Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector

A lentiviral vector and lentivirus technology, applied in the field of RNAi research and antiviral, can solve the problems of inability to achieve long-term expression, inability to integrate into the host chromosome, easy to cause immune response, etc., and achieve the effect of solving low transfer efficiency and controlling virus infection.

Inactive Publication Date: 2011-10-05
成都康珞生物科技有限公司
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Problems solved by technology

However, retrovirus only infects dividing cells, and the DNA fragment that accommodates foreign genes does not exceed 8kb; when adenovirus vectors infect cells, the viral DNA is free in the nucleus and cannot be integrated into the host chromosome, and cannot achieve stable long-term in vivo Expression, and repeated application is easy to cause immune response, so the lentiviral vector (Lentiviral vector) derived from human immunodeficiency virus-1 (HIV-1) has attracted more and more attention

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  • Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector
  • Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector
  • Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector

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[0042] Below in conjunction with example and accompanying drawing, the present invention will be further described:

[0043] The method for mediating RNAi using doxycycline-controlled lentiviral vector system provided by the present invention comprises the following steps:

[0044] 1. Use bioinformatics to screen out siRNAs targeting different conserved regions of HBV. First, according to the HBV gene sequence U95551 in GeneBank, refer to the principles of siRNA design, and use the shRNA design website http: / / rnadesigner.classic.invitrogen.com / rnaiexpress / index.jsp selects a conserved nucleotide sequence with a length of 21nt at the beginning of the gene coding region AA and a GC% between 45% and 55%, avoiding the non-coding regions at the 5' and 3' ends, and then performing BLAST on the selected sequence Homology analysis, avoiding interference sequences that are homologous to the human genome; then, according to the requirements of the pSuper vector, design 60-64nt oligos t...

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Abstract

The present invention relates to a mediating method of RNAi (ribonucleic acid interference) utilizing a lentiviral vector. The method comprises the following specific steps of: firstly carrying out homology analysis on the nucleic acid sequence of relevant viruses to find out conserved regions and designing siRNA (small interfering ribonucleic acid) corresponding to the conserved regions of the related viruses; and cloning the siRNA to an expression plasmid pLVTH, then co-transfecting 293T cells together with pCMV-dR8.91 and pMD2.G, collecting the supernatant of the transfected cells, carrying out ultracentrifugation, purification and concentration to obtain a high-titer lentiviral vector expressing the siRNA, then carrying out cotransduction into target cells together with LV-tTR-KRAB, and strictly controlling the acting time and dose of siRNA drugs utilizing tetracycline analogue DOX. Because of high transduction rate and a stringent control system, the mediating method can actually evaluate the in-vivo antiviral action of the siRNA and can track the whole process of drug interference action, thus having clinical application prospects.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and molecular virology, in particular to a method for antiviral research based on doxycycline-controlled lentiviral vector-mediated RNAi. Background technique [0002] In recent years, many laboratories in the world have used siRNA interference to carry out anti-virus research, and many results are either contradictory or the interference effect is not obvious. The key problem is that the models they work with are mostly limited to the use of transfected cell lines. Due to the extremely limited transfection efficiency, it is difficult to truly evaluate the antiviral effect of siRNA entering cells. Later, RNAi often utilizes viral vectors to express siRNA, among which retroviral vectors and adenoviral vectors are common. However, retrovirus only infects dividing cells, and the DNA fragment that accommodates foreign genes does not exceed 8kb; when adenovirus vectors infect cells, the viral...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12Q1/70C12Q1/68G01N33/569
Inventor 胡康洪王薇薇彭洪泉
Owner 成都康珞生物科技有限公司
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