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Hantavirus-like particles fused with RGD for tumor gene therapy and preparation method of hantavirus-like particles fused with RGD

A hantavirus and virus-like technology, applied in the field of RGD-fused hantavirus-like particles and their preparation, can solve the problems of serious harm, high fatality rate, no specific and effective therapeutic drugs, etc., to reduce damage and reduce operation. Difficulty, effect suitable for industrial production

Inactive Publication Date: 2018-04-13
宫丽丽
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease is very harmful and has a high fatality rate. China is the country with the most serious epidemic situation of hemorrhagic fever with renal syndrome in the world. At present, there is no specific and effective treatment drug for this disease, and vaccines are mainly used for prevention.

Method used

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  • Hantavirus-like particles fused with RGD for tumor gene therapy and preparation method of hantavirus-like particles fused with RGD
  • Hantavirus-like particles fused with RGD for tumor gene therapy and preparation method of hantavirus-like particles fused with RGD
  • Hantavirus-like particles fused with RGD for tumor gene therapy and preparation method of hantavirus-like particles fused with RGD

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The extraction of embodiment 1.mRNA

[0040] Follow the instructions of the mRNA miniprep kit, and the specific operations are as follows:

[0041] 1) Add 750 μL Tripure LS Reagent to 250 μL anticoagulated serum sample, repeatedly pump with a pipette gun and shake to mix, and lyse at room temperature for 5 minutes;

[0042] 2) Add chloroform to the lysate at a ratio of 0.2mL per milliliter, and oscillate to fully mix the sample;

[0043] 3) The mixture was centrifuged at 12 000 r / min for 15 minutes at 4° C. to separate the mixture into two phases, DNA and protein were extracted into the organic phase, and RNA remained in the aqueous phase. Transfer the upper aqueous phase to a new centrifuge tube;

[0044] 4) Add isopropanol and RNA precipitation solution to the water phase, mix well, and place at room temperature for 25 minutes to precipitate RNA;

[0045] 5) Centrifuge at 12 000 r / min at 4°C for 15 min, remove the supernatant, and collect the precipitated RNA;

[...

Embodiment 2

[0050] Example 2. Synthesis of cDNA by reverse transcription reaction

[0051] According to the instructions of the RT-PCR kit, the reverse transcription reaction system was prepared on ice, and the operation was performed in a 0.2 mL microcentrifuge tube. The specific ingredients are as follows:

[0052]

[0053] In the PCR instrument, put the microcentrifuge tube into the corresponding well, react at 42°C for 1 hour, then bathe in a water bath at 70°C for 10 minutes to inactivate the RT enzyme, and store the obtained RT product cDNA at -80°C Save for later.

Embodiment 3

[0054] Embodiment 3.PCR amplification HFRS target fragment

[0055] According to the instructions of the PCR kit, the PCR reaction system was prepared in a 0.5 mL microcentrifuge tube on ice. in,

[0056] Upstream primer P1: 5'-ATGGACATCGACCACTAC-3';

[0057] Downstream primer P2: 5'-TTAGTGGTGGTGGTGGTGGTGCGGCAGAGTGG-3'.

[0058] The specific ingredients are as follows:

[0059]

[0060] After preparing the system, cover the cap of the centrifuge tube, mix it upside down several times, and immediately place it on the PCR machine to perform amplification. The reaction conditions are as follows:

[0061]

[0062] After finishing the reaction, store the PCR product at 4°C for electrophoresis detection or store it at -20°C for long-term storage.

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Abstract

The invention relates to the field of virus gene engineering, and particularly relates to hantavirus-like particles fused with RGD for tumor gene therapy and a preparation method of the hantavirus-like particles fused with RGD. According to the hantavirus-like particles fused with RGD provided by the invention, RGD oligopeptide is inserted into an M segment region of HFRS by the gene cloning technology; the obtained target sequence is connected to a prokaryotic expression vector pET-30a to successfully establish a prokaryotic expression vector pET30a-RGD-HFRS; the expression vector is appliedto prokaryotic protein expression; after successful expression, HFRS virus-like particles fused with RGD oligopeptide can be obtained; so, when the hantavirus-like particles are adopted as a transfervector for gene therapy, the vector can better identify the tumor cells on a target position, so as to reduce harm to normal cells of a human body and lay a foundation for follow-up study and development of drugs targeting kidney diseases.

Description

technical field [0001] The invention relates to the field of viral genetic engineering, in particular to a hantavirus-like particle fused with RGD and a preparation method thereof. Background technique [0002] Virus-like particles (VLPs) are nanoparticles that do not contain a viral genome and can be self-assembled from all or part of viral structural proteins, and are a non-viral vector. Many viral structural proteins have the ability to automatically assemble into VLPs, which are similar in shape and structure to natural virus particles, and have strong immunogenicity and biological activity. The structure of VLPs allows the insertion of foreign genes or gene fragments to form chimeric VLPs and display foreign antigens on their surface. At present, at least 30 kinds of VLPs have been reported in the literature. These VLPs play an important role in various fields of biomedicine. Most of them are used as vaccine vectors, virus infection pathways or nanoparticles. [0003]...

Claims

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Application Information

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IPC IPC(8): C07K14/175C12N15/70
CPCC07K14/005C07K2319/01C12N15/70C12N2760/12123
Inventor 宫丽丽郝恩超张友林吴迪周文静
Owner 宫丽丽
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