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Virus with surface modified with calcium phosphate and preparing method of virus

A calcium phosphate and virus technology, applied in botany equipment and methods, biochemical equipment and methods, pharmaceutical formulations, etc., can solve problems affecting clinical effects, adenovirus titer, etc., to improve transfection efficiency, transfection Efficiency enhancement, solving the effect of vaccine inactivation

Active Publication Date: 2013-06-19
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has an impact on the titer of the adenovirus in vivo, potentially affecting its clinical efficacy

Method used

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  • Virus with surface modified with calcium phosphate and preparing method of virus
  • Virus with surface modified with calcium phosphate and preparing method of virus
  • Virus with surface modified with calcium phosphate and preparing method of virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, in situ mineralization of adenovirus

[0043] Schematic diagram of the flow chart for the in situ mineralization of adenovirus, see figure 1 .

[0044] 1. In situ mineralization of Ad5-EGFP virus

[0045] 1. Adjust the pH of the Ad5-EGFP virus solution to 7.2 with ammonia water. At this point, the surface Zeta potential of the virus particles was measured and found to be -28mV. It has been determined that the surface Zeta potential of virus particles in the pH range of 6.8-8.0 is negatively charged, which can adsorb cations.

[0046] 2. The virus liquid obtained in 10mL step 1 (2×10 8 PFU / ml) by adding CaCl 2 , so that CaCl 2 The final concentration of the solution reached 20mM, adjusted the pH to 7.2 with ammonia water, and stood at 4°C for 12 hours (the purpose is to make Ca 2+ Fully enriched around the virus particles); Slowly add 10mL 20mM Na 2 HPO 4 aqueous solution and keep stirring, adjust the pH to 9.0 with ammonia water, and stir at 4°C f...

Embodiment 2

[0059] Embodiment 2, the dot hybridization experiment of virus after mineralization

[0060] Anti-adenovirus hexon (Hexon) monoclonal antibody: purchased from Abcam, article number ab8249, name Mousemonoclonal to Adenovirus hexon.

[0061] Alkaline Phosphatase-conjugated goat anti-mouse IgG: purchased from Jackson, Cat. No. 515-055-003, name AlkalinePhosphatase-conjugated AffiniPure Sheep Anti-Mouse IgG (H+L).

[0062] BCIP / NBT (dye solution): purchased from Sigma, Cat. No. B1911-100ML.

[0063] Adenovirus mouse polyclonal antibodies were prepared by the following method: select 4-week-old Balb / C female mice, dilute the Ad5-EGFP virus solution in 100 μl PBS buffer, and immunize by subcutaneous multi-point injection; the immunization process is as follows: initial immunization, Each mouse was injected with 200 μl of the dilution of the virus solution (containing 1.28×10 7 PFU); booster immunization once every 2 weeks, a total of 3 times of booster immunization, and each mouse...

Embodiment 3

[0087] Example 3, the transfection efficiency of the virus at the cellular level and in vivo after mineralization

[0088] 1. Transfection efficiency of Ad5-Luc2-CaPi virus at the cellular level

[0089] The Ad5-Luc2-CaPi virus liquid prepared in Example 1 and the Ad5-Luc2 virus liquid were carried out to the following experiments respectively:

[0090] 1. Infect HEK293 cells with virus solution at doses of infection (MOI) of 100, 10, and 1, respectively, and collect cells at 12h, 24h, 48h, and 72h after infection.

[0091] 2. Lyse the cells collected in step 1 in the cell lysate for 15 minutes, and centrifuge to get the supernatant.

[0092] 3. Take the supernatant from step 2, and use NanoDrop 1000 to detect the protein content, the unit is mg.

[0093] 4. Add the supernatant from step 2 to the three auxiliary wells of a fluorescence-specific 96-well plate, 20 μl per well, and place it in a GloMax96 microplate luminescence detector. Add 100 μl luciferase substrate to each ...

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Abstract

The invention discloses a virus with the surface modified with calcium phosphate and a preparing method of the virus. The virus with the surface modified with the calcium phosphate is obtained by the fact that the virus is subjected to biomineralization with CaCl2 and Na2HPO4. The virus with the surface modified with the calcium phosphate has the advantages that quick and efficient enrichment can be achieved; transfection efficiency in a cell and an organism is greatly improved; the virus can be adaptive to dosing in an intravenous injection way, a calcium phosphate outer shell on the surface of the virus can protect adenovirus vector particles which then cannot be restrained by existing immunity or antibodies, and accordingly the function of the adenovirus vector particles as drug carriers can be well achieved. The Virus with the surface modified with the calcium phosphate and the preparing method of the virus are widely used in fields such as new generation vaccines and gene therapy.

Description

technical field [0001] The invention relates to a virus whose surface is modified with calcium phosphate and a preparation method thereof. Background technique [0002] Adenovirus is a non-enveloped animal virus with double-stranded DNA, and its genome size is about 36 kb. There are nearly 50 serotypes of adenovirus, among which human adenovirus type 2 and type 5 are commonly used as vectors. As a vector, adenovirus has the advantages of wide host range, stable genome, and high transfection efficiency, and has been widely used in the fields of gene therapy and vaccine research. At present, a number of products using recombinant adenoviruses as vectors have entered the clinical research stage. [0003] However, the clinical application of recombinant adenoviruses as vectors still faces many problems, the most important of which is that due to the history of adenovirus infection, most people have a certain level of adenovirus antibodies, and these pre-existing antibodies can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00
Inventor 王晓雨秦成峰邓永强唐睿康
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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