| Hypodermin C is a kind of serine enzyme secreated by the first stage larva of warble fly during migration in the host. It is highly antigenic and specific to Hypoderma.spp, so it is an ideal antigen for diagnoseing.An approach of expression and optimization of Hypodermin C (HC) in Pichia pastoris system was established, and identified the character of recombinant protein. At first, HC gene and plasmid pPIC9k were combined together to construct the recombinant plasmid pPIC9k-HC. After electroporated into the GS115 cells, the recombinant genes were screened out by His- medium and G418. The protein was induced in the medium contains methanol. The supernatant was collected on the 60 hours, and then purified by anion exchange. SDS-PAGE indicated that the target protein was around 28KD. Western-blotting showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed its enzyme activity. The optimization experiments indicated that highest yield could be gotten by 1.0% methanol, 0.25% glycerol, 6.0 pH value and 60 hours. The yield is around 121mg/L. The producton is more than the other expression system.Conditions were optimized for using these preparations for an HC specific ELISA. The result was shown that the optimal concentration of HC for coating of plate was 1μg/ml, blocked with 5% dry milk powder at 37℃for 3h, the dilution of serum sample was 1:50, the working concentration of HRP-labeled rabbit-anti-bovine IgG was 1:2000, serum sample for detecting and HRP-labeled rabbit-anti- bovine IgG should be incubated at 37℃for 2h and 1h respectively, the substrate for ELISA was incubated at 37℃for 10min before reading the OD450. Following the determination of conditions of ELISA, the cut-off value was 0.21. It showed that the purified protein had good antigenicity, the ELISA had good specificity for the detection of HC antibody in serum. The study above facilitates the further research on ELISA kit of hypodermosis.
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