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Application of HMGCR gene in improvement of LV packaging efficiency and infectivity

An infectivity and gene technology, applied in the field of genetic engineering, can solve the problem of not meeting the requirements of cGMP, and achieve the effects of improving titer and infectivity, low cost and simple operation steps.

Pending Publication Date: 2019-06-25
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some cytotoxicity occurs in the presence of methyl-β-cyclodextrin (one of the components of cholesterol supplements)
Additionally, cholesterol used in cell culture is often derived from animal sources and does not meet cGMP requirements

Method used

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  • Application of HMGCR gene in improvement of LV packaging efficiency and infectivity
  • Application of HMGCR gene in improvement of LV packaging efficiency and infectivity
  • Application of HMGCR gene in improvement of LV packaging efficiency and infectivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 pcDNA4-HMGCR recombinant overexpression vector construction

[0074] This embodiment discloses a method for constructing a pcDNA4-HMGCR recombinant overexpression vector, which specifically includes the following steps:

[0075] 1.1 Synthesis of HMGCR gene sequence

[0076] Look up the gene sequence of NM_000859 in the NCBI database, artificially synthesize the sequence, and design an Apa I restriction site upstream of the sequence, and a Pme I restriction site downstream to obtain the HMGCR gene sequence, and its nucleotide sequence is as shown in SEQ ID NO: 1 shown.

[0077] 1.2 Vector construction

[0078] 1.2.1 Use Apa I and Pme I to digest the pcDNA4-Myc-6His plasmid respectively (the plasmid map is as follows figure 1 shown) and the synthesized HMGCR DNA were subjected to agarose gel electrophoresis after digestion at 37°C for 2 hours. The digestion system was as follows:

[0079] 2μg (2μl)

pcDNA4-Myc-6His or HMGCR DNA

1μl

Apa...

Embodiment 2

[0091] This embodiment provides a method for increasing lentivirus titer, comprising the following steps:

[0092] 1.1 Inoculate 293T cells (purchased from ATCC cell bank in the United States) on cell culture plates.

[0093] 1.2 Carry out the transfection test when the cell confluency reaches 80%; the specific steps are:

[0094] 1.2.1 Preparation of DNA and transfection reagents:

[0095] When the cell culture plate is a 6-well plate, the ratio of each well is plasmid pcDNA4-HMGCR (overexpression well) or PBS (control well): pLenti-CMV-mCherry-3FLAG-PGK-Puro: psPAX2: pHCMV-VSV-G: Transfection reagent (lipofectmine 2000)=lug: lug: lug: lug: 8ul. Among them, the plasmid maps of the lentiviral vector plasmid pLenti-CMV-mCherry-3FLAG-PGK-Puro, the viral packaging auxiliary plasmid psPAX2 and the lentiviral packaging auxiliary plasmid pHCMV-VSV-G are as follows Figure 2-Figure 4 shown.

[0096] 1.2.2 Transfection

[0097] 1.2.2.1 Incubate the diluted DNA and transfection re...

Embodiment 3

[0104] This embodiment provides a method for improving lentivirus infectivity, wherein the steps of the experimental group are as follows:

[0105] S1: co-transfect cells with eukaryotic expression vector and lentiviral packaging system plasmid;

[0106] S2: Collect lentivirus stock solution and concentrate and purify;

[0107] S3: Infect the cells with the concentrated and purified lentivirus stock solution.

[0108] Explanation of terms:

[0109] The MOI value (Multiplicity Of Infection, multiplicity of infection) usually has two meanings: 1) refers to the average number of active units infected with the virus per cell; 2) expresses the number of viruses in terms of virus particles or genome numbers, such as v.p. or v.g., at this time MOI The meaning of refers to the average number of virus particles or genomes per cell infected with the virus.

[0110] The formula for calculating the amount of virus added: (number of cells × MOI value / titer of virus stock solution) × 1...

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Abstract

The invention belongs to the field of genetic engineering, and particularly relates to application of an HMGCR(3-hydroxy-3-methylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase1) gene in improvement of titer and infectivity of lentivirus. The invention discloses an HMGCR gene and further discloses a eukaryotic expression vector comprising the HMGCR gene. The nucleotide sequence of the HMGCR gene is shown as SEQ ID NO:1. A pcDNA4-HMGCR vector and the plasmid of a lentivirus packaging system jointly transfect cells, so that the titer and infectivity of the lentivirus canbe greatly improved while the capability of the lentivirus produced by cells is obviously improved, and a novel large-scale high-infectivity lentivirus packaging method is provided for basic researchand clinical application fields. In addition, application has the advantages of simple operation steps, low cost, non-animal source production and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to the application of HMGCR gene in improving LV packaging efficiency and infectivity. Background technique [0002] Lenti virus (LV) is a kind of viral vector modified from human immunodeficiency virus (HIV), with RNA genome and the ability to infect both dividing and non-dividing cells. After the lentiviral genome enters the cell, it is reverse-transcribed into DNA in the cytoplasm to form a pre-DNA integration complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA, which returns to the cytoplasm to express the target protein; or produces small RNA. Lentivirus-mediated gene expression or small RNA interference is sustained and stable, and divides as the cellular genome divides. Lentivirus has the advantages of wide host range, low immunogenicity, large gene capacity, and long-term expression,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/867C12N15/53C12R1/93
Inventor 夏清梅鲁济真杨兴林马佩敏刘晓芬
Owner OBIO TECH SHANGHAI CORP LTD
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