Study On Cloning, Expression Of OmpK Genes From Vibrio Spp. And Their Applications

Vibrio is an ubiquitous genus of Gram-negative bacteria in the marine environments. Many species of Vibrio, such as Vibrio harveyi, Vibrio parahaemolyticus and Vibrio alginolyticus are important pathogens of maricultural fishes. Recent studies have indicated that the outer membrane proteins (OMPs) of the pathogenic bacteria might function as protective antigens in host organisms, hence, as potential vaccine candidates, OMPs have received increasing atteition. Among those OMPs, OmpK(outer membrane protein K), a receptor for a broad-host-range vibriophage KVP40 , especially caused our attention due to its wide distribution among Vibrio spp. and luminescence bacteria.To investigate the possibility of OmpK as vaccine candidate, the primes were designed according to OmpK and GAPDH gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1, which is a vector used for protein surface display on Saccharomyces cerevisiae cells. The genes encoding OmpK and OmpK-GAPDH were amplified by PCR using the genomic DNA of V. harveyi and the plasmid pET-OmpK-GAPDH as templates. Both the PCR products were inserted into the plasmid pYD1. Then the constructs were transformed into the yeast strain EBY100. After the target genes in the transformants were induced with 2% galactose for 36 hours, the display of the OmpK and OmpK-GAPDH protein on the yeast surface were confirmed by immnofluorescence with fluorescence microscope with yeast cells containing no vector and empty vector as controls.The gene encoding OmpK of Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus was cloned into expression vector pQE-30. Then the constructs were transformed into E.coli M15 for expression of the recombinant OMPKs. The engineered E.coli strains were proved to be capable of overexpression of fusion proteins, induced by IPIG for 4 hours. All of the recombinant OMPKs were purified with nickel-chelated column. The expressed fusion protein were analyzed by SDS-PAGE of about 29 kDa,30 kDa,30 kDa were found。.The yeast cells displaying the OMPKs were administered to turbot(Scophthalmus maximus)by injection .The antibody in blood serum of fish was detected with the purified recombinant OMPK of Vibrio harveyi by indirect ELISA. The total antibody against the specific antigen of fish by indirect ELISA indicated the experimental group had the titer, but the average titer was low. As a conclusion, our study suggested that the live yeast displaying OMPK can induce a certain antibody to the fish.