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Simulating retrovirus of target tumor, and preparation and use thereof

A retrovirus, targeting technology, applied in the field of genetic engineering, can solve problems such as tumor-free cell targeting, and achieve the effects of improving safety and effectiveness, simple preparation method, and unlimited capacity

Inactive Publication Date: 2009-01-28
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned gene carriers all have the defect of no tumor cell targeting

Method used

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  • Simulating retrovirus of target tumor, and preparation and use thereof
  • Simulating retrovirus of target tumor, and preparation and use thereof
  • Simulating retrovirus of target tumor, and preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1, Preparation of Simulated Retroviruses Targeting Tumors

[0039] 1. Preparation of fusion polypeptide

[0040] The fusion polypeptide of this example is formed by linking polylysine and the amino terminal of the RGD peptide through a flexible linker, using K 18 -linker-RGD indicates that the amino acid sequence is: K 18 -Xaa-Ser-Xaa-Ser-Xaa-Ser-Ala-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-Ala (Xaa=Acp), this fusion polypeptide entrusts Gil Biochemical (Shanghai) Co., Ltd. to adopt The solid-phase peptide synthesis method is used to prepare the product, and the product is identified by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and the purity is 97.6%.

[0041] 2. Preparation of siRNA retrovirus recombinant expression vector

[0042] Utilize the online tool (www.ambion.com / techlib / misc / siRNAfinder.html) provided by American Ambion Company, select the siRNA target sequence of Pokemon gene (Genbank accession number is NM_015898), accordin...

Embodiment 2

[0049] Example 2, Preparation of Simulated Retroviruses Targeting Tumors

[0050] 1. Preparation of fusion polypeptide

[0051] Preparation method is identical with embodiment 1;

[0052] 2. Preparation of siRNA retrovirus recombinant expression vector

[0053] Preparation method is identical with embodiment 1;

[0054] 3. Preparation of simulated retrovirus

[0055] In 60 μL of HBS buffer (consisting of 140 mmol / L NaCl and 10 mmol / L Hepes) with a pH value of 7.4, 2 μL of 500 ng / μL of the siRNA target sequence I obtained in step 2 was added. , II, III or IV siRNA retrovirus recombinant expression vector solution, under vortex conditions, add 1 μL of the fusion polypeptide solution obtained in step 1 with a concentration of 2.65 μg / μL dropwise, after the titration is completed, continue at room temperature Vortex for 40 minutes, and then stand still for 50 minutes. The fusion polypeptide can wrap the siRNA retroviral recombinant expression vector containing the siRNA target...

Embodiment 3

[0056] Example 3, Preparation of Simulated Retroviruses Targeting Tumors

[0057] 1. Preparation of fusion polypeptide

[0058] The fusion polypeptide of this embodiment is formed by directly linking polylysine to the amino terminal of the RGD peptide, and K 18 -RGD said, the amino acid sequence is: K 18 -Ala-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-Ala, this fusion polypeptide was entrusted to Gill Biochemical (Shanghai) Co., Ltd. to prepare by solid-phase peptide synthesis. The product was identified by HPLC and MS with a purity of 98.1 %;

[0059] 2. Preparation of siRNA retrovirus recombinant expression vector

[0060] Preparation method is identical with embodiment 1;

[0061] 3. Preparation of simulated retrovirus

[0062] In 60 μL of HBS buffer (consisting of 150 mmol / L NaCl and 10 mmol / L Hepes) with a pH value of 7.4, 2 μL of 500 ng / μL of the siRNA target sequence I obtained in step 2 was added. , II, III or IV siRNA retrovirus recombinant expression vector solution, unde...

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Abstract

The invention discloses a simulated retrovirus of a targeting tumor; the simulated retrovirus adopts fusion polypeptide consisting of poly-D-lysine and RGD peptide as a genomic vector, packs siRNA retrovirus recombinant and expression vector of gene which is related to the tumor and is formed by self-assembling; a preparation method thereof comprises three steps of the preparation of the fusion polypeptide, the preparation of the siRNA retrovirus recombinant and expression vector and the preparation of the simulated retrovirus; the simulated retrovirus of the targeting tumor of the invention can be targeted and positioned into tumor cells, mediates gene transfection effectively, inhibits the generation and propagation of the tumor obviously, and improves the safety and effectiveness of the tumor gene therapy; and the preparation method is simple and convenient, can be used for preparing anti-tumor drugs and has good application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a tumor-targeting simulated retrovirus (mimoretrovirus) and its preparation method and application. Background technique [0002] Modern medicine still lacks effective prevention and treatment measures for tumors. The occurrence of most tumors is closely related to gene variation, mainly manifested in the mutation, amplification, overexpression of oncogenes and the mutation and inactivation of tumor suppressor genes. An important idea in cancer therapy. RNA interference (RNA interference, RNAi) technology, as a new tool for gene silencing, can quickly, efficiently and specifically inhibit target genes through small interfering RNA (small interfering RNA, siRNA) that specifically binds homologous mRNA and promotes its degradation. It has a very bright prospect for its application in the field of tumor gene therapy. [0003] At present, the primary problem of tumor gene therap...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/11C12N15/867A61K48/00A61K47/42A61P35/00A61K47/34C12N15/113
Inventor 倪兵石晶磊
Owner ARMY MEDICAL UNIV
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