Bullfrog lectin gene, encoded lectin and uses thereof
A technology of lectin and bullfrog, applied in the field of genetic engineering, can solve problems such as toxicity and immunogenic side effects, and achieve the effects of inhibiting bacterial growth, convenient artificial synthesis and simple structure
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Embodiment 1
[0040] Bullfrog lectin gene clone:
[0041] I. Extraction of Total RNA from Bullfrog Skin
[0042] A. The live bullfrog was cleaned with double-distilled water, and the medullary cavity of the bullfrog was destroyed with a sterilized needle. 50-100 mg of bullfrog skin tissue was put into a dry-baked mortar, and 1 mL of TRIZOL Reagent (product of Invitrogen Company) was added to quickly Grind in an ice water bath.
[0043] B. After fully mixing, transfer to a 1.5mL centrifuge tube (DEPC tube) and place at 15-30°C for 5min, add an equal volume of chloroform, vortex mix for 15s, and centrifuge at 12000×g for 15min at 4°C.
[0044] C. Add 500 μL of isopropanol to the supernatant, place at 15-30°C for 5 minutes, vortex for 15 seconds to mix well, and centrifuge at 12,000×g for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to rinse the precipitate, centrifuge at 7500×g for 5 min, and repeat once. Dry in an ultra-clean workbench for 90 seconds, dissolve with ...
Embodiment 2
[0090] I. Sample preparation method: the amino acid sequence of bullfrog lectin was deduced according to the gene encoding bullfrog lectin, and its complete sequence was synthesized with an automatic polypeptide synthesizer APEX396. Desalted and purified by HPLC reverse phase C18 column chromatography.
[0091] II. Molecular weight was determined by electrospray ionization (ESI) with an emission voltage of 4.5Kv.
[0092] III. The purity of the purified bullfrog lectin was identified by high performance liquid chromatography HPLC (flow rate 1.0mL / min; mobile phase acetonitrile+water+TFA0.01%, gradient elution; chromatographic column C18, detection wavelength 220nm), molecular weight determination The isoelectric point was determined by fast atom bombardment mass spectrometry and isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.
[0093] Bullfrog lectin is a single-chain po...
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