small fragment were ligated at 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 were connected through the NotI restriction site.

[0087] The obtained vector pBK-CMVΔ-SNAT1-HA-EGFP recombinant plasmid is as follows figure 1 A...

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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, and discloses a fusion protein expression vector of glutamine transporter 1 (SNAT1), HA tag protein and enhanced green fluorescent protein (EGFP) and its construction for fusion protein carrier construction and expression detection technology methods and applications. 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 or more 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] The HA-tagged protein has only 9 amino acids, and generally does not affect the structure and function of the target protein, and the HA antibody has strong specificity and high sensitivity, and is a commonly used tag. [0004] Enhanced green fluorescent protein (EG...

Claims

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Application Information

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Patent Type & Authority Applications(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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