Lentivirus freeze-drying protective agent and lentivirus freeze-dried powder

A technology of drying protective agent and lentivirus, which is applied in the preservation of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve the problem of no lentivirus freeze-dried powder, etc., and achieve the effect of good application value, reduced activity loss, and easy transportation.

Inactive Publication Date: 2018-10-09
SHANGHAI TELEBIO BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no stable lentiviral freeze-dried powder

Method used

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  • Lentivirus freeze-drying protective agent and lentivirus freeze-dried powder
  • Lentivirus freeze-drying protective agent and lentivirus freeze-dried powder
  • Lentivirus freeze-drying protective agent and lentivirus freeze-dried powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation of lentiviral freeze-drying protective agent

[0055] (1) Weigh an appropriate amount of disodium hydrogen phosphate and sodium dihydrogen phosphate powder, fully mix and dissolve with deionized water, adjust the pH value to 7.0-7.6 with 2M sodium hydroxide solution, and filter with a 0.45µm filter membrane to obtain phosphate Buffer solution (50mmol / L, pH7.0~7.6) at room temperature for later use.

[0056] (2) Weigh glycerin, sucrose, mannitol, L-arginine, glycine, PEG6000, and gelatin according to the following ingredients and final concentration requirements, add them to phosphate buffer, mix and dissolve thoroughly, and measure the pH value of the solution again and adjust Between 7.0 and 7.6, filter with a 0.22 μm filter membrane to obtain a lentiviral freeze-dried protective agent.

[0057] Protective agent components and content:

[0058] Glycerin 1.0% (V / V)

[0059] Sucrose 20mg / mL

[0060] Mannitol 25mg / mL

[0061] L-Arginine 1mg / mL ...

Embodiment 2

[0066] Example 2 Preparation of lentivirus freeze-dried powder

[0067] (1) The present invention prepares RCR-deficient lentivirus stock solution according to conventional methods: prepare auxiliary packaging plasmid (pspax2), envelope plasmid (pMD), expression plasmid containing GFP protein (pCDHGFP) and 293T cells; prepare the three plasmids Mix according to a certain ratio (pspax2:pMD:pCDHGFP=2:1:3) and transfect into 293T cells with PEI reagent; after 12h, replace the fresh medium to continue culturing, collect the supernatant at 48h and 72h respectively to obtain the stock solution containing lentivirus and Filtered through a 0.45 µm membrane filter and stored at 4°C for later use.

[0068](2) The present invention uses anion exchange chromatography to purify the lentivirus stock solution: weigh an appropriate amount of tris base, and prepare equilibration buffer A (25mM Tris, pH8.0) and elution buffer B (1M Tris, pH 8.0) with deionized water, respectively. pH8.0), take...

Embodiment 3

[0071] Example 3 Comparative analysis of activity of lentiviral freeze-dried powder

[0072] According to the formula described in Example 1 and the production process described in Example 2, three batches of lentivirus lyophilized powder were produced, and the transduction activity (TU / ML) was measured before and after lyophilization. The test results are as follows:

[0073] Table 1 The results of transduction activity determination of lentiviral lyophilized powder before and after lyophilization

[0074]

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PUM

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Abstract

The invention provides a lentivirus freeze-drying protective agent and lentivirus freeze-dried powder. The protective agent comprises ingredients of glycerinum, saccharose, mannitol, L-arginine, glycine, PEG6000, gelatin and a phosphate buffer solution (disodium hydrogen phosphate/monosodium phosphate); the lentivirus freeze-dried powder is formed by freeze-drying a lentivirus and the protective agent at a freeze-drying state; the stability of the lentivirus freeze-dried powder is better at the temperature of minus 80 DEG C, and the activity of a lentivirus particle can be effectively kept.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a lentivirus freeze-dried protective agent, that is, a lentivirus freeze-dried powder. . Background technique [0002] Lentivirus is a kind of artificially modified retrovirus derived from human immunodeficiency virus. It is composed of envelope protein (such as VSVG) and capsid protein (such as p24) encapsulating genetic material DNA nucleic acid. DNA nucleic acid coding structure Protein gag, polymerase Pol, transport regulation protein Rev and other key molecules. The advantage of lentivirus is that it can mediate the integration of foreign genes into the host genome through LTR elements, so that the foreign genes can be stably inherited together with the host genes. Therefore, since its development, lentivirus has been mainly used in the field of basic research, that is, as a transport tool to carry foreign genes into host cells or animals, enhance or weaken the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12R1/91
CPCC12N1/04
Inventor 杨光华何东洋
Owner SHANGHAI TELEBIO BIOMEDICAL
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