Expression Of Hypodermin A Gene In Pichia Pastoris Expression System

Hypodermin A (HA) is a kind of serine enzyme secreated by the first stage larva of warble fly during migration in the host. It can cause anti-inflammatory activity, delay immune response at primary infestation, and might be one of the vaccine that can be used to control Hypodermosis.An approach of optimized expression of Hypodermin A (HA) in Pichia Pastoris was established, and immunoassay was done by the use of purified protein. At first, HA gene and plasmid pPIC9k were combined together to construct the recombinant plasmid pPIC9k-HA. After electroporated into the GS115 cells, the recombinant plasmids were screened out by His" medium and G418. The protein was induced in the medium containing 5% methanol. The supernatant was collected on the 3rd day, and then purified by FPLC. SDS-PAGE indicated that the target protein is around 30KD. Western-blotting shows it can react with rabbit-anti HA serum. Gelatin substrate SDS-PAGE displayed its enzyme activity. PAS stain of the 50KD protein came out after 3 days, which indicated it's an over-glycosylated protein. The amino acid analyze showed that the amino acid content of 50KD protein is as same as that of the 30KD ones. The optimization experiments indicated that highest yield can be achieved by incubation in 0.5% methanol, 70% cubage at least, and for 56 hours. The maximal yield is around 1500mg/L.The rabbits were vaccined by the 30KD protein and the 50K.D protein appeared 3 days after inducement, the level of antibody reached the highest titer on the 3rd week, and came down slowly afterwards. The immunization effect of the 50KD protein is better than that of the 30KD one, and the difference is very obvious (P<0.01).The protective effect of the 50K.D protein as a vaccine on naive yaks is quiet good. The infection rate of groups vaccined separately on July and September is 83.3% and 36.3% (55.5% as control); and the average infection intensity of these 2 groups is 6.5 and 4.2 (13.0 as control), the difference is not obvious (P>0.05). The level of antibody reached the highest titer on the 3rd month post vaccination, and decreased slowly afterwards. The immune effect of group vaccined on September is better than that of the group vaccined on July, the difference of the antibody level of 3 groups is very obvious (P<0.01).This study facilitates further investigation on HA as a vaccine and the use of Pichia Pastoris in parasite domain.