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Method for detecting purity of superhelix structure of lentivirus packaging system helper plasmid

A detection method and supercoil technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low accuracy, not suitable for use, and easy to cause errors, etc., and achieve high sensitivity, simple analysis method, and high resolution. Effect

Active Publication Date: 2021-05-14
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the process of plasmid purification, the supercoiled structure of the plasmid DNA will be destroyed, and a variety of plasmid DNAs with different configurations will be formed, thereby affecting the packaging efficiency of the plasmid. Therefore, it is necessary to make an accurate quantitative analysis of the supercoiled content of the plasmid DNA.
[0008] Existing techniques for detecting supercoiled DNA content include agarose gel electrophoresis and capillary gel electrophoresis, but both have shortcomings in detection methods. For example, capillary gel electrophoresis requires tedious sample pretreatment and is not suitable for used in each step of plasmid DNA production; agarose gel electrophoresis at OD 260 The detection range is too narrow under the wavelength, and it is easy to cause errors in the sample dilution process, the accuracy is very low, and it is not suitable for use in every step of plasmid DNA production
More importantly, neither of the above two methods can quantitatively detect the content of supercoiled DNA

Method used

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  • Method for detecting purity of superhelix structure of lentivirus packaging system helper plasmid
  • Method for detecting purity of superhelix structure of lentivirus packaging system helper plasmid
  • Method for detecting purity of superhelix structure of lentivirus packaging system helper plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Screening and optimization of detection conditions

[0041] The BZ2K plasmid is one of the necessary helper plasmids in the lentiviral packaging process, and its structure is as follows: figure 1 shown. The structures of BZ2K plasmid were separated by using Waters e2695 high performance liquid chromatography and TSKgel DNA-NPR column.

[0042] Experiment 1:

[0043] Detection wavelength 260nm;

[0044] Flow rate 0.5ml / min;

[0045] Column temperature 35°C;

[0046] Loading volume 20μL (2μg);

[0047] Mobile phase A: 20mmol / L Tris pH8.8 solution;

[0048] Mobile phase B: 20mmol / L Tris 1mol / L NaCl pH8.8 solution;

[0049] Elution gradient 1% (Table 1).

[0050] Table 1

[0051] time (minutes) Mobile phase A% Mobile phase B% 0 50 50 5 50 50 45 10 90 50 50 50

[0052] The result is as figure 2 As shown, it is found that the separation effect of the different components of the BZ2K plasmid (supercoiled struc...

Embodiment 2

[0070] Example 2: Preparation of working standards for each configuration of BZ2K plasmid

[0071] 1. Preparation of BZ2K plasmid linear structure working standard

[0072] Take 2 μg to 5 μg of BZ2K plasmid, perform single enzyme digestion with MluI-HF, and alcoholize the single enzyme digestion product. After alcoholization, the product is dissolved in mobile phase A to 50 μL, which is the BZ2K plasmid linear plasmid working standard solution.

[0073] 2. Preparation of BZ2K plasmid open-circle structure working standard

[0074] Using the optimal chromatographic detection conditions determined in Example 1 (the detection wavelength is 260nm; the flow rate is 0.5ml / min; the column temperature is 35°C; the loading volume is 20 μL; the mobile phase A is 20mmol / L Tris pH8.0 solution; Phase B is 20mmol / L Tris1mol / L NaCl pH8.0 solution; gradient elution is 1%), the BZ2K plasmid stock solution is injected, and the miscellaneous peaks in the chromatogram (the miscellaneous peaks ab...

Embodiment 3

[0083] Embodiment 3: system suitability solution preparation

[0084] Dissolve 50 μL of the linearized plasmid working standard, 50 μL of the open-loop plasmid working standard and 20 μg of the supercoiled plasmid working standard with mobile phase A to 200 μL and mix well to obtain the system suitability solution.

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Abstract

The invention relates to a method for detecting the content of helper plasmid superhelix of a lentivirus packaging system by high performance liquid chromatography, the high performance liquid chromatography is used for determination, and the helper plasmid superhelix can be used as a working standard substance of a plasmid superhelix structure by recovering and identifying a main peak of the high performance liquid chromatography. Results show that when the method is used for detecting the superhelical structure of the BZK2 plasmid, the purity of the superhelical structure reaches 84.71%, and the method is high in specificity, easy to operate, high in sensitivity, accurate in result and suitable for detecting the purity of the superhelical structure of the plasmid product.

Description

technical field [0001] The invention relates to the field of drug analysis, in particular to a method for detecting the purity of the supercoiled structure of plasmid DNA by using high performance liquid chromatography. Background technique [0002] Plasmids exist widely in the biological world, ranging from bacteria, actinomycetes, filamentous fungi, macrofungi, yeast to plants, and even human organisms. From the perspective of molecular composition, there are DNA plasmids and RNA plasmids; from the perspective of molecular configuration, there are linear plasmids and circular plasmids, and their phenotypes are also diverse. Bacterial plasmids are the most commonly used vectors in genetic engineering. Plasmid is a genetic component outside of bacterial or cell chromatin, capable of autonomous replication, and symbiotic with bacteria or cells. It is a double-stranded covalently closed circular DNA outside of chromatin, which can naturally form a supercoiled structure. [0...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/047
Inventor 张同存张琴星马传艳彭波
Owner WUHAN BIO RAID BIOTECH CO LTD
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