Construction method and application of chicken AID overexpression vector plasmid
A technology of overexpression vector and construction method, applied in the field of chicken AID overexpression vector plasmid construction
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Embodiment 1
[0026] Example 1 Construction of ch-AID-Pcdna3.1 overexpression vector
[0027] (1) Design of AID primers. The mRNA sequence of chicken AID was searched in NCBI (sequence number: NM_001243222.1). The primers for the full-length cloning of AID mRNA were designed using Primer Premier 5.0 software. The upstream primer ch-AID-F1 had a HamIII restriction site, and the downstream primer ch-AID-R1 had a XbaⅠ restriction site. AID was amplified according to the high-fidelity enzyme provided by Vazyme and the primers designed by Jinweizhi. Wherein, the nucleotide sequences of primers ch-AID-F1 and ch-AID-R1 used are as follows:
[0028] ch-AID-F1: 5'-CCCaagcttATGGACAGCCTCTTGATGA-3' (SEQ ID NO.1)
[0029] ch-AID-R1: 5'-GCtctagaATTGCTGATGACTTCCCCTT-3' (SEQ ID NO.2)
[0030] (2) Amplification of AID fragments. use Reagent extracted total RNA from chicken macrophage HD-11 treated with AZA for 48 hours, and after the concentration was determined, it was reverse-transcribed into cDNA ...
Embodiment 2
[0034] Example 2 Chicken AID RT-qPCR detection
[0035] In Example 2, the pcDNA3.1-AID high expression vector obtained in Example 1 was used to transfect into DF-1 cells, and RT-qPCR detection was performed on them.
[0036] Specifically include the following steps:
[0037] (1) Resuscitate DF-1 cells, spread the cells in a 12-well plate, and transfer the pcDNA3.1-AID vector into the cells when they grow to about 80%, collect the cells after 36 hours, and use Reagent extracts total RNA and measures its concentration.
[0038] (2) Use PrimeScript TM Reagents provided in the RT reagent Kit with gDNA Eraser kit remove genome.
[0039] The reaction system includes: 1 μg total RNA obtained in step (1) of Example 2 as a template, 2 μL 5×gDNA Eraser Buffer, 1 μL gDNA Eraser, RNase Free dH 2 O to make up to 10 μL.
[0040] The reaction conditions are: 42°C for 2 minutes; 4°C for maintenance.
[0041](3) Use PrimeScript TM The reagents provided in the RT reagent KitwithgDNA Era...
Embodiment 3
[0050] Example 3 Analysis of pcDNA3.1-AID-induced antiviral innate immunity
[0051] In Example 3, using the pcDNA3.1-AID overexpression plasmid obtained in Example 2, the ability of pcDNA3.1-AID to induce antiviral innate immunity was evaluated by fluorescent quantitative PCR.
[0052] Specifically include the following steps:
[0053] (1) Chicken embryo fibroblast DF-1 was inoculated into a 6-well cell culture plate, and then transfected with pcDNA3.1-AID overexpression plasmid. At the same time, the GFP overexpression plasmid was set as the vector control group.
[0054] (2) 36 hours after transfection, the cells were collected;
[0055] (3) Total RNA was extracted, and the genes related to interference signal pathway were detected by fluorescent quantitative PCR method.
[0056] (4) Figure 5 It can be seen that the expression of interferon-related genes tends to be up-regulated, indicating that the high expression of AID may be due to changes in the expression of inna...
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