Lentivirus vectors expressing exosome markers and building method and application of lentivirus vectors

A lentiviral vector and exosome technology, applied in the field of genetic engineering, can solve problems such as hindering research progress and weak brightness, and achieve the effect of strong fluorescence and easy instant observation.

Active Publication Date: 2016-06-15
武汉淼灵生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these fluorescent proteins have hindered research progress to some extent due to their weak brightness.

Method used

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  • Lentivirus vectors expressing exosome markers and building method and application of lentivirus vectors
  • Lentivirus vectors expressing exosome markers and building method and application of lentivirus vectors
  • Lentivirus vectors expressing exosome markers and building method and application of lentivirus vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro vector:

[0034] The pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro vector was constructed on the basis of pCDH-CMV-MCS-EF1-Puro.

[0035] The pCDH-CMV-MCS-EF1-Puro vector has a CMV promoter, a multiple cloning site MCS, a selective marker Puromycin, an ampicillin resistance gene, etc., forming a 7.4kb DNA fragment. The CD63-SBP-ZsGreen1 sequence was added to the multiple cloning site between the CMV promoter and Puromycin to construct the vector pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro.

[0036] The specific construction process of the carrier is:

[0037] 1. According to the genetic information of the CDS sequence, SBP tag sequence, and ZsGreen1 tag protein sequence on human CD63 mRNA in GeneBank, the XbaI-CD63-SBP-ZsGreen1-NotI gene DNA fragment was designed and sent to Shanghai Invitrogen Company for synthesis.

[0038] 2. Use restriction enzymes XbaI and NotI to digest the double-stranded DNA molecules synthesized in step 1 (sy...

Embodiment 2

[0042] Construction of pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro vector:

[0043] The pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro vector was constructed on the basis of pCDH-CMV-MCS-EF1-Puro.

[0044] The pCDH-CMV-MCS-EF1-Puro vector has a CMV promoter, a multiple cloning site MCS, a selective marker Puromycin, an ampicillin resistance gene, etc., forming a 7.4kb DNA fragment. The CD9-SBP-ZsGreen1 sequence was added to the multiple cloning site between the CMV promoter and Puromycin to construct the vector pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro.

[0045] The specific construction process of the carrier is:

[0046] 1. According to the genetic information of the CDS sequence, SBP tag sequence, and ZsGreen1 tag protein sequence on human CD9 mRNA in GeneBank, the DNA fragment of XbaI-CD9-SBP-ZsGreen1-NotI was designed and sent to Shanghai Invitrogen Company for synthesis.

[0047] 2. Use restriction enzymes XbaI and NotI to digest the double-stranded DNA molecules synthesized in step 1 (synthesize...

Embodiment 3

[0051] Application of pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro and pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro vectors in cell infection and cell line construction

[0052] 1. Transient transfection

[0053] (1) Cells: Take out cryopreserved HEK293T cells from liquid nitrogen, quickly dissolve them in a 37°C water bath, then collect the cells by centrifugation, and culture them in poly-lysine-coated cell culture dishes after dilution with medium . When the density reaches 60%-70%, it can be used for subsequent transfection experiments.

[0054] (2) Transfection: 30–40 μg DNA (pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro or pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro vector: 15 μg without endotoxin, pLP-1 : 10 μg, pLP-2: 10 μg, pLP-VSVG: 5 μg) were transfected into the above cells. Add the DNA-calcium phosphate mixture to the cultured cells, and replace the medium after culturing for 8-12 hours. After continuing to cultivate for 60 hours, collect the cell culture medium, centrifuge at 4000g for 5 minutes, ...

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Abstract

The invention discloses lentivirus vectors expressing exosome markers and a building method and application of the lentivirus vectors.The lentivirus vectors are characterized in that CD63 and CD9 genes with SBP labels are introduced into the multiple cloning sites XbaI and NotI regions of pCDH-CMV-MCS-EF1-Puro, green fluorescent protein genes ZsGreen1 with extremely high fluorescence ability are added to the rear of the introduced CD63 and CD9 genes at the same time, and the recombinant lentivirus vectors pCDH-CMV-CD63-SBP-ZsGreen1-EF1-Puro and pCDH-CMV-CD9-SBP-ZsGreen1-EF1-Puro are built.The lentivirus vectors expressing the exosome markers have the advantages that green fluorescent protein genes ZsGreen1 fast in maturation has a high fluorescent effect which facilitates real-time observation, the expressed exosome carries the SBP labels, and exosome observation and purification can be integrated.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the construction of exosome marker plasmid expressed by lentivirus and its application. Background technique [0002] Exosomes are cell-derived vesicles that exist in biological fluids and cell culture media, with diameters ranging from 30 nm to 100 nm. exosomes are also called [0003] Microvesicles, epididimosomes, argosomes, exosome-likevesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes and doncosomes. There are two ways to form exosomes: one is the fusion of vesicles and cell membranes in the cell; the other is directly formed by the cell membrane. (Wikipedia). Exosomes contain various cell-derived components, such as proteins, RNA, double-stranded DNA, fats, and metabolic molecules. But different exosomes contain the same components. Exosomes can play a role by fusing with other cell membranes and endocytosis, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N15/65
CPCC12N15/65C12N15/66C12N15/86C12N2740/15043C12N2800/107
Inventor 金卫林殷楚
Owner 武汉淼灵生物科技有限公司
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