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

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

Inactive Publication Date: 2009-11-11
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 of recombined lentivirus vector aiming at PKC gamma gene RNA interference and application thereof
  • Construction of recombined lentivirus vector aiming at PKC gamma gene RNA interference and application thereof
  • Construction of recombined lentivirus vector aiming at PKC gamma gene RNA interference and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Design and synthesis of DNA targeting ShRNA

[0078] According to the PKCγmRNA sequence (NCBI 012628), the sense and antisense strands of the following nucleotide sequences were designed and synthesized according to Ambion online software.

[0079]PSCSI625:

[0080] Sense strand: 5'-CCGGCAGAAGACAAAGACCGTGAAATTCAAGAGATTTCACGGTCTTTGTCTTCTGTTTTTG-3'

[0081] Antisense strand: 3'-GTCTTCTGTTTCTGGCACTTTAAGTTTCTCTAAAGTGCCAGAAACAGAAGACAAAAACTTAA-5'

[0082] PSCSI626:

[0083] Sense strand: 5'-CCGGGAAGTTTGAGGCCTGTAATTATTCAAGAGATAATTACAGGCCTCAAACTTCTTTTTG-3'

[0084] Antisense strand: 3'-CTTCAAACTCCGGACATTAATAAGTTCTCTATTAATGTCCGGAGTTTGAAGAAAAACTTAA-5'

[0085] PSCSI627:

[0086] Sense strand: 5'-CCGGAACTCTATGCCATCAAGATACTTCAAGAGAGTATCTTGATGGCATAGAGTTTTTTTG-3'

[0087] Antisense strand: 3'-TTGAGATACGGTAGTTCTATGAAGTTCTCTCATAGAACTACCGTATCTCAAAAAAACTTAA-5'

[0088] PSCSI628:

[0089] Sense strand: 5'-CCGGCAGACTACATAGCACCTGAGATTCAAGAGATCTCAGGTGCTATGTAGTCTGTTTTTG-3' ...

Embodiment 2

[0096] Embodiment 2: Construction of recombinant plasmid

[0097] 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.) was digested with AgeI and EcoRI To linearize them, large fragments were recovered 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 ...

Embodiment 3

[0101] Embodiment 3: RNAi lentiviral packaging

[0102] 1. Virus packaging (see Figure 12 )

[0103] 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.

[0104] Cell line 293T, the packaging cell o...

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Abstract

The invention relates to a recombined lentivirus vector aiming at PKC gamma gene RNA interference, construction thereof and application thereof. A lentivirus vector of ShRNA aiming at PKC gamma gene is constructed by experiments; a synthesized DNA fragment aiming at the ShRNA is mediated through the lentivirus vector, and transfects a 293T cell with pHelper 1.0 vectors and pHelper 2.0 vectors for culturing; and after the recombined lentivirus vector is acquired, a target cell is transfected to realize RNA interference aiming at the PKC gamma gene. The self-inactivated third generation lentivirus (SIN) is adopted, and the recombined lentivirus vector has the advantages of safety and reliability, unseparated cell infection, long-term expression for the target gene integrated to a target cell gene group, small immunity reaction and the like, and is an ideal vector. The lentivirus vector has interference effect on rat primary culture neuron over 85 percent, so that the recombined lentivirus vector can become a powerful tool for treating gene 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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IPC IPC(8): C12N15/867A61K48/00A61P29/00
Inventor 邹望远郭曲练宋宗斌张重刘畅赵媛
Owner CENT SOUTH UNIV
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