Design and application for lentiviral expression vector

An expression vector and lentivirus technology, which is applied in the fields of molecular biology and cell biology, can solve the problems of not having the advantages of pCDH vector, failing to meet the experimental requirements, and the expression level of inserted genes is not very high, so as to achieve transfection efficiency and virus The effect of small packaging efficiency, small impact, and small carrier length

Active Publication Date: 2020-04-10
SHANXI UNIV
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Problems solved by technology

However, the inventors found in the experiment that the expression level of the inserted gene of the pCDH series vector is not very high, which canno

Method used

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  • Design and application for lentiviral expression vector
  • Design and application for lentiviral expression vector
  • Design and application for lentiviral expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Vector design and construction:

[0045] According to the carrier map ( figure 1 ), each element required by the vector is arranged in map order, and designed as a pRSV-CMV-Flag-EF1-Puro-T2A-GFP vector. According to the name of the element, the sequence of each element is queried in the NCBI database, and the sequence of each element is copied into the corresponding position according to the carrier map information to form the sequence information of the carrier (SEQ ID NO.1). Then, the nucleic acid sequence information of the vector is handed over to a third-party company, which is synthesized according to the vector sequence, and the vector construction is completed.

Embodiment 2

[0047]The application of the vector, according to the vector designed in Example 1, four restriction sites (BamHI, SalI, XhoI, XbaI) and two Flag tags were inserted after the CMV promoter, and N-terminal Flag and C-terminal could be expressed as required Insertion of Flag, Flag at both ends or other tags into the gene expression vector. For the connection of recombinant vectors, it is recommended to use various DNA cloning and assembly techniques developed from the principles of atypical enzyme-cut ligation, PCR, homologous recombination, and single-strand annealing splicing.

[0048] After the recombinant vector is successfully constructed, the recombinant lentiviral vector and the packaging vector are co-transfected into 293T cells. The packaging vectors can be psPAX2 and PMD2.G, or REV, VSV-G and PMDL. After 72 hours of transfection, the virus was collected, transfected into target cells, and the transfection rate was observed by fluorescence microscope. In the case of a h...

Embodiment 3

[0050] In order to verify the advantages of the lentiviral vector designed in the present invention in high-efficiency expression of inserted genes, the most popular vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (pCDH) with the same type of characteristics is selected for comparison.

[0051] By RT-PCR, the sequence of the inserted gene Insert was obtained from the target cell HCT116, and the HA tag was added to the N-terminal of the Insert by PCR technology to obtain the HA-insert sequence, and the HA-insert was inserted into pCDH-CMV- by the method of Gibson splicing. Multiple cloning sites for MCS-EF1-copGFP-T2A-Puro(pCDH) and pRSV-CMV-Flag-EF1-Puro-T2A-GFP(pRSV).

[0052] After the sequencing is correct, transfect 293T cells with lentiviral expression vectors and packaging vectors (psPAX2, PMD2.G). After 72 hours of transfection, collect the virus, concentrate the virus with PEG8000 / NaCl, and transfect HCT116 cells. After 48 hours of transfection, take A part of the cells was us...

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Abstract

The invention belongs to the fields of molecular biology and cytobiology, and specifically relates to a design and an application for a lentiviral expression vector. According to the vector designed by the invention, a CMV enhancer and a CMV promoter are used as transcription starting elements of an insertion gene; an EF1a-core promoter is adopted as a transcription starting element for screeningand marking puromycin and green fluorescent protein (GFP), and a puromycin resistance gene and a GFP sequence are connected and expressed in series through T2A; a lentiviral vector is adopted as a vector skeleton, and two above-mentioned elements are inserted into the skeleton vector; thus, the lentiviral expression vector is obtained by reasonable design and optimization. By adoption of the lentiviral vector designed by the invention, the inserted gene can be efficiently expressed, and the expression efficiency of the lentiviral expression vector is superior to the expression efficiency of conventional vectors with the same type of characteristics.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and in particular relates to the design and application of a lentiviral expression vector. Background technique [0002] Lentiviral vector is a virus vector system modified on the basis of HIV-1 virus, which can efficiently introduce target genes into animal and human primary cells or cell lines. Lentiviral vector-mediated gene expression is sustained and stable due to the integration of the inserted gene into the host cell genome and division as the cell genome divides. In addition, lentivirus has a wide range of hosts, can infect dividing and non-dividing cells, and can infect almost all types of cells, especially for cells with low transfection efficiency of plasmid vectors. [0003] At present, there are various forms of lentiviral expression vectors, all of which are designed based on the lentiviral vector backbone and loaded with different gene manipulation elements. Whe...

Claims

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Application Information

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IPC IPC(8): C12N15/867G01N33/68G01N33/58G01N33/542
CPCC12N15/86G01N33/68G01N33/582G01N33/542C12N2740/15043G01N2333/43595Y02A50/30
Inventor 武兴康鲁玉凡韩晨晨秦雪梅
Owner SHANXI UNIV
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