Lentiviral mutiple promoter stable expression carrier constructed through insulator combination and construction method

A lentiviral vector and stable expression technology, applied in the field of biomedical engineering, can solve the problems of stable expression and multi-gene transfection with few breakthroughs, and no commercial vectors.

Active Publication Date: 2020-02-07
CHANGZHOU NO 2 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the randomness of exogenous gene insertion into genomic DNA, there are few breakthroughs in its stable expression and multi-gene transfection
Although some scientists have tried to construct a multi-promoter vector in adenovirus, and it has been reported that they have received relatively satisfactory results, but in terms of lentivirus, many efforts have failed because the mutual interference between the promoters affects its expression function. Not to mention the commercial carrier in this regard

Method used

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  • Lentiviral mutiple promoter stable expression carrier constructed through insulator combination and construction method
  • Lentiviral mutiple promoter stable expression carrier constructed through insulator combination and construction method
  • Lentiviral mutiple promoter stable expression carrier constructed through insulator combination and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Basic lentiviral vector construction

[0070] (1) Using the viral vector (pLenti6V5-GW-LaZ) from Invitrogen Company as a template, as shown in Figure 9, Figure 9 Indicates the restriction endonuclease and PCR primer sites. Most of the fragments of the viral backbone were generated by PCR, and the viral backbone was as follows Figure 10 As shown, the 5' primer (Bone-F) has an NsiI site. M: Marke: 14, 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.5, 0.2kb; lane 1: backbone DNA fragment obtained by PCR, 4280bp. The PCR reaction program is shown in Table 2.

[0071] table 3

[0072]

[0073] (2) cPPT was produced from plasmid pLL3.7 by PCR. The 5' and 3' primers (cPPT-F; cPPT-R) contained NotI and EcoRI restriction sites, respectively (see Table 1). The specific system and conditions of the PCR reaction are shown in Tables 2 and 4. RRE-cPPT elements such as Figure 11 As shown, M: Marker 14, 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.5, 0.2kb; Lane 1: RRE-cPPT elemen...

Embodiment 2

[0085] Example 2 Construction of RNAi lentiviral vector containing U6 promoter

[0086] (1) Generation of U6 promoter fragment: PCR uses plasmid pLL3.7 as template, 5' primer (U6-F) has EcoRI site; 3' primer (U6-R) has BamHI / PmlI / MluI / SalI site.

[0087] Table 7. PCR reaction program to generate U6

[0088]

[0089] Such as Figure 15 As shown, mU6 promoter elements M were obtained by PCR: Marker 14, 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.5, 0.2 kb. A. Lane 1: The 314bp mU6 promoter element was obtained by PCR.

[0090] (2) PGK1-GFP-WPRE was produced from plasmid pLL 3.7 using high-fidelity Taq enzyme as shown in Table 1. Its 5' end primer (PGK-WPRE-F) has a SalI / BsiWI / XbaI site, and the 3' end primer (with PGK-WPRE-R) has a KpnI site. The PCR reaction system and conditions are shown in Tables 2 and 4.

[0091] Such as Figure 16 Indicated, PGK1-GFP-WPRE element. Lane 2: 2087bp PGK-GFP-WPRE element was obtained by PCR. M: Marker 14, 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1,...

Embodiment 3

[0094] Example 3 Construction of double-promoter RNAi lentiviral vector

[0095] IS22.3-CMV / H1 fragment was developed by the inventors with the pLenti-IS22.3-CMV / H1 lentiviral vector ( Figure 18 ) as a template, digested with MluI and SalI to generate a sticky-end DNA fragment, and then cloned into the corresponding site of the LV-U6-IS2 plasmid ( Figure 13 B). Finally, the double-promoter lentiviral vector LV-U6-H1-IS2 plasmid was obtained (Fig. 13C).

[0096] Such as Figure 18 As shown, the pLenti-IS22·3-CMV / H1 lentiviral vector contains the insulator IS22·3 and the fusion promoter CMV / H1.

[0097] Such as Figure 19 As indicated, the LV-U6-H1-IS2 plasmid was identified. A. Enzyme digestion identification, M: Marker 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1000bp; lane 1: a single band of 9725bp formed by NdeI single digestion plasmid; lane 2: 2556bp and 7169bp band. B. Plasmid map of SnapGene software.

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Abstract

The invention relates to a lentiviral mutiple promoter stable expression carrier constructed through insulator combination and a construction method, and belongs to the technical field of biomedical engineering. The insulator IS22.3 and an insulator IS2 are integrated into the lentiviral carrier, the sequence of the IS22.3 is shown as SEQID No.1, and the sequence of the insulator IS2 is shown as SEQID No.2. The lentiviral carrier is used for RNA interference experiments. In accordance with some blanks of a conventional lentiviral carrier in functions, the insulator IS22.3 and the IS2 are integrated into the lentiviral carrier, on one hand, mutual interference of insulators can be isolated, and on the other hand, gene silencing can be avoided. The carrier can realize polygene expression, and can also guarantee the expression stability.

Description

technical field [0001] The invention relates to a lentiviral multi-promoter stable expression vector and a construction method constructed by combining insulators, belonging to the technical field of biomedical engineering. Background technique [0002] Lentiviral vectors (Lentiviral vectors, LVs) are generally favored by researchers because they can effectively transfect various types of cells, especially non-dividing cells. At present, it has become an effective carrier for exogenous gene transfer and is widely used in the fields of basic experimental research and gene therapy. [0003] During the development and growth of eukaryotes, there is an orderly opening and closing of genes. In this process, specific genes or gene clusters in the genome are specifically expressed at different times and in different spaces. In addition to gene transcription regulators such as cis-acting elements and trans-acting elements, the promoter is the fundamental element that determines the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66
CPCC12N15/86C12N15/66C12N2740/15043
Inventor 孙逸武卢绪章王芳周民
Owner CHANGZHOU NO 2 PEOPLES HOSPITAL
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