A method for detecting vaccinia virus capping enzyme activity
A technology of vaccinia virus and capping enzyme, applied in the field of bioengineering, can solve the problem of high cost
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Embodiment 1
[0098] Example 1: 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 Mix), Novoprotein (Nearshore Protein Technology Co., Ltd.), Cat. No.: E035; T7 High Yield RNA Transcription kit (T7 High Yield RNA Transcription Kit) , Novoprotein (Nearshore Protein Technology Co., Ltd.), Item No.: E131; Vaccinia Capping System (vaccinia virus capping system), Novoprotein (Nearshore Protein Technology Co., Ltd.), Item No.: M062-YH01; RNA purification magnetic beads; 0.2ml EP Tubes, magnetic stands, PCR machines, Qubit nucleic acid quantifiers, 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 carries the T7 promoter sequence, and the specific sequence is as follows:
[0104] T7-F:
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Embodiment 2
[0124] Embodiment 2: a kind of method for detecting vaccinia virus capping enzyme activity
[0125] 1. Sample and material preparation
[0126] a. Sample: The starting 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 Quantitator, LightCycler 480 Quantitator, etc.
[0128] 2. RNA capping reaction. The RNA transcribed in vitro was placed in a PCR machine at 65 ° C for 5 min, and the reaction was immediately placed on ice for 5 min, and the following configuration reaction system (Table 2) was added (wherein the vaccinia virus capping enzyme used in the positive control was the one produced by the applicant. Identification of qualified control enzymes by LC-MS method):
[01...
Embodiment 3
[0161] Embodiment 3: a kind of method for quantitative detection of capping enzyme activity of vaccinia virus
[0162] The positive control in Example 2 was diluted with a 5-fold concentration gradient, and the method in Example 2 was used to measure the enzymatic activity, and the results were as follows (Table 4):
[0163] Table 4 Quantitative determination of enzyme activity
[0164]
[0165] The RT-qPCR calculation results are shown in Table 4. ΔCT = CT value of streptavidin-adsorbed RNA - CT value of capped RNA. The average value of three replicates for each sample was taken to calculate the ΔCT before and after streptavidin treatment.
[0166] The method can distinguish capping enzymes with different activities and has good repeatability. Taking the logarithmic value of the enzyme concentration of the gradient dilution as the X-axis and the corresponding ΔCT as the Y-axis, the standard curve can be drawn ( Figure 4 ), and then used for quantitative analysis of capp...
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