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LAMP1 eukaryotic expression vector having Flag label, and applications of LAMP1 eukaryotic expression vector having Flag label in lysosome separation

A eukaryotic expression vector and lysosome technology, applied in the field of molecular biology, can solve the problems that are not separated from the efficient separation of lysosomes, the specificity and titer of antibodies are not high, and the purity of lysosomes cannot be obtained. Simple operation, low cost, good separation effect

Inactive Publication Date: 2015-05-20
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity and potency of the existing antibodies targeting endogenous LAMP1 in cells are not high, which is inseparable from the efficient separation of lysosomes
Separating lysosomes using the traditional density-concentration gradient method takes a long time, requires a large amount of samples, and cannot obtain lysosomes with high purity

Method used

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  • LAMP1 eukaryotic expression vector having Flag label, and applications of LAMP1 eukaryotic expression vector having Flag label in lysosome separation
  • LAMP1 eukaryotic expression vector having Flag label, and applications of LAMP1 eukaryotic expression vector having Flag label in lysosome separation
  • LAMP1 eukaryotic expression vector having Flag label, and applications of LAMP1 eukaryotic expression vector having Flag label in lysosome separation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Cloning of LAMP1-Flag Gene and Construction of pcDNA3.1-LAMP1-Flag Recombinant Plasmid

[0024] (1) RNA extraction and cDNA preparation of human cervical cancer cell Hela

[0025] will be 10 5 Hela cells were inoculated in a 6-well plate with 2 ml of DMEM complete medium containing 10% FBS, placed at 37 °C, and the volume fraction was 5% CO 2 When the cell density reaches 80%-90% confluence, treat with 1mL TRIzol, extract total RNA, and reverse transcribe into cDNA according to the instructions of Fermentas reverse transcriptase (RevertAidTM M-MuLV).

[0026](2) Cloning of LAMP1 gene and construction of pcDNA3.1(+)-LAMP2-Flag recombinant plasmid

[0027] Use the cDNA of human cervical cancer cell Hela cells as a template and the DNA sequences of SEQ ID NO.2 and SEQ ID NO.3 in Sequence Table 1 as two primers to carry out PCR amplification of the target gene, and obtain the DNA containing BamH I after purification. site and the LAMP1-Flag gene of the Xho I re...

Embodiment 2

[0030] Example 2 Establishment and screening of a cell line stably expressing LAMP1-Flag fusion protein

[0031] Human cervical cancer cells Hela were cultured in DMEM complete medium containing 10% fetal calf serum at 37°C in 5% CO 2 Cultured in medium and subcultured every 2-3 days. When the cell density in the culture dish reaches 80%-90% confluence, digest with trypsin and inoculate (0.5-1)×10 5 The cells were placed in a 6-well plate, and the next day, 2 μg of pcDNA3.1(+)-LAMP1-Flag was transfected with Lipofectamine2000 transfection reagent from Invitrogen Company. After 24 hours, the transfected cells were transferred to a 100mm culture dish, screened with 800ng / mL G418, single clones were picked out, cultured in different plates, and the single clone with the highest expression of LAMP1-Flag was screened by semi-quantitative PCR and then used 400ng / mL G418 was maintained in culture to obtain a cell line stably expressing the LAMP1-Flag fusion protein.

Embodiment 3

[0032] Example 3 Co-immunoprecipitation

[0033] (1) Collect Hela cells expressing LAMP1-Flag from five 100mm culture dishes by trypsinization, and use non-deformable cell lysate containing protease inhibitor PMSF (10mM HEPES, pH7.9, 1, 5mM MgCl 2 , 10mM KCl) and disrupt the cells with a homogenizer. The cell lysate was centrifuged at 1000g / min to remove cell nuclei and cell membranes, and the supernatant was recovered. The supernatant was washed with IP buffer (20mM HEPES, pH7.9, 150mM NaCl, 1.5mM MgCl 2 , 10mM KCl), add 1ug of anti-Flag M2 antibody and 25ul of protein A / G, and incubate overnight at 4°C. The precipitate was collected by centrifugation at 300g, and washed 4 times with 1ml IP buffer, and the final product obtained was the immunoenrichment product of LAMP1-Flag.

[0034] (2) Western blot detection: Add 5×protein loading buffer to the sample, put it in a 95°C water bath for 5 minutes to denature. Load 10%input, 10%anti-IgG IP, 10%anti-Flag IP. Protein sample...

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Abstract

The present invention belongs to the field of molecular biology, and specifically discloes an LAMP1 eukaryotic expression vector having Flag label, and applications of the LAMP1 eukaryotic expression vector having the Flag label in lysosome separation. According to the present invention, the nucleotide sequence of the LAMP1 having the Flag label in the LAMP1 eukaryotic expression vector having the Flag label is represented by SEQNO. 1, the LAMP1 gene and the label protein gene form the fusion gene, the promoter and the signal sequence on the vector are adopted to control the expression of the fusion gene, and the immunoprecipitation way is adopted to separate lysosome; and advantages of simple operation, high enrichment efficiency, easy result detection, low cost, and efficient lysosome separation are provided.

Description

【Technical field】 [0001] The invention belongs to the technical field of molecular biology, in particular to a eukaryotic expression vector of LAMP1 (lysosomal membrane protein 1) with a Flag tag and its application in lysosome separation. 【Background technique】 [0002] Lysosome (lysosome) is a small body surrounded by a single layer of lipoprotein membrane in eukaryotic cells and contains a series of acid hydrolytic enzymes. It is the common end point of the two metabolic pathways of endocytosis and autophagy. Lysosomes can not only fuse with phagosomes, acidify and hydrolyze biomacromolecules that enter cells through the endocytic pathway, regulate signal transmission at the cellular level, but also fuse with autophagosomes, degrade them and wrap them into autophagy through the autophagic pathway. The organelles and endogenous biomacromolecules in the body provide new raw materials for biosynthesis for cells. The isolation of lysosomes by molecular biochemical means is o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/07
Inventor 张磊李红昌杨诗涵
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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