Recombinant vector, strain, and expression and purification method of Rhizoctonia solani effector protein
A rice sheath blight bacteria and effector protein technology, applied in the biological field, can solve the problems of protein non-expression and insolubility
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Embodiment 1
[0029] This embodiment provides a recombinant vector and a bacterial strain containing the recombinant vector, and the recombinant vector and bacterial strain are used to express the rice sheath blight effector protein; wherein, the recombinant vector is obtained by introducing the rice sheath blight The gene encoding the effector protein is inserted between the EcoRI and XhoI restriction sites of the pET-32a vector. The map of the pET-32a vector is attached figure 1 As shown; the rice sheath blight effector protein is named AGLIP1, and its amino acid sequence is shown in the sequence listing SEQID NO:1; the nucleotide sequence of the gene encoding the rice sheath blight effector protein is as in the sequence listing Shown in SEQ ID NO:2.
[0030] In addition, the bacterial strain comprising the above-mentioned recombinant vector is obtained through the following steps:
[0031] (1) Take 50 μL of Escherichia coli BL21 competent cell suspension from the -80°C refrigerator, tha...
Embodiment 2
[0036] This embodiment provides a method for expression and purification of rice sheath blight effector protein, which comprises the following steps:
[0037] (1) Place 1 mL of the bacterial strain containing the recombinant vector provided in Example 1 above into 5 mL of LB liquid medium, add ampicillin antibiotic, and then place it at 37°C and 200 rpm for overnight shake flask activation culture to obtain cultured liquid.
[0038] (2) Add all the above-mentioned culture solution to 100mL LB liquid medium, add ampicillin, and then place it at 37°C and 200rpm for expanded culture until OD600=0.4, then add 100μL with a concentration of 0.1mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG), and placed under the conditions of 20°C and 140rpm to induce expression for 4h, to obtain the induced bacterial liquid.
[0039] (3) Put all the above-mentioned induced bacteria liquid into a 250mL centrifuge bottle, centrifuge at 10000rpm for 5min, discard the supernatant, resuspend the bact...
Embodiment 3
[0043] This embodiment provides a method for expression and purification of rice sheath blight effector protein, which comprises the following steps:
[0044] (1) Place 1 mL of the bacterial strain containing the recombinant vector provided in Example 1 above into 5 mL of LB liquid medium, add ampicillin antibiotic, and then place it at 37°C and 200 rpm for overnight shake flask activation culture to obtain cultured liquid.
[0045] (2) Add all the above-mentioned culture solution to 100mL LB liquid medium, add ampicillin, and then place it at 37°C and 200rpm for expanded culture until OD600=0.8, then add 100μL with a concentration of 0.2mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG), and induced expression under the conditions of 20-25°C and 140rpm for 4h to obtain the induced bacterial liquid.
[0046] (3) Put all the above-mentioned induced bacteria liquid into a 250mL centrifuge bottle, centrifuge at 10000rpm for 5min, discard the supernatant, resuspend the bacteria wi...
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