Method for producing periplaneta americana allergen protein Per a 5 in baculovirus-insect expression system
An allergen protein, Periplaneta americana technology, applied in the field of genetic engineering, can solve problems such as poor curative effect, side effects, and hindering the detection of special allergens in patients
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Embodiment 1
[0020] 1. The steps of this embodiment are as follows:
[0021] (1) Total RNA was extracted from Periplaneta americana tissue; artificially raised and frozen Periplaneta americana (from Jiangsu Provincial Center for Disease Control) was taken, ground with liquid nitrogen, and Trizol reagent was added at 100 mg tissue / mL Trizol reagent. Let stand for 10 minutes, then add chloroform at 300ul / mL, mix well, let stand for 10min, centrifuge at 12000g for 10min, discard the supernatant, and add isopropanol at 500ul / mL. Mix well, let stand for 10 minutes, centrifuge at 12000g for 10 minutes, discard the supernatant, and the sediment at the bottom of the tube is the total RNA of Periplaneta americana. Add 75% ethanol to suspend the precipitate, centrifuge at 12000 g for 10 min, and discard the supernatant. Dry at room temperature for 5 min, add ribonuclease-free double distilled water to dissolve.
[0022] (2) Using the extracted total RNA of Periplaneta americana as a template, the ...
Embodiment 2
[0027] The specific IgE binding activity of the expressed Per a 5 protein is determined:
[0028] The specific IgE binding activity of the Per a 5 protein produced by the baculovirus-insect expression system and Escherichia coli (prepared for our laboratory) was measured by indirect ELISA method. The Per a 5 protein produced by the baculovirus-insect expression system is the purified Per a 5 protein after the embodiment one step (4); the source of the Per a 5 protein produced by Escherichia coli is the embodiment one step ( 2) The resulting Periplaneta americana allergen Per a 5 gene shown in SEQ ID No: 1 is cloned into the pCold II plasmid, transformed Escherichia coli Origami (DE3), induced and expressed overnight with 0.5mMIPTG, and the thalline is collected , supernatant was collected by centrifugation after sonication, and purified by Nickel affinity column.
[0029] Dilute the Per a 5 protein produced by the baculovirus-insect expression system and Escherichia coli to 1...
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