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Construction and application of recombined lentivirus vector aiming at RNA interference of PKC gamma genes

A technology of recombinant lentivirus and RNA interference, which is applied in the field of molecular biology and biomedicine, can solve the problems of gene expression silencing and inability to guide the synthesis of proteins, etc., and achieve good results, no toxic side effects, and low cost

Inactive Publication Date: 2012-07-25
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi interference (RNA interference, RNAi) technology is a new technology that blocks the expression of a specific gene, so that it cannot guide the synthesis of the corresponding protein, resulting in the silence of the gene expression

Method used

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  • Construction and application of recombined lentivirus vector aiming at RNA interference of PKC gamma genes
  • Construction and application of recombined lentivirus vector aiming at RNA interference of PKC gamma genes
  • Construction and application of recombined lentivirus vector aiming at RNA interference of PKC gamma genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Design and synthesis of DNA targeting ShRNA

[0071] According to the PKCγ mRNA sequence (NCBI 012628), the sense and antisense strands of the following nucleotide sequences were designed and synthesized according to Ambion online software.

[0072] PSCSI625:

[0073] Sense strand: 5'-CCGGCAGAAGACAAAGACCGTGAAATTCAAGAGATTTCACGGTCTTTGTCTTCTGTTTTTG-3'

[0074]Antisense strand: 3'-GTCTTCTGTTTCTGGCACTTTAAGTTTCTCTAAAGTGCCAGAAACAGAAGACAAAAACTTAA-5'PSCSI626:

[0075] Sense strand: 5'-CCGGGAAGTTTGAGGCCTGTAATTATTCAAGAGATAATTACAGGCCTCAAACTTCTTTTTG-3'

[0076] Antisense strand: 3'-CTTCAAACTCCGGACATTAATAAGTTCTCTATTAATGTCCGGAGTTTGAAGAAAAACTTAA-5'PSCSI627:

[0077] Sense strand: 5'-CCGGAACTCTATGCCATCAAGATACTTCAAGAGAGTATCTTGATGGCATAGAGTTTTTTTG-3'

[0078] Antisense strand: 3'-TTGAGATACGGTAGTTCTATGAAGTTCTCTCATAGAACTACCGTATCTCAAAAAAACTTAA-5'PSCSI628:

[0079] Sense strand: 5'-CCGGCAGACTACATAGCACCTGAGATTCAAGAGATCTCAGGTGCTATGTAGTCTGTTTTTG-3'

[0080] Antisense strand: 3'-G...

Embodiment 2

[0086] Embodiment 2: Construction of recombinant plasmid

[0087] The process of constructing recombinant plasmids is as follows Figure 11 As shown, take 5 μg each of the sense chain and antisense chain synthesized and purified in Example 1, and anneal to form complementary double chains. Any of the above complementary double chains can be connected to the vector pGCSIL-GFP. 15mL / L non-denaturing polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis PAGE) gel was used to detect the double-strand formation efficiency, and the pGCSIL-GFP / U6 vector (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.) enzyme Cut to linearize and recover large fragments by gel electrophoresis. The large fragments recovered from the plasmid vector were ligated with the synthesized DNA with T4 phage DNA ligase, transformed into competent bacteria DH5α, and the recombinant positive clones were picked for PCR and sequencing identification (ABI 3730 sequencer was used by...

Embodiment 3

[0091] Embodiment 3: RNAi lentiviral packaging

[0092] 1. Virus packaging (see Figure 12 )

[0093] Prepare recombinant viral plasmids encoding lentiviral particles and their two auxiliary packaging element vector plasmids, namely pGCSIL-GFP / U6-Sh PKCγ recombinant vector, pHelper 1.0 (gag / pol element) vector, pHelper 2.0 (VSVG element). The three kinds of plasmid vectors were extracted with high purity and no endotoxin respectively, and were co-transfected into 293T cells according to the instructions of Invitrogen Lipofectamine2000. After 8 hours of transfection, the complete medium was replaced. After 48 hours of culture, the cells rich in lentiviral particles were collected. The supernatant was concentrated to obtain a high-titer lentivirus concentrate, and the virus titer was measured and calibrated in 293T cells. Lentiviral particles within a certain titer range can meet the needs of most in vivo and in vitro experiments.

[0094] Cell line 293T, the packaging cell o...

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Abstract

The invention relates to construction and application of a recombined lentivirus vector aiming at RNA interference of PKC gamma genes. The construction comprises the following steps of: experimentally constructing the lentivirus vector of ShRNA aiming at the PKC gamma genes, mediating the synthetic DNA fragments aiming at ShRNA by the lentivirus vector, performing cotransfection of 293T cells with two kinds of vectors of pHelper 1.0 and pHelper 2.0 and then culturing the cells, and performing transfection of target cells after the lentivirus vector is recombined to realize the RNA interference aiming at the PKC gamma genes. Because the third generation lentivirus vector (SIN) which is self-inactivated is used, the recombined lentivirus vector of the invention has the advantages of safety and reliability, long-term expression for the integration of the target genets into the target cell genomes, little immune response, the infection of non-dividing cells and the like and is a kind of ideal vector. The interference effect of the lentivirus vector of the invention on primary culture neurons of rats is over 85 percent, so that the recombined lentivirus vector possibly becomes a powerful means for gene therapy of chronic neuropathic pain.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine. It specifically relates to a recombinant lentiviral vector for PKCγ gene RNA interference and a construction method thereof, and the viral vector is used to prepare medicine for treating chronic neuropathic pain. Background technique [0002] (1) PKCγ gene is an important molecular target for the treatment of chronic neuropathic pain. [0003] Chronic pain is not only a difficult problem in the medical field, but also has become a major burden on the social economy. According to statistics, more than 320 million people in the world suffer from chronic pain. Neuropathic pain (Neuropathic Pain) is a group of symptoms associated with a variety of peripheral nerve disorders, including diabetes, hypothyroidism, uremia, nutritional deficiency and chemotherapy drugs (vincristine, cisplatin, etc.) neurological disorders; also include: Guillain-Barr syndrome, postherpetic neur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/113C12N15/66A61K48/00A61P25/02A61P29/00
Inventor 邹望远郭曲练宋宗斌张重刘畅赵媛
Owner CENT SOUTH UNIV
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