Recombinant adeno-associated virus capable of silencing CDK2 genes in targeted manner

A virus, silent technology, applied in the fields of molecular biology and biomedicine, to achieve the effect of inhibiting growth

Inactive Publication Date: 2015-11-25
于英君 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no literature report on the application of recombinant adeno-associated virus targeting CDK2 gene for in vivo targeted therapy of liver cancer

Method used

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  • Recombinant adeno-associated virus capable of silencing CDK2 genes in targeted manner
  • Recombinant adeno-associated virus capable of silencing CDK2 genes in targeted manner
  • Recombinant adeno-associated virus capable of silencing CDK2 genes in targeted manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of recombinant adeno-associated virus targeting the silenced CDK2 gene

[0033] 1. Construction of recombinant plasmid pAKD-shRNA-CDK2

[0034] According to the sequence of human liver cancer cell CDK2 gene (GenBankAccessionNumber: NM_001798) and referring to the siRNA target sequence selection principle proposed by Tuschl et al., the specific siRNA sequence against CDK2 was designed: GACCCTAACAAGCGGATTTCG. Add KpnI (-GTAC-) and BgIII (-GATC-) endonuclease restriction site sequences and transcription termination sequences on the basis of the selected target sequence, and entrust Shanghai Yingjun Biotechnology Co., Ltd. to synthesize the complete shDNA sequence :

[0035]

[0036] Perform annealing of the shRNA (shorthairpinRNA) template, and form a double-stranded oligonucleotide fragment with KpnI and BgIII restriction sites at both ends after the annealing reaction, labeled as CDK2-shRNA.

[0037] Take the pAKD-shRNA plasmid vector, use Kp...

Embodiment 2

[0057] Example 2: Animal experiment of recombinant adeno-associated virus targeting silenced CDK2 gene

[0058] 1. Establishment of subcutaneous xenograft tumor model in nude mice

[0059] BALB / c-nu nude mice, female, were inoculated after 3 days of adaptive feeding, and the concentration of HepG2 cell suspension was diluted to 2×10 with PBS buffer. 7 cells / ml, take 0.2ml / cell suspension and inoculate subcutaneously. Nude mice were randomly divided into three groups 15 days after inoculation: tumor group, NC (non-related sequence) control group, and rAAV-shRNA-CDK2 group. The three groups were administered 0.2ml / mouse once by tail vein injection, and the tumor group was injected with PBS buffer solution. Measure the long diameter (a) and short diameter (b) of the tumor with a vernier caliper every five days after inoculation, according to the formula V=a×b 2 / 2 to calculate the tumor volume (see Figure 9 ). According to the average value of the transplanted tumor volume ...

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Abstract

The invention relates to a recombinant adeno-associated virus capable of silencing CDK2 genes in a targeted manner. The experiment shows that the recombinant adeno-associated virus rAAV-shRNA-CDK2 can stably express in vivo for a long term, so as to silence the expression of the CDK2 genes and inhibit the growth of hepatoma carcinoma cells, and meanwhile, the recombinant adeno-associated virus rAAV-shRNA-CDK2 is small in toxic and side effects. A means and a method for effectively treating liver cancer are provided, and the application prospect is good.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine, and specifically relates to a recombinant adeno-associated virus targeting the silenced CDK2 gene. technical background [0002] Liver cancer is the second cause of death among malignant tumor diseases in my country due to its high incidence, difficulty in treatment, and high mortality. The number of people who die from liver cancer every year accounts for 53% of the total number of deaths from liver cancer in the world. Its pathogenesis is still unclear. The methods mainly include radiotherapy and chemotherapy, surgery and liver transplantation. However, more than 80% of the patients with liver cancer have reached the advanced stage and cannot apply the above treatment methods. Even if surgical resection can be performed, the postoperative recurrence rate is relatively high, and the long-term postoperative The prognosis is unsatisfactory, so effective early diagnosis met...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/113
Inventor 于英君于水澜李和伟姜颖张梅宋高臣刘厚广韩晶晶肖景仁韩松阳
Owner 于英君
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