Recombinant adeno-associated virus vector capable of regulating and controlling genetic expression

A virus vector and gene regulation technology, applied in the field of genetic engineering, can solve the problem of inability to up-regulate or down-regulate the target gene, and achieve the effect of obvious regulation and good application prospects

Inactive Publication Date: 2016-09-21
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the simple transgenic operation, the expression of the target gene Kal is not artificially controlled, so that the expression of the target gene cannot be up-regulated or down-regulated as required

Method used

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  • Recombinant adeno-associated virus vector capable of regulating and controlling genetic expression
  • Recombinant adeno-associated virus vector capable of regulating and controlling genetic expression
  • Recombinant adeno-associated virus vector capable of regulating and controlling genetic expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of pAM-kal-rs recombinant plasmid

[0021] (1) Amplification of the riboswitch gene: The riboswitch element sequence (as shown in SEQ ID NO: 1) is obtained through NCBI search, and the gene sequence is synthesized, and a spacer sequence is used to synthesize 2-4 complete switch sequences. Tandem riboswitch sequence, design specific primer pair F1 / F2 (as shown in SEQ ID NO: 2 and 3) according to the riboswitch element, that is, riboswitch gene inserted into the 5'end UTR of Kal gene:

[0022] F1: 5’-GGATCCAAACAAACAAAGCTGTCACC;

[0023] F2: 5’-GAATTCAAAAAATTTTTATTTTTCTTT.

[0024] Using the riboswitch gene as a template and using the above primers (F1 / F2) as a primer pair for PCR amplification:

[0025] The PCR reaction system is shown in Table 1;

[0026] Table 1 PCR reaction system

[0027]

[0028]

[0029] PCR reaction program: 94°C pre-denaturation 5min; 94°C denaturation 30s, 55°C annealing 45s, 72°C 20s (each additional series switch increases by 20s), ...

Embodiment 2

[0039] Example 2 Identification of the obtained pAM-kal-rs recombinant plasmid expression

[0040] Use a commercial plasmid extraction kit (Beijing Zhuangmeng International Biogene Technology Co., Ltd.) to extract the pAM-kal-rs recombinant plasmid obtained in Example 1; use liposome transient transfection method to recombine the pAM-kal-rs The plasmid was introduced into mammalian cells, and the supernatant of the cell culture medium was obtained after 48 hours, and the content of KAl in the sample was detected using a commercial ELISA kit (R&D).

Embodiment 3

[0041] Example 3 Packaging and construction of rAAV vectors

[0042] The pAM-kal-rs recombinant plasmid obtained in Example 1 was extracted in a large amount, and then the rAAV vector was packaged and constructed by the three-plasmid co-transfection method; the specific operation of the large-scale extraction of the pAM-kal-rs recombinant plasmid is as follows:

[0043] (1) Take 10μL of cryopreserved bacteria and inoculate it into a 10mL test tube of LB liquid medium containing ampicillin (Amp), place it on a shaker, and incubate with shaking at 37°C and 220rpm for 10-12 hours to reach the OD of the bacterial solution. 600 The value is equal to 0.6;

[0044] (2) Inoculate the activated strains into a 1L Erlenmeyer flask containing Amp-containing LB liquid medium, place it on a shaker, and culture with shaking at 37°C and 220rpm for 10-12 hours;

[0045] (3) Transfer the bacterial solution to a 500 mL centrifuge tube, centrifuge at 4°C at 4700 rpm for 20 minutes, discard the supernatant...

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Abstract

The invention provides a recombinant adeno-associated virus vector capable of regulating and controlling genetic expression. Construction operation of the recombinant adeno-associated virus vector includes the following steps that riboswitch elements composed of theophylline aptamer and tabacco ringsport virus hammerhead ribozyme are recombined into a Kal gene sequence, a specific primer pair is designed to amplify the sequence according to the insertion position of the riboswitch elements in the Kal gene, and the rAAV vector is packaged; the mode of recombining the riboswitch element into the Kal gene sequence includes the steps that the single switch element is inserted into the 5'tail end and / or 3'tail end UTR of the Kal gene, or the multiple switch elements are inserted into the 5'tail end and / or 3'tail end UTR of the Kal gene in an end-to-end mode. The vector can regulate and control transgenosis expression in a mammal and has an obvious regulating and controlling function.

Description

【Technical Field】 [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant adeno-associated virus vector capable of regulating gene expression. 【Background technique】 [0002] Most of the traditional methods of regulating gene expression use transcription factors to interfere with target genes, which are more common but have obvious limitations. Transcription factors need to be translated into proteins to act, which is not only time-consuming and energy-consuming, but also immunogenic, which is not conducive to clinical applications. [0003] Riboswitch is a primitive non-coding RNA, which is a self-regulating element mostly derived from prokaryotes; its main structure includes two parts: aptamer domain and expression platform domain. The combination of ligand and aptamer will cause the secondary structure of the entire switch element The change in the above, which causes the change of the mRNA chain where the riboswitch...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/66
CPCC07K14/811C12N15/66C12N15/86C12N2750/14143C12N2800/107
Inventor 冯婧娴王佳稳林俊生刁勇
Owner HUAQIAO UNIVERSITY
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