Study On The Expression Of Recombinant Receptor Binding Subunit Gene Of The Extoxin A Of P.aeruginosa In E.coli And Its Immunogenecity

Pseudoinonas aeruginosa is the main pathogen caused infection after surgery and burn, and extoxin A is one of the principle pathogenic factor of PA. PEA consists of 3 functional domains. One of them, domain 1. the receptor-binding subunit plays an important role in the process of PA infection of target cells.For high-level expression of the receptor-binding subunit of PEA in E. coli, an expression plasmid pET-28(c)+B 10 was constructed by insertion of the subunit gene from the expression vector pET-20(b)+B10 into pET28(C) downstream of T7lac promoter. After transforming the expression plasmid into the expression hosts BL2I(DE3), BL2I(DE3) pLysS, the transformed hosts were induced with IPTG. By SDS-PAGE and Western blot analysis of the host protein, the expression of the subunit was detected, and it could account for 58% of the total host protein.The mice inoculated with the expressed PEA subunit 3 times at 0.5mg/mice each time were challenged with 3 LD50 of PEA at 0~ 7, 14, 21, 28, 35 days after the final inoculation. The mice were 100% protected during at 14 days, 50% protection at 2ldays and no protection at 28 and 35 days. The principle histopathological lesion was degeneration and necrosis in the liver and kidney cells.After purification the expressed subunit was emulsificated with Freund complete adjuvant and then inoculated rabbits 3 times with 14 day interval. By analysis of the anti-PEA antibody level in the rabbit serum by indirect ELISA, the highest antibody level was observed during 7-l4days after the final inoculation, and dropped gradually from 21 days after the last inoculation. No difference was observed before inoculation and 28 days after the final inoculation. Goat anti-PEA serum with 1:3200 ELISA titre was prepared by inoculating goat with the expressed subunit 8 times with 14 day interval, and harvested serum at 14 days after the final inoculation.