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Novel lentivirus packaging method

A lentiviral packaging, a new type of technology, applied in the direction of virus/bacteriophage, biochemical equipment and methods, microorganisms, etc., can solve the problems of low degree of transfection fusion, failure to satisfy customers, long packaging time, etc., to achieve transfection The effect of high fusion, increased virus titer, and reduced steps

Active Publication Date: 2017-03-15
杭州倍沃医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0029] This existing virus packaging method has problems such as uncertain passage, low degree of transfection fusion, relatively long time to complete packaging, and relatively low titer of purified virus, which may lead to failure to meet the requirements of customers' downstream experiments

Method used

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Embodiment 1

[0059] The novel lentiviral packaging method of the present embodiment consists of the following steps:

[0060] (1) On a 10cm tissue culture plate, 12ml medium was inoculated with 2×10 6 293T cells, cultured at 37 degrees, 5% carbon dioxide, ready to transfect cells with plasmids;

[0061] (2) The transfected mixture was cultured for 12-15 hours;

[0062] (3) In a polypropylene centrifuge tube, add the following plasmids, then mix well,

[0063] 10ug small fragment RNA plasmid,

[0064] 40ug packaging plasmid,

[0065] 20ug coated protein particles,

[0066]Make up the volume to 100 μL with 100 mM sodium chloride;

[0067] (4) Mix Biomiga GenetranⅢ transfection reagent with sodium chloride, and prepare a mixture of Biomiga GenetranⅢ and sodium chloride;

[0068] (5) Add 80 μL of Biomiga Genetran III and sodium chloride mixture to the transfection centrifuge tube, with a total volume of 180 μL, and gently rotate the centrifuge tube;

[0069] (6) Place at room temperatur...

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Abstract

The invention relates to a novel lentivirus packaging method. The lentivirus packaging method comprises the following steps: (1) inoculating 2*10<6>293T cells; (2) cultivating transfected mixture for 12-15 hours; (3) mixing each transfection centrifugal tube, wherein virus vectors are 10 microg of small-fragment RNA plasmid, 40 microg of package plasmid and 20 microg of enveloped protein plasmid, making up to 100 microL with 100mM sodium chloride; (4) preparing mixed solution of Biomiga Genetran III and sodium chloride; (5) adding 80 microL Biomiga Genetran III and sodium chloride mixed solution into the transfection centrifugal tube, wherein the total volume is 180 microL; (6) standing for 20-30 minutes at room temperature; (7) sucking culture medium from cells, then adding 8ml of fresh culture medium, adding 50 microL of sec-butyl babian within half an hour and mixing evenly; (8) culturing for 48-60 hours; and (9) collecting viruses after 72 hours.

Description

technical field [0001] The invention relates to a novel lentivirus packaging method, which belongs to the field of biotechnology. Background technique [0002] Lentivirus is a genus under the Retroviridae family, including 8 viruses that can infect humans and vertebrates. The primary infected cells are mainly lymphocytes and macrophages, and the infected individuals eventually develop disease. The salient feature of lentivirus infection is that most infected individuals go through a incubation period of several years before typical clinical symptoms appear, and then develop slowly, so these pathogens are called lentiviruses. For example, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), and feline immunodeficiency virus (FIV) are all lentiviruses, and the original text of lentivirus is Lentivirus, where Lenti- is It means slow in Latin. [0003] The research on lentiviral vectors is developing rapidly, and the research...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02
Inventor 史镜宇章宏远刘艳红言国英王敬红赵林军刘师岐
Owner 杭州倍沃医学科技有限公司