Method for detecting activity of capsidase of vaccinia virus

A technology of vaccinia virus and capping enzyme, applied in the field of bioengineering, can solve the problem of high cost

Active Publication Date: 2022-04-26
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has high sensitivity and high precision, but there are certain requirements for the instrument and the cost is high

Method used

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  • Method for detecting activity of capsidase of vaccinia virus
  • Method for detecting activity of capsidase of vaccinia virus
  • Method for detecting activity of capsidase of vaccinia virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1: The establishment of RNA reaction system

[0099] 1. Sample and material preparation

[0100] a. Materials, reagents and instruments: 2x Fast Pfu Master Mix (2x Fast Pfu Polymerase Mixture), Novoprotein (Inshore Protein Technology Co., Ltd.), Cat. No.: E035; T7 High Yield RNA Transcription Kit (T7 High Yield RNA Transcription Kit) , Novoprotein (Inshore Protein Technology Co., Ltd.), product number: E131; Vaccinia Capping System (Vaccinia Capping System), Novoprotein (Inshore Protein Technology Co., Ltd.), product number: M062-YH01; RNA purification magnetic beads; 0.2ml EP tube, magnetic stand, PCR instrument, Qubit nucleic acid quantifier, etc.

[0101] b. Sample: a plasmid containing the gene of interest.

[0102] 2. Design of primers T7-F and T7-R

[0103] In this experiment, the CLDN2G gene was selected as the target gene, and primers were designed, in which T7-F had a T7 promoter sequence, and the specific sequence was as follows:

[0104] T7-F: ...

Embodiment 2

[0124] Example 2: A method for detecting the activity of vaccinia virus capping enzyme

[0125] 1. Sample and material preparation

[0126] a. Sample: The initial sample is 50 μg of in vitro transcribed RNA dissolved in Nuclease Free Water; the capping enzyme of vaccinia virus to be tested.

[0127] b. Materials, reagents and instruments: NovoScript® Probe One-Step qRT-PCR kit (Novoprotein, E094-LPAA), streptavidin magnetic beads, 3' Biotin GTP (all common commercial reagents), 0.2ml EP tube, Qubit nucleic acid quantifier, LightCycler 480 quantifier, etc.

[0128] 2. RNA capping reaction. Place the in vitro transcribed RNA in a PCR instrument at 65°C for 5 minutes, place it on ice for 5 minutes immediately after the reaction, and add the following configuration reaction system (Table 2) (wherein the vaccinia virus capping enzyme used in the positive control is the already produced vaccinia virus produced by the applicant. Qualified control enzymes were identified by LC-MS...

Embodiment 3

[0161] Example 3: A method for quantitative detection of vaccinia virus capping enzyme activity

[0162] The positive control in embodiment 2 is carried out 5 times of concentration gradient dilutions, adopts the method in embodiment 2 to carry out the mensuration of enzyme activity, and the results are as follows (table 4):

[0163] Table 4 Quantitative determination of enzyme activity

[0164]

[0165] The RT-qPCR calculation results are shown in Table 4. △CT=CT value of streptavidin-adsorbed RNA-CT value of capped RNA. Three replicates of each sample were averaged to calculate the ΔCT before and after streptavidin treatment.

[0166] The method can distinguish capping enzymes with different activities, and has good repeatability. The logarithmic value of the enzyme concentration diluted by gradient is the X-axis, and the corresponding △CT is the Y-axis, and the standard curve can be drawn ( Figure 4 ), and then used for quantitative analysis of capping enzyme activity...

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Abstract

The invention provides a method for in-vitro detection of the activity of vaccinia virus capping enzyme. Specifically, the method comprises the following steps: A, transcribing target gene RNA by using T7 RNA polymerase; b, carrying out capping reaction on the RNA in the step A by using 3 'biotin-GTP and a vaccinia virus capping enzyme; c, carrying out capping RNA adsorption reaction on the RNA in the step B by using streptavidin magnetic beads; and D, detecting the difference of the CT values in the steps B and C by using an RT-qPCR method so as to judge the activity of the vaccinia virus capping enzyme in the step B. The method disclosed by the invention is good in repeatability, the measured capped mRNA yield and non-capped mRNA yield are obviously different, and the capped enzyme activity of the vaccinia virus can be successfully and quantitatively detected.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a method for detecting the activity of vaccinia virus capping enzyme. Background technique [0002] The rapid development of mRNA vaccine technology has led to an increasing demand for RNA biology, and the key step in the preparation of mRNA vaccines is the preparation of mRNA samples. The most basic structure of an mRNA sample includes 5'cap, 5'UTR, ORF, 3'UTR, and 3'tail. Among them, the function of the 5' cap structure is very important. First, it can protect the mRNA from being rapidly degraded by exonucleases; secondly, the 5' cap structure plays an indispensable role in the translation process, because eukaryotes play Initiator recognizes and binds to the cap structure of mRNA. In addition, the 5' cap structure plays a further role in preventing mRNA recognition by innate immune sensors. [0003] Currently, there are two different approaches to capping in vitro transcribed ...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/44C12Q1/686
CPCC12Q1/48C12Q1/44C12Q1/686C12Q2521/107
Inventor郑紫君李亚丽汤玉洁周玥赵曼曼张清仪王明明朱化星
OwnerNOVOPROTEIN SCI INC