Polygene interferential shRNA plasmid expression vector, construction method and application

A vector and plasmid technology, which is applied in gene therapy, recombinant DNA technology, and the introduction of foreign genetic material using vectors, etc., can solve the problems of cumbersome directional connection, strong inhibitory effect on target gene expression, and inferior expression of shRNA/siRNA, etc., to achieve Easy to operate, expensive, stable effect

Inactive Publication Date: 2008-08-20
马义才
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  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of this scheme is that before the construction of multiple shRNA expression vectors, the U6 promoter needs to be isolated and prepared by complex molecular biological means; when the vector is constructed, the isolated U6 promoter needs to be further directionally connected to each of the shRNA expression vectors by enzymatic means. Synthetic "shRNA+U6 termination signal" double-stranded DNA structure, if this operation involves expressing more shRNAs, this directional connection will be more cumbersome
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  • Polygene interferential shRNA plasmid expression vector, construction method and application
  • Polygene interferential shRNA plasmid expression vector, construction method and application
  • Polygene interferential shRNA plasmid expression vector, construction method and application

Examples

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Embodiment 1

[0048] Example 1: Construction of shRNA plasmid expression vector pSilencer-U6 / H1-(shRNA)n based on polygenic interference

[0049] In order to realize the concatenation of multiple shRNA transcription units on a single vector, it is necessary to design and introduce two sets of multiple cloning sites on both sides of the shRNA transcription units of the initial vector pSilencer2.0-U6 or pSilencer3.0-H1 (purchased from Ambion) Sequence (MCS), make the two sets of multiple cloning site sequences and shRNA transcription unit, plasmid sequence orderly combined, construct two sets of multiple cloning site sequences (MCS 1 and MC s ) between the base vector pSilencer-U6 / H1-2MCS with multiple shRNA transcription units connected in series. Afterwards, the coding sequences of multiple target gene interference shRNA transcription units are serially inserted between the two MCSs of the pSilencer-U6 / H1-2MCS vector.

[0050] 1. Construction of pSilcencer-U6 / H1-2MCS basic plasmid vector ...

Embodiment 2

[0078] Example 2: Construction of shRNA plasmid expression vector pSilencer-U6 / H1-HIV-(shRNA)n based on HIV polygenic interference

[0079] 1. Construction of pSilcencer-U6 / H1-HIV-2MCS basic plasmid vector

[0080] Method according to Example 1.

[0081] 2. Selection and design of shRNA target sequences

[0082] The selection of HIV-1 env, gag, pol, vif gene shRNA target sequences mainly follows the Tushl principle and refers to some other standards. After the target sequence is selected, Blast search is carried out so that the target sequence has no homology with other genes.

[0083] After the target sequence is determined, the shRNA template DNA of each target sequence is synthesized according to the rules (see Thijn R. Brummelkamp, ​​et al. Science, 2002, 296: 550-553). Each shRNA template DNA includes sense and antisense two complementary short DNA single strands, and its basic units are: BamHI site, target sequence sense strand, 9bp loop sequence, target sequence anti...

Embodiment 3

[0129] Example 3: Application of the shRNA plasmid expression vector of HIV polygene interference in the preparation of AIDS treatment drugs

[0130] The shRNA plasmid expression vector pSilencer-U6 / H1-HIV-(shRNA)n constructed above was transformed into Escherichia coli, and the Escherichia coli was used for high-density fermentation (in a fermenter), the cells were collected by centrifugation, and pSilencer was extracted and purified according to the conventional plasmid extraction method -U6 / H1-HIV-(shRNA)n plasmid, measure OD 260 Plasmid concentration, sequencing to identify the correctness of the product. The pure grains are prepared into injections or oral capsules, which can be used for the treatment of HIV-infected and AIDS patients. Plasmid expression vectors can transcribe and synthesize corresponding shRNAs targeting HIV-1env, gag, pol, and vif genes in human cells. mRNA degradation, thereby inhibiting the expression of multiple HIV genes and killing HIV.

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Abstract

The invention discloses a plasmid vector pSilencer-U6/H1-(shRNA)n which can be used for simultaneously expressing a plurality of shRNA, a construction method and application thereof. The vector is composed of two groups of sequences of multiple cloning sites (MCS), a plurality of target gene shRNA transcription units and plasmid sequences. The shRNA transcription units of a plurality of different or same target genes can be connected in series between the two groups of the sequences of the multiple cloning sites of the vector, a plurality of shRNA transcription units thereon can express one target gene for many times or express a plurality of different sites of one target gene or express different target genes after the transcription of cells or animals, so as to specifically inhibit the expressions of two or more target genes. The vector can be easily used in the gene function research and the research and development of the gene therapeutic drugs for diseases.

Description

1. Technical field [0001] The invention relates to an shRNA plasmid expression vector pSilencer-U6 / H1-(shRNA)n based on polygene interference in the field of gene medicine, a construction method and application thereof. 2. Background technology [0002] RNA interference (RNAi) refers to the use of double-stranded RNA (double-stranded RNA, dsRNA) to specifically induce the degradation of mRNA homologous to its sequence, resulting in post-transcriptional gene silencing (PTGS) that specifically inhibits the expression of the corresponding target gene Phenomenon. RNA interference was discovered in 1998 in the genetic analysis of C. elegans. In 1999, it was detected in plants that 21-25nt RNA fragments were necessary for plant RNAi, and they were called small interfering RNA (short or sallinterfering RNA, siRNA). Quickly applied to anti-virus, anti-tumor and gene function research. From 2001 to 2003, RNAi technology was rated as one of the world's top ten scientific achievemen...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66A61K48/00
Inventor 马义才顾敏马灵
Owner 马义才
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