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Freeze-drying technology of M-MLV reverse transcriptase

A reverse transcriptase and freeze-drying technology, applied in the field of freeze-drying technology of M-MLV reverse transcriptase, can solve the problems of not more than 1 year, inconvenient transportation, storage and application of reverse transcriptase, and achieve the effect of broad conditions

Inactive Publication Date: 2017-11-21
GUANGZHOU HEAS BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the activity and stability of M-MLV reverse transcriptase are also affected by factors such as temperature, pH value, and storage time. The transportation, storage and application of reverse transcriptase have brought inconvenience. Therefore, explore and improve the freeze-drying process of M-MLV reverse transcriptase, strengthen the stability of reverse transcriptase dry powder, extend its storage period and maintain M-MLV reverse transcriptase. The activity of the transcriptase after lyophilization is very necessary

Method used

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  • Freeze-drying technology of M-MLV reverse transcriptase
  • Freeze-drying technology of M-MLV reverse transcriptase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The lyophilization process step of embodiment 1 M-MLV reverse transcriptase

[0047] A. Pretreatment: Dissolve M-MLV reverse transcriptase in 10% trehalose solution and distribute it in screw-cap cryopreservation tubes as samples to be freeze-dried;

[0048] B. Pre-freezing: Turn on the freeze dryer, adjust the temperature in the freeze-drying chamber to -60°C, put the pretreated M-MLV reverse transcriptase into the freeze-dryer for quick freezing; adjust the temperature in the freeze-drying chamber to -50°C, Pre-freeze the product for 2 hours; adjust the temperature in the freeze-drying room to -40°C, and pre-freeze the product for 2 hours;

[0049] C. Under the condition of vacuum degree of 500Pa, adjust the temperature in the freeze-drying chamber to -40°C and maintain it for 5 hours;

[0050] D. Under the condition of vacuum degree of 490Pa, adjust the temperature in the freeze-drying chamber to -25°C and maintain it for 10 hours;

[0051] E. Under the condition o...

Embodiment 2

[0054] Embodiment 2 M-MLV reverse transcriptase freeze-dried product stability comparison experiment

[0055] 1. Use the nucleic acid extraction or purification kit produced by Guangzhou Heshi Biotechnology Co., Ltd. to extract the nucleic acid of two influenza A virus samples, namely sample 1 and sample 2.

[0056] 2. Prepare the newly prepared M-MLV reverse transcriptase solution stored at -20±5°C, the freeze-dried product of M-MLV reverse transcriptase prepared under the conditions of Example 1, and the conditions of Example 1 for 2 years at room temperature Two freshly prepared freeze-dried products of M-MLV reverse transcriptase were prepared.

[0057] 3. Use the newly prepared M-MLV reverse transcriptase solution stored at -20±5°C, the M-MLV reverse transcriptase freeze-dried product prepared under the conditions of Example 1 and Example 1, which were stored at room temperature for 2 years. The newly prepared M-MLV reverse transcriptase freeze-dried product and the infl...

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Abstract

The invention discloses a freeze-drying technology of M-MLV reverse transcriptase. The freeze-drying technology comprises the following steps: (1) pretreatment: dissolving M-MLV reverse transcriptase into trehalose solution at certain concentration, and subpackaging as a to-be-freeze dried sample; (2) pre-freezing: starting a freeze drier, adjusting the interior temperature of a freeze drying chamber to be -50DEG C below, and putting the pretreated M-MLV reverse transcriptase into the freeze dryer; (3) under vacuum condition, adjusting the interior temperature of the freeze drying chamber to be -40+ / -3DEG C, and maintaining for 5+ / -0.5 hours; (4) under vacuum condition, adjusting the interior temperature of the freeze drying chamber to be -25+ / -3DEG C, and maintaining for 10+ / -0.5 hours; (5) under vacuum condition, adjusting the interior temperature of the freeze drying chamber to be 0+ / -3DEG C, returning temperature to be less than or equal to 2DEG C every hour, and maintaining for 5+ / -0.5 hours; (6) under vacuum condition, adjusting the interior temperature of the freeze drying chamber to be 10+ / -3DEG C, returning temperature to be less than or equal to 7.5DEG C every hour, and maintaining for 2+ / -0.5 hours; and (7) closing the freeze dryer, and taking out the freeze-dried M-MLV reverse transcriptase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a freeze-drying process of M-MLV reverse transcriptase. Background technique [0002] M-MLV reverse transcriptase is a Moloney murine leukemia virus (Moloneymurineleukeminvirus) reverse transcriptase (M-MLVRTase for short). The wild type is the same, while its elongation ability is also significantly improved. One unit of reverse transcriptase (M-MLV) activity is defined as the amount of enzyme required to incorporate 1 nmol of deoxyribonucleic acid into acidic precipitation at 37°C for 10 minutes. [0003] M-MLV reverse transcriptase is used for the first-strand synthesis of cDNA and the extension of primers. This enzyme requires magnesium ions or manganese ions as cofactors. When mRNA is used as a template, single-stranded DNA (ssDNA) is first synthesized, and then under the action of reverse transcriptase and DNA polymerase I, single-stranded DNA is synthesized as a template. "...

Claims

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Application Information

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IPC IPC(8): C12N9/12
CPCC12N9/1276C12Y207/07049
Inventor 肖湘文陈华云刘淑园叶映玲
Owner GUANGZHOU HEAS BIOTECH CO LTD
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