Apolygus lucorum cell nucleus hormone receptor E75, coding sequence thereof, carrier and bacterial strain
A technology of nuclear hormones and green lygus, applied in the field of agricultural science, can solve problems such as the rise of green lygus
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Embodiment 1
[0029] Embodiment 1: Acquisition of the nuclear hormone receptor E75 gene (AlE75 gene) of Lygus green bug
[0030] The TRIzol method was used to extract the total RNA of Lygus japonica, and the total RNA samples with good electrophoretic pattern and OD260 / OD280 value between 1.8 and 2.0 were selected and combined for mRNA purification, and the first-strand cDNA was synthesized by reverse transcription. The PCR reaction system was: 10 μl Total RNA, 4.0 μl 5X reaction buffer (Sigma), 2 μl 10 mmol dNTP, 1 μl ribonucleotide inhibitor (Sigma), 2 μl reverse transcriptase (Sigma), add ddH 2 O to a reaction volume of 20 μl. The parameters of the PCR reaction were: warm bath at 42°C for 60 minutes, and warm bath at 72°C for 10 minutes. Primers AlE75-F (5'-CATTATGGWGTYTAYAGYTGTG-3') and AlE75-R (5' -AGHAGTTCATTCCAVCCTGCTC-3'), the above-mentioned primers are degenerate primers to obtain the conserved sequence of Lygus nuclei hormone receptor E75, the conserved sequence is shown in SEQ...
Embodiment 2
[0039] Embodiment 2: Construction of AlE75 gene prokaryotic expression vector
[0040] The method for constructing the prokaryotic expression vector containing the AlE75 gene T vector is as follows: the above sequenced correct fragment of Lygus lucidum nuclear hormone receptor E75 is connected to the target gene of the pMD18-T vector and the pCzn1 vector with the restriction endonuclease Ndel and Xbal double enzyme digestion, for large fragment ligation ( figure 2 ). The enzyme digestion system is: 0.25 μl of each of the two restriction endonucleases (Sigma Company), 1.0 μl of 10×Buffer buffer, 3 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 10 μl, and bathe in water at 37°C for 3h. The ligation system is: 5.0 μl of the recovered vector after enzyme digestion, 10.0 μl of the gene fragment, 1.0 μl of T4 DNA ligase (Sigma Company), 2.0 μl of 10×Buffer buffer, and use ddH 2 Make up O to 20μl, and react at 16°C for 12-16h. After the ligation ...
Embodiment 3
[0042] Example 3: Denaturation, renaturation and protein purification of AlE75 fusion protein
[0043] Denaturation and renaturation of inclusion body proteins were performed according to the following steps: resuspend the bacterial cell pellet in 20ml lysis buffer (Sigma Company, 20mM Tris-HCl containing 1mM PMSF and bacteria protease inhibitorcocktail, pH 8.0), sonicate (power 400W , working for 4 sec, intermittent for 8 sec, 20 min in total); centrifuge the sonicated cell lysate at 10,000 g for 20 min at 4°C to collect the precipitate; use inclusion body washing solution (20 mM Tris, 1 mM EDTA, 2M urea, 1M NaCl, 1% Triton X- 100, pH8.0) to wash the inclusion body 3 times; dissolve the inclusion body in a certain proportion with a dissolution buffer (20mM Tris, 5mMDTT, 8M urea pH8.0), and place it at 4 degrees overnight; room temperature, 15000rpm centrifugation for 15min; Add dropwise 20mM Tris-HCl 100mM NaCl Buffer pH8.0 (Sigma Company) buffer solution, gradually double th...
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