RNAi vector and its application

A carrier and carrier skeleton technology, applied in recombinant DNA technology, using vectors to introduce foreign genetic material, gene therapy, etc., can solve problems such as low risk, unfavorable long-term expression, RNA replicase lack of DNA polymerase supervision and error correction mechanism, etc.

Active Publication Date: 2007-11-07
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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AI Technical Summary

Problems solved by technology

[0020] (2) Stability of siRNA: RNA is easily degraded in vitro and in cells, how to store and deliver siRNA efficiently and specifically to the appropriate position in the patient's body is a big problem
[0026] The most important feature of the existing SFV vector is that the transfection and expression efficiency are extremely high and it does not cause antiviral immunity, but the expression time is relatively short. This characteristic becomes a disadvantage when the body needs long-term expression, which is not as good as lentivirus, etc.
But from the perspective of immunology and safety, this is also an advantage, because short-term high-intensity stimulation meets the requirements of the immune system, and if it is not integrated into the host cell genome, the risk of carcinogenesis is lower; on the other hand, RNA Replicase lacks the supervisory error-correction mechanism of DNA polymerases and is itself not conducive to long-term expression

Method used

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, the construction of RNAi vector pSFV-RNAi Ready

[0045] Taking pSFV-1 as the starting vector, the RNAi vector pSFV-RNAi Ready of the present invention is constructed, and the specific construction method includes the following steps:

[0046] 1) Replace the SP6 promoter with the CMVIE promoter+Sv40 poly A system (see ① in Figure 1). The specific method is:

[0047] Synthetic primer P1 (the underlined base is the Spe I recognition site): 5'-caa Actagt caaacatgataagatac-3', P2 (the underlined base is the Xba I recognition site): 5'-ggc tctaga taccacatttgtagagg-3', P3 (the underlined base is the Sph I recognition site): 5'-gccg gcatgc tagttattaataggtaa tcaattacggggtc-3', P4: 5-catccgccatcgttagccagaggatctgacg-3' and P5: 5'-ctctggctaacgatggcggatgtgtgacatac-3' and P6 (underlined bases are EcoR V recognition sites): 5'-cagt gatatc caagatg agtgtgtctttg-3'. First add Sv40 poly A downstream of SFV3'-UTR: with pIRES 2 -EGFP plasmid (Clontech Company) was us...

Embodiment 2

[0058] Embodiment 2, using the RNAi carrier of the present invention to inhibit the expression of HPV16E6 gene in human cervical cancer cell Caski

[0059] Taking the HPV16E6 gene as an example, the RNAi effect of the carrier pSFV-RNAi Ready constructed in Example 1 is detected, and the specific method includes the following steps:

[0060] 1. Design of small interfering RNA that inhibits HPV16E6 gene expression and acquisition of its coding gene

[0061] According to the HPV16E6 gene sequence (GenBank No.: AF536179, 83-559), a 21bp sequence located at 104-124 downstream of the ATG transcription start site of the HPV16E6 gene open reading frame (ORTs) sequence was selected as the target sequence for design, and the inhibitory The small interfering RNA expressed by the HPV16E6 gene has the following sequence:

[0062] Justice Strand: 5'- UGUGUGUACUGCAAGCAAC UU-3';

[0063] Antisense strand: 5'- GUUGCUUGCAGUACACACA UU-3'.

[0064] Non-specific homologous sequences were ex...

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Abstract

The present invention discloses one kind of RNAi vector and its application, and provides one kind of recombinant SFV expression vector based on SFV virus replicon vector and its application in preparing treating medicine for inhibiting the target gene expression. The RNAi vector is recombinant SFV expression vector with SFV vector skeleton containing CMV IE promoter and Sv40 poly A, the CMV IE promoter has GenBank number of U57607.1(1-589), and Sv40 poly A has GenBank number of AM697713(2404-2644). The vector is suitable for use in research needing short time and high efficiency silence of target gene, especially in preparing treating gene medicine for inhibiting target gene expression. Therefore, the present invention is significant in biomedicine and gene function research.

Description

technical field [0001] The present invention relates to vectors and applications thereof, in particular to an RNAi vector constructed on the basis of SFV viral replicon vectors and its application in the preparation of gene therapy drugs that inhibit the expression of target genes. Background technique [0002] RNA interference (RNA interference, RNAi) is one of the important scientific research achievements in the field of life science in recent years. It is a gene silencing mechanism at the post-transcriptional level, through complementary base pairing of double-stranded RNA (dsRNA) and homologous mRNA, and guiding nuclease to rapidly degrade the mRNA at the binding site, thereby blocking the expression of the corresponding gene, with high Stable, high efficiency, high specificity and high penetration characteristics, so it is widely used to silence specific genes. It is more efficient, specific and easy to implement than all previous methods of inhibiting gene expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86A61K48/00
Inventor 杨峥嵘黄来强王海峰
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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