Application of recombinant plasmid with high expression of dnmt2 gene in preventing piglets from weaning stress
A recombinant plasmid and high-expression technology, applied in the field of animal genetic engineering, can solve serious problems such as antibiotic resistance, achieve the effects of no toxic side effects, relieve diarrhea symptoms, and easy preparation
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Embodiment 1
[0053] Construction and verification of embodiment 1 recombinant plasmid pcDNA3.1-DNMT2
[0054] (1) Total RNA was extracted from pig spleen and cDNA was reverse transcribed, and the coding sequence of DNMT2 was amplified using cDNA as a template. The primer sequences for amplifying the gene DNMT2 are as follows:
[0055] Forward primer F: 5′CTA CTA GCT AGC ATG GAG CCC CTG CGG GTC 3′,
[0056] Reverse primer R: 5′CGA CTG CTC GAG TTT GGA TAT GAT GGA ATA CTG GA 3′;
[0057] The PCR reaction system is shown in Table 1
[0058] Table 1 PCR reaction system
[0059]
[0060] The reaction conditions were 95°C for 5min; 30 cycles of 95°C for 30s, 60°C for 30s, and 72°C for 1min; 72°C for 10min.
[0061] (2) The target fragment was recovered using an agarose gel recovery kit, and 1 μL was taken to determine the purity and concentration of the target fragment with Nano Drop 2000.
[0062] (3) First digest an appropriate amount of the target fragment and the pcDNA3.1 vector with ...
Embodiment 2
[0071] Example 2 Large-scale preparation of recombinant plasmid pcDNA3.1-DNMT2
[0072] 1. Preparation of Escherichia coli Competent Cells
[0073] (1) Take Escherichia coli DH5α strains frozen at -80°C and inoculate them on LB agar plates without antibiotics. After incubating in a 37°C incubator for 12-16 hours, pick a single colony with good growth and inoculate it in 5 mL In LB culture medium, culture at 37°C for 5-6h, shaking speed of 250-300rpm;
[0074] (2) Transfer the activated Escherichia coli liquid into a conical flask filled with 50mL LB under aseptic conditions, continue to shake and cultivate for about 2.5-3h, until the OD value of the bacterial liquid at 600nm reaches 0.5-0.6, Under aseptic conditions, transfer the bacteria to a 50mL sterile centrifuge tube pre-cooled with ice, and place on ice for 30min;
[0075] (3) Centrifuge at 4000rpm for 10min at 4°C, discard the supernatant under sterile conditions, invert the centrifuge tube for 1min to drain the resid...
Embodiment 3
[0100] Embodiment 3 Recombinant plasmid of the present invention makes its DNMT2 overexpression verification in animal body
[0101] 1. Feeding the Mice
[0102] Select 6 SPF male Kunming mice (purchased from Hubei Provincial Center for Disease Control and Prevention) with the same body weight and age, and randomly divide them into two groups, with 3 mice in each group. Another group was intraperitoneally injected with 25 μL of eukaryotic expression recombinant plasmid pcDNA3.1-DNMT2 prepared by the present invention at a concentration of 1 μg / μL. SPF male Kunming mice were fed for 3 days, then killed by asphyxiation with carbon dioxide, and the kidneys were taken.
[0103] 2. Detection of DNMT2 expression in kidney by real-time fluorescent quantitative PCR
[0104] (1) Extract tissue total RNA
[0105] Operation in the ultra-clean workbench: take 100 mg of the above-mentioned mouse kidney tissue sample, cut it into pieces, put it in a pre-cooled grinder, add 1 mL of PBS, u...
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